Health, Medicine & Nanobio 1. Therapeutics ...........................................................................................2

1.1 Definition...........................................................................................2 1.2 Short Description..................................................................................2 1.3 State of R&D .......................................................................................2 1.4 Additional Demands for Research: ........................................................... 14 1.5 Applications and Perspectives ................................................................ 15

2. Sensors & Diagnostics............................................................................... 17 2.1 Definition......................................................................................... 17 2.2 Short Description................................................................................ 17 2.3 State of R&D ..................................................................................... 18 2.4 Additional Demand for Research ............................................................. 34 2.5 Applications and Perspectives ................................................................ 34

3. Regenerative Medicine ............................................................................. 36 3.1 Description ....................................................................................... 36 3.2 Short Description................................................................................ 36 3.3 State of R&D ..................................................................................... 36 3.4 Additional Demand for Research ............................................................. 45 3.5 Applications and Perspectives ................................................................ 46

4. Implants, Surgery & Coatings...................................................................... 47 4.1 Definition......................................................................................... 47 4.2 Short Description................................................................................ 47 4.3 State of R&D ..................................................................................... 47 4.4 Additional Demand for Research ............................................................. 58 4.5 Applications & Perspectives ................................................................... 59

5. Novel Bionanostructures ........................................................................... 60 5.1 Definition......................................................................................... 60 5.2 Short Description................................................................................ 60 5.3 State of R&D ..................................................................................... 61 5.4 Additional Demand for Research ............................................................. 70

6. Cosmetics............................................................................................. 71 6.1 Definition......................................................................................... 71 6.2 Short Description................................................................................ 71 6.3 State of R&D ..................................................................................... 72 6.4 Additional Demand for Research ............................................................. 78 6.5 Applications and Perspectives ................................................................ 78

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7. References ........................................................................................... 80

1. Therapeutics

Keywords: nano-emulsion, nano-gel, solid lipid nanoparticle, liposome, micelles, dendrimer, hyperbranched polymer, carbon nanotube, carbon nanohorn, nano-sponge, thin film, nanoshell

1.1 Definition

For the purpose of this report, therapeutics are defined as drugs and their associated delivery systems used for the treatment of disease.

1.2 Short Description

The pharmaceutical industry is currently facing a crisis. The high increase in development costs, competition from generic manufacturers, complex patent management systems and increased failure rates are putting immense pressure on the profitability and future survival of the leading companies. There is also a shift away from the “one size fits all” blockbuster drug model towards personalised medicine, which will severely impact on the revenue of therapies.

New tools and techniques enabled by nanotechnology can help in better understanding intracellular functions. However it is the targeting of drugs, which can reduce toxicity and improve efficiency, where nanotechnology can make a huge impact. Nanotechnology-based delivery systems can also protect drugs from degradation. These properties can help reduce the number of doses required, make treatment a better experience and reduce treatment expenses. A number of nano-based systems allow delivery of insoluble drugs, allowing the use of previously rejected drugs or drugs which are difficult to administer e.g. paclitaxel. At present these systems are generally used for existing, fully developed off-patent drugs, the so-called “low-hanging fruit” of nanotechnology-based delivery. However, the success of these paves the way for new drugs being delivered in the same way.

The market for nanotechnology enabled drug delivery is set to have a cumulative growth rate of 48% over the next four years to increase to US $20.1 billion by 2012, an 11% share of the global drug delivery market. In 2008, the nanotechnology-enabled drug delivery market is estimated at US $4.1 billion, or 4% of the global drug delivery market1.

1.3 State of R&D

Currently, there are few commercially available therapeutic products based on nanotechnology. A number are in clinical development and are expected to appear on the market. Many of these are based on existing, off-patent drugs (e.g. paclitaxel), therefore eliminating part of the costly drug development process.

1.3.1 Polymer therapeutics

Polymer therapeutics is an umbrella term to describe polymeric drugs, polymer-drug conjugates, polymer-protein conjugates, polymeric micelles to which drug is covalently bound, and multi-component polyplexes being developed as non-viral vectors2.

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One of the biggest advantages of using polymers in drug delivery is that it is possible to manipulate their properties (e.g. molecular weight, linkers etc.) to adapt to the drug delivery requirements. In addition, polymers are easy to scale up, provide high biocompatibility and increase the stability of volatile pharmaceutical agents. These particles and their conjugates can be used to deliver a higher concentration of pharmaceutical agents to a desired location with potential applications in antibiotics targeting and cancer therapy. Conjugating nanoparticles and other nanomaterials to polyethylene glycol (PEG), known as PEGylation, is used widely as it offers a number of advantages3. These include increased protein solubility and stability, reduced immunogenicity, prevention of clearance by the reticuloendothelial system (RES) and increased plasma half-life - leading to less frequent dosing.

1.3.1.1 Polymer-protein conjugates

Polymers conjugated with proteins can then be administered parenterally can increase protein solubility and stability. PEGylation has been used to treat several diseases including hepatitis B and C (PEG–IFNα 2a)4, acute lymphoblastic leukaemia (PEG–L-Asparaginase)5, neutropaenia associated with cancer chemotherapy (PEG–GCSF)6 and different cancers [PEG–glutaminase combined with a glutamine anti-metabolite 6-diazo-5-oxo-norleucine (DON]7.

1.3.1.2 Polymer-drug conjugates

Polymer drug conjugates can improve the targeting ability, reduce the associated toxicity and overcome drug resistance. Linking drugs to polymers limits the cellular uptake to endocrine route and enhances the EPR Effecti. Hydrophilic polymers can be conjugated with hydrophobic drugs to increase their solubility.

Duncan outlined the major features required for the design of polymer-drug conjugates8. She suggests that it should be non-toxic and non-immunogenic, must be stable during transport, the drug should be released at an optimum rate on arrival within tumour cells and be able to carry an adequate drug payload in relation to its potency.

Different types of polymer have been proposed for drug conjugation. Kumazawa et al.9 report the use of a dextran-linked camptothecin analogue to shrink tumours in mice. PEG has also been proposed as a suitable candidate to attach drugs to10. HPMA (N-(2-hydroxypropyl) methacrylamide) copolymer conjugates have also been used for targeted drug delivery in the inhibition of a number of tumour types11-17.

1.3.1.3 Polyketal nanoparticles

Polyketals are readily-synthesized, biocompatible, hydrophobic polymers with biodegradable ketal linkages in their backbone. They can form nanoparticles for encapsulating hydrophobic drugs or proteins. They can undergo acid-catalysed hydrolysis to release their therapeutic payload in acidic environments such as tumours, inflammatory tissues etc. There are no acidic byproducts produced during degradation, unlike other polymers (e.g. polyester), that can cause inflammation.

Nanoparticles of poly(1,4-phenyleneacetone dimethylene ketal) (PPADK) have been used therapeutically18. Applications include the treatment of acute lung diseases19, the delivery and release of superoxide dismutase (SOD), in vivo imaging of hydrogen peroxide and it’s detection in atherosclerotic plaques20.

i EPR Effect: Enhanced Permeability and Retention (EPR) is the ability of drug conjugates to circulate within the blood for several hours more than the conventional low molecular weight drugs which leaves the blood stream within minutes. The higher drug availability facilitates better passive tumour adsorption through the permeability offered by tumour blood vessel.

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1.3.1.4 Nanogels

Nanogels are cross-linked nanoscale particles made of flexible hydrophilic polymers. They are soluble in water and allow spontaneous loading of drugs in aqueous media. The nanogel collapses to form dense nanoparticles after adding the drug molecules. Nanogels possess large surface area, tuneable size and a network to allow incorporation of molecules. They have been used to incorporate drugs, DNA/RNA and inorganic molecules such as quantum dots.

Nanogel particles comprised of PEG and polyethylenimine (>100nm) have been used to cross the blood-brain barrier (BBB) and deliver oligonucleotides to the brain21. Nanogels have also been used for pH-dependant release of doxorubicin22 and incorporation of an insoluble small molecule anticancer drug, AQ1023.

To be used safely within the body they must be stable in blood and biodegrade sufficiently. For a recent review of synthetic methods and applications in nanogel-based drug delivery, see Oh et al.24.

1.3.1.5 Dendrimers & Hyperbranched Polymers

Dendrimers are unimolecular, monodisperse, micellar nanostructures with a well-defined, regularly branched symmetrical structure and a high density of functional end groups. They are robust, covalently fixed, 3D structures possessing both a solvent-filled interior core (nanoscale container) that can carry molecules, e.g. drugs, and a homogenous, defined, exterior surface functionality (nano-scaffold) that can be functionalised25. The first and most widely studied dendrimers are poly(amidoamine) (PAMAM) dendrimers.

Dendrimers can be created using a divergent method where the dendron originates from a central core and branches out. Alternatively, a convergent method where the dendrimers grow inwards to a focal point may be used. A review on dendrimers states that over one hundred compositionally different dendrimer families have been synthesized with over 1000 differentiated chemical surface modifications26. Recently alternative synthetic strategies have been utilised.

One of the advantages of dendrimers is that they are similar in size to many proteins and biomolecules like insulin, cytochrome C and haemoglobin. 2nd generation dendrimers have a width similar to that of DNA (2.4 nm), and 5th and 6th generation PAMAM dendrimers have similar widths to cellular lipid membranes (~5.5 nm).

The loading capacity of dendrimers can be manipulated by the addition of different guest molecules onto the surface of dendrimers. Diederich et al.27 have developed dendrimers modified to bind hydrophobic molecules to the core, called dendrophanes, and those ones which bind to the polar bioactive compounds called dendroclefts28. Dendrophanes made with a cyclophane core have shown excellent steroid carrying properties and water solublity.

Several studies have demonstrated the use of dendrimers for targeted drug delivery, examples include cisplatin29, nifedipine30, doxorubicin31 and methotrexate32. The size of the dendrimer, the type of functional groups and the pH of the medium all appear to play an important role in determining the amount of solubility. An antibody-dendrimer conjugate that targeted the prostate specific membrane antigen was reported for prostate cancer treatement33.

Dendrimers have also been used as for in vitro gene delivery as they can complex DNA34. Dendrimer-DNA complexes have been locally administered to eyes35, lungs36, heart37 and tumours38. Generally larger dendrimers are more stable and better at binding DNA. However, one study39 found that 2nd generation PAMAM dendrimers bind DNA better than 6th generation due to the more fluid structure of small dendrimers. Dendrimers can also accommodate and deliver large DNA constructs40,41.

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Dendrimers have also been effective against bacterial and viral infection. Dendrimers hybridised with chitosan have useful antibacterial properties as well as potentially acting as drug delivery agents42. The ability of dendrimers modified with sulfonated naphthyl groups to inhibit viral activity has been shown by Bourne et al.43. They found that modified PAMAM dendrimers were highly effective in vitro against herpes simplex virus types 1 and 2.

The Australian company Starpharma (www.starpharma.com) has produced Vivagel®, a dendrimer-based vaginal microbicide, for the prevention of STIs and also acts as a contraceptive. It effectively prevents transmission of HIV and HSV-2 with no inflammation or irritation44. Vivagel® is currently in clinical trials but has been granted Fast Track status by the FDA as a product for prevention of HIV infection, so is expected to enter the market in 2009-201045. Starpharma is also developing Vivagel® for the prevention of HPV and as a condom coating in collaboration with SSL International, manufacturers of Durex® condoms.

Hyperbranched polymers (HBPs) are essentially irregular dendrimers. They are imperfectly branched, have an average (rather than precise) number of terminal functional groups and are chemically polydisperse. Due to their irregularity they can be prepared in a single step, making them much more cost effective than dendrimers. The large numbers of external groups are suitable for multifunctionalisation, advantageous for use as a drug carrier. Some HBPs are biocompatible and biodegradable with non-toxic degradation products.

HBPs have been conjugated to ibuprofen for delivery46. The conjugates gave rapid suppression of PGE2 synthesis when localised in the cytosol compared with no activity using free ibuprofen. A novel class of HBP based on polyglycerol (PG) and PEG was synthesised and the blood compatibility of these polymers was assessed at various concentrations47. They had no significant effect on factors including complement activation, platelet activation, coagulation, erythrocyte aggregation and hemolysis compared to branched cationic polyethyleneimine (PEI). The polymers had high affinity for DNA and could condense it into highly compact, stable, water soluble nanoparticles in the range of 60–80 nm. The method offers potential applications in polymer therapeutics and could be used as a more biocompatible approach for high affinity DNA binding than a dendrimer based approach.

Dendrimers and HBPs have good potential as therapeutics as they are made from biocompatible materials and can have high drug loading. HBPS are cheaper to synthesise so may be more suited to these applications.

1.3.2 Lipids in Drug Delivery

Lipid-based structures and formulations have been used as delivery systems for many years. Liposomes and micelles are used in cosmetics (see ‘Cosmetics subsector report) and foods. Lipid nanostructures are capable of protecting their contents from the conditions within the body that could potentially cause degradation. They can be used to deliver insoluble drugs and targeting ligands can be attached.

1.3.2.1 Liposomes & Niosomes

Liposomes are vesicular structures with an aqueous core surrounded by a lipid bilayer. They were first prepared artificially in the mid 1960s48 and their first practical application in drug delivery emerged in the 1970s. They are normally created by the extrusion of phospholipids. Solutes, such as a drug in the core, cannot pass through the hydrophobic bilayer although hydrophobic molecules can be adsorbed into the bilayer, enabling the liposome to carry both hydrophilic and hydrophobic molecules. They have also been used in the cosmetic industry (see Cosmetics subsector report). The size of the liposomes can vary from 15 nm to several µm. Liposomes with nanometre sized cavities are also called nanoliposomes. Solutions with high or low pH (e.g. dissolved drugs in solution) can be carried via the liposomes as the drugs get diffused out at the targeted site when the pH is neutralised in the body.

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PEGylated liposomes that avoid clearance by the RES are known as “stealth liposomes”49. Liposomes exploit the leaky nature of tumour vesicles which allow particles of less than 400 nm to pass through. Early research in this area has shown that liposomes remain in the tumour interstitial fluid in close vicinity to tumour vessels50. Surface modification of liposomes with ligands like vitamins, antigens and antibodies for improved endocytosis by other cell types has also been proposed51.

A variety of biological and drug compounds have been delivered using liposomes. These include antibiotics, antioxidants (retinoids, carotenoids, tamoxifen, urate, glutathione etc.), vitamins (Vitamins A, C and E), haemoglobin, ATP, NSAIDs (indomethacin and naproxen) and genetic materials (plasmid DNA). Detailed lists of compounds that can be delivered via liposomes are mentioned elsewhere1. Several formulations including amphotericin B52 and daunorubicin53 have been successfully commercialised. Doxorubicin, a highly effective cancer drug with toxic side effects, has been encapsulated in liposomes and tested for treatment54. A good overview of the different methods of encapsulating doxorubicin into liposomes can be found in the review by Abraham et al.55.

Liposomes can also be modified to incorporate a magnetic element for use in monitoring their movement within the body using MRI. Such liposomes enhance the efficacy of their use by allowing more external control on its movement. De Cuyper et al.56 have shown that magnetoliposomes with nanometre sized magnetite cores, enwrapped by a bilayer of phospholipid molecules can be used as biocompatible MRI agents with potential applications in combined imaging and drug delivery.

Liposomes can be used to entrap gases and drugs for ultrasound-controlled drug delivery and release, reviewed in this reference57. This technique has also been used to entrap photosensitisers. Ultrasound can enhance the pharmacological activity of certain drugs, improve drug transport through tissues and cell membranes, and can create a hyperthermic condition that promotes the destruction of cancerous tissue1. However, techniques using perflutren-containing ultrasound contrast agents have been given a ‘black box’ warning by FDA after 4 patient deaths. A detailed study of the side effects of microbubble-based delivery is necessary for the success of this novel approach. It is also important to find the optimum lipid composition for prolonged circulation and stable gas retention, methods of conjugating ligands to liposomes without compromising their echogenicity.

Niosomes

Niosomes are non-ionic surfactant vesicles with a similar structure to liposomes. The name Niosome is a trademark of L’Oreal.

They can encapsulate aqueous solutes and act as drug carriers. Niosomes are formed by the self assembly of non-ionic amphiphiles in aqueous media. The application of heat or physical agitation helps the process to attain a closed bilayer structure58. Their uptake by organs such as the liver and spleen make niosomes best suited as drug delivery agents in diseases affecting these organs. They are also used in targeting cancer cells59. Since niosomal antigens are potent stimulators of the cellular and humoral immune responses they are also useful as adjuvants in vaccine delivery.

Niosomes for drug delivery have been reported since 1997. They are stable and retain their contents at low temperatures. Administration of methotrexate60, DOX61 and DOX N(2-hydroxypropylmethacrylamide) copolymer conjugate62 have been reported using niosomes. High levels of drugs were found in the target location when administered via niosomes compared to conventional routes. They have also been used with anti-inflammatory agents63 and anti-infective agents64. PEGylated cationic niosomes have been used for the cellular delivery of oligonucleotides65.

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Niosomes can enhance transdermal drug delivery. Vanhal et al.66 reported that niosome encapsulated drugs have been delivered through the stratum corneum which is normally considered as highly impermeable. Niosomes made from a novel surfactant (Bola surfactant), have been found highly effective for percutaneous drug delivery applications67. Studies have shown that they improve percutaneous passage of 5-fluorouracil (5-FU) through human stratum corneum and epidermis, and are non-toxic68. Niosomes of frusamide have been reported, that increased skin permeability and sustained drug levels69.

1.3.2.2 Micelles

Micelles are also spherical lipid nanostructures but they do not have a bilayer or inner cavity. The hydrophobic ends of the phospholipids point inwards and the hydrophilic ends face the outside, forming a spherical structure. Reverse micelles have this polarity the opposite way. The typical size of micelles for pharmaceutical applications ranges from 10- 80 nm.

Compared to liposomes, micelles have a short circulation time within the body due to their smaller size. However, this gives them the advantage of being able to enter tumour cells more easily, because of the EPR effect.

Micelles can also be made from polymers. Polymeric micelles are formed by block-copolymers consisting of hydrophilic (e.g. PEG) and hydrophobic monomer units with longer hydrophilic blocks and shorter hydrophobic blocks70. They have a hydrophobic core stabilised by hydrophilic units. These micelles are more stable than conventional micelles and are preferred for drug delivery applications as the circulation time is longer and they offer better biodistribution71.

Lipid-polymer conjugate micelles can also be made. They can carry different types of chemicals like paclitaxel72, diazepam73 and captothecin 74. They also exhibit good longevity and stability. Micelles with improved solubility and intracellular delivery have been prepared using PEG-phosphatidylethanolamine (PEG-PE) conjugates. In recent studies PEG-PE micelles were targeted to tumours in mice75 and to damaged heart cells in rabbits with myocardial infarction76. Micelles with shorter PEG have been found to be better and more efficient carriers of soluble drugs due to their high hydrophobic to hydrophilic phase ratio.

Conjugating polymer micelles with ligands has been used for efficient delivery. Micelles conjugated with transferrin can target cancer cells and deliver DNA77. Similarly folate residues attached to micelles have been used to deliver adriamycin to cancer cells78. Positively charged nanoparticles have been shown to enhance drug uptake by cancer cells. However, many of the PEG-PE micelles carry a net negative charge, so positively charged lipids have been attached to these micelles to enhance uptake.

Micelles of thermal or pH sensitive polymers have been prepared. pH-sensitive micelles have been proposed for oral delivery applications. Temperature dependent micelles are reported to have increased drug release capacity79. Ultrasound has been suggested as a non-invasive stimulus to micelles for triggering drug release80.

Micelles have also been proposed as contrast agents in diagnostic applications to be used along with drug delivery. Paramagnetic metals, such as gadolinium (Gd) or manganese (Mn), normally used in contrast agents can easily be incorporated into micelles for imaging applications. The advantage of such agents is enhanced target penetration due to the smaller size and easy movement to the target location. Chelated Gd in PEG-PE based micelles has been used for experimental percutaneous lymphography in rabbits by gamma-scintigraphy and MRI81.

1.3.2.3 Lipid Nanoparticles (SLNs, NLCs & lipid drug conjugates)

Solid Lipid Nanoparticles

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Solid lipid nanoparticles (SLNs) are particles of nanometre dimensions with a solid lipid matrix. They are oily droplets made from lipids which are solid at room temperature and stabilised by surfactants. The advantage of SLNs is that there is no need for organic solvents in the preparation, they provide protection from water and can be used for controlled drug release. They also have applications in cosmetics (see Cosmetics subsector report). Stealth and non-stealth SLNs have been used to deliver paclitaxel82. The drug accumulated up to 2.8 %, did not crystallise and was stable over time. Sustained release of doxorubicin has been reported using SLCs83.

Although SLNs are promising they suffer some drawbacks. Their loading capacity is low and there is a tendency to expel the contents during storage. These problems are caused by the tendency for the particle matrix to form a perfect crystal lattice when solid lipids are used. The high water content of SLN dispersions can also be problematic.

Nanostructured lipid carriers (NLCs)

In order to overcome some of the drawbacks of SLNs, a second generation of lipid particles have been developed by mixing solid lipids with liquid lipids. They are called nanostructured lipid carriers (NLCs). Compared to SLNs, NLCs usually have a distorted structure which makes the matrix structure imperfect, creating spaces to accommodate active compounds. They have been investigated for the topical delivery of drugs, including anti-fungals and non-steroidal anti-inflammatories and have been recently reviewed84. These structures also have applications in cosmetics and are discussed in the Cosmetics subsector report.

Lipid-drug conjugate nanoparticles

In order to overcome the limitations of SLNs, drug-lipid conjugates have been developed with an observed loading capacity of up to 33 % 85.

Mehnert et al.86 investigated the structure of lipid based nanoparticles and reported that SLNs and other nanostructured lipid carriers did not show any advantage with respect to incorporation rate compared to conventional nanoemulsions.

1.3.2.4 Nanoemulsions

Nanoemulsions are dispersions of nanoscale droplets of one liquid within another. There are a number of high and low-energy methods of formation87.

Nanoemulsions have a number of advantages over larger scale emulsions. They can be stabilised to increase the time before creaming occurs88. They are transparent or translucent, and have a larger surface area due to the small particle size. They are currently used in commercially available cosmetics (see Cosmetics subsector report) and have been used for imaging and delivery of poorly soluble drugs.

When used for drug delivery, nanoemulsions have significant advantages over traditional formulations for poorly soluble drugs. They eliminate the need for harmful co-solvents and can be administered at a much higher dose (approx. 10-fold), cutting down on the administration volume and time. The components of nanoemulsions are usually GRAS compounds (generally recognised as safe), therefore they are considered relatively safe systems which can break down to their safe components.

Cornerstone Pharmaceuticals (www.cornerstonepharma.com), have developed a stable oil water lipid nanoemulsion called Emulsiphan for tumour targeting. Their lead product, an Emulsiphan nanoemulsion containing paclitaxel (EmPAC), showed increased efficacy and uptake by tumour cells compared to Taxol® and is currently in clinical development.

Nanoemulsions allow transport through membranes so have been suggested for use in patches for administering drugs across the skin.

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1.3.2.5 Lipid nanocapsules (LNCs)

These systems can be thought of as a cross between liposomes and nanoemulsion particles. Their outer wall is thicker than a traditional nanoemulsion particle allowing functionalisation and more controlled delivery. LNCs are composed of a liquid, oily core (medium-chain triglycerides) surrounded by hydrophilic and lipophilic surfactants. They were patented in 2001 for therapeutic applications89 and an efficient, solvent free phase-inversion method for their preparation has been developed90. Stealth LNCs have also been synthesised using PEG to improve circulation time. LNCs have been used to deliver anticancer drugs91,92,93. LNCs have been used to deliver therapeutic molecules and radionuclides across the blood brain barrier by conjugation of antibodies or antibody fragments94,95,96.

1.3.3 Nanoparticles in Drug Delivery

The interest in using nanoparticles for drug delivery has increased at an exponential rate in the past few years. Nanoparticles can offer significant advantages over the traditional delivery mechanisms in terms of high stability, high specificity, high drug carrying capacity, ability for controlled release, possibility to use in different types of drug administration and the capability to transport both hydrophilic and hydrophobic molecules.

The drugs may be enclosed inside the sphere of the nanoparticle or linked to the surface. Once they are at the target site, the drug payload may be released from the nanoparticle by diffusion, swelling, erosion or degradation. Active systems are also possible, e.g. drug release in response to the input of external energy such as targeted ultrasound, light or magnetic field.

1.3.3.1 Protein nanoparticles

Albumin nanoparticles

The protein albumin has been modified to create novel nanostructures for applications in drug delivery. The surface of albumin has several groups available for covalent conjugation of biomolecules and drugs. Albumin-DNA–polyethylenimine (PEI) conjugates have been used for gene delivery, with reduced irritation, damage and toxicity97.

Albumin can also form nanoparticles which can be modified to alter size, polydispersity, surface charge, drug loading and release. Functionalised albumin nanoparticles have been shown to cross the BBB98. Similarly, bovine serum albumin nanoparticles loaded with sodium ferulate have been targeted to the liver99. These experiments showed that cross-linking with varying amounts of glutaraldehyde can alter the drug release rate.

The most advanced use of albumin nanoparticles has been Abraxane®, a solvent-free formulation of paclitaxel (Abraxis Biosciences Ltd.)100, which has been approved for breast cancer treatment101 in 36 countries across Europe, North America, Asia and Australia. The drug is conjugated to the nanoparticles (130 nm) and administered as a saline suspension at up to 10-fold higher concentration than previous formulations. It eliminates the solvent-derived side effects and the need for pre-medication against these, and shows reduced toxicity. The increased dose means there is a much lower administration time. There are a number of clinical trials planned to demonstrate the use of Abraxane® in other types of cancer102.

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Chitosan and Lectin nanoparticles

Chitosan is a natural linear polysaccharide derived from the shells of crustaceans. Chitosan has the ability to clot blood and is used in bandages and other haemostatic agents. Its derivatives such as trimethylchitosan are used in non-viral gene delivery. It has also been used for the production of nanoparticles by ionotropic gelation with tripolyphosphate103. Nanoparticles of chitosan and egg phosphatidylcholine (ePC) have been reported for the delivery of anticancer drug paclitaxel104. The chitosan-ePC structure was found to be highly stable and biocompatible with these properties dependent on the ratio of the two materials. The activity of this system was confirmed in mice105 but an accumulation of paclitaxel was seen in the heart which may cause complications106.

Lecithin is a lipid mixture of phospholipids mainly comprising phosphatidylcholine which is normally extracted from egg yolk or soy beans, and is widely used as a food additive. It is also used for liposome and micelle formation107. Chitosan is normally coated on the surface of lipid based nanostructures to improve the adhesive properties and increase stability.

Self-assembled spherical nanoparticles were formed by injection of an alcoholic lecithin solution into an aqueous chitosan solution108. The particles could encapsulate progesterone with an efficiency of ~60 %, however they had poor loading capabilities with hydrophilic drugs.

1.3.3.2 Gold nanoparticles

Colloidal gold nanoparticles have been used for a relatively long time for the treatment of diseases including cancer, rheumatoid arthritis, multiple sclerosis and neurodegenerative conditions such as Alzheimer’s disease. The advantages of gold nanoparticles are their ease of preparation in a range of sizes, good biocompatibility, easily functionalised and their ability to conjugate with other biomolecules without altering their biological properties109. They also have unique optical properties, making them suitable for various spectroscopic applications and as photo-thermal agents in hyperthermia. Gold nanoparticles with diameters ≤ 50 nm have been shown to cross the BBB110.

PEGylated gold nanoparticles conjugated with TNF (tumour necrosis factor) can enter tumour cells through their leaky vasculature. The treatment, called Aurimune™, is being developed by CytImmume Sciences Inc. (www.cytimmune.com) and is currently in clinical trials. Similar systems which also have a chemotherapeutic drug attached are being developed. Functionalised gold nanoparticles have been used for effective oral and intranasal insulin delivery in a rat diabetes model111.

1.3.3.3 Magnetic nanoparticles

Magnetic nanoparticles have become one of the most studied and applied nanotechnology in the past few years. Applications involving magnetic nanoparticles include targeted drug delivery, as contrast agents in MRI (e.g. Feridex), gene delivery and cell separation/cell labelling. Iron oxide nanoparticles are widely studied due to their biodegradable nature, biocompatibility and superparamagnetic properties suited for MRI applications112.

Magforce Nanotechnologies (www.magforce.de) are currently undertaking phase II clinical trials for a magnetic nanoparticle-based cancer therapy (Nano-Cancer® therapy). Iron oxide nanoparticles coated with aminosilane are injected into a tumour and an alternating magnetic field is applied. The particles oscillate and can produce a range of temperatures, from 41 C° to 70° C. At temperatures up to 46 °C (hyperthermia) the effect of radiation or chemotherapy is enhanced. At temperatures over 46 °C the tumour cells are destroyed (thermoablation). Current clinical trials are being undertaken for a number of cancers113.

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Magnetic nanoparticles that can be loaded with drugs and still retain their MRI properties have been reported114. The iron oxide nanoparticles were coated with oleic acid and loaded with anticancer agents doxorubicin and paclitaxel with a loading efficiency of up to 95%. Experiments in breast cancer cells showed high antiproliferative activity, greater sensitivity in T2 weighted imaging and a higher circulation half life than Feridex. The drug release can be enabled by inducing hypothermia through an applied magnetic field.

1.3.3.4 Ceramic nanoparticles

Nanoparticles of silica, titania, alumina etc. are normally classified under the heading ceramic nanoparticles. One of the advantages of these particles is that their preparation is very simple. They are unaffected by changes in pH or temperature. It is possible to manipulate many features of these nanoparticles, including size, shape, porosity, inertness etc., and they can easily be modified to attach different biomolecules. Their typical size is around 50 nm.

Ceramic nanoparticles have been used to encapsulate hydrophobic drug molecules115, the acid-labile model enzyme, serratiopeptidase116 and increase the transfection efficiency of DNA (used with a DNA-dendrimer conjugate)117.

1.3.3.5 Nanoshells

A nanoshell comprises a spherical core made from silica or other similar materials, surrounded by a coating a few nms thick. Typically the coatings comprise a metal such as gold or silver. One of the advantages of gold-coated nanoshells is that they possess plasmon resonance. This is the property of the electrons to oscillate collectively at a particular frequency when the incident light wave vector matches that of the electrons. By changing the thickness of the gold layers, the surface plasmon resonance can be tuned for various frequencies. This property has also been utilised in biosensing applications.

In cancer applications, antibodies or other biomolecules are attached to the gold surface to target it to the tumour site. By applying moderately low levels of near infra-red radiation in vitro, an appropriate dose of heat can be applied to kill the cancer cells (thermoablation). This technique has been used to kill prostate cancer cells118. The nanoshell-based heating mechanism can also be used for increasing tumour perfusion or to shut down perfusion completely for thermal ablative therapy. Recently, Zasadzinski et al.119 showed that hollow gold nanoshells thethered to liposomes achieved near complete liposome release using a near infra-red (NIR) pulsed laser.

1.3.3.6 Aptamer-nanoparticle conjugates

Aptamers are nucleic acid ligands (single-stranded DNA or RNA) with high affinity and specificity towards target receptors or molecules like phospholipids, sugars and proteins. They offer a number of advantages over antibodies, although many of these are also offered by other ligands e.g. peptides. Aptamers have been reported for many targets and can be used as inhibitors themselves. An aptamer-based therapeutic is available (known as pegaptanib or Macugen®) for the treatment of age-related macular degeneration (AMD). Others are also in clinical trials.

Recently, there has been increasing interest in using aptamer-nanoparticle conjugates for targeted drug delivery and other therapeutic applications. The first example of this was reported in 2004 using rhodamine-labelled dextran as a model drug120. RNA aptamers specific to the prostate-specific membrane antigen (PSMA), a protein over-expressed in prostate cancer epithelial cells, were attached to polymer nanoparticles. The conjugate delivered drugs selectively to cells expressing the marker. This system was extended to deliver the anti-cancer drug docetaxel in an in vivo model of prostate carcinoma and reduced tumour size effectively following a single intratumour injection121.

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Aptamer have also been conjugated to gold nanoparticles, liposomes, quantum dots and carbon nanotube field-effect transistors for delivery, imaging and sensing applications. A recent review has been published reviewing the use of aptamers122.

1.3.4 Nanosuspensions & Nanocrystals

Nanosuspensions are colloidal dispersions of nanoparticles of an insoluble molecule, which are stabilized by surfactants. Nanosuspensions can be used to maintain these drugs in a preferred crystalline state of sufficiently small size for intravenous administration123. Their advantages are similar to those of nanoemulsions. They can also achieve even higher levels of drug loading because the drug is in the solid state. Several studies have demonstrated the use of nanosuspensions for drug delivery with improved efficacy and release124,125. A good review of the recent developments in this field is available126.

Nanocrystals are aggregates comprising several hundred to tens of thousands of atoms that combine into a "cluster". Typical sizes of these aggregates are between 10-400 nm and they exhibit physical and chemical properties somewhere between that of bulk solids and molecules. By controlling the size and surface area, other properties such as bandgap, charge conductivity, crystalline structure and melting temperature can be altered. The crystals must be stabilised to prevent larger aggregates from forming.

Nanocrystals are produced by nanosonication. First, a nanosuspension is formed by high speed stirring, followed by wet milling, high pressure homogenisation, nanocrystallisation and spray drying to create nanosized crystals127. The advantages of nanocrystallisation are the ability to solubilise poorly soluble drugs, high bioavailability, major decrease in dosage volume (up to 4-fold), and an increase in tolerated dose (up to 10-fold for one cancer compound).

Elan Corporation (http://www.elan.com) have successfully commercialised and licensed their NanoCrystal® technology to produce nanocrystals of drug molecules that can be used in a number of dosage forms. Four products using this technology are currently available and another nine are in clinical development128.

Baxter have also developed a similar technology called Nanoedge (www.baxterbiopharmasolutions.com) to solve drug formulation problems by reducing the size of particles to 100 nm and coating them with an excipient to increase the solubility129.

1.3.5 Carbon Nanostructures in Drug Delivery

1.3.5.1 Carbon nanotubes

CNTs have the ability to transport drug molecules, proteins and nucleotides. Due to their size and shape, carbon nanotubes can enter living cells without causing cell death or obvious damage. Molecules can be covalently or non-covalently attached to the surface. The hollow structure of CNTs allows encapsulation of molecules but as yet there are very few examples of this for drug delivery130.

For biological applications CNTs require covalent131-137 or non-covalent138-146 functionalisation to prevent aggregation and increase their solubility. Several drugs have been successfully delivered, including amphotericin B147, which is normally insoluble and toxic due to its tendency to aggregate. When delivered using CNTs there was increased solubility, low aggregation (and therefore lower toxicity) and increased anti-fungal action.

A number of therapeutic applications of CNTs have been reported, including boron neutron capture therapy (BNCT)148, inducing immunoresponse149, gene150 and siRNA delivery151. A recent review details biological applications of CNTs152.

Recently a ‘smart bio nanotube’ was reported which has ends that can open and close through altering the charge153. This opens up possibilities for controlled encapsulation and release.

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Functionalisation needs to be efficient and cost effective. However, effective procedures for the production and/or purification to isolate CNTs of uniform size and shape are required before they can be used for medical purposes. There is debate about the toxicity of CNTs and much research is required in this area before medical applications can move from the lab to the clinic.

1.3.5.2 Carbon nanohorns

Carbon nanohorns have a structure similar to CNTs except they are closed at one end, forming a cone-shaped cap, or ‘horn’. They have a tendency to form spherical dahlia-flowerlike aggregates, roughly <100 nm. They have been used to deliver cisplatin154 and dexamethasone

155. Of the different carbon structures used in delivery, single walled nanohorns have been suggested as the most suitable due to their size, ease of synthesis (no metal catalyst required) and initial results showing that functionalised nanohorns are non-toxic to cells156.

1.3.5.3 Nanodiamonds

Diamond nanoparticles, or nanodiamonds have the capability for surface functionalisation. This has been used to immobilise proteins and deliver drug molecules157. Recently, nanodiamonds bound to doxorubicin were embedded into a polymer microfilm to achieve slow release of the drug over one month158. This system could potentially be used for tumour patches. Fluorescent nanodiamonds can enter cells, and may have applications in cell tracking and imaging. Currently, functionalised nanodiamonds are considered as biocompatible but very few studies have been conducted on this.

A recent review159 on carbon-based nanomaterials for cancer therapy highlights excellently the pros and cons of using such materials. In summary, all of the materials show promise as drug delivery systems. However, there are a number of toxicity issues, particularly with CNTs, that have not been fully investigated and remain an area of concern. There is currently a lot of research ongoing in this area but so far there have been no conclusive studies on the effects of CNTs160-164. Carbon nanohorns and nanodiamonds are relatively new discoveries and have even less safety data.

Due to these issues, at present carbon nanostructures do not seem ideal candidates for a general delivery system. Other systems, such as nanoemulsions, do not contain components with these toxicity issues and at present appear more likely to be accepted. This may change if conclusive safety data is produced and made available although it may prove difficult to predict the long-term effects of allowing these materials to enter the body.

1.3.6 Other Nanotechnologies for Drug Delivery

1.3.6.1 Cyclodextrin Nanosponges

Cyclodextrin nanosponges are complex networks of cross-linked cyclodextrins cross-linked and formed into a roughly spherical structure, about the size of a protein, with channels and pores inside. The surface charge density, porosity and pore sizes of sponges can be controlled to attach different molecules. Nanosponges have been used for removal of organic impurities in water165. Few studies have been conducted on their drug delivery capabilities, but in 2006 Trotta et al. showed that lipophilic and hydrophilic drugs can be solubilised and carried166. Researchers at Vanderbilt University are developing nanosponges attached to dendrimers for drug delivery167.

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1.3.6.2 Drug Carrying Implantable Thin films

These are nanoscale thin films that can be precisely controlled to release chemical agents by applying an electrostatic field. Hammond et al.168 reported the development of a thin film of approx. 150 nm thickness using a layer by layer approach. It is made up of the negatively charged material Prussian Blue and a positively charged drug molecule, or a positively charged molecule enclosing a drug. The pigment sandwiches the drug molecules and holds them in place. When a voltage is applied the pigment disintegrates and delivers the drugs. A very small voltage (in the range of 1.25 V) can be used to release multiple doses from one or more films in a single solution. By changing the voltage, the time for delivery and quantity of drug can be tuned.

The advantages of the layer by layer approach include ease of preparation, versatility, capability of incorporating high loading of biomolecules into films, fine control over the structure, and robustness of the products under ambient and physiological conditions169. The film can be implanted in the body and can carry discrete packets of drugs that can be released separately, which could be particularly useful for chemotherapy. This electrostatic system can be used to deliver drugs for a variety of diseases like diabetes, cancer, epilepsy etc. In addition, it has potential applications in tissue engineering, diagnostics and chemical detection. The films are easy to mass-produce using a variety of techniques, and can be directly applied or patterned onto surfaces irrespective of size, shape or chemical composition

Lynn et al.170 made thin films for the localized delivery of fDNA into cells. Multilayered polyelectrolyte films of thickness 100nm were created by alternative layer by layer deposition of plasmid DNA and a synthetic degradable cationic polymer. Quartz crystals coated with the thin films were put in contact with COS-7 cells. The introduction of quartz resulted in localised gene expression in cells under the film, without the aid of a secondary transfection agent. By incorporating plasmid DNA into thin films, it is possible to control the location and distribution of these molecules from implantable materials or other biodevices.

1.4 Additional Demands for Research:

Understanding the mechanism of nanostructure transport across cell membranes and the interactions between these novel structures and cells, particularly at the sub-cellular level.

Tumour targeting strategies other than EPR effect.

Development of self assembling polymers to create novel structures for drug encapsulation and delivery.

Development of novel structures that can release drugs in response to change (pH, temperature, enzyme interaction etc).

Development of coatings that can enable nanostructures to respond to change.

Novel triggers for the controlled release of drugs from nanostructures.

Development of implantable nanodevices for drug delivery.

Incorporation of nanosensors in implantable drug delivery devices.

Virus-like particles for gene delivery171.

Methods to improve the drug loading efficiency of nanostructures.

Methods to prevent the accumulation of drugs in organs when used with nanostructures for delivery (seen in many studies)172,173.

Development of new nanoformulations which can be administered orally.

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Development of new structures that can cross the blood brain barrier for treatment of CNS diseases.

Development of methods to cross specialized neural barriers (eye, ear, and peripheral nerves etc.).

Developing effective scalable processes to attach small drug molecules, proteins, peptides and genes inside and onto carbon nanotubes.

Manufacturing and scale up of nanostructures.

Studies on the fate of delivery systems and their degradation pathways (including environmental fate).

Toxicity studies (in vitro and in vivo) of the nanomaterials used in drug delivery.

Development of new risk management plan for nanotechnology based medical products.

1.5 Applications and Perspectives

The technologies reviewed in this document underline the importance of nanotechnology in therapeutics and the role played by nanotechnology in combating some of the chronic diseases, such as cancer. Areas in drug delivery where nanotechnology can make a difference include1:

developing systems that improve the solubility and bioavailability of hydrophobic drugs

designing delivery vehicles that can improve the circulatory presence of drugs

eliminating or minimising toxicity

increasing specificity

targeting drugs to specific cells or tissues

developing delivery systems for slow release

improving vaccine adjuvants and delivery

developing novel nanostructures that can be used in specific applications, e.g. ocular, cancer therapy, neurology, orthopaedics

Nanotechnology-based therapeutics have the potential to significantly impact on the pharmaceutical industry. Smaller companies can afford to develop novel formulations of off-patent drugs, but may not have the capital to invest in a costly and lengthy drug discovery and approval process. This may encourage more partnerships, licensing and co-development of products. Also, until relatively recently the pharmaceutical industry generated revenue from huge blockbuster drugs that sold many millions of units worldwide. With a trend towards personalised medicine emerging, in part enabled through nanotechnology, this huge market for a single product may no longer be there. Therefore, a new approach to the drug discovery and development process may be required. However, many novel nanotechnology-based delivery systems enable the delivery of insoluble drugs, opening up larger regions of ‘chemical space’ for exploration. Lead compounds previously rejected due to solubility issues may now be feasible and useful drug candidates.

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However, the novel properties and characteristics that bring new benefits in drug delivery also bring new challenges in risk management and toxicity. Some of these novel characteristics are poorly understood or studies. Presently, systems made from natural or GRAS components, such as liposomes or albumin nanoparticles, appear to have the most potential. This is both due to their minimal toxicity concerns and the precedent set by existing products. Other systems such as CNTs, while promising, raise a number of toxicity issues. Until these are addressed CNT-based delivery systems are not likely to appear as therapies.

In order to take nanotechnology-based medical products forward to the clinic, it is necessary to address the risk issues simultaneously, including any novel risks resulting from the nanoscale properties of the materials used.

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2. Sensors & Diagnostics Keywords: sensor, magnetic nanoparticle, nanowire, nanoshell, carbon nanotube, cantilever, Surface Enhanced Raman Scattering, lab-on-a-chip, theranostics, dendrimer, microscopy

2.1 Definition

For the purpose of this report, diagnostics applies to nanomaterials and tools for measuring nanoscale phenomena both in vivo and in vitro.

2.2 Short Description

Advances in nanotechnology have significantly improved the ability to detect and diagnose disease. The technology has facilitated the development of small, highly sensitive, inexpensive devices for the manipulation and analysis of cells. Improvements in existing techniques due to novel nanomaterials and surface modification methods have enabled us to see and analyse intracellular operations in real time. They are also being used to develop parallel assays which can detect various analytes simultaneously. More detailed and in-depth analysis of cellular functions is helping scientists and researchers to detect diseases at a very early stage and find suitable cures. Quantum dots for imaging is one of the most advanced applications of nanotechnology. They also have applications in many areas including biomarking, genomic sensing, multiplexed arrays, nanobarcodes, immunoassays and live cell imaging. Carbon nanotube-based glucose sensors which change infrared fluorescence according to the glucose level have been proposed. Gold nanoparticles have been used for single nucleotide polymorphism (SNP) genotyping and nanowire field-effect transistors (FETs) are used for the real time electrical detection of single virus particles.

This report reviews some of the important developments in in vitro and in vivo diagnostics and how nanotechnology enables scientists to understand the cellular functions in a better fashion.

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2.3 State of R&D

2.3.1 Nanosensors

Nanotechnology plays an important role in the development of highly sensitive sensors, particularly in biosensing applications. Nanosensors are sensors of nanoscale size with high sensitivity. They have found applications in different areas such as medical diagnosis, homeland security, agrifood, construction and transport. Nanoscale sensors have a number of advantages and potential advantages over conventional sensors such as their small size, low or zero power consumption, better selectivity and low weight. Nanosensors with a high surface area to volume ratio can be very sensitive, with a larger signal per volume of sensor. The minimally invasive nature of nanosensors is desirable in diagnostics applications.

Different types of nanomaterials and nanostructures are being used to improve the sensitivity of biosensors. Fullerenes and carbon nanotubes are seen as promising for electrochemical mediators and enzyme stabilizers for glucose biosensors174. Different sensors which utilise nanotechnology are discussed below.

2.3.1.1 Nanowire sensors

Nanowires are conducting or semiconducting nanofibres175. The basic principle of a nanowire sensor is that binding of a macromolecule onto the surface of the wire changes its conductivity. This change in conductivity can be measured and used to identify the molecule. The output from a nanowire sensor can be measured optically (i.e. using fluorescence based assays) or electrically by configuring them as field-effect transistors176. Electrical output sensors eliminate the need to label target molecules. These sensors are very small and offer potential for integration into hand held devices or other electronic devices to process the data. They can be combined into arrays for multiple molecule sensing.

Silicon and carbon nanowires are typically used for these sensors and boron-doped silicon nanowires for electrical biosensors were reported as early as 2001177. Sensors using semiconducting nanowires have been reported that can detect single virus particles at ultra-low concentrations178. Methodology to mass manufacture these devices using similar processes to the semiconductor industry has been proposed. Recently, sensing using conducting polymer (CP) nanowires has been reported179,180. These are easier to synthesise and functionalise than silicon or carbon nanowires.

Detection of different biomolecules has been reported, including antibodies181, proteins, RNA, DNA182 and viruses183. DNA detection has significant applications in genetic screening, bioterrorism and basic biological research. Vista Therapeutics are developing a universal nanowire array chip with applications in medicine and monitoring of bio-terrorism agents (http://www.vistatherapeutics.org/).

2.3.1.2 Carbon Nanotube sensors

Carbon nanotubes (CNTs) are of interest for sensing due to their unusual electrical, mechanical and thermal properties. Both single-walled (SWCNTs) and multi-walled CNTs (MWCNTs) are used for sensing184.

They can be used in applications which need detection within a short time span. Certain microorganisms whose life span is very short become ineffective within a couple of minutes after exposure to contaminants. To discover drugs against such organisms, it is important to understand their behaviours and properties, and nanotube sensors are of great use in such situations. Optical or electronic detection methods can be used and CNTs can also be configured as field effect transistors (FETs).

Dai et al.185 have shown that carbon nanotubes can be used as glucose sensors. They used aligned carbon nanotubes produced from FePc (iron(II) phthalocyanine) to make novel conducting polymer-carbon nanotube (CP-NT) coaxial nanowires.

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Jeykumari and Narayanan186 report the development of a glucose biosensor using MWCNTs. They developed a sensor by functionalising MWCNTs with neutral red (NR) and used Nafion (Nf) as a binder with glucose oxidase (GOx) acting as a biocatalyst. The formed nanobiocomposite MWCNT–NR–GOx–Nafion showed superior performance in the selective detection of glucose from different biological fluids. The enzymatic reaction of glucose oxidase upon glucose with NR functionalized nanotubes liberated hydrogen peroxide and led to the selective detection of glucose. The sensor benefits from a large detection range varying from 1 x 10-8 to 1 x 10-3 M with a detection limit of 3 x 10-9 glucose. The response time was less than 4 seconds and it displayed good stability as well as anti-interferent ability.

Sotiropoulou et al.187 have shown that aligned MWCNTs can be used to develop amperometric biosensors. They have shown that carbon nanotubes are promising materials as electrochemical mediators and enzyme stabilizers.

The Stoddart group have developed a sensor for a number of organic molecules using an FET made from pyrene-modified β-cyclodextrin functionalised SWCNTs. The cyclodextrin complex can recognise and complex a number of molecules in solution, enabling them to be detected. NTFETs can be tuned for sensing of breath components188. A device using this technology has superior performance capabilities in detecting asthma compared to existing methods. The device showed higher sensitivity, the ability to measure in real time and can be incorporated into handheld devices, a step towards personalised medicine.

A novel CNT nanosensor has been reported that exploits the fact that CNTs can glow when exposed to light189. Single strands of DNA were attached to the sensor and when a complimentary DNA strand was bound, the sensor changed colour. More than 50 different DNA sequences were screened at once by tuning the colours emitted by the CNTs190.

A large number of chemical and biological sensors using CNTs for applications, including glucose sensing, cholesterol monitoring191 and antigen antibody interactions192 are reported. Such a high interest in the use of CNTs as biosensors indicates their importance and potential.

2.3.1.3 Carbon Nanofibre sensors

Carbon nanofibres have similar properties to carbon nanotubes, but with a slightly larger diameter. One advantage of nanofibres over nanotubes is that they can be grown on a silicon substrate in the exact structure desired. The application of nanofibres in biosensing was first reported in 1996193. Since then several techniques have been reported for sensing single molecules.

Recently, a nanofibre based sensor that can detect trace amounts of 25 different microorganisms simultaneously within minutes has been developed. The technology, developed by NASA, is being commercialised by a company called Early Warning (http://www.earlywarninginc.com). The first product is primarily aimed at detecting micro-organisms associated with waterborne illnesses. The sensor they have developed can be incorporated on a lab on a chip device for remote use, with potential applications in military and medical diagnosis194.

Optical nanofibres can be used as bioprobes. These fibres have applications in near field optical microscopy. These technical advances have been used in areas like single molecular detection and single dye labelled DNA molecules195. Carbon nanofibre arrays functionalised with silver/silver chloride core-shell nanoparticles have been utilised as dry electrophysiological sensors for bio-potential monitoring applications196. The presence of nanoparticles on the fibre surface enhanced transduction in the ionic media and enabled accurate measurement of slow changing bio-potentials.

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2.3.1.4 Nanoshells/Nanoparticle sensors

Nanoshells are hollow nanoparticles that have been used for imaging and drug delivery. However, recent research has shown that they can be used to detect biological analytes. Nanoshells with a non-conducting core and metal shell are considered as the most suitable particles for sensing applications. The sensing technology is built on the principle that the ratio of the thickness (i.e. radius) of the core to the thickness of the metal shell is determinative of the wavelength of maximum absorbance of the particle. By controlling the relative core and shell thicknesses, biosensing metal nanoshells are fabricated which absorb light at any desired wavelength across the ultraviolet to infrared range of the electromagnetic spectrum197. The particles will emit signals characteristic to the analytes and the subject is identified. Metal nanoparticles can also be used in electrochemical labelling without affecting the function of these molecules.

A study comparing molecular sensors found that using gold nanoshells rather than nanoparticles increased sensitivity198. Colloidal gold nanoparticles can also be used as fluorescence quenchers in optical biosensors199. Oligonucleotide molecules were attached to the gold nanoparticles and this attachment caused the hybrid structure to assemble in an arch-like fashion on the nanoparticle surface. The binding of a target molecule resulted in a change in conformation. The probe’s background fluorescence increased in proportion to the change in temperature.

2.3.1.5 Magnetic nanoparticles

Magnetic nanoparticles have applications in many areas of medicine. They are used in targeted drug delivery, as MRI contrast agents, gene delivery and cell separation/cell labelling.

The use of superparamagnetic particles with a SQUID microscope (superconducting quantum interference device) for the rapid detection of biological targets has been reported200. The target is bound to a mylar film and a suspension of magnetic nanoparticles coated with antibodies to the target was added. Magnetic field pulses were applied and the relaxation of the particles studied after the pulse. The unbound nanoparticles relaxed quickly without producing a signal while the nanoparticles that bound itself into the target underwent Neel relaxation. This relaxation produces a slow decaying magnetic field which was detected by SQUID. The technique allows the selective detection of bound and unbound magnetic labels and a method for developing homogeneous assays. It eliminates the need for washing and requires only a small sample volume.

A magnetic nanoparticle dye to detect breast cancer, developed by University College London and the University of Houston, is being commercialised by Endomagnetics Inc. (www.endomagnetics.com/). The magnetic nanoparticle dye is injected into the tumour, which then naturally drains into the lymphatic system. After about 30 minutes, some of the dye accumulates in the sentinel node. A handheld probe device called SentiMAG™ is used to locate this node, both before and after surgical incision and can give information about the location of the node, its direction and proximity.

Semiconductor nanoparticles have also been used in biosensing applications. Curri et al.201 demonstrate that nanocrystalline CdS increased the efficiency of photochemical reactions. They have also shown that it is possible to couple these particles with biomolecules such as enzymes to develop novel photoelectrochemical systems.

2.3.1.6 Biomimetic sensors

Biomimetics is the concept of mimicking natural systems to develop novel structures. Techniques based on molecular self assembly are widely used to develop novel biomimetic structures. Self assembly is the process of assembling components to create a new level of organization without an external input202. Recent research shows that these self assembled structures can be used for sensing applications.

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Prashar et al.203 report the development of a novel lipid membrane that can remain stable at room temperature for three months. The membrane has a gate ion channel and when a target molecule binds to the surface, it results in a measurable change in the flow of ions through the channel. The technology based on this membrane is now being commercialised by Ambri Biosensors (www.ambri.com) under the name Ion Channel Switch (ICS™). The technology can be incorporated into handheld devices and can reduce diagnosis time from hours to minutes.

2.3.1.7 Viral Nanosensors

Virus particles can be considered as biological nanoparticles. In a viral induced self assembly, magnetic and optical properties of nanoparticles get distorted. A sensor capable of detecting up to 5 virus particles in low concentrations was developed to exploit this204.

The sensor was created by coating iron oxide nanoparticles with dextran and coupling with a linker (for virus attachment). When added to the magnetic nanosensor in serum, the virus particles (~100 nm) were observed, along with magnetic nanoparticles (46 nm). After 30 min viral particles attached to the nanosensors created a nanoassembly sized 550 nm. The effect of the nanoassembly on MRI T2 relaxation times revealed the number of virus particles in the solution. It detected 50 viral particles in 100 µl and up to 5 particles in 10 µl.

A novel method of detecting Dengue virus using nanoscale optofluidic sensor arrays (NOSA) has been reported205. The technology, which has optofluidic architecture, is based on the principle that a positive binding between a surface bound molecule and its solution phase target will create a change in refractive index which will shift the resonant wavelength.

2.3.1.8 Pebble Nanosensor (Probe Encapsulated By Biologically Localised Embedding)

PEBBLEs are nanometre-sized optical sensors trapped in an inert matrix206. They can be inserted non-invasively into living cells to monitor small analytes like H+, Ca2+, Mg2+ 207, Zn2+ 208, O2, K+, Na+, Cl-, OH- and glucose209. PEBBLEs for detecting reactive oxygen species in bovine oviducts have also been reported210.

They typically have two fluorescent dyes; one sensitive to the analyte and the other which acts as a reference. The semi-permeable and transparent nature of the matrix allows the analyte to interact with the indicator dye, which reports this interaction via a change in emitted fluorescence211.

The advantage of these devices is the minimal distortion to cells, even when a large number are inserted. The response time is also shortened so they can be used for real time cell imaging. The presence of the matrix protects the cell from toxic dyes and protects the dyes the cellular environment. The matrix allows ions or neutral analyte species to diffuse through and bind with the indicator, but precludes the diffusion of the indicator dyes, thus avoiding sequestration and self-quenching212.

2.3.1.9 Optical Biosensors

Fibre optics has found applications in high resolution molecular imaging. Tapered optical fibres coated with metallic films can be used for high resolution imaging of biomolecules213. These fibres can confine excitation light to sub-wavelength linear dimensions and cubic nanometer excitation volumes to probe highly localised and specific cellular functions and obtain high resolution, low noise signals. Surface enhanced Raman scattering (SERS) probes of 30-80 nm were made by immobilising silver nanoparticle colloids on these fibres and were capable of detecting molecules at picomolar solutions. The technique has been used to detect intracellular proteins of the interleukin-5 system in cellular environments using SERS and quantum dots.

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A recent review on optical biosensors214 highlights that the smallest molecule to be non-destructively probed with a fibre-optic nanobiosensor is the carcinogen benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon215. The detection of BaP transport inside single cells is useful for monitoring exposure which can lead to DNA damage. The advantage of a fibre optic nanosensor is that photons can only travel the length of the fibre used. After that only evanescent fields continue to travel, and then only a very short distance, providing excitation for only the fluorescent species of interest adjacent to the biosensing layer. The advantage is that only species in extreme close proximity to the fibre's nanoprobe will get excited.

2.3.1.10 Immunosensors

An immunosensor is a device comprising an antigen or antibody species coupled to a signal transducer, which detects binding of the complementary species216. By monitoring the antigen-antibody interactions in real time, these sensors are capable of detecting different types of molecules at varying ranges. Several different approaches have been proposed to improve the sensitivity and capability of these sensors.

Lin et al.217 propose the development of a particle-based renewable electrochemical magnetic immunosensor using magnetic beads and gold nanoparticle labels. The sensor has a magnet fixed inside which attach magnetic particles to the renewable carbon paste transducer. Gold nanoparticles were encapsulated on to the surface of the magnetic particles which are modified by anti-immunoglobulin G antibodies by sandwich immunoassay. Electrochemical stripping analysis is conducted and the stripping signals of gold nanoparticles are monitored to measure the concentration of immunoglobulin G in the solution. The detection limit of 0.02 µg ml-1 of IgG was obtained under optimum experimental conditions. The technique has the potential to be developed further for the simultaneous detection of multiple proteins using multiple nanoparticles in a parallel assay.

Carbon nanotubes have been used in immunosensors for highly sensitive detection218. It has been suggested that the technology is not advanced enough to be called nanosensors but should be called ‘immuno-electrodes’. One of the main issues faced by these nanosensors is the difficulty in fabrication. However, the fabrication of a carbon nanotube based FET immunosensor through patterned catalyst growth techniques combined with conventional photolithographic methods has been reported219. This type of device can give improvements in sensitivity when the Schottky contact area is increased. The detection limit can be improved by as much as 104 times compared to conventional devices. Protein adsorptions were increased and specific protein-protein interactions were detected at 1 pM concentrations220.

2.3.1.11 Enzymatic sensors

The properties of carbon nanotubes, including large surface area, electrical properties and electrocatalytic effects, make them ideal candidates for enzymatic sensors221.

Carbon nanotubes have been reported for glucose biosensing222. A carbon nanotube paste (CNTP) electrode and carbon nanotube paste/glucose oxidase (CNTP/GOx) electrode have been used to develop electrochemiluminescent sensors for detecting glucose. Compared to the carbon paste sensors, the CNTP sensors offer increased sensitivity due to the electrocatalytic activity of the carbon nanotubes and the specificity of the enzymatic reaction.

Several approaches to increase the sensitivity and robustness of CNTs have been proposed. These include the incorporation of nanocrystals in the CNT-paste matrix and the development of nanosensors in epoxy matrix223. Other approaches like coating multiple layers of enzymes layer by layer on to the nanotubes have also been proposed224.

Quantum dots have also been reported as electron mediators for glucose oxidase225.

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2.3.1.12 Genosensors

Nanomaterials can be used for DNA sensing applications. One of the earliest findings was the use of CNTs for the label free detection of DNA226. Further research has shown that MW or bamboo like MWCNTs are better at sensing than SWCNTs227. CNTs have been reported for monitoring the efficiency of anti-tumour drugs in leukaemia K562 cells228. An array of SiO2-insulated nanotubes has been used for ultrasensitive DNA detection229. The addition of platinum nanoparticles to MWCNTs was found to increase the detection limits230. Colloidal gold (diameter ~16 nm) has been used to enhance the amount of immobilised DNA in gold electrodes, resulting in the easy attachment of oligonucleotides and improved the nucleic acid detection capability 231. Gold nanoparticles have been used for SNP genotyping without the need for target amplification232. They have also been conjugated with thiol modified oligonucleotide probes to distinguish sequences with a single-base mismatch233.

The development of hybridisation assays which can probe multiple DNAs simultaneously has received increased attention. Hybridisation probes which can simultaneously detect five metals with a minimum peak overlap have been developed234. Quantum dots of ZnS, CdS and PbS were used as encoding nanoparticles, providing distinctive signals related to the corresponding DNA targets. Stripping voltammetry, a highly powerful electroanalytical technique for trace metal measurements, has been used in the development of a nanoparticle based bioassay. Future developments in this area include the use of nanotubes as load carriers to increase the detection limits, the addition of more quantum dots to scale up genosensing and the tailoring of these sensors for other types of electrochemical detection methods like immunoassay.

2.3.1.13 Cantilever Sensors

A nanometre scale cantilever can be used as a very sensitive, simple and cheap biochemical sensor in ambient and aqueous environments. The basic principle is that bending of the cantilever is an indicator of the biochemical reaction happening at the cantilever surface. Different types of biochemical reaction and protein detection can be carried out using micrometer sized cantilevers. Nanosized ones give high sensitivity detection. Advantageously, the molecules that are being detected usually do not require any labelling.

An array of nanocantilevers has been used to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations, within minutes235. Cantilever arrays can detect femtomoles of DNA using multiple binding assays in parallel and the nanomechanical motion depends on the concentration of DNA molecules in the solution.

Cantilever based sensors with integrated read-out have been reported236. The detection technique involves no labelling of the molecules and avoids bulky detection schemes like laser scans, CCD imaging or radiography. The technology has the capability to integrate arrays of cantilevers in small channels for maximum sensitivity. Cantilever sensors have been made from the polymer SU-8, which is negative resist with excellent mechanical, thermal and chemical properties. The advantage of using polymers is that it makes the fabrication process simple, flexible and cheap. They can also be tuned to increase sensitivity. Silicon based nanocantilevers with integrated capacitive read-out and electrostatic actuations have also been proposed for mass detection. One of the biggest advantages of cantilever based technologies is that it is compatible with silicon technology and can be integrated into hand held devices. Researchers at the London Centre for Nanotechnology have recently been awarded a £2 million EPSRC Nanotechnology for Healthcare grant to develop a nanocantilever array device for the rapid diagnosis of HIV237.

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Quartz crystal microbalance (QCM) sensors work on the principle of piezoelectricity. A QCM comprises a thin quartz crystal disk with electrodes plated on either side. The application of an external electrical potential across the device causes the piezoelectric crystal to vibrate at its resonant frequency. This resonant frequency is sensitive to mass changes (and other factors) of the crystal and its electrodes and have been used in detection for many years, reviewed in these references238,239. QCMs have been used for DNA sensing, particularly for detection of E. coli O157:H7. However, these sensors currently require PCR amplification. Ideally, this will be eliminated which would shorten the detection time and equipment required.

Nanotechnology has been used to improve the sensitivity and detection limits of these DNA sensors, with the eventual aim of completely eliminating the need for pre-detection PCR amplification. A sensor with nanosilver-coated electrodes improved detection sensitivity more than 3-fold when used to detect E. coli in drinking water240. Nanoparticles can be used with QCM detection as ‘mass enhancers’ to enhance the change in frequency, therefore increasing sensitivity. Streptavidin-conjugated Fe3O4 nanoparticles have been used in this way to detect E. coli241. Gold nanoparticles have also been used to detect oligonucleotides242, single base

mismatches243 and Staphylococcus epidermidis244.

2.3.1.15 Nanobarcodes

Nanobarcodes are metallic barcodes prepared by the electrochemical deposition of sub micron level particles in a striped format. The stripes can be manufactured from different types of metals with different widths and composition to make a large number of unique codes on a single strip. The different reflectivities of adjacent metal strips are then read using optical microscopes and other microscopic techniques. These barcodes can be used for multiplex biological assays and only need a small amount of sample.

A nanobarcode for multiplexed single nucleotide polymorphism (SNP) genotyping was made by electroplating inert metals (gold or silver) onto templates and subsequently releasing them to create striped nanoparticles245. These particle encoded submicron metallic nanowires in striped format form the nanobarcodes. These nanowires are then conjugated to labelled probes (single stranded DNA). When the target meets a complementary DNA strand, the corresponding nanowire exhibits fluorescence. The system provides highly accurate data and the design offers the flexibility to introduce different SNPs to the existing master mix, something not as straightforward in other array systems. The possibility to generate 4169 unique barcodes with a strip of just thirteen particles makes nanobarcodes ideal for multiplexing.

Magnetic microbeads packed with nanocrystal quantum dots at controlled ratios to have been used to develop highly multiplexed nanobarcodes that can encode a flexible panel of genes246. Four colours of quantum dots with different emission wavelength to were used to create a combination possibility of 20736 unique barcodes. The nanobarcodes were formed by incorporating different quantum dots into a polymer. Each nanobarcode was then conjugated with an oligonucleotide probe. Compared with Affymetrix Genechip® microarray, a widely used gene probing system, the nanobarcode system offered improved hybridization speed, sensitivity, flexibility and a reduction in sample amount. The technology also has the potential to provide new labelling tools for small and efficient diagnostic devices for quick analysis of radiation and microgravity induced diseases in space conditions.

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2.3.1.16 Key Challenges

In addition to some of the applications mentioned earlier in this report, nanobiosensors have found various applications in new areas like stent monitoring for plaque build up or artery thickening, as a fibrillation detector in smart catheters, dynamic dose control or micro delivery in radiation therapy, drug delivery and in post operative patient monitoring. However, the sector faces some major challenges, primarily the difficulty in integrating these novel sensors into devices. In order to maximise the sensitivity, arrays of sensors need to be printed on devices. Creating such nanoarrays and manufacturing them on a mass scale is considered as a big challenge.

Recently developed methodology that replicates semiconductor industry practices looks promising in addressing this issue and helping to create sensors that support device integration into signal processing and information systems247. The complementary metal oxide semiconductor (CMOS) technology was used to develop nanowire sensors that allow label free detection at femtomolar concentrations from commercially available silicon wafers. This compatibility offers the ability to fabricate thousands of sensors in arrays for high sensitivity.

Other challenges in biosensing include making the sensor usable in situations outside the laboratory. Additionally, biosensing applications require the precision flow control which will progress only with advances in nanofluidics. Similar to other new technologies, novel biosensing technologies rely on new software and processors to simulate the nanoscale reactions. There are also significant challenges in transferring this technology into commercially viable products due to the high costs of nanomaterials.

2.3.2 Molecular Imaging

The molecular imaging sector has grown significantly in the past few years due to the advances in nanoscience and nanotechnology. Molecular imaging is defined as the non-invasive, quantitative, and repetitive imaging of biomolecules and biological processes in living organisms248. The ability to create novel nanoparticles of varying size has made significant changes in the way molecular imaging is conducted. Some of these new technologies have been commercialised and the trend is towards developing personalised medicines and point of care devices. Some important developments in molecular imaging supported by nanotechnology are discussed in this section.

2.3.2.1 Nanoparticles

The development of targeted contrast agents such as fluorescent probes have made it possible to selectively view specific biological events and processes with improved detection limits, imaging modalities and engineered biomarker functionality249. These agents are now widely used for imaging living cells in research. Various methods have been proposed for non invasive imaging. These include MRI, computed tomography, positron emission tomography and ultra sonography. In all the techniques, the diseased tissues are identified by injecting contrast agents into the body of the patient which will illuminate under scanning.

Similarly in other imaging processes like optical imaging, the property of the fluorophore determines the quality and accuracy of the images. Organic dyes are commonly used as fluorophores in optical fluorescence methods. Imaging agents, both endogenous (agents that uses enzyme mediated processes to generate visible light, e.g. green fluorescent protein) and exogenous (conventional agents like dyes), suffer several drawbacks. These include rapid photo bleaching250, unsuitability for simultaneous multicolour imaging, excitation issues due to changes in the environment and overlap with autofluorescence from tissues. MRI agents also have issues related to dose, timing251 and contrast enhancement by surgical manipulation. Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) is one of the most commonly used MRI contrast agents. However, the toxicity of gadolinium is a concern.

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Different types of nanomaterials have been proposed for use in molecular imaging to alleviate some of the difficulties and nanoparticle based optical contrast agents are considered as a better alternative than conventional ones.

The design and development of these nanoparticles for bio imaging follows the following steps252.

Synthesis of an optical core which encapsulates fluorophores like dyes or quantum dots

Synthesis of the shell to protect the optical core to improve photostability

Surface modification to avoid coagulation and to make them suitably dispersed

Bioconjugation and targeting: attachment of suitable biomolecules for targeted delivery of nanoparticles to the desired location.

Gold Nanoparticles

Gold nanoparticles are used as contrast agents due to their optical properties induced by surface plasmon resonance. Depending on their size, shape and agglomeration, gold nanoparticles can appear in different colours. Stable gold colloids (diameter ~15 nm) appear as red when not agglomerated. If agglomeration occurs there is a shift in surface plasmon resonance, inducing a colour change to violet or blue. One of the advantages of colloidal gold nanoparticles is that they are photostable and are less toxic compared to quantum dot semiconductors.

Their optical properties can also be controlled to suit the requirements of the application. The plasmon resonance of nanoshells with a dielectric core of silica surrounded by a thin gold shell can be tuned by controlling the ratio of the core radius to the total radius 253. Gold nanoshells have been used to detect sub ng ml-1 quantities of various analytes in different media in 10-30 min. Aggregation of nanoshell conjugates is seen in the near infrared spectra and this can be used to detect immunoglobulins in saline, serum, and whole blood254.

Gold nanoparticles enhanced by silver (for signal amplification) can be used for labelling255. Functionalised gold nanoparticles with thiol-modified oligonucleotides were used to detect DNA at femtomolar levels. Gold nanoparticle tags can be used to analyze signals on protein microarrays using surface plasmon resonance256. They have a greater increase in angle shift making them a better sensor than other nanoparticles. The surface of these nanoparticles can be tailored to capture different proteins and they can be incorporated in chips for various sensing applications.

Silica Nanoparticles

When silica nanoparticles are used for molecular imaging, a dye is encapsulated inside the particle to increase chemical stability and reduce photobleaching by limiting the oxygen access. The advantages of using silica is that it is optically transparent so light can pass through it efficiently, it is non toxic and biocompatible. It is also resistant to swelling, can be tuned for interaction with different particles and the surface can be easily modified to attach biomolecules257.

Fluorescent silica nanoparticles (FSNP) have been used to detect over-expressed folate receptors typical for certain malignancies258.

Magnetic Nanoparticles

Functionalised magnetic nanoparticles functionalised are being increasingly used for imaging, specifically MRI. Feridex is a nanoparticle-based MRI contrast agent. A typical supermagnetic nanoparticle consists of a metal core (commonly iron) surrounded by an organic coating that can be functionalised for various applications. Magnetic nanoparticles have the advantage that they are non toxic, biocompatible and easy to target and accumulate.

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When exposed to magnetic field, antibodies labelled with magnetic nanoparticles exhibit magnetic signals that can be screened to get highly sensitive images. It has also been found that localised supermagnetic nanoparticles can create localised hyperthermia which can be utilised for the treatment of cancer.

Due to the magnetic sensitivity of these particles, they are also used as contrast agents in MRI. Current MRI techniques suffer from the low signal sensitivity although it is the most popular non invasive imaging method. Magnetic nanoparticles can enhance the MRI signal. A stable, biocompatible magnetic nanoparticle with an iron core and a polymer coating has been developed and can increase sensitivity in MRI while being stable to long-term storage and pH variation259.

Iron nanoparticles conjugated with herceptin, a cancer targeting antibody, have been used to selectively target in vivo human cancer cells implanted in live mice260. Magnetic nanocrystals were produced using thermal decomposition and then coated with dimercaptosuccinic acid (DMSA) for biocompatibility and water solubility. When conjugated with herceptin there was a noticeable difference in particle accumulation at the targeted site within 5 min of injection and a 10% drop in the T2 value.

2.3.2.2 Quantum Dots

Quantum dots are nanometer sized semiconductor crystals that fluoresce when exited by a light source. They are usually composed of atoms from groups II–VI or III–V elements e.g. cadmium (Cd). At this scale (around 1-10 nm) the excitation depends directly on the size of the crystal. This phenomenon is known as quantum confinement.

Quantum dots are widely used in biological applications that require fluorescence, including DNA array technology, cell biology and immunofluoresence assays, particularly in the immunostaining of proteins, microtubules, actins and nuclear antigens261. Q dots are resistant to photobleaching which results in high quality 3D images of cell sections with the need for only a small number for high quality imaging. Q dots have also found applications in bioelectronics, particularly in neural engineering as nanoparticle-neuronal receptor interfaces262.

Quantum dots have the advantage over other labeling agents, like organic dyes, due to their narrow emission spectra and broad excitation spectra. The broad excitation spectra allow it to be tuned for specific applications, while the narrow emission spectra allow the parallel analysis of different emissions with minimum overlap. Changing the particle size and composition for the precise control of emission wavelength and the use of a single light source for simultaneous excitation makes q dots suitable for complex multiplex bioassays. Quantum dots exhibit strongly size-dependent optical and electrical properties and the size can be controlled by the duration, temperature and ligand molecules used in its synthesis263. Another advantage of quantum dots is that they can be covalently bonded to other molecules for targeted delivery. They are also highly resistant to metabolic degradation due to the inorganic nature of the quantum dots and the passive coating over them.

Chan et al.264 have shown that q dots have high molar extinction coefficients and high quantum yields making them bright fluorescent probes. This property makes them ideal for use in aqueous solutions and photon emitted in vivo conditions where scattering and absorption of light are high265. They report the use of quantum dots covalently coupled to biomolecules for use in ultrasensitive biological detection. The biomolecule coupled conjugates were found to be biocompatible, water soluble, 20 times as bright and 100 times more stable against photobleaching when compared to organic dyes such as Rhodamine.

Reiss et al.266 have reported that coating q dots with a material which has larger band gap than the core material can increase the photoluminescence. In their work, they coated CdSe nanocrystals with ZnSe shell. The resulting core/shell nanocrystals exhibited high room temperature photoluminescence efficiencies of 60-85% in organic solvents and in water after functionalisation.

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Being inorganic, q dots require functionalisation before being used for biological imaging. Several approaches have been proposed to functionalise the dots, including covalent bonding to create bioconjugates. Other approaches such as connecting positively-charged linked proteins like avidin, streptavidin or biotin have been reported267. Histidine residues have also been corodinated to Zn metal ions on the shell of the CdSe/ZnS nanoparticles268. Peptide based functionalisation has advantages over other approaches as it protects the shell core from losing its photoluminescence capabilities, provides excellent colloidal properties and makes it biocompatible269. It is possible to functionalise individual quantum dots with different functionalities which makes them ideal candidates for multiplex immunoassays.

One of the advantages of using q dots for in vivo applications is their small size but multifunctional capability. Large size q dots can have difficulties in accessing neuronal synapses in hippocampal neurons270 but the size can be reduced by linking the dots with ligands271. Quantum dots with epidermal growth factors are highly specific and potent in the binding and activation of the EGF receptor (erbB1). However, early methods of linking ligands with q dots have affected the stability of the dots in biological buffers272.

Another challenge is targeting quantum dots to cytoplasmic molecules. Most research has concentrated on the use of q dots as biomarkers. However, in the cytoplasm, the dots must reach the target without being accumulated in vesicles. A possible approach is to inject the dots into the nucleus273. Q dots have been injected intravenously into mice for multiphoton imaging of blood vessels274. Similarly Kim et al.275 report the injection of dots in mice through tail vein. On screening the movement of dots, they found that the q dots localised in liver and lymph nodes on two different occasions. They have shown that quantum dots can be used in major cancer surgery and in sentinel lymph node mapping. This indicates the potential use of q dots as a biological probe and their use in other medical applications like surgery. However this approach has a weakness as the process is time consuming when it comes to labelling large number of cells with dots.

Q dots wrapped in chaperone proteins showed high stability in aqueous media276. The q dots can be released using ATP.

Although quantum dots offer a lot of advantages in sensing, imaging and as contrast agents in various techniques like MRI, PET, IR fluorescent imaging and computed tomography, there is uncertainty surrounding the toxicity of the materials used. For example, cadmium has a half life of 20 years in human body and there is no specific mechanism to excrete it. Also, quantum dots can be unpredictable as they can have different properties even when made of the same material as the surface to volume ratio, size, shape, charge etc. determines the property of the dots.

2.3.2.3 Surface Enhanced Raman Scattering

Surface enhanced Raman spectroscopy (SERS) is a method used for the sensitive analysis of multiple analytes. Probes for this generally contain a metal core of gold or silver, with a silica shell and a reporter molecule. The metal core helps in the optical enhancement, the silica shell is used for protein conjugation, while the reporter molecule is used as a spectroscopic signature. The principle is that when excited with laser light, the reporter molecule emits a highly specific Raman signature which is significantly amplified by the gold/silver colloid thereby facilitating ultra-sensitive detection.

An assay has been developed, using surface-enhanced resonance Raman scattering (SERRS) with silver nanoparticles, to simultaneously detect the activity of multiple hydrolases at ultra-low concentrations277.

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2.3.3 Nanotechnology on a Chip

Devices designed to detect a single or class of (bio)chemicals, or have system level analytical capabilities for broad range of (bio)chemical species and have the advantage of incorporating sample handling, separation, detection, and data analysis onto one platform are called ‘Lab on a Chip’ devices278. One of the most important requirements for a diagnostic lab on a chip device is the precise control of fluids through tiny channels. In sensing applications it is vital that the flow is controlled so as to measure the concentration of molecules or ions which are available in very low quantities. In order to detect molecules at the lowest level, the liquids are forced to flow through channels of nanoscale diameters. Nanotechnology on a chip is an umbrella term which incorporates different technologies that enhance the diagnostic capabilities of various lab on a chip technologies with a huge number of potential applications in in vitro and in vivo diagnostics.

2.3.3.1 Nanopore

A nanopore is a nanoscale pore through which highly sensitive biomolecules are pushed. It can be a hole in a solid state membrane or a protein channel in a lipid bilayer. When molecules move through these holes the properties of liquids can be studied by noting the changes in the electrical properties of nanopores. Nanopores can be used as single molecule detectors.

Using a bottom up manufacturing process, the pore size can be controlled. A method using UV light has been developed and used to control the diameter of self assembling nanopores down to diameters of less than 1 nm279. However, producing symmetrical nanopores remains a challenge.

An array of nanopores that allowed parallel detection of DNA at the single molecule level was made in an aluminium/silicon nitride membrane. Simultaneous optical detection was used for analysis, therefore speeding up the process280. Polymeric nanopores with fixed charges can show ionic selectivity when immersed in aqueous electrolyte solutions281.

Using a protein pore of 1 nm diameter highly specific analyte detection at molecular level is possible282. Oxford Nanopore Technologies (www.nanoporetech.com) are commercially developing this using nanopore protein α-hemolysin to detect a wide range of molecules, metal ions, reactive compounds, proteins and DNA. The frequency of occurrence of events reveals the concentration of the analyte, and the current signature (the mean duration and amplitude of the events) reveals its identity.

A new method for rapid, sensitive, and high-throughput detection of colon cancer cells in response to differentiation therapy, was developed using a novel electrochemical lab on a chip system283. Colon cancer cells were miniaturised in nanovolume chip membranes, differentiation inducing agents were introduced and the efficacies of these agents were evaluated using electrochemical detection of enzyme activity. A high correlation between the number of cancer cells in the nanovolume chambers and induced current signals were observed. This may allow creation of an array of nanovolume chambers that can be integrated into a lab on a chip device to detect multiple drug agents.

2.3.3.2 Lab in a Cell

Studies have shown that a cell can simultaneously perform up to 104 different chemical operations. The concept of a laboratory-in-a-cell (LIC) is using a cell as a laboratory to perform complex biochemical operations, and employing advanced micro- and nanotechnological tools to access and analyse this laboratory284. Although it is difficult to mimic complex properties of cells, the idea is to put a single cell on a chip and use nanotechnology to understand the complex cellular properties. In fact, cells have been used for sensing applications for a long time; the lack of tools hindered the progress of single cell analysis. It has been suggested that that LICs should provide the following functions284:

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Cell manipulation and Immobilisation: Can be done using dielectrophoresis (DEP), electrophoresis or using an optical trap which is essentially a microfluidic device that integrates laser trapping force and DEP.

Characterisation: Can be done using mechanical, electrical, optical or biochemical mechanisms.

External and internal communication: Electroporation, nanoneedles, nanotubes as well as nanofluidic bubble pumps can be used. The nanofluidic pumps are used for the precise delivery and withdrawal of subpicolitres to and from the cells. These novel tools allow non contactable transient permeability.

The technology can be used in the study of intracellular functions and as a single cell sensor. It can also be applied in drug testing and as a nanoreactor in combinational chemistry.

2.3.3.3 Nanobioreactors

Individual cells operate in picolitre volumes at the cellular level. However, culture techniques used in industry are micro or milli-litre levels. This disparity in environments inhibits our ability to understand some of the cellular operations with accuracy. The scaled up environment with fixed parameters can damage some of the sensitive cells and their properties. However, the scaling down of reactor systems gives the advantage of recreating an environment which better mimics the real cellular mechanisms. This comparable environment will provide valuable information about the fast reactions happening at the nanoscale. In addition it will reduce assay response times, analyte volume requirement and allow parallel assay operation. Such an environment and the development of nanobioreactors for cell culturing are highly desirable for the pharmaceutical industry.

A nanobioreactor which can fill this gap in the cellular bioanalysis sector has been designed285. The reactor has a culture chamber, inlet and outlet ports and microfluidic passages and was developed using soft lithography. Three cell lines (fibroblast, CHO cells, and hepatocytes) were tested and cell growth was successfully studied. However, the reactors were non-reusable and cell debris and other components were left behind. If nanosensors are incorporated in nanobioreactors, the parallel monitoring of different cell lines in real time may be possible.

2.3.3.4 Cell on a Chip/Single drop analysis

High Content Screening (HCS) is a method widely used in the pharmaceutical industry to understand the impact of chemical stimulation on an entire population of cells. To understand the behaviour of individual cells upon chemical stimulation, it is desirable to have either a smaller population of cells or a single cell available for analysis. However, this has been difficult to achieve.

A new technique for analysing cells, called NanoDrop, aims to address this problem286. The plate or chip used, Drop Chip, is a glass substrate with the cells cultured in nanolitre droplets. The technique allows the analysis of less than 100 cells per assay and testing of drugs in nano volumes. In addition it allows single cell phenotyping. The effects of siRNA and cisplatin on cancerous cells have been analysed and cell behaviours have been detected at the individual cell level using high-resolution fluorescence microscopy and automated image analysis.

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The major advantage of this method over others is that the shape and volume of the drops can be controlled easily by manipulating the glass characteristics (particularly surface tension). The number of cells per drop can be easily adjusted depending on the requirement of the study. Also the liquid convection within each drop provides excellent conditions for cell-based screening as well as mixing cells and molecules in a homogeneous and continuous manner. Due to the confinement of the cell-based assay within the drop, the NanoDrop technology is suitable for parallel analysis of various cells in nanomolar quantities in a single experiment making each culture a unique nanobioreactor. The EC FP6 project TOXDROP was set up to develop a ‘Cell on a Chip’ technology able to characterise various phenotypes illustrating cytotoxicity in parallel cell based reactions on a single chip.

2.3.3.5 Nanofluidics

Nanofluidics is the manipulation and control of liquids through nanoscale channels. When confined to nanoscale diameters, liquids show different properties compared with their flow in macroscale dimensions and nanofluidics can be used to investigate these novel properties. It has applications in identifying biomolecules, diagnostic tools, lab on a chip devices and in situations that demand sample handling in lower quantities.

There are a number of applications ranging from self cleaning surfaces to polymers for lubrication to filtration. One of the advantages of nanofluidics is that it confines the liquid, forcing complex molecules like DNA to stretch enabling better measurement. In an attempt to push the DNA through tiny pores Timp et al.287 report the use of strong electric field. They pushed the DNA though the pores of less than 2.5 nm diameters forcing it to stretch thereby increasing the chances of reading the sequence of DNA nucleotides. The method developed has potential applications in understanding the mechanical properties of DNA-protein assemblies.

One of the key issues in lab on a chip devices is how to control the flow of liquids through the channels so that the technology can be used in large scale nanofluidic circuits. However, a recent development could be a significant advance in nanofluidics288. A device, made of nanoscale silicon, that acts like a transistor can start and stop the flow of fluids by applying a voltage. The nanofluidic transistor can also be used to tune the ionic environment and to control the transport and concentrations of ions or charged biomolecular species. The channels can be lined with receptors for various molecules and the transistor like mechanism for precise fluid control. Another method developed for controlling the flow of the liquid through nanofluidic channels has been reported289. A backpressure regulation system through a microchannel that is connected to nanochannels was used to regulate the pressure. Large arrays of nanofluidic channels with controlled dimensions have also been fabricated290.

BioNanoMatrix have developed a chip using patented nanofluidic (nanochannel) technology to sequence long strands of DNA in parallel291. This research is part of a five-year US federally funded project which aims to sequence the human genome in eight hours for $100.

The nanofluidics/nanochip technology suffers from the lack of a better fabrication technique to make it commercially viable. Alternative methods to the non-uniform film deposition process such as multiple vacuum evaporation or shadow sputtering have been used successfully. However, most of these processes are complex, elaborate and time consuming. Improvements in the areas like nano imprint lithography might provide a possible solution for creating channels that can be incorporated into other devices. There is also a possibility that larger molecules in the liquid can block the channel. Novel nanomaterials for coatings to reduce friction have been suggested as a suitable solution. Channel clogging due to debris is another challenge. However, at present the biggest challenge is the scalability and reproducibility of the technology.

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2.3.3.6 Capillary Electrophoresis

Capillary electrophoresis (CE) is used to separate ionic species by their charge and frictional forces. The technique was introduced in the 1960s to separate species based on their size to charge ratio, in the interior of a small capillary filled with an electrolyte. The process offers unparalleled resolution and selectivity allowing for separation of analytes with very little physical difference292. Due to the better reproducibility, large surface-to-volume ratio and favourable surface chemistry, nanoparticles are used in CE for improved separation and high selectivity. These particles are normally used as an inner surface coating or added to the buffer to be used in partial or continuous filling form.

Gold nanoparticles can be used as a modifier of stacking and separation buffers for on-chip trace analysis of DNA293. A simple and sensitive on-chip preconcentration, separation, and electrochemical detection (ED) method was developed for the trace analysis of DNA. The gold nanoparticles improved the sensitivity by approximately 25,000-fold when compared with a conventional MGE-ED analysis. Liu et al.294 report the use of gold nanoparticles in normal and chip based capillary electrophoresis. They used gold nanoparticles as an anionic surface coating with poly diallyldimethylammonium chloride as the first cationic layer. They point out that nanoparticles can be used as a pseudo-stationary phase in the presence of poly ethylene oxide (PEO) for double-stranded DNA separation. Gold nanoparticles can enhance interactions between DNA and PEO adsorbed on the nanoparticle surface which improved the sieving ability of PEO without significant changes in viscosity.

2.3.4 Advances in Microscopy

The development of various microscopic tools which can see and manipulate atoms played an important role in the emergence of nanotechnology. Different types of scanning probes that can pick and place atoms at the nanometre scale with high precision have significant advantages in biology and cellular science. Some of the main microscopic techniques used are scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and scanning tunnelling microscopy (STM). A group of IBM researchers led by Binning and Rohrer developed AFM and STM in the 1980s and they have since emerged as significant tools in optical microscopy and nanotechnology. Since then several advances have been made in improving the imaging capability of these tools.

Carbon nanotubes have been proposed as scanning probe tips to manipulate the surface with high accuracy and as a suitable material to engrave patterns on silicon surfaces. SWNTs can be used as AFM tips for high resolution electrochemical and topographical imaging295. These nanotube tips have been used to manufacture nanoelectrodes whose shape, size and composition can be controlled.

Another important technique is Dip Pen Lithography. It is a direct-write soft lithography technique that is used to create nanostructures on a substrate of interest by delivering collections of molecules via capillary transport from an AFM tip to a surface296. The technique can be used to tailor the chemical composition and structure of a surface on the 1-100 nm length scale297. One of the advantages of using these instruments in bio-imaging is that it can produce molecular level images with high resolution in aqueous environments. The nanotubes can also be used for chemically sensitive surface recognition298. AFM tips made of chemically modified nanotubes provided high resolution imaging compared to the commercially available Si and SiN tips.

Recently, a high resolution 3D light microscope with a resolution of 40 nm was developed299. The microscope has been used to image individual fluorescently labelled protein clusters in the mitochondria of live cells.

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In high resolution imaging, point spread function (PSF), the response of an imaging system to a point source or point object dictates the resolution of the image. If PSF is sharper, the resolution is high. The difficulty with conventional microscopy is that due to the principles of diffraction it is difficult to focus light to a size less than half of its wavelength. Several methods, like molecular switching, have been useful in increasing resolution. However there is difficulty extending it to the 3D scale if not combined with multiphoton excitation.

New research adopted a different approach of spot size reduction to sharpen the PSF. Instead of one light as used in conventional microscopy, they used two lights to allow the coherent addition of wave fronts at the focal point. The first one probed the fluorescent labelled cells and created excitation on the labelling particles while the second one inhibited the fluorescence to confine the spot size in the region of 40 nm in diameter. If a second colour channel is added, it can be used to image the spatial relationship between two or more molecules. The microscope they have developed has significant applications in monitoring and analysing the functions of living cells.

Another interesting technology is dual polarisation interferometry (DPI)which offers a method for label free optical analysis300. The structure and density of a protein can be measured to subatomic dimensions by coupling it onto a glass slide and probing with the non-diffractive optical method. The technology has been commercialised by the UK company Farfield Scientific (www.farfield-group.com) and can be used to measure proteins, DNA, lipids, lectins, carbohydrates, surfactants and polymers. It can also be used for interfacial studies and surface characterization. Its main advantages are that it provides real time, high-resolution, dynamic measurements of molecular size, density and mass301.

A novel scanning probe microscopy technique based on scanned nanopipettes has been developed to image the surface of living cells302. The main advantage of the technique is that it provides a non contact mode of imaging and the hollow pipette can be used for targeted and controlled delivery of molecules onto the surface. Although AFMs offer non-contactable mode of imaging living cells, a small force can change the topography of the cells or result in distortion of the highly responsive soft cells. The pipette scans the surface further away from the cell compared to an AFM thereby avoiding any cell deformation. The method is based on the principles of scanning ion conductance microscopy, a scanning probe method in which the pipette scans the subject in a conducting solution. Similar to other existing scanning methods, the distance of the nanopipette from the surface is adjusted by applying a voltage between electrodes in the bath and pipette. In this way a constant ionic current is maintained. For imaging, the pipette is scanned over the surface and the position of the pipette is recorded to obtain the topographic image. The reverse method of keeping the pipette fixed and moving the sample to maintain the distance has also been used for fluorescence measurements and topographic imaging. The images were found to have the same level of resolution as obtained from conventional scanning probe methods.

The technique has been used to monitor cellular motion in labelled cardiac myocytes by observing the change in cell height due to changes in intracellular calcium. This allowed mapping of the position of ATP dependent potassium channels in these cells. It has also been used to deposit fluorophore labelled DNA on the surface of streptavidin coated glass surface with precision control, showing potential as an alternative method to dip pen techniques. Cell communication mechanisms have been studied by the injection of alpha toxin through a nanopipette onto a cardiac myocyte membrane and recording the changes in the whole cell. Adjacent cells in the cluster lost synchronicity due to these changes.

The nanopipette based scanner has shown potential in terms of controlled deposition and local delivery of probes for cell mapping although it require improvements in scanning speed, closer surface scanning, precision delivery and higher resolution.

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2.4 Additional Demand for Research

Development of novel coatings to reduce clogging in nanofluidic channels

Improvements in fabrication techniques to develop nanochips and models for scale up

Sensor that can function in high noise level situations (e.g. war)

The precision flow control of fluids through nanofluidic channels

New software and processors that can simulate nanoscale reactions and manage the data from diagnostic tools

Development of probes that can cross the blood brain barrier and can penetrate into the cells without damage

Methods to improve the stability of quantum dots and other nanomaterials used in diagnostics and imaging

Alternatives to heavy metals in quantum dots

Studies on the toxicity of materials used in quantum dots and other devices

Better small animal models, and adapted imaging techniques for these models, for the development of new in vivo techniques and more accurate probe development303

Incorporation of nanosensors in nanobioreactors

2.5 Applications and Perspectives

Nanotechnology has found applications in many areas of diagnostics and imaging. In in vivo diagnostics the technology is used in the detection of pathogens to early detection of chronic diseases like cancer. Quantum dots have been widely tested for applications in diagnostics and imaging. Streptavidin-coated quantum dots have been reported for detecting bacteria304. They can detect as little as 10 bacterial cells per ml with an approximate 100-fold amplification of the signal over the background in 1 In. These techniques are expected to be highly useful in detecting slow growing bacteria like mycobacterium or bacillus anthracis.

In sensing, glucose sensing has advanced a lot due to nanotechnology. Carbon nanotube-based glucose sensors which will change the nanotubes infrared fluorescence according to the glucose level have been proposed305. When energized by infrared light, electrons are excited from the nanotubes coated with glucose oxidase. The higher the glucose content, higher the fluorescence and vice versa. Efficient techniques using MWNTs for glucose sensing have been reported. Micromechanical detection of glucose using cantilevers has been studied by Thundat et al.306. Glucose oxidase is coated on to the surface of microcantilever and the cantilever bends due to changes in surface stress created by the reaction between the coated surface and glucose in solution.

Nanowire FETs are used for the real time electrical detection of single virus particles307. Electrical and optical measurements were conducted on nanowire arrays modified with antibodies for Influenza A which changed conductance when a binding event occurred. Gupta et al.308 report the use of arrays of silicon cantilever beams as microresonator sensors with nanoscale thickness to detect virus particles. The change in the resonant frequency as a function of the virus particle mass binding on the cantilever has been measured to detect single vaccinia virus particle with an average mass of 9.5 fg.

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Cancer diagnosis is another area benefiting from advances in nanotechnology. In in vivo diagnostics, different types of nanoparticles have been proposed for targeted imaging, increased sensitivity and high specificity. Quantum dots and gold nanoparticles have been identified as better alternatives for labeling than conventional dyes. Super-paramagnetic nanoparticles have been found to provide high resolution imaging in MRI. However, the big challenges here are how to increase the imaging/sensitization capabilities and how to deliver the particles onto the diseased cells without much damage to the neighbouring cells. Some of the heavy metals used in q dots have toxic side effects and this must also be addressed.

Theranostics has been mentioned many times as an important future medical development that will come from nanotechnology however, it a poorly defined term. Broadly speaking it refers to the combination of imaging or a diagnostic test, with delivery of a therapy. Some examples of nanoparticles which may have the capacity to perform this function have been reported and theranostic devices could be hugely beneficial in the future. However, further research to develop sensors and diagnostic tests is required before this can be truly realised.

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3. Regenerative Medicine

3.1 Description

For the purpose of this report, regenerative medicine describes those nanomaterials and techniques employed to repair or replace damaged soft and hard tissue.

3.2 Short Description

Tissue engineering is the use of cells and their molecules in artificial constructs that compensate for lost or impaired body functions309. Scaffolds made of porous biomaterials which mimic the cellular environment are seeded with cells and allowed to grow there. The grown tissue construct is then implanted into the body of the patient where it replaces the diseased tissues and the scaffold degrades. Since its inception in the 1980s, the technology has grown to a stage where it has been used to replace pulmonary arteries310 and to regenerate human thumb tissue311.

With the emergence of nanotechnology and new characterisation tools, it has become easier to synthesise and characterise materials at the nanoscale to enhance the activities of biological molecules and to mimic the biological functions. The advantage of nanotechnology based methods and materials in regenerative medicine is that tissues and associated extracellular matrix (ECM) which help in the regeneration of tissues in biological systems are also nanostructured materials. The interaction between ECM and the cells determines the cell growth, mobility and behaviour. The use of nanomaterials in tissue regeneration can help to create an environment which mimics the natural conditions promoting cell adhesion, cell differentiation and cell growth.

The delivery of proteins, peptides, genes and other growth factors in a sequential manner is also important in assisting cell growth. These bioactive signalling molecules trigger the regenerative activities by their entrance into the cellular matrix at the appropriate time. Methodologies used to incorporate these molecules into the artificial cellular matrix using nanotechnology have been proposed. These include the use of polymers in ECM to immobilise these triggering agents. Functionalisation of scaffolds with different biomolecules to target different types of cell has been proposed as a strategy to improve tissue adhesion and growth. This report examines some of the recent advances in regenerative medicine enabled by the use of novel materials, developments in bioreactor technologies and nanotechnology.

3.3 State of R&D

The greatest advantage of using nanotechnology in tissue engineering is that the novel properties of nanomaterials make the cell interaction and other cellular functions much more efficient than traditional materials. The building blocks of tissues have a nanoscale structure which makes nanomaterials best placed to assist in their regeneration. For example, non collageneous proteins, fibrillar collagen and embedded mineral crystals in human femur have a nanometre level structure312. Nanomaterials have a larger surface area than other materials which allows high protein absorption. In tissue generation processes, proteins normally get absorbed onto the surface of materials more quickly than the cells and they have the capability of enhancing or inhibiting the cellular growth processes. Vitronection, a protein which enhances osteoblasts adhesion, can adsorb better on nanophase alumina than conventional, therefore enhancing cell adhesion313. It has also been recently reported that nanotopography has greater influence on cellular functions and impacts activities like cell adhesion, proliferation and differentiation314.

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3.3.1 Nanophase Materials

The main requirements for a scaffold for regenerative applications are that it should have a highly porous structure, and be non-toxic and biodegradable. It should also have good mechanical integrity to maintain the pre-designed tissue structure, good pore size and large surface area. Finally, the scaffold should interact positively with cells enhancing cell growth, adhesion, migration, and differentiation315. Nanophase materials (which have grain sizes less than 100 nm) can be used to enhance tissue regeneration and to improve cell adhesion, cell spread and migration. The ability of these novel materials to replicate the characteristics and simulate the functions of several body tissues has been studied.

Nanophase titania and alumina ceramics show increased cell adhesion and could have applications in orthopaedics and dentistry.316. These materials were also found to enhance osteoclast and osteoblast functions317.

Nanophase Ti, Ti6Al4V and CoCrMo alloys were synthesized to examine whether nanostructured surfaces enhanced osteoblast metabolic functions318. Initial studies showed deposition of calcium and phosphorous on metal surfaces, which is an indicator of high osteoblast activity. However, it is yet to be confirmed whether this increased deposition on metal surfaces is due to the increased osteoblast metabolic activity or simply precipitation. Nevertheless, it is an indicator that nanophase metals may promote bone regeneration.

It has been reported that high pressure torsion (HPT) processed titanium nanograins improve osteoblast adhesion319. The HPT technique that exerts the highest hydrostatic pressure is of particular interest, since it can most effectively refine metal grains and produce the finest nanostructures of all SPD (severe plastic deformation) techniques. It also reduces porosity, cracks, and other macroscopic defects320. Using HPT, titanium grains less than 50 nm were found to increase adhesion and cellular growth in pre-osteoblasts attached to the nanophase. It could also effectively absorb fibronectin for assisting in cellular adhesion, growth, and spreading. Preferential growth of osteoblasts over fibroblasts was observed, which is desirable for bone regeneration as fibroblasts may cause detrimental implant loosening321. However, further investigation is required to understand this preference.

Nanophase coatings on the surface of biomaterials have been used to improve their biocompatibility and bioactivity. Nanophase hydroxyapatite (HA) has been coated onto titanium322 and tanatalum323 to improve osteointegration and promote bone growth. Soboyejo et al.324 report the deposition of titanium on silica which was then functionalised with argenine-glycine-aspartic acid for improved biocompatibility. Human osteosarcoma (HOS) cells cultured on the coated surfaces showed increased cell spreading and improved cell adhesion. The method has potential applications in implantable BioMEMS devices (mainly used in in vivo applications). Nanocrystalline organoapatite (a biomimetic hydroxyapatite-based material) was coated on titanium mesh to increase proliferation and improve migration of osteogenic cells325.

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3.3.2 Nanocomposite Scaffolds

Polymers are used widely in drug delivery applications to enhance the hydrophilicity of the non-soluble drug carriers and are functionalised with biomolecules for targeting specific cells. One of the most widely used polymers in drug delivery is polyethylene glycol (PEG). Attaching PEG to other molecules helps to increase the solubility and stability and prevents the rapid clearance of drug conjugates by the reticuloendothelial (RES) system. In tissue engineering, similar techniques have been used to deliver genes, peptides and other growth factors for biosignalling purposes. Conjugating polymers with other nanomaterials help in controlling and monitoring the cell growth while providing timely triggers to accelerate the process. The most useful of these are stimuli response polymers which respond to changes in pH, temperature, magnetic field and other external factors to enable external cell growth manipulation. The most commonly used polymers are PEG, polylactic acid (PLA), polyglycolic acid (PGA), poly (N-isopropylacrylamide) (PNIPAAm), polyelectrolytes, such as polymethacrylic acid (PMAA), polyacrylic acid (PAA) and poly(lactide-co-glicolide) (PLGA). PNIPAAm has been identified as the most temperature sensitive polymer used326.

Several studies have shown that the use of polymers in tissue engineering is desirable due to the increased biodegradability. During the natural tissue regeneration process, they degrade in vivo by hydrolysis into non-toxic products which enter into normal metabolic pathways and are excreted from the body as carbon dioxide and water327. The use of PLGA in bone regeneration increased osteoblastic functions and reduce fibroblast activities328.

Combining nanophase materials with polymers has been shown to improve the mechanical properties in biocomposites, and improve adhesion and calcium deposition in bone regeneration processes. PLGA with nanophase titania has been used to improve osteoblast and chondrocyte adhesion in vitro329. The use of PLA with high porous nanophase HA (hydroxyapatite) scaffolds can increase the mechanical properties and osteoblast functions330.

Large improvements in mechanical properties such as hardness and tensile strength, as well as bending capabilities, have been reported due to the decrease in material size to nanosize. Composite scaffolds of needle-like hydroxyapatite (HA) nanoparticles and PLA have been reported, that mimic the mechanical strength and microstructure of bone331. Compared to scaffolds made of pure PLA, the nanocomposite scaffolds were found to have high cell affinity and biocompatibility. A PLGA/HA composite has been used for cartilage regeneration332.

3.3.3 Nanofibre Scaffolds

Novel nanobiomaterials and the associated triggering mechanisms have the ability to mimic the ECM, the complex structure which enables tissue regeneration. Nanofibres, made of synthetic or natural materials, have been proposed as one of the most promising materials to develop scaffolds. These fibres have a high surface area and very good porosity which makes them suitable for enhanced cell colonisation and efficient nutrient exchange between cells and the external environment. In order to be used for scaffolding purposes the material should be able to interact with the cells in three dimensions and facilitate the communication between cells and the stimulants333.

Proteins that make up the natural ECM have sizes in the nanometre range, making it vital when mimicking this synthetically to create a matrix that has dimensions in the nanoscale. The use of polymers in tissue engineering and gene delivery is well known. However, the problem with conventional methods in polymer processing is that it is not possible to achieve fibres less than 10 µm. This makes it undesirable for creating a perfect ECM that can replace the natural one. However, three methods have been found to be useful for producing fibres in the nanometre scale. They are self assembly, phase separation and electrospinning.

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Self-assembly is the process in which molecules and supramolecular aggregates organise themselves into an ordered structure through weak and non-covalent bonds334. The challenge is controlling this spontaneous organisation of components to form structures that can dynamically change and reorganise as required. Self-assembly creates the smallest nanofibres, the low productivity and scale-up difficulty of this method make it currently undesirable for commercial applications.

pH-induced self-assembly of a peptide-amphiphile was used to produce nanofibres of 5-8 nm diameter335. The nanofibres were then cross linked and used to form an HA composite material which mimicked the matrix arrangement that exists between collagen fibrils and HA crystals in bone. A self-assembled peptide hydrogel as a scaffold to encapsulate chondrocytes for cartilage tissue repair and regeneration has also been reported336. Recent developments in peptide self assembly with potential applications in nanobiotechnology have been reviewed by Lu et al.337.

The second method used for the production of nanofibres is phase separation. It is the thermodynamic separation of a polymer solution into a polymer-rich component and a polymer-poor/solvent-rich component338. The polymer is dissolved in a solution and is induced thermally to create a gel. Non-solvent is also used for phase separation. The solvent is then extracted from the gel by adding water and the gel is cooled and freeze dried to produce nanofibres, typically from 50-500 nm339. Biodegradable aliphatic polymers are normally used in this technique.

This method allows control of the pore size and scaffold structure for various applications by the addition of various porogens or by varying polymer concentrations. Additionally, this method doesn’t need specialised equipment and is easy to carry out. However, similar to self assembly, this process is restricted to laboratory use and can only be used with a limited number of polymers.

The third and the most widely used method for the preparation of nanofibres is electrospinning. One of the oldest methods, it uses an electric field to create nanofibres. An electrostatic force is applied between the positively charged polymeric solution in a capillary tube and the substrate. When the charge becomes higher than the surface tension of the capillary tip, a fine polymer jet is created which is deposited on the substrate. One of the advantages of this process is that a variety of synthetic and natural polymers can be used. It is also possible to mix different types of polymers to create nanofibres and the thickness of the fibres can be controlled by changing the amount of polymer in the solution. By controlling the mechanical characteristics and scaffold geometry, this method can be used to produce fibres for a variety of applications. A wide range of polymer thickness from several microns to less than 100 nm can be produced with this method. One of the biggest advantages of this technology is that it is simple and scalable. However, it has been argued that the technique creates random voids in the scaffold and could result in micron sized scaffolds, depending on the type of polymer used. Due to this there is no suitable way of making a uniform, controlled, 3D pore structure in scaffolds created through this method340. However, several studies suggest that both the phase separation and electrospinning can be used to produce fibres of less than 100 nm341.

Living cells have been incorporated in cellular matrices using electrospinning. Simultaneous electrospraying and electrospinning has been used to incorporate living cells in extra cellular matrices in a one step process342. Vascular smooth muscle cells (SMCs) were electrosprayed while electrospinning poly (ester urethane) urea (PEUU) to incorporate them into the scaffolds. This method offers a quick alternative to create tissues and other structures which takes long time to develop in the traditional bioreactor based methods. There was no decrease in cell viability and proliferation was similar to the cultures without SMCs. Muscle cell integrated PEUU fibres were found to be stronger and flexible with high tensile strength.

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3.3.3.1 Polymers used in Nanofibre Scaffolds

Different types of polymers, both synthetic and natural, have been used to create nanofibres. The advantage of natural polymers is that they can contain several growth factors and biosignalling molecules that can trigger cell growth343. The polymers that are used to make nanofibres and their potential uses are reviewed in this section.

PGA (polyglycolic acid) and PLA (polylactic acid) are two of the most commonly used polymers. One of the advantages of PGA is that it has a predictable bioabsorption344. However, it has high degradability which may change the pH, creating unwanted tissue responses. Using PLA is advantageous because it is highly soluble in organic solvents, reduces hydrolysis and increases the time before degradation. A PLA scaffold for nerve tissue regeneration has been reported345. A mixture of both polymers has been proposed to obtain desirable scaffold qualities.

The polymer PLGA has been widely used for tissue engineering purposes. Although the fibre dimensions of nanofibres created through this process are similar to PGA, the ability to control the mechanical properties gives it an advantage over solely PGA based nanofibres. PLGA-based nanofibres have been used for cardiac tissue engineering applications346,347.

Polydioxanone (PDO or PDS) is a colourless, crystalline, biodegradable polymer used mainly in the preparation of sutures. It has been proposed as a suitable material for tissue regeneration and particularly for engineering vascular grafts348. The mechanical properties of nanofibres of PDO resemble that of natural soft tissues like collagen and elastin349.

Polycaprolactone (PCL) is another polymer which has been successfully electrospun and useful for scaffolding. This elastic polymer has low toxicity and slow degradation, and has been used to create scaffolds for bone regeneration350 as well as cardiac graft engineering351. PCL is also blended with other polymers, such as PGA and PLA, to incorporate the desirable qualities of both the polymers. For example, PCL mixed with PLA has shown increased flexibility and elasticity while maintaining the tensile strength of PLA fibres352. This mixture is ideal for creating scaffolds for vascular grafts as these require both these qualities to withstand pressures created by blood flows.

Natural polymers have also been used to create nanofibres that mimic the ECM. Elastin has been electrospun to produce nanofibre matrices. Being one of the most important proteins that constitute artery walls, elastin has huge potential applications in vascular tissue engineering. Nanofibres of PDO and elastin were used to mimic cardiovascular grafts353. The grafts had mechanical properties similar to that of native arteries and the bioactive nature of the elastin allowed the cells to migrate the full thickness in 7 days. Nanofibres and fibre networks of elastin-mimetic peptide polymers have also been reported354.

Fibres of globular proteins like fibrinogen, haemoglobin and myoglobin have also been reported for applications in wound dressings, hemostatic products and scaffolding. The high surface area offered by these fibres make them ideal for hemostatic applications, particularly in enhancing resistance to infection in wounds. Electrospun fibrogenin fibre mats have been used as scaffolds for growing human bladder smooth muscle cells355.

Collagens and derived materials e.g. gelatins, have also been used to form fibres to produce matrixes for various applications356. Electrospun collagen was used to produce scaffolds for cartilage tissue engineering357. Collagen nanofibres have also been used to grow mesenchymal stem cells with high amounts of osteogemic gene expression358. Scaffolds made from electrospun fibres of gelatin have shown similar properties (cell adhesion, proliferation, attachment etc.) to that of nanofibre scaffolds made of other proteins like collagen, solubilised alpha-elastin, and recombinant human tropoelastin359.

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Collagen blends, created by mixing collagen with other polymers, have also been reported to produce fibres for scaffolds. Nanofibres and non-woven fibre networks made from a mix of type I collagen and PEO (polyethylene oxide) have been reported for applications in tissue engineering, wound healing and as hemostatic agents360. The tensile strength and elasticity of the fabrics was dependent upon the weight ratio of the collagen-PEO blend. A mix of collagen and elastin was used to produce fibres for creating three layered vascular tubes. Studies showed that seeded endothelial cells, fibroblasts and muscle cells tended to grow and proliferate361.

3.3.4 Bioactive Scaffolds

As mentioned earlier in this report, the topography of the scaffolds plays an important role in determining the cellular growth and other properties such as cell adhesion and proliferation. The use of polymers to create nanosized fibres help the process by making the scaffolds more hydrophilic and providing larger surface areas to grow and penetrate. Several techniques like soft lithography, polymer etching, demixing, dip-pen lithography, colloidal lithography, electron beam lithography etc. have been used for nanopatterning the surfaces to make them more bioactive. In addition to the above mentioned processes, functionalisation of the polymers with biomolecules and nanoparticles has been proposed to improve cell adhesion, migration and differentiation. Attaching growth factors to the surface of polymers helps to increase the cellular growth properties by triggering signals at the correct time. The bulk modification of the surfaces can also be achieved by co-polymerisation of the polymer chains.

Cross linked hydrophilic polymers that can absorb water without dissolving are widely used to create matrixes for tissue engineering. The porosity, easy modification and biocompatibility make these hydrogels attractive in regenerative medicine. Addition of certain ligands on the polymer surface has been shown to offer better control over cell behaviour in the ECM.

Lindermann et al.362 have shown that coupling of the ligand arginine-glycine-aspartic acid (RGD) as high density islands can be used to alter cell behaviour in tissue culture. In their study, they controlled cell spreading, osteogenic differentiation and focal adhesion kinase (FAK) Y397 phosphorylation of MC3T3-E1 preosteoblasts by spacing the ligand islands in the hydrogel. If the ligand islands were closely distributed, it favoured FAK Y397 phosphorylation and cell spreading. But, when they were more widely spaced, it favoured differentiation while proliferation depended on the RGD bulk density. Functionalised peptides on nanocomposites can also enhance cell adhesion and regeneration363.

Albumin nanoparticles functionalised with fibronectin were found to enhance cell secreted protein deposition on extracellular matrix fibrils364. Antibody CD34 coated polytetrafluoroethylene (ePTFE) grafts have been shown to increase endothelialisation compared with bare grafts365.

Another novel strategy used in tissue engineering is the use of magnetic nanoparticles to control the development of multilayered cell sheet-like structures, ECM and tubular structures. Liposomes can be used as carriers. The magnetic nanoparticles can be attached to the cells by endocytosis. The magnetically labelled cells can be then controlled externally using magnetic forces. It is also possible to conjugate different types of biomolecules like antibodies with magnetic nanoparticles to target specific cells. This method of manipulating the cell functions using magnetic force is called magnetic force-based tissue engineering (Mag-TE)366. It has been found that the presence of magnetic force enhances cell adhesion, seeding and proliferation. Stem cells and progenitor cells can be magnetically tracked by labelling with iron oxide nanoparticles.

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Bulte et al.367 report the use of iron oxide nanoparticles to develop magnetodendrimers that can be used to label human neural stem cells (NSCs) and mesenchymal stem cells (MSCs) through nonspecific membrane adsorption processes. Sasaki et al.368 report that coating magnetic nanoparticles with chitosan enhanced cell invasion efficiency and propose this method as a useful strategy to avoid tissue necrosis. The degree of magnetic force determined the efficacy of invasion. The system was found to enhance the cell seeding, cell-cell interactions and shortened the cell proliferation time. Perea et al.369 seeded human smooth muscle cells labelled with magnetic nanoparticles onto the luminal surface of a tubular shaped collagen membrane to create vascular grafts. Vascular grafts are multilayered structures and their tubular geometry makes conventional engineering practices almost ineffective. After 5 hrs they seeded human umbilical vein endothelial cells labelled with magnetic particles. Using a magnetic force for cell seeding (20-40 min) improved the seeding efficiency to 90%. Histological examination after five days of incubation have revealed densely packed multilayers of smooth muscle cells covered by a monolayer of endothelial cells.

However, one of the issues of using magnetic force in cell functional enhancement is that it may trigger the undesirable growth of endothelial and smooth muscle cells creating intimal thickening. The labelling technique has also been used to visualise and track cell migration after implanting. Noth et al.370 report the use of super paramagnetic iron oxide particles to label human mesenchymal stem cells to track their migration using MRI after transplanting it for cartilage repair.

3.3.5 Carbon Nanotubes

The unique capabilities and properties of carbon nanotubes (CNTs) make them potentially useful for various applications from drug delivery to sensing. These capabilities can also be used for tissue engineering. The sensing capabilities of CNTs can be used to monitor and evaluate the cellular interactions and the environmental changes once the tissues have been implanted on the body. The large surface area of CNTs makes them suitable for immobilising a variety of biomolecules and the small size makes allows for cellular sensing, tracking and labelling. The optical labelling of cells with CNTs helps in tracking cellular migration pathways, understanding the cell biodistribution as well as the evaluation of engineered tissues371. Studies have shown that nanotubes can remain in the cells for a prolonged time during cell division implying that they can be used for studying cell proliferation and stem cell differentiation372. They can also be functionalised to monitor cell functions using MRI. Functionalised nanotubes have been used for delivering drugs and nucleic acids. This capability can be utilised to deliver growth factors to cells to manipulate the cell growth.

However the most important use of nanotubes is in enhancing the capabilities of extracellular matrixes. Carbon nanotubes blended with polymers can be used to grow cells. A nanocomposite formed by blending collagen with SWNT has been used to grow living smooth muscle cells373. A nanocomposite made of ultra short SWCNTs and poly propylene fumarate (PPF) has been used for bone tissue scaffold374. Studies on a rabbit model using porous nanocomposite and comparing it with PPF polymer control scaffolds have shown that the nanocomposite enhanced bone tissue growth three times greater than the control. It also increased connective tissue organisation and reduced inflammatory cell density while the biodegradability was found to be similar to that of PPF.

Nanotubes can also be used to provide structural support to matrices made of polymers such as PLA, PGA or chitosan375. Carbon nanotubes have also been cross-linked in hydrogels to increase the rigidity and to enhance cell growth. Alginate is a viscous gum used for mould-making in dentistry, prosthetics and lifecastingii. CNTs have been used to increase the mechanical strength of alginate hydrogel376. The CNT-Alg gel also displayed faster gelling while the saline sorption was comparable to that of conventional gel.

ii http://www.wordwebonline.com/search.pl?ww=5&w=alginate

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Polymer-functionalised CNTs have been used to grow neurons377,378. Sheets and yarns derived from MWCNTs have been used to grow skin fibroblasts and Schwann cells. Carbon nanotube fibres prepared by a particle-coagulation spinning process have also been shown to promote mammalian and neuron cell growth379.

It was recently reported that coating CNTs onto electrical devices implanted in the nervous system improved electrical stimulation in rats and monkeys380. Conventional electrodes coated with CNTs showed increased charge transfer and decreased electrical impedance.

CNTs have been proposed as an excellent material in tissue engineering. Their rigidity and strength make them appealing in bone regeneration and their electrical properties are attractive for neuronal growth. However, the risks associated with CNTs are the subject of debate. The toxicity of nanotubes was found to be reduced or eliminated to a large extent by functionalising them with various polymers, chemicals and molecules. However, the biodegradability of the materials still remains an issue. The ideal situation is that these novel materials are removed by the body through normal metabolic processes. However, this has not yet been confirmed. While nanotubes promise a wealth of possibilities in tissue engineering, the risk of these materials should be assessed thoroughly and comprehensively before purposefully introducing them into the body.

3.3.6 Cell Sheet Engineering

In tissue engineering, the cultured cells are detached from the ECM using proteolytic enzymes such as trypsin. However, in this process cell-cell junctions are broken and cells are harvested as single cells. There is also the possibility of damaging cell membrane proteins. These single cell suspensions injected into animals for the transplantation of tissues may not work in large or hard tissues. To address these issues, temperature sensitive polymers have been proposed as an alternative to harvest the cultured cells381. A poly (N-isopropylacrylamide) sheet (~ 20nm thick) was covalently attached to the surface of the culture dish. Cells were allowed to grow, migrate and proliferate at 37°C. At this temperature the polymer is hydrophobic, enhancing cell regeneration. However, when the temperature was reduced below 32°C, the polymer became hydrophilic, spontaneously lifting the cells from the surface as a single sheet, without the use of any proteolytic enzymes.

The method has been used for various tissue engineering applications including corneal reconstruction382, periodontal regeneration, bladder augmentation, and cardiac patches.

However, one of the problems with this method is that the amount of ECM attached to the cell sheet is very low compared to other harvesting methods, so it is not ideal for creating cell-sparse tissues like bone or cartilage. But cell sheets composed of periosteum or perichondrium can be used for developing hard tissues382.

3.3.7 Stem Cells

Nanotechnology can be used to encourage the growth and influence the differentiation of stem cells. As mentioned in section 3.3.4, magnetic nanoparticles can be used to label stem cells, as can quantum dots. Nanotechnology-based delivery systems could also be used to delivery of biomolecules required for differentiation.

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Cells can respond to the shape of their environment. Mesenchymal stem cells (MSCs) can be grown on surfaces with nanoscale topographical features. Changes in these features can alter the differentiation pathway that stem cells can take by mimicking the complex structure of natural ECM. Initially, surfaces were roughened at random in an irreproducible fashion. This led to conflicting results due to the lack of consistency between studies. The development of new methods for nanopatterning surfaces have led to ordered, reproducible nanotopography being studied. For example, MSCs from bone marrow usually produce soft tissue, rather than bone, on metal implants. However, nanopatterned polymer surfaces created using electron beam lithography have been used to stimulate MSCs to produce bone mineral in vitro, in the absence of osteogenic supplements383. MSCs grown on polymer nanogratings have been induced to follow a neuronal lineage384.

Other uses of stem cells are covered in the Surgery, Implants and Coatings subsector report. A recent review also covers this area in further detail385.

3.3.7 Bioreactors, Biocapsules and Biochips

Bioreactors are conventionally used to grow tissues in vitro by providing optimum conditions like temperature, pH, pressure etc. The reactors currently used in tissue engineering are mostly on the µl scale and are called micro bioreactors. However, cellular actions take place at the nano or pico level. Scaling down reactors will allow understanding of the cellular operations at their functional level and enable the creation of an optimum environment to speed up cell regeneration. Nanobiosensors can be used in these bioreactors to provide information on the changes taking place. This can be utilised to create a feedback mechanism to change the conditions automatically to suit the cell growth. These sensors can also be integrated with ‘Lab on a chip’ devices to monitor and manipulate the conditions inside the reactor to maximise cell adhesion, migration and proliferation. Additionally, the presence of nanosensors can reduce assay response times, reduce the analyte volume and allow parallel assay operation.

Prokop et al.386 have developed such as system for cellular bioanalysis. They designed three types of bioreactors each having a culture chamber, inlet and outlet ports and microfluidic passages. The incorporation of nanosensors allowed the in situ measurement of physiological events in real time, opening up possibilities to create better bioreactors. The system they have developed has been applied to measure the reactions of three different cell lines in parallel which are of particular significance to pharmaceutical industry.

Microfluidic devices which can mimic the functions of several organs and the creation of an in vivo environment on a chip are becoming one of the most studied areas. These devices will reduce the complexity and huge expenses associated with in vivo testing. These microfluidic devices contain microchannels and microwells which mimic the complex tissue architecture. The micro chambers inside the cell biochips contain cell cultures or engineered tissues which are connected through a microfluidic network that transport nutrients and remove waste similar to in the body. Shuler et al.387 reported the development and use of such a biochip system for the in vitro analysis of chronic liver toxicity of engineered tissues. The system they have developed is called microscale cell culture analog (µCCA) and consists of four chambers which mimic the pharmacokinetic model of a rat. L2 was selected to mimic lung, HepG2/C3A for liver while differentiated 3T3-L1 adipocytes were selected to mimic the functions of fat. Studies were conducted to analyse the bioaccumulation, distribution and toxicity of various compounds like fluoranthene, naphthalene etc.

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The system is a starting point in developing microfluidic devices that can be used to mimic the in vivo functionalities in vitro and as an alternative to conducting expensive and complicated tests prior to animal trials. One of the additional benefits of a cell biochip is that it helps to preserve the cellular functions over a long term. This solves the problems with the use of Petri-dish methods for toxicology studies that need a long time frame for experiments. In Petri-dishes it is virtually impossible to keep their specific activity over a longer term due to the dedifferentiation of the cell types388.

In cell based therapies, immunoisolation of implanted cells are very important for sustained release of therapeutics. However, the rejection of implanted cells has been high in past experiments conducted in this area. This may be due to many reasons including the biomechanical instability, anomalies in the pore size distribution, mechanical rupture of the membrane or due to the compatibility issues of the cell islets389. In addition to that, failures have also been attributed to the increased reaction of the grafted cells with cytokines and the leakage of antigens out of the biocapsules stimulating macrophage activation390. However, the key in the success of such implanted cells for sustained and controlled drug release is that it should be designed in such a way to allow the transfer of essential nutrients in and prevent the movement of macrophages, cytotoxic cells and antibodies into the system.

Desai et al.391 report the development of such a biocapsule which allows the movement of molecules less than 7 nm and prevents those larger than 15 nm. Based on BioMEMS fabrication methods, they created membrane surfaces with uniform pore sizes as low as 7 nm. The membrane thickness was in the microscale (100-200 µm) to improve the dynamic responses. Perhaps one of the most important features of this novel approach is that it allows the replacement of islets even after implanting it on the body. This is particularly useful if the cells are required for a long term. The system was successfully tested for cell delivery in diabetic rats392. The insulinoma cells encapsulated in the capsule were implanted into rats and diabetes was reverted from day 1. They also showed that a pore size of less than 20 nm is capable of retaining the insulin stimulation capability of the islets in vivo393. The technology, which is capable of immunoisolation and biomolecular separation, also offers future potential in terms of incorporating nanosensors into the system for better control over release and real time monitoring, localised release of drugs and the immobilisation of growth factors.

3.4 Additional Demand for Research

Design and development of novel nanophase materials which can enhance tissue engineering properties utilising nanotopography

Methods to control and manipulate the nanomaterial topography for specific requirements.

Development of intelligent polymers which can respond to changes in the environment

Development of novel biomaterials and polymers which can be used as coatings to improve biocompatibility.

Design and development of nanocomposite scaffolds that promote cell growth and proliferation.

Scaffolds that can recruit cells instead of being seeded.

Methods to create uniform, controlled, 3D pore structure in scaffolds

New methods to incorporate living cells and other growth factors in scaffolds to trigger growth signals in a timely fashion.

Techniques for the controlled release of growth factors

Study into the potential of natural polymers as scaffolds.

Potential of carbon nanofibres and nanotubes in neural engineering.

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Development of nanosensors which can be incorporated into nanobioreactors.

Methods for ensuring their stability in the system

Development of nanosensors which can monitor the tissue regeneration process in situ

Development of novel materials which can self assemble in situ to form ECMs

Suitable encapsulation techniques for the immunoisolation of implanted cells.

3.5 Applications and Perspectives

The growth of nanotechnology and associated characterisation techniques has increased the hope that tissues which can mimic the cellular functions can be regenerated more efficiently and be grafted into the body without rejection. Nanomaterials, due to their larger surface area and smaller thickness, offer the possibility of enhancing cell adhesion, migration and proliferation. Advances in various methods of nanopatterning polymers to make them suitable to grow different cells played an important role in the current growth of tissue engineering. It has been reported that nanotopography plays an important role in the functional behaviour of scaffolds and affects the various properties like cell adhesion and cell differentiation. The scaffolds that are made out of nanophase composites or from nanofibres are promising biodegradable platforms which can be used to grow cells in vivo or in vitro. Functionalising these scaffolds with several biomolecules can be used to trigger the cellular activities at the right time to enhance cellular regeneration. Cell sheet engineering, which allows grafting the cells along with the scaffold without the loss of cell-cell junctions using temperature sensitive polymers are particularly useful in cardiac engineering and corneal tissue regeneration.

Carbon nanotubes and carbon nanofibres have also been found to be extremely useful in creating neural networks. The unique properties of nanotubes have also been used to offer increased structural strength to the scaffolds. The application of alternating current on MWCNT composites can increase the osteoblastic proliferation by over 45 % and calcium deposition by over 300 %394. Several different methods and materials based on nanotechnology offers hope that creating an artificial organ which can be similar in function to the natural organs are not too far away. Readers are advised to read this report along with the Implants, Surgery and Coatings subsector report, to gain an understanding of the role of nanotechnology in tissue engineering, novel implants and wound care management.

As a final note it is very important to consider the toxicity of many of these novel biomaterials as there is no universal agreement in the scientific world about the potential hazards of many nanomaterials.

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4. Implants, Surgery & Coatings Keywords: active implant, passive implant, surgical blade, tweezers, needle, catheter, coating, stent, bone, dental, nanostructure.

4.1 Definition

For the purpose of this report, implants, surgery and coatings describes nanomaterials that can be used in both active and passive implants and in surgical procedures.

4.2 Short Description

The major issues with placing implants in living tissue are rejection by the body and the need for replacement after a few years. This is not only costly but also puts the patient through another set of complicated procedures and their associated problems. In order to be successful, implants should be biocompatible and control the growth of tissues.

Nanotechnology has the capability to improve implant biocompatibility, either by coating implants with nanomaterials or by using nanomaterials as implant materials. Different types of coatings can be applied to improve the sustainability of the implants and protect them against bacterial and fungal infection. Coatings have also been applied in synthetic vascular grafts to avoid the deposition of biological material, thereby reducing the chances of occlusion395. Nanoscale materials can be used to make lighter and stronger implants that last longer. Some nanoscale materials can also accelerate cell growth after implantation.

In addition to supporting the basic requirements of implants, nanotechnology can also help to improve the monitoring and control of factors that help the growth of tissues in vivo with the use of sensors. This also allows the localised application of stimuli to encourage cell adhesion and growth. Nanotechnology can also be used to create smaller, rechargeable batteries for use in active implants. The technology has also been applied to create corrosion free suture needles with improved strength and ductility. Nanoparticles have also been incorporated into fibres to be used for wound dressings.

The ultimate aim of using these novel technologies in implants, surgery or wound care is to heal the body quickly and efficiently without creating excessive pain or irritation. It may also allow development of biomimetic cures for some of the chronic and degenerative diseases.

This report is aimed at highlighting some of the important developments in the area of passive and active implants, surgery and the use of textiles in wound care. Tissue engineering applications are covered in the regenerative medicine subsector report in this series.

4.3 State of R&D

4.3.1 Bone and Dental Implants

The use of nanomaterials and nanocomposites has made significant advances in the area of bone and dental implants. Nanophase materials have been successfully used on their own and alongside several other polymers to create biocompatible extracellular matrices (ECM) that can enhance osteointegration. These novel materials can provide a porous surface with large surface area and good mechanical integrity and are found to improve cell adhesion, cell spread and migration. In addition, nanofibres of several polymers have been highly useful in creating matrices that can grow living cells and incorporate growth factors to provide stimulation at the appropriate times.

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The main reasons for implant failure are poor osseointegration, issues with the bonding of an orthopaedic implant to juxtaposed bone, and the inability of implants to match the physical properties of surrounding bone396. In fact, osseointegration is the single most important factor that determines the success of a bone implant. Several studies have shown that when an implant is placed to replace the natural bone, the level of integration and the fastness of healing will depend on the bone-to-implant contact area. If the contact area is higher, it will result in enhanced implant integration into the host bone. The advantage of nanotechnology is that it offers a higher surface area and thereby higher osseointegration potential.

Coating dental implants with nano TiO2/HA (hydroxyapatite) encouraged nerve regeneration in cultured Schwann cells397. No changes in cell morphology or function were observed, indicating the biocompatibility of the material. However, coating materials with nanoscale TiO2 alone has shown no significant difference to microscale TiO2

398.

Nanostructured Ti can be used for bone re-engineering, giving increased surface area and osseointegration399. Studies using nanophase Ti, Ti6Al4V, and CoCrMo alloys saw high deposition of calcium and phosphorus compared with conventional phase metals, indicating that nanophase promotes osteoblast metabolic functions and can be used as materials for orthopaedic applications400. Human mesenchymal stem cells (MSCs) have been grown on Ti surfaces with nanoscale TiO2 patterning401. Recently this technique has been used to induce MSC differentiation into bone matrix on titanium surfaces with 15 nm TiO2 nanopillars402.

Growth factors and biomolecules can also be immobilised onto implants to enhance growth and integration. TiO2 nanotubes produced by anodisation have been proposed as drug eluting coatings for implantable devices403. The surface of the tubes can be functionalised to attach biomolecules, such as bovine serum albumin. Bone morphogenic protein (BMP) has been immobilised on the surface of Ti based implants to enhance bioactivity and bone formation404. The advantage of immobilising BMP is that it allows controlled administration and avoids the common issues associated with overdosing. Implants have been coated with nanocrystalline diamonds to increase the surface area and facilitate immobilisation of BMP405. The differentiation and proliferation of cells406 without changing the overall texture of the specimen can be achieved using these diamonds.

Nanocomposites and nanomaterial coatings have been developed commercially for bone and dental implants. Biomet (http://www.biomet.com/) has recently introduced a new dental implant called NanoTite which deposits nanoscale calcium phosphate crystals to approximately 50% of the implant surface. Angstrom Medica (www.angstrommedica.com) has a product called NanOss™ Bone Void Filler, made of HA nanocrystals for high osteoconductivity and strength in bone replacement. Although nanocomposites and other coatings offer great potential, the cost and difficulty of scale up hinders its fast commercialisation.

Biocompatible oxide ceramic based nanostructured composites have been developed by the EC funded Bioker project, in an attempt to develop new hip and knee replacements as well as for dental implants407. The nanocomposites developed offer similar crack resistance to the covalent materials used in implants while avoiding the associated drawbacks due to processing and machining. Some of the composites (containing zirconia nanoparticles) show superior mechanical resistance to commercially available products, better biocompatibility than alumina and the wear test demonstrated that the material can be used for biomedical applications.

The process used to create these nanocomposites used a single step without nanoparticle handling and the particle size could be controlled by changing the processing parameters. This makes the method highly desirable. Currently the continuation of the Bioker project, now called IP-Nanoker (http://www.nanoker-society.org/index.aspx?id_page=121), aims to provide nanoceramics (<100 nm) and nanocomposites (second phases <10 nm) for industrial applications.

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High dispersion hydroxyapatite (HA) and zirconia nanocrystal nanocomposites which can mimic the mechanical strength and bioactivity of natural bones have been developed by IBN (http://www.ibn.a-star.edu.sg/)408. Enhanced attachment and proliferation of MC3T3 osteoblast cells was seen when a nanocomposite foam of collagen fibres and apatite nanocrystals was used409. In vivo bone, tissue and blood vessel formation using this scaffold has been demonstrated. The tuneable pore size, high osteoinductivity and high mechanical strength make it ideal for use in load bearing tissue implants.

HA nanoparticles have been widely used for re-engineering due to their enhanced mechanical properties and ability to improve cell growth and proliferation. Needle-like HA nanoparticles with PLLA [poly (L-lactide acid)] have been used to develop composite scaffolds to create bone structures with mechanical strength of natural bones, high porosity, high cell affinity and biocompatibility 410. Tantalum coated with nanophase HA has shown increased bone growth411.

4.3.2 Cartilage Implants

One of the biggest problems with cartilage tissue is its’ inability to repair itself. Several approaches have been proposed to overcome this. One of the most widely used methods is the regeneration of cartilage tissues in vitro on a 3D scaffold and transplanting it to the damaged location. Several nanomaterials and polymers have been proposed as suitable materials to create cartilage tissues.

A PLGA/HA nanocomposite for cartilage regeneration enhanced chondrocyte (cartilage cell) attachment, proliferation and response and doubled the tensile strength412. A self-assembled peptide hydrogel was used as a scaffold to encapsulate chondrocytes for cartilage tissue repair and regeneration413. Anodised Ti with nanosized pores has been found to increase chondrocyte adhesion and migration414.

Recently, implants made from carbon nanotube composites were used to grow cartilage cells415. Electrical pulses were applied to the composites and accelerated cell growth and production, while the presence of the CNTs gave increased mechanical strength. The biocompatibility of CNTs must be addressed before this application can be realised.

Adult mesenchymal stem cells (MSCs) grown on a nanofibrous scaffold have shown differentiation into chondrocytes416. Electrospun nanofibres of a synthetic biodegradable polymer, poly(e-caprolactone) (PCL), were used to construct the scaffold and MSCs were cultured on this scaffold in the presence of TGF-b1. The cells differentiated to a chondrocytic phenotype at a level comparable to that of MSCs maintained as cell aggregates or pellets. The nanofibre scaffolds can be constructed to any size and offer better physical support for cells. These properties make them good candidates for stem cell-based cartilage repair. (For more information on nanotechnology and stem cells please see the Regenerative Medicine subsector report).

4.3.3 Oesophageal, Tracheal and Bladder Implants

Advances in tissue engineering are creating opportunities to do more complex transplants. In the case of tracheal and oesophageal prosthesis, one of the biggest problems is the post-operation side effects like scarring, inflammation and oesophageal constriction. Also, the removal of damaged epithelial cells results in the loss of normal epithelial barrier function.

In the case of tracheal replacement, most existing methods are surgical and the mortality rates are high. It normally requires replacing the tissues with tracheal allografts or autologous portions of gastrointestinal segments. Cell sheet engineering has been used to grow epithelial cells in vitro and replace damaged cells using non-invasive methods. Tracheal replacement studies conducted on rabbits have shown successful migration of implanted cells onto the host trachea while the epithelial cell sheets formed a fully functional lumen417.

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For oesophageal transplantation, autologous oral mucosal epithelial cells have been seeded to create cell sheets. Wound healing improved remarkably causing very little inflammation418.

Bladder cancer can result in removal of a large part of the bladder wall and in some cases the entire wall. Nanotopography, nanoscale features on a surface, plays an important part in improving cell adhesion, proliferation etc. Recent in vitro developments using this technique appear promising for future bladder repair.

Nanostructured polymers PLGA, PLA and poly-ether-urethane (PU) were chemically treated to increase surface roughness and used to grow bladder smooth muscle cells419. Enhanced cell adhesion was seen and is thought to be due to the roughness and nanotopography of the surface.

Electrospun polystyrene scaffolds have been used to support the growth of smooth bladder cells420. Fibrinogen421 and cellulose acetate422 have also been used to create nanofibre scaffolds that promote cell adhesion and proliferation of bladder cells. Harrington et al.423 reported coating self assembling peptide-amphiphile (PA) molecules onto PGA and electrospinning to produce nanofibres. The advantage of incorporating PA is that it ends up in creating positively charged lysine moieties on the surface of scaffolds which enhances cell adhesion and matrix deposition. The PAs can also be used to encapsulate cells and other growth factors which can trigger the cell growth.

For a detailed review of nanotechnology in bladder replacement see Cheng et al.424.

4.3.4 Vascular Implants and Stents

One of the biggest challenges in vessel fabrication is the complex multilayer architecture of the tubules. Different approaches based on tissue engineering have been proposed to overcome this. These include seeding cells in scaffolds to produce a 3D structure, cell sheet engineering and the use of hydrogels for cell growth. Electrospinning polymers to produce nanofibre scaffolds has been widely demonstrated as a method to create this complex vascular structure.

A collagen and elastin mix was used to produce fibres that can create three layered vascular tubes425. Human smooth muscle cells labelled with magnetic nanoparticles have been seeded onto the luminal surface of a tubular shaped collagen membrane to create vascular grafts426. Magnetic force was used to improve the seeding efficiency to 90%. Polydioxanone (PDO or PDS) a colourless, crystalline, biodegradable polymer used mainly in the preparation of sutures has been proposed as a suitable material for engineering vascular grafts427. Studies on the mechanical properties of PDO nanofibres show that they resemble that of natural soft tissues like collagen and elastin428. Fibres made using PCL/PLA mxtures have shown increased flexibility and elasticity while maintaining the tensile strength of PLA fibres, making them ideal for create vascular graft scaffolds which need to withstand pressures created by blood flow429.

One of the most widely used implantable devices in vascular intervention is a stent. Stents are thin tubular devices implanted into arteries to support the blood vessels following angioplasty. They are commonly made of stainless steel, cobalt chromium alloys, or more recently Nitinol, an alloy of nickel and titanium with shape memory properties, which means they can be inserted in a collapsed form and expand once in place. While stents are extremely useful in restoring blood flow, their long term use is associated with several compatibility issues often requiring further surgery. Narrowing of arteries at the stent implant location can occur, leading to stent restenosis. One of the reasons for this problem is venous neointimal hyperplasia, aggressive growth of smooth muscle cells, in the vessel wall430. Often restenosis leads to the blood clots in the blood vessels, or thrombosis.

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Stents are also used for urinary tract blockages and to extract bile from the gall bladder to diagnose diseases like gall stones. One of the issues associated with the use of plastic stents is the formation of sludge, followed by a blockage. This occurs because proteins, glycoproteins and bacteria adhere easily on the surface of the stents and the bile flow is not sufficient to clean the surface431. The use of nanotechnology has been suggested as the best possible solution to some of these problems.

The formation of endothelium on stent surfaces is considered as a suitable solution to restenosis. The creation of nanostructured features on the surface of widely used stent materials can promote endothelial cell growth and proliferation432. Nanostructured Ti and CoCrMo showed increased endothelial and vascular smooth muscle cell adhesion compared to conventional Ti and CoCrMo. Higher endothelial cell growth compared to muscle cell growth was also seen433. Endothelial cells loaded with polymeric magnetic nanoparticles have been targeted to the surface of stents, both in vitro and in vivo (rats), using a magnetic field434. Coating stents with nanoporous ceramic coatings has also been found useful against thrombogenesis. Hydroxyapatite and alumina are being experimented as stent coatings.

Nanoscale sol-gel coatings have been used successfully to reduce sludge accumulation in biliary Teflon stents435.

Layer by layer deposition of nanocoatings on the surface of arteries have also been reported as a way of treating damaged blood vessels. Self-assembled polysaccharide-based nanocoatings were deposited onto damaged arteries, preventing platelet adhesion and thrombogenesis436.

Drugs may also be incorporated into stent coatings for controlled delivery when implanted and could be used to treat conditions such as hyperplasia. Deposition of self-assembled monolayers (SAMs) on stent surfaces has been used to create drug eluting stents with excellent stability437. PLGA nanoparticle-paclitaxel conjugates were loaded onto vascular grafts and delivered the drug while reducing the initial burst release438.

4.3.5 Neural Implants

The process of neuron regeneration is extremely difficult, as is developing neural implants Nerve cells require the correct environment and growth factors at the right time to grow and proliferate. They also need inductive scaffolds.

Carbon nanotubes (CNTs) or nanofibres have been proposed as ideal materials to replace or annex axons due to their electrical properties. Carbon nanotube fibres have been used to promote mammalian and neuron cell growth, migration and proliferation439. Carbon nanofibres have also been used to reduce scar tissue formation440. Astrocytes (glial cells that form scar tissue) adhered less on carbon nanofibres in the lower nanometre range.

Biodegradable polymer scaffolds for nerve tissue generation have been reported441.

Scaffolds that can overcome the difficulties of axonal regeneration after injury to the central nervous system (CNS) have also been reported442. Scaffolds from self-assembling peptide nanofibres were developed in order to avoid the common problems in neural implants like scar tissue formation, gaps in the nervous tissues formed during phagocytosis of dying cells, and the difficulties in axonal extension by adult neurons. Studies on adult hamsters with their eyes disconnected from their brain showed that the use of peptide scaffolds allowed axon growth while inhibiting/discouraging the scar formation. They were also able to conform to the injury site. As the environment changes, the material will remodel to fit the new environment, allowing for regeneration. 75 % of animals treated with nanofibre scaffolds for injury in the optic tract had restored functional vision compared to none without the scaffolds, showing that regenerated axons can support visual behaviour. The fibres mimic the natural ECM and can be can be broken down safely into natural amino acids, thereby reducing the chances of rejection.

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Biocompatible self-assembled monolayers, containing 16-mercaptohexadecanoic acid (MHA) and end-thiolated poly(3-(2-ethylhexyl)thiophene) (EHPT) in varying proportions, were formed on gold. When coated on neuron electrodes, the presence of EHPT in the SAM increased biocompatibility and reduced impedance443. This method is significant over other types of coatings as it does not increase the separation between electrodes and neurons, so does not reduce the signal strength. It also offers the flexibility to add dopants to improve the conductivity. Nanofibrils fabricated on neural prosthetic devices with a poly(3,4-dioxyethylenethiophene) (PEDOT) coating to improve conductivity and reduce impedance444. These conducting polymer coatings offer huge opportunities in neural implants and biosensors.

Layer by layer assembly has also been used to create coatings on the surface of electrodes. Nanoscale coatings were formed on Si/SiO2 substrates by alternating polycations [polyethyleneimine (PEI) or chitosan (CH)], with polyanions (gelatin or laminin). The coatings didn’t alter the impedance, were stable for nearly 7 days and promoted neuron growth445.

Electrical stimulation is considered as a promising way to restore the functionalities of damaged brain cells. Many techniques have been tried in the past to provide electrical impulses to stimulate the cells. Insertion of micro-machined devices or electrodes of nanotubes and nanowires have been proposed for brain stimulation.

Nanowire arrays have been used as contact sites for neural stimulation and for analysing isolated single unit activities446. An array offers higher surface area and is an ideal alternative to the existing neural implants. Also, nanotubes and nanowires have high conductivity making them ideal for electrical stimulation of brain.

Micro-machined devices made from single crystal silicon wafers and silicon on insulator wafers were inserted into the brain and their effects on brain stimulation were studied447. Two types of response were seen in animal models. One proportional to the size of the device which was due to insertion of the devices, and a second , a sustained response due to the tissue-device interaction. All animals survived the experimentation period and recovered well from the surgery without any side effects. Micro-machined devices have been used for deep-brain stimulation of the subthalamic nucleus without damaging the neighbouring tissues448. This type of stimulation using small devices has significant possibilities in the treatment of degenerative diseases such as Parkinson’s and Alzheimer’s, and for blindness, spinal injuries etc.

4.3.6 Pacemakers, Retinal and Cochlear Implants

Nanotechnology also has applications in pacemakers. One of the main problems with existing pacemakers is that they are not energy efficient. In most of the cases, the battery needs replaced every two years and even the newest Li batteries only have a lifetime of 7-10 years. Another problem is the pacemakers’ intolerance to high electromagnetic fields such as those used in MRI. This may lead to damaged or destruction of the pacemaker and/or the adjacent tissues. Optical fibres have been used instead of electrical leads to solve this problem. However, the need for a constant power source is still necessary. The NASA Ames Research Centre for Nanotechnology (http://www.nasa.gov/centers/ames/) and Biophan (http://www.biophan.com/) have developed a battery suitable for implantable devices such as pacemakers which can convert body heat into electricity449.

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Spintronic technology, which is based on the spins of electrons rather than charge, has applications in medical devices like pacemakers, neurostimulators and cochlear implants. The most common spintronic device is a spintronic sensor which uses the principle of giant magneto resistance (GMR). GMR is a phenomenon of high signal production in relation to small changes in magnetic field when a nonmagnetic conduction layer is sandwiched between two ferromagnetic layers. The optimum thickness of the middle layer for maximum signal production is typically around 3 nm. Copper is often used as the middle layer due to its high resistance at this thickness. Spintronic sensors have been hailed as the next generation sensor devices due to their high sensitivity and precision over other sensors. They can withstand large magnetic fields, making it ideal to use with MRI. However, the key advantage is that it integrates well with the existing devices and conventional semiconductor processing can be used to develop them450. Spintronic devices can be mainly used for the replacement of ampoule and MEMS reed switches for the magnetic activation of programming and special modes in implantable devices. The sensors fitted on the implants allow high speed data communication between a remote computer and the implant, allowing real time data monitoring. In the case of cochlear implants, GMR-based sensors can switch signal processing modes without user intervention451. NVE Corporation (www.nve.com ) makes spintronic devices for the pacemaker and hearing aid market.

Tissue engineering has been used to restore damaged endothelial and epithelial cells in the human cornea. Cell sheet engineering can create corneal epithelial and endothelial cell sheets which can be transplanted to restore vision452. Genetically modified corneal epithelial stem cell sheets have been used to treat hereditary corneal diseases453. Modified versions of corneal endothelium, which exists as a monolayer under the stroma to maintain its hydration and thickness, have been constructed in vitro.

One of the problems with endothelium is that it will not regenerate easily in vivo. Using the cell sheet engineering method, endothelial cells have been cultivated and grown on collagen-coated sheets, obtaining a density similar to that of normal tissue454. Increased collagen and fibronectin deposition was also observed in ECMs. When tested in rabbit models, the endothelium maintained the stromal hydration and thickness similar to natural cells and reduced the amount of swelling.

A recent clinical study implanted a 4 mm square of retinal tissue containing retinal progenitor cells and the retinal pigment epithelium in the sub-retinal space under the fovea. A large improvement in vision of patients was seen, however this deteriorated over 6 years post-implantation455. This demonstrates that tissues can be implanted in sensitive locations without any adverse reactions.

One of the challenges in the field of active implants is how to stimulate nerves without damaging the surrounding tissues. Nerves can also take at least three months to recover after implanting a device. One possible way to avoid this damage is to integrate the nerves with the electrodes and allow them to grow on these. Such a high level of integration would allow packing of a large number of electrodes into a small space, providing a higher level of stimulation. MEMS have been already utilised to create device platforms for integrating a large number of electrodes. However, integrating nerves has proved difficult. Electrodes with polymer coatings have been synthesised to help the nerves to grow and connect faster456. These coatings can be designed to release drugs and biomolecules for a prolonged period of time. When tested on retinal implants from rabbits, the neurons grew and extended into the electrodes. The coatings released neutrophins for a period of three months. By using these coatings, a much more efficient electrode-tissue interface can be achieved, which could be used to reduce the high stimulation thresholds required to enhance neuron growth.

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60 electrodes have been integrated on a chip and implanted for retinal stimulation. In this system, the patient wears a pair of glasses fitted with a camera which sends wireless signals to the chip implanted on the retina. The electrodes receive these signals, convert them to electrical pulses and send them to the neural cells in the eye, mimicking the role of light sensitive cells. These signals are then relayed to the brain. It offers a significant improvement to those who lost vision due to retinitis pigmentosa or macular degeneration457. However, in the future the key will be the ability to manipulate the electrodes to stimulate the cells in such a way that it is recognised by the brain as natural stimulation. Artificial synapse chips and their ability to be used as neurotransmitters in retinal implants have been studied earlier. A prototype device containing 150nm gold electrodes for neuron stimulation has been developed for chemical neurotransmission458.

Silicon nanowires have been proposed as alternatives to conventional electrodes as they have the ability to detect multiple signals from neurons. Conventional electrodes can only detect 1 or 2 electrical signals but nanowires can detect signals from 50 different points in a neuron459. This ability to form multiple junctions offers the capability of simultaneous measurement of the rate, amplitude, and shape of signals propagating along individual axons and dendrites. Another advantage of nanowires is their similar size to axons and so the signal transfer between neurons using them as an interface will be similar to axon-axon transfer. Nanowires can act as field effect transistors and amplify very small signals from neuron-nanowire junctions. They have potential applications in cochlear implants and can also be used to replace damaged nerves or in the treatment of other CNS diseases. Up to 50 nanowires have been incorporated into a small chip, offering another opportunity to use it as a device for diagnostic and drug discovery applications.

4.3.7 Other Developments

Sensors are getting smaller and smaller, but with high sensitivity, due to the unique properties at the nanoscale. These properties have been combined with other technologies, such as artificial intelligence, to create artificial knees which can learn from experiences to reduce the chance of fall in the future. Microprocessors which control smart implant systems have been developed by Ossur (http://www.ossur.com/). The proprietary Bionic Technology allows their RheoKnee™ to learn the users walking patterns and to go beyond the defined parameters to adapt to constant changes in the surroundings to reduce failures.

In therapies which require sustained release of drugs, immunoisolation of implants is very important. Desai et al.460 report the development of a biocapsule which allow the movement of molecules of less than 7 nm while preventing those larger than 15 nm between the capsule and the external environment. Using BioMEMS fabrication methods, they created uniform pore sizes on the surface of membranes with sizes as low as 7 nm. The system was successfully tested for cell delivery in diabetic rats461. Diabetes was reverted in rats from day 1 after insulinoma cells encapsulated in the capsule were implanted.

Implantable nanoscale thin films that can be precisely controlled to release chemical agents have been proposed for drug delivery, gene delivery, tissue engineering, diagnostics and chemical detection.

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4.3.8 Nanotechnology in Surgery

4.3.8.1 Surgical blades

Advances in novel manufacturing methods have enabled the production of surgical blades with a cutting edge diameter in the region of 5nm -1 µm. Ophthalmic surgical blades which offer a blade edge radii of 5-500 nm have been manufactured from either a crystalline or polycrystalline material462. The blades are prepared from crystalline or polycrystalline silicon wafers by mounting them and machining trenches into the wafers. With this method, any required angle can be obtained and the resulting blade has a cutting edge of that of a diamond edge blade. Such blades can be used for various complex surgical operations such as cataract surgery.

However, these blades can bend on contact with tissues and this forces the surgeons to use more cutting force, increasing the chances of tissue damage. Lingenfelder et al.463 report the development of trephines with nanostructured carbon coatings to obtain cutting edges of higher stability and properties like diamonds. Studies conducted on pig cornea using the carbon coated blade revealed smooth surfaces compared to unmodified cutting edges. Force measurements observed a reduction in cutting forces for the modified blades and the cornea treated with the modified trephines showed a smoother surface. This is mainly due to the diamond-like properties of the carbon coated trephines that lowered the frictional coefficient. Additionally, these properties create biological inertness on the surfaces of blades which reduce the physical adhesion to tissues464.

Diamond-coated surgical blades with an approximate surface roughness of 20 - 40 nm have been developed by GFD Gmbh (http://en.blades.diamaze-gfd.com/).

4.3.8.2 Nanoneedle

Nanoneedles have a typical diameter of 200-300 nm and can penetrate into the cells without causing cell damage or death. Typically they are made from silicon using existing fabrication methods and are used as AFM tips to carry out cell surgery or molecule delivery. The advantage of the needles over existing capillaries is that they can manipulate cell activity and monitor reactions simultaneously without damaging the cells.

Nanoneedles with a length of 6-8µm have been used to penetrate human epidermal melanocytes and HEK293 (human embryonic kidney) cells465. There was no shape change in plasma membrane and the nucleus remained intact. In this system the displacement of the needle is accurate and it is possible to monitor and control the force on the needle, potentially enabling single cell manipulation. The position of the needle can be estimated by monitoring the force on the needle. The surface of the needle can also be modified to load and deliver various biomolecules like proteins and nucleic acids.

Pyramid-type AFM tips with an estimated radius of curvature of 50 nm have been used to remove large patches of the outer cell wall of the bacterium Lactobacillus helveticus466. Recently, CNTs were functionalised to behave as nanoneedles to penetrate plasma membranes and translocate directly into the cytoplasm for drug delivery applications467.

4.3.8.3 Nanotweezers

Nanotweezers are surgical tools which can be used to grab and move single biological molecules within cells. These tweezers, typically with a thickness in the nanometer range and controlled by electrostatic forces, are developed by attaching carbon nanotubes to the end of electrodes. These nanotubes are then manipulated by electric forces which bends the nanotubes inwards to grab the molecule. The first nanotweezers had a diameter of 50 nm and began functioning at 8.5 V468. The physics behind the technique is that the nanotweezer tries to balance the elastic energy cost with the electrostatic energy gain allowing it to close beyond a certain voltage.

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Since carbon nanotubes are used as nanotweezers, it is also possible to measure the electrical properties of the materials grabbed. The technique can be applied to manipulate and move biological molecules like DNA. One of the potential applications of nanotweezers is conducting surgery on single cells. Silicon nanotweezers with an initial tip gap of 20 µm were used to perform static and dynamic mechanical manipulations on DNA molecules. The technique was used to study the viscoelastic behaviour of DNA bundles and obtained a resolution better than 0.2 nm in static mode469.

Nanotweezers have shown that the drug topotecan kills cancer cells by preventing the enzyme DNA topoisomerase I from uncoiling double-stranded DNA in cells. Nanotweezers were used to monitor changes in the length of an individual DNA molecule caused by the action of a single topoisomerase I enzyme. The technique was also used to study how the binding of the drug to this enzyme-DNA complex changed DNA uncoiling470.

Optical tweezers which use lasers to manipulate biological cells have also been suggested as a method of non-invasive surgery. The intensity of the light and momentum created by the continuous application of laser beam can be used to move and manipulate biomolecules.

4.3.8.4 Femtosecond Lasers

A femtosecond laser is a laser which emits ultrashort optical pulses with durations in the range of femtoseconds (1 fs = 10-15 s). These lasers belong to the category of ultrafast lasers or ultrashort pulse lasers capable of creating intensities in the range of 1013 W/cm2. Their ease of use, precision, and ability to localise light make them excellent tools for the manipulation of structures and biological molecules.

Femtosecond lasers have been used to cut and reshape the cornea to correct vision in humans. Using a femtosecond laser, the damage caused to the endothelial tissues on the surface of the cornea has been eliminiated471. These lasers have also been used to cut single actin stress fibres in living cells and study the changes in cell shape472, and to cut chromosomes473. They have also been used to remove mitochondria from living cells while retaining cell division474. This demonstrates the use of lasers for nanosurgery to remove specific organelles without affecting long term viability.

Due to their precision, femtosecond laser ablation has found several applications in the medicine area. Irradiating targets with repetitive pulses to create cumulative effects has been tailored for specific applications. Femtosecond laser ablation has been used to study the factors that affect nerve cell regeneration and growth475. The advantage of this technique is the ability to cut individual axons. The laser system was also used to find out which specific neuron responded to changes in temperature476. They have also been used to create artificial occlusion and haemorrhages in rodents to study the effects of strokes on the health of older people477. A good review of femtosecond lasers by Nishimura et al. is available478.

4.3.8.5 Catheters

Catheters are small tubes which are inserted into the body cavity to inject or drain fluids or to keep a passageway clear. One of the issues associated with catheters is thrombus formation on the surface of these devices. CNTs have been studied as a suitable material for developing catheters. A polyamide (nylon) catheter reinforced with CNTs was developed for insertion into blood vessels in the brain or heart. The flexibility was increased, the damage upon bending was decreased and thrombus formation was reduced479.

Catheters can also be coated with silver nanoparticles to give them antibacterial properties and prevent surface biofilm formation480. Other antibacterial coatings have been reported in attempts to reduce bacterial colonisation on catheter surfaces481.

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Verimetra Inc. (www.verimetra.com) is developing a ‘smart catheter’ that incorporates nano biosensors to provide surgeons with real time data on tissue density, temperature and other environmental factors at the site. This will allow them to develop faster methods for preparing, cutting and extracting tissues and fluids. It will also be useful in a variety of surgical areas like cardiovascular surgery, stent insertion, percutaneous transluminal coronary angioplasty, cerebrovascular surgery etc482.

4.3.9 Wound care and Smart Textiles

4.3.9.1 Silver

Silver, known for its antibacterial properties, has been used for centuries for antimicrobial applications. However, silver nanoparticles smaller than 100 nm are now widely used in consumer products, biomedical equipments, textiles and wound care483. The increased surface area offers a larger contact area and hence increased antimicrobial properties. It can also allow silver coatings to be incorporated into new areas e.g. textile incorporation or coating on medical devices. The silver particles’ ability to kill microbes rapidly by blocking the respiratory pathway or by breaking the outer walls have made it useful for dressing scars, wounds, acne etc.

The antibacterial properties of silver are utilised in wound dressings. Several methods for incorporating AgNPs into dressings are used. These include padding or spraying, surface treatments on hydrogels, adding it onto material compounds and methods like chemical vapour deposition or ionic plasma deposition484. In a multilayer wound care structure, AgNPs are added onto the wound contact layer, normally made of polymers. Actisorbiii, by Johnson & Johnson, is an activated charcoal cloth which incorporates AgNPs for wound dressing. Smith and Nephew’s nanosilver dressing, Acticoativ, has a broad antimicrobial spectrum effective against most common wound pathogens485. Nanosilver dressings are also effective against MRSA486. These dressings protect and cover the wound and provide a moist environment for faster healing. They can also reduce the number of dressing changes required. Nanosilver has been shown to increase the speed of wound healing487.

An eco-friendly and cost effective method for the mass production of nanosilver coated fabrics have been reported488 and socks containing 0.3 % w/w nanosilver are produced by JR Nanotech (http://www.jrnanotech.com/) to protect against foot infections.

A powder of TiO2 nanoparticles coated with silver, developed by the German company ItN Nanovation GmbH (www.itn-nanovation.com), is effective against the SARS virus489. The powder, marketed as Nanozid, can be added to paints and lacquers to coat operating tables, door knobs or door handles in hospitals to deactivate pathogens like bacteria, virus or fungi.

There are concerns surrounding AgNPs. Currently little is know about their fate within the body or the environment. Nanoparticles may react and interact very differently within the body and in the environment. However, ionic silver is known to be toxic to aquatic organisms, raising concerns for the washing of textiles. More toxicity studies are needed, especially due to the number of products currently available using this technology.

A good report on the issues surrounding nanosilver has been published by the Project on Emerging Nanotechnologies490. However, nanosilver adds value to many products and is likely to continue being used extensively in the future.

iii http://www.jnjgateway.com/home.jhtml?loc=USENG&page=viewContent&contentId=09008b9880ec8c74 iv http://wound.smith-nephew.com/UK/Standard.asp?NodeId=2792

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Self-assembling peptides have been used to create a nanofibre mesh extracellular matrix to stop bleeding491. Hemostasis was achieved in different types of wounds and multiple tissues in less than 15 seconds. The matrix is made of biodegradable, synthetic materials which do not contain any blood products, collagens, or biological contaminants and can be applied directly onto the wound. The material can then break down and be used as building blocks for repair, or excreted. It does not expand once applied, thereby reducing the risk of secondary tissue damage as well as problems caused by constricted blood flow.

Electrospun nanofibrous polyurethane membranes have been used in wound care 492. The porous nature of the nanofibres allows oxygen permeability, promotes fluid drainage and controls evaporative loss. The use of the fibres increased epithelialisation and the dermis became well organised. Similarly, electrospun silk fibroin nanofibres were reported as suitable materials for use in wound dressings493.

The concept of smart textiles which can respond to the changes in the environment has been explored to provide new functionalities like self cleaning, sensing and communication. Textiles that incorporate sensors which can measure and monitor changes in the body mechanism have been explored for healthcare applications. In normal cases, sensors are considered separate components which are attached to the textiles to do the monitoring functions. However, the shift is towards functionalised fabrics.

Electrospun CNT fibres have also been proposed for creating smart textiles utilising its strain sensing ability. Piezopolymers electrospun into smart fabrics showed a 35 times increase in strain sensing capability494. CNTs have also been incorporated into the fabrics by immersing textiles in aqueous sulfonated polyaniline-carbon nanotube dispersion. The formed textiles doubled the capacitance and increased the conductivity by four times compared to the sulfonated polyaniline dyed textiles. Stretching experiments have shown that the textiles retained their textile behaviour even after repeated stretching indicating that it can be used as wearable strain gauges495. The EU Biotex project is exploring the possibility of incorporating biosensors into wound dressings to monitor wound healing. A biosensor is programmed to monitor pH and C-reactive protein expression to measure the degree of inflammation. This monitoring and the capability to signal warnings at an early stage will enable clinicians to detect inflammations and take appropriate action. The technology could also be used in other areas such as skin grafts and ulcer treatments.

More information on nanotechnologies in textiles is available in the observatoryNANO Textiles sector report.

4.4 Additional Demand for Research

Development of novel biocompatible composites for bone replacements

Developing biocompatible instruments by better functionalisation.

Developing ‘smart stents’ which can respond to changes in the environment.

Surface modification techniques to enhance implant integration.

Development of novel coatings to immobilise functional molecules on implant surfaces.

Methods for large scale production of ceramic nanocomposites suitable for bone and dental implants.

Studies on the potential of nanotubes in implants.

Development of animal models to test the efficiency and toxicity of various biomaterials.

Development of novel polymers, polymer mixes and self assembled compounds to improve the stability, biocompatibility and integration of implants.

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Methods to prevent implant related infections.

Coatings to improve the stability and surface smoothness of surgical blades.

Development of novel power generating devices which can be implanted to support active implants like pacemakers and cochlear devices.

Methods for the selective stimulation of nerve cells.

Methods for further miniaturisation of implantable active devices.

Techniques to improve the sensitivity of nanosensors and methods for their incorporation in devices.

Development of functionalised fabrics which can sense environmental changes and respond accordingly.

Active vs. Passive biomaterials

Degradation measures

Active indicators of functionality of implant

A reachable standard for purity or impurity of an implant

Shedding standards for various implanted devices

Molecular medical devices

4.5 Applications & Perspectives

Novel materials, techniques and coatings for implants and stents have enabled the creation of implants with much higher biocompatibility and integration. The ability to control and encourage effective cell growth and differentiation using novel nanostructured materials or nanotopography significantly improves the function and lifetime of implants. More recently there has been interest in ‘smart’ implants that are dual purpose. For example, implants that can also deliver anti-infection medication or bone implants seeded with magnetic nanoparticles to treat bone cancer. These will allow improved treatment of diseases and significantly reduce patient discomfort. Reproducible, accurate and scaleable methods of patterning surfaces with nanoscale features are required for the production of new implants.

The anti-bacterial properties of silver are well known; however the safety of using silver nanoparticles, particularly on open wounds, should be addressed.

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5. Novel Bionanostructures Keywords: synthetic cells, nano-emulsion, self-assembly, DNA, molecular motors, nano-cages, bio-nanostructure.

5.1 Definition

For the purpose of this report bionanostructures are defined as engineered nanoscale structures made from biological material or for biological purposes.

5.2 Short Description

Self assembling molecules which form novel nanostructures have already found applications in different areas ranging from drug delivery to cosmetics. Lipid-based nanostructures like liposomes and nanosomes have been used in the cosmetic industry for the last two decades. Proteins e.g. albumin have been modified to create nanoparticles for applications in cancer therapeutics. While the applications of self assembling molecules are slowly reaching the market, fundamental research on the self assembly of molecules to understand the complex cellular processes as well as next generation electric circuits are gaining pace. Synthetic cells which replicate the cellular functions enable to understand the biological processes taking place have implications in drug discovery. Liposomes, polymers and nanoemulsions have been used to create synthetic cells.

In addition, novel fabrication techniques along with nanomaterials have been used to create synthetic molecules by a bottom-up approach. To overcome the constraints in IC manufacturing as explained by Moore’s law, the efforts are now on developing molecular switches. Chemically self assembling molecules which can be switched on and off have been proposed as molecular switches. Carbon nanotubes and nanowires have been used to link together different molecules in logic circuits. Complex molecules like catenanes and rotaxanes have been used to create molecular motors. DNA which can be programmed to self assemble, has been used to develop biosensors. RNA has also been used to produce devices which can be organised to detect molecules and perform drug delivery. They have also been used to build logic gates (AND, NOR, NAND, or OR gates) as well as signal filters.

DNA motors which can transport cargo (like natural molecular motors myosin and kinesin) have been developed. Different types of powering strategies have been developed for molecular motors. Models have been developed for DNA nanomotors powered by energy from DNA and RNA hydrolysis, ATP hydrolysis and DNA hybridisation. The report looks into the development of these novel bionanostructures and its manipulation for different applications.

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5.3 State of R&D

5.3.1 Synthetic Cells

Cells are very complex structures. Understanding the complexity in cellular operations has been a challenge since the concept of cell was incorporated and still is the case. Significant advances have been made in this respect due to novel tools and techniques which have enabled scientists to look inside the cell with much more precision and to manipulate the cytoplasmic molecules to learn how one impacts the other. However explaining this is very complex as these molecules are heterogeneous in nature. Different factors affect the dynamic nature of molecules in the cells and understanding their behaviour to external stimuli as well as other factors will provide valuable information to prevent diseases as well as discover new drugs. Several proposals have been made to mimic the cellular structure and to create a model which is capable of carrying out normal cell operations including self replication. The concept of artificial cells or synthetic cells has received an additional momentum due to the tools and techniques provided by nanotechnology. It is essentially assembling synthetic materials to create a cell which can function similar to a normal cell. The design of such a structure benefits from the tools which are available due to nanotechnology. These tools will help the scientists to work at the biological level (nanoscale) and help them to create cells parts by parts using a bottom up methodology. However, the difficulty lies in the fact that cells are capable of carrying out thousands of functions at a time with high efficiency which may never be matched by an artificial cell.

Several approaches to create containers that can incorporate fluids and molecules as in a natural cell have been proposed. The advantage of such as small container is that small volumes help to understand the molecular reaction systems and self organization at the cellular scale with high efficiency496. It also avoids the requirement for mixing and the small number of molecules in the artificial system enables the study of reactions involving single molecules497. These containers have a small internal volume and may be used for single cell analysis, high throughput screening, protein synthesis or single molecule enzymology498.

5.3.1.1 Liposomes

Phospholipids have been used to create a variety of nanostructures called liposomes, niosomes, nanosomes, cubosomes, solid lipid nanoparticles, nanostructured lipid particles, etc. These structures have already found applications in drug delivery and some of them have been commercialised for use in the cosmetic industry to carry cosmetic ingredients. More information about lipid based nanobiostructures and their applications can be found in the Therapeutics and Cosmetics subsector reports.

Enzymes have been encapsulating inside liposomes499 and have subsequently been used in diagnostics, metabolising toxic reagents and as catalysts500. Successful reactions have been performed inside liposomes to produce proteins. Expression of green florescent protein (GFP) was used as an indicator in these reactions to verify that proteins have been produced501.

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5.3.1.2 Polymers

Polymers have found applications in drug delivery as polymer nanoparticles to carry drugs and to create conjugates to target certain molecules, and can also be used to create synthetic cells. The advantage of using polymers is that it is possible to change the characteristics and properties of these polymers to create vesicles of choice. In addition, polymers are easy to scale up, provide high biocompatibility and increase the stability of volatile materials that are encapsulated. Synthetic polymers provide much more stability to the structure compared to the amphiphilic molecules when used to create synthetic cells. The polymers can also be manipulated to self assemble to form novel structures. These polymers are also called polymersomes. Polymer-protein hybrid membranes that can act as a selective filter have been developed. Such hybrids can incorporate proteins even though the membrane is much thicker than lipid based membranes502.

Recently, energy-transducing proteins bacteriorhodopsins (BR) from Halobacterium halobium and cytochrome c oxidase (COX) from Rhodobacter sphaeroides have been used to create hybrid polymers503. Light-driven transmembrane pH gradients and pH gradient-induced electron release were observed in these polymers which created µA level currents without voltage. The technique has potential applications in high power density devices. More information about polymers and their applications are detailed in the Therapeutics and Regenerative Medicine subsector reports. Polymers have also been used to study the internal movement of molecules inside the cell. A polyethylene glycol (PEG) and dextran system was used to study how molecules behave inside the cell504. The polymers were mixed to form an aqueous two-phase polymer system (ATPS) inside a test tube coated with a dry lipid film. The lipid film got wet in the process and developed an elliptical two molecule-thick cell vesicle containing ATPS. When heat was applied the PEG and dextran separated to form two discrete phases. Dextran, which is heavier than PEG, sank to the bottom of the test tube while PEG went to the top. PEG collected on the inside perimeter of the vesicle wall and dextran collected at the centre forming a phase-separated microcompartment. Different responses can be studied by varying heat and osmotic pressure of the solution.

5.3.1.3 Nanoemulsions

Nanoemulsions are dispersions of nanoscale droplets of one liquid within another505. Nanoemulsions have a number of advantages over emulsions of larger particle size. Unlike microemulsions, nanoemulsions are kinetically stable due to their small droplet size506.

The simple methods used in creating water-in-oil (W/O) emulsions can be used to create water droplets in the femto litre volume range. More sophisticated methods like ultrasonication have also been used to create droplets of smaller volumes. Droplets of the order of 1010/ml can be created. Another advantage is the possibility of testing an entire DNA sequence or a single strand using nanoemulsions.

W/O emulsions have been used for high throughput screening of enzymes507. The process, known as in vitro compartmentalisation, creates droplets which each contain a single gene. These droplets can act as single cells, facilitating the processes of transcription and translation. The oil phase is inert and prevents the diffusion of genes and proteins between different droplets that act as compartments. The advantage of such a system is that it enables the selection of enzymes based on the phenotype or the product due to the genotype activity. The high number of compartments created enables analysis of a larger number of gene libraries quickly and simultaneously.

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Recently, a double emulsion IVC system was developed to sort single genes. Fluorescent markers were used and later sorted with FACS (fluorescence activated cell sorter). In this process w/o emulsions containing single genes were created with one gene per droplet. They were then transcribed and translated using compartmentalisation. The w/o emulsion is then converted into w/o/w emulsion, and the proteins with enzymatic activity change the non-fluorescent substrates into fluorescent products. FACS sorting isolated the genes that encoded active enzymes. These recovered genes can be used for further levels of selection process if required. Studies have shown that creating double emulsions does not disrupt the contents encapsulated inside the emulsions.

Current efforts are focussed on creating a system where the exchange of reagents between compartments becomes possible. Other applications of nanoemulsions are discussed in the Cosmetics and Therapeutics subsector reports.

5.3.1.4 Novel Fabrication techniques and Nanomaterials

The use of synthetic nanomaterials combined with novel fabrication technologies has been explored to create cell like structures. A variety of fabrication techniques have been successfully used to create synthetic membranes. These include surface micromachining, laser interference lithography, nanoimprint lithography, track etching, backside etching and phase separation. The advantage of these fabrication techniques is that by changing the parameters, it is possible to control the characteristics of the cells formed. By changing the thickness of the membrane, internal volume, catalyst used, crystallographic properties etc. it is possible to create a structure that closely resembles natural cells with membranes that can communicate each other. Another advantage is that these cells offer more robustness and rigidity compared to other types of synthetic cells. It can also store the contents for a long time in comparison with cells formed using phospholipids.

Carbon nanofibres (CNF) have been used to create synthetic cells that are able to transfer their contents between each other508. Vertically aligned carbon nanofibres (VACNF) grown using plasma-enhanced chemical vapour deposition (PECVD) were used in combination with silicon micromachining techniques to create synthetic membranes. Microfluidic channels were created using photolithography and etching. The catalyst was lifted off and CNF forests were grown on the surface of the substrate forming a complex interconnected cell mesh. The formed cells were then filled with molecules of interest and sealed with a transparent lid to create a synthetic cell completely isolated from the external environment. The nanosized pores created on the membrane supported the diffusion of fluids between cells thus enabling cell-to-cell communications. A similar approach was used by Fletcher et al.509.

However, controlling the pore size and membrane properties has been challenging. Coating the surface of VACNF with SiO2 was found to be a better way of controlling the membrane characteristics.

Coating polymers on the surface of nanomaterials has also been explored to create active membranes and bionanostructures. Electrically conductive polymers such as polypyrrole (PPy) have been used to control the membrane properties of nanofibre based structures510. The polymer coatings were deposited on the sidewalls of nanofibres using electropolymerization. The ability to control the polymer characteristics helped to modify the properties of nanofibres and the structures. Carbon nanofibres have also been used as an interface in neural engineering and carbon nanotube fibres can promote neuronal cell growth511. The ‘hair like’ conductive wires incorporate the properties of electrodes, permeable microfluidic conduits and the porosity of the CNTs and were found to promote cell growth, migration and proliferation. CNFs have also been used for axonal regeneration by forming a novel nanofibre network which mimics the natural extracellular matrix to promote cell growth, adhesion and proliferation. The main advantage of these fibres are that they are immunologically inert thereby reducing the chances of rejection.

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More information about the use of carbon nanofibres can be found in the subsector reports Regenerative Medicine and Implants, Surgery and Coatings.

5.3.2 Other Self Assembled Nanostructures

5.3.2.1 Albumin, chitosan, lecithin

5.3.2.2 Niosomes

Niosomes are non-ionic surfactant based vesicles that have a similar structure to that of phospholipid vesicles like liposomes and can be used to encapsulate aqueous solutes and act as drug carriers. They are formed by the self assembly of non-ionic amphiphiles in aqueous media. The application of heat or physical agitation helps the process to attain a closed bilayer structure512. The hydrophobic parts are shielded from the aqueous solvent while the hydrophilic head groups are in contact with it. They are used as anti-inflammatory agents513 as well as anti-infective agents514. They have also been used to enhance transdermal drug delivery. Recently, Paolino et al.515 reported the development of a new type of niosome made of α,ω-hexadecyl-bis-(1-aza-18-crown-6) (Bola C16), Span 80® and cholesterol (2:5:2 molar ratio) called Bola-niosomes for percutaneous drug delivery applications. Studies have shown that these niosomes improves percutaneous passage of drugs through human stratum corneum and epidermis and are non toxic. This property has been exploited by the cosmetic industry. The first cosmetic products containing niosomes were developed and marketed by L’Oreal (www.loreal.com) in 1975. They hold the patent for the process of preparing compositions containing niosomes and a water-soluble polyamide for cosmetic and pharmaceutical applications516. The product also had its successors like ‘Niosome Plus’ anti-ageing cream by Lancome (www.lancome.com) which reached the market in the early 1990s.

5.3.2.3 Solid Lipid Nanoparticles

Solid lipid nanoparticles (SLNs) are particles of nanometre dimensions with a solid lipid matrix. They are oily droplets made from lipids which are solid at room temperature and stabilised by surfactants. Their production is a simple process where the liquid lipid (oil) in an emulsion is exchanged by solid lipids, i.e. lipids that are solid at body temperature517. The advantage of SLNs is that there is no for need organic solvents in the preparation, they provide protection from water and can be used for controlled drug release. Two different methods are generally used to prepare SLNs. First, the hot homogenization process in which melted lipids at high temperatures are dispersed in hot surfactant solutions. The second method is called cold homogenisation in which the lipid melt is cooled and dispersed in cold surfactant solution. When these suspensions are subjected to high-pressure, micoroparticles is broken down into SLNs518. These nanoparticles have been used as colloidal carriers to deliver paclitaxel519. Due to their occlusive properties, SLNs are considered as ideal for use in day creams. The compound coenzyme Q10 has been incorporated into an SLN for cosmetic applications520.

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5.3.2.4 Nanostructured Lipid Carriers

In order to overcome the drug loading inefficiency associated with SLNs, a second generation of lipid particles have been developed by mixing solid lipids with liquid lipids. They are called nanostructured lipid carriers (NLC). Different methods have been proposed for creating NLCs. The first is by mixing solid lipids with small amounts of oil (liquid lipids) to improve the drug loading capacity. These are called imperfect type NLCs. When mixed with large amount of oil, the lipid was found to solubilise certain drugs which were not soluble otherwise. They are called multiple type NLCs. A third type called amorphous NLC was created to reduce drug expulsion by mixing special lipids like hydroxyoctacosanylhydroxystearate and isopropylmyristate. Compared to SLNs, NLCs have a distorted structure which makes the matrix structure imperfect creating spaces to accommodate active compounds. The high loading capacity and long term stability offered by the NLCs makes it a superior structure in many of the cosmetic applications. The first product utilising the lipid nanoparticle characteristics reached the market in 2005. The products called NanoRepair Q 10 Cream and NanoRepair Q 10 Serum are marketed by the German company Dr. Rimpler (http://www.rimpler.de/intro/ ).

5.3.2.5 Cubosomes

Cubosomes are discrete, sub-micron, nanostructured particles of bicontinuous cubic liquid crystalline phase521. Bicontinuous cubic liquid crystalline phase is an optically clear, very viscous material that has a unique structure at the nanometre scale522. They are formed by the self assembly of liquid crystalline particles of certain surfactants when mixed with water and a microstructure in a certain ratio. What makes cubosomes unique compared to the parent surfactants is that they offer a large surface area and lower viscosity. The relative insolubility of cubic phase-forming lipid in water allows them to exist at almost any dilution level making them an attractive structure to incorporate into many formulations. Produced by high-energy dispersion of bulk cubic phase, they have high heat stability and are capable of loading hydrophilic and hydrophobic molecules523. Combined with the low cost of raw materials required, they are an attractive choice for cosmetic applications. The technology has been widely investigated for applications in the cosmetic industry. For more information see the Cosmetics subsector report.

5.3.2.6 DNA Nanocages

DNA nanocages are novel structures formed by self assembly of DNA molecules under certain conditions. The structures are hollow inside and can be used to encapsulate nanoparticles and other protein drugs which can be released at a target site using external stimuli. Typical cages sizes range from 2 nm to 200 nm. Although it is possible to change the size depending on the type of applications required, it is preferred to keep the diameter in the range of 1 to 50 nm and the length from 100 nm to 10µm to keep the nanolevel advantages. Kazunori et al.524 have developed a process to produce DNA nanocages in a single step. The cages are formed by the self assembly of three types of two dimensional oligonucleotides by hybridisation. The advantage is that the process doesn’t consume energy and the structure, which has tridirectionally branched DNAs with self-complementary chains, can be obtained by the simple process of mixing oligonucleotides. Highly symmetrical cages (spherical) are obtained if the lengths of the nucleotides are same. However, different shapes (tube, egg etc.) can be obtained by changing parameters such as length of the assembling components, concentration of the DNA etc.

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DNA nanocages offer potential as drug carriers for pharmaceutical applications. The advisable size of the nanoparticle for incorporation into the hollow space of the nanocages is between 2 nm to 200 nm. There is a high probability of particle leakage if the particles are smaller than 2 nm. Different types of particles and molecules can be incorporated into the nanocages, including metal nanoparticles, semiconductive nanoparticles, photocatalytic nanoparticles, magnetic nanoparticles, biological molecules like viruses, proteins, peptides, polysaccharides and other DNA molecules. The desirability of the cages in delivery applications is increased by their ability to bind cell-targeting molecules onto the surface in addition to the slow release capability. The drug release can be controlled by making it to respond to changes in conditions like temperature. It also offers potential in DNA molecular recognition.

5.3.3 Molecular Switches and Molecular Motors

Self assembly of individual molecules to form complex structures is a common phenomenon that takes place in biological systems. The individual molecules self assemble under certain conditions to form higher order structures to carry out various biological operations. The technique has been adopted by researchers to create novel nanobiostructures to create models that mimic the living environment.

Similar principles have been adopted to create molecular switches and motors by altering the conditions and environment. Molecular switches that switch reversibly between two different positions and synthetic molecular motors which functions in a similar manner by utilising the energy from ATP have been developed. Though at the very early stage of development, these novel structures have significance in areas like electronics, drug delivery, drug discovery and drug design. Researchers are already aware that Moore’s law won’t be applicable after a certain period in the future and there won’t be any room at the bottom to incorporate more transistors. In the current environment it is possible to produce integrated circuits with transistors of resolution in the range of 100 nm525. The peak in the number of transistors implies that new methods are required to continue the process of miniaturisation. One of the possibilities lies in manipulating molecules to create novel devices and switches.

The method of ‘bottom-up’ manufacturing is considered as the best possible approach to create molecular devices from individual molecular components. These molecular components can be controlled and manipulated to create logic gates and circuits which are able to communicate to each other and with the external environment526. Unlike a switch which returns to its original state after the switching process, a molecular motor is a device which can do work repeatedly and progressively on a system527. So developing a molecular motor is a much more complex procedure. These devices should also be controllable, reversible and readable at the molecular level.

5.3.3.1 Self Assembling Molecular Switches

A molecular switch has been developed which has an electrically adjustable tunnel junction between the two connecting wires528. The device operates using the oxidation and reduction of the molecules sandwiched between the wires. The redox reaction of the molecules affects the tunnelling height of the two wires and thereby controls the rate of charge flow through the junctions. The tunnelling resistance between the two wires depends on the chemical state of the molecular switches. Monolayers of molecular switches are connected by nanowires at each of the junctions of the grid. However, one of the major problems with this device was that the switch was irreversible and repeated logic operations were impossible. Another issue was that the wires used in the switches were made using conventional lithographic process, limiting the ability to shrink the devices.

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To overcome this problem, CNTs and nanowires were used as links between different molecules in logic circuits. Nanowires of carbon or slilicon can also chemically self-assemble like the molecules used in these switches, and a similar device was synthesised using these529. Rotoxane was used as the molecular monolayer and by applying -2V the switch was closed and a current flow was observed. However, when the voltage was raised to +.7V the switch opened due to oxidation of the monolayer molecules. Repeated switching was observed in these molecular switches and the system was configured to create logic gates.

Molecular devices that can display hysteretic switching between two metastable states can also act as memory cells530. As well as redox activity, light has been used as a stimulus to reverse the states of switches. Dithienylethene derivatives have been widely explored as photoswitches. The switching behaviour of self assembled molecules of dithienylethene on gold has been investigated using STM531. Light of a certain wavelength was used to induce a change in the molecule that resulted in a lengthening of ~0.1 nm. The two different lengths were observed by STM. The switching behaviour was found to depend on the nature, length and position of the spacer linking switching unit to the anchor group. These differences can be explored to create a unidirectional or bidirectional switching.

A detailed review of integrating molecular switches into electronic circuits is available by Mayor et al.532.

A molecular switch that can change states without a change in geometry has been developed. The molecule used is naphthalocyanine, which has two opposing hydrogen atoms which can flip in opposite directions without a change in the geometry of the structure. When the voltage is low, the atoms won’t switch and the state of the molecule can be read. By changing the applied voltage, flip of hydrogen atoms (tautomerisation) can be induced533. This flip can be measured using STM as a change in conductivity of the molecules. It has also been demonstrated that a charge injection in one molecule induces tautomerization in an adjacent molecule. However, the system currently works only at very low temperatures (5K) and requires vacuum preventing its practical use.

Rotaxanes and catenanes are examples of interlocked molecules. They consist of two or more component molecules that are not covalently bonded, but are intrinsically linked, through a mechanical bond. Catenanes consist of two or more interlocked rings. Rotaxanes have a central rod and rings are trapped on this rod by bulky stoppers at the ends. Once complicated to synthesise, they are much more readily accessible through modern templating strategies.

These structures have been used for a number of applications, including molecular switches, molecular motors and nanovalves. The Stoddart group have advanced this field significantly since their first published rotaxane ‘molecular shuttle’ in 1991534. They have used rotaxanes as molecular switches, moving a ring from one position (‘ON’) to another (‘OFF’) using a number of control methods, including pH, electrical and chemical control535. Recently one of their rotaxanes has been used to construct a 160,000-bit molecular electronic memory circuit with a density of 1011 bits per square centimetre, using the rotaxane as the data storage element536. Rotaxanes attached to mesoporous silica nanoparticles to form nanovalves that can be opened with the appropriate stimulus have been reported537. They envisage trapping drug molecules within the pores of the silica and only allowing release under appropriate conditions, for controlled drug delivery.

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Leigh and co-workers have built upon the use of catenanes and rotaxanes to create molecular machines538. One of their rotaxanes works by attaching and removing silyl groups. The presence of silyl ether makes the systems unbalanced and the removal of the group makes the system balanced. The process, called linking, ‘switches on’ the exchange of the macrocycle between the destinations. The linking process moves the macrocyle into the destination. This removal restores the balance by a biased Brownian motion to a new equilibrium state. When the silyl group is attached again (unlinking), the system moves towards an unbalanced state again. The operational cycle is continued to move the macrocycles. Using this method, they have moved 56% of the macrocycle to its destination. The operation is irreversible and the state of machine does not determine the position of the substrate. They have also developed a catenane-based reversible synthetic rotary molecular motor that moves unidirectionally between four positions in a ring, controlled by hydrogen bonding539.

5.3.3.2 DNA Switches

The simplicity of the structure and the interactions that are allowed by DNA has enabled researchers to control the assembly of these structures. Although RNA and proteins are more suitable to create molecular devices due to their structural versatility, the ease of controlling and manipulating DNA has made it an attractive choice to create synthetic devices. This control over the structure has been exploited to create self assembled molecular motors, molecular switches, electronic circuits and enzyme networks540.

Some of the simplest devices created by manipulating DNA are molecular switches. Switching has been obtained by changing external conditions like pH, temperature, ionisation, by inducing chemical reactions, or through the binding of signalling molecules. DNA switches have been made using the i-motif, a three dimensional DNA structure formed by the folding of single strands with correctly spaced cytosine bases. This structure can be closed and opened, effectively turning on and off, by changing the pH541.

Addition of DNA or RNA control strands can induce conformational changes in DNA for use as switches. This use of RNA is significant as it demonstrates that such molecular devices can be controlled by transcriptional circuits542. Some DNA nanomachines have been placed into 2D crystalline DNA arrays while retaining full functionality543.

Self-assembled DNA has been used to develop biosensors. Typically, a gene chip works by incorporating single stranded DNAs that search for its complementary fragments. The transduction process is performed by conventional electrochemical or optical methods.

However, a DNA biosensor which can also perform the function of a transducer has been reported544. The principle is based on hybridization chain reactions where DNA molecules self assemble to form new structures when exposed to a target DNA fragment. When hybridised by a target, a DNA hairpin loop breaks to form a nicked double-helix. This change in conformation can be utilised for biosensing applications. An inverse relationship between the concentration of initiator and the average molecular weight of the resulting structures was seen and may allow for quantitative biosensing. Incorporation of aptamers into the DNA hairpins has been proposed to sense more complex biomolecules like protein, ATP or other smaller molecules.

Ribozymes, catalytic RNA molecules, have been manipulated by control strands to produce logic circuits for the detection of oligonucleotides545. Changes can be configured into the system to create multilayered operational circuits.

An RNA device which can be used in drug delivery or diagnostic applications has been reported546. The programmable nature of RNA has been used to produce a device which can be manipulated to detect thousands of molecules simultaneously and perform cellular information processing operations. The three main components of the device - sensor, actuator, and transmitter - are made of RNA. When the input sensors detect a target, the transmitter is activated and triggers the actuator ribozyme molecules. RNA devices have been used to detect the drugs tetracycline and theophylline within yeast cells547.

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5.3.3.3 DNA Nanomotors

Biological molecular motors, like myosin and kinesin, use the energy from the hydrolysis of ATP to initiate mechanical movements. These motors are essential molecular machines for movement in living organisms. Synthetic molecular motors which derive their operating concept from natural motors have been designed in a similar way using ATP as the energy source. Additionally, studies have been conducted on motors which use energy from the hydrolysis of the DNA backbone and DNA hybridisation. Innovations in synthetic chemistry and genetic engineering enabled by nanotechnology are creating opportunities for scientist to create new motors which can complete a fully cycle of motion without external intervention. Although in the laboratory stage, some of the advances in the area are promising.

Two models of molecular motors powered by DNA and RNA hydrolysis have been developed548,549. In one model the cargo strand is hybridised into an anchorage. The track for the movement of the motor is made of identical single stranded anchorages attached to the double stranded backbone. Once it is hybridised, the enzymes contained in the cargo cut the anchorage, releasing a small fragment. This allows the cargo to stick to the next anchorage. The cargo can be then transferred fully to the next anchorage by a branch migration reaction. The cycle is repeated to complete the operation. In a second system a similar track was used to carry out the movement of the device. However, instead of the ‘10-23’ catalytic enzyme used in the first motor, a recognition site in the cargo-anchorage duplex for a restriction enzyme was present. Unidirectional motion was obtained by the destruction of the track once the motor has moved on. However, this reduces the potential use of these devices. The probability of the cargo moving out of the track can be reduced if the interaction between the cargo and anchorages are strong.

ATP hydrolysis has also been used to move DNA nanomotors and has also been developed to move cargo. Compared with other systems the probability of cargo moving out of the track is minimal as there is covalent anchorage rather than hybridisation. The track is made of four double stranded anchorages tied together by single strands with a double stranded DNA backbone. The cargo has two short fragments of DNA that are attached covalently to one set of anchorages. The second sets of anchorages have sticky ends which are complementary to that of the free ends of the cargo. The gap between the two sets of anchorages is closed by enzymatic ligation and the base pairs are matched by a restriction enzyme. Subsequently, the bonding is cleaved, the cargo is transferred to the second set of anchorages through a covalent attachment and the cycle continues. However, it has drawbacks. The simultaneous use of three enzymes makes the process complicated and backward motion is not possible.

DNA hybridisation has also been proposed as a suitable method to fuel molecular motors. The advantage is that the reaction can be controlled by manipulating the base pairs and concentrations of the control strands. Turberfield et al.550 have developed a hairpin loop design to fuel DNA motors. The DNA hairpin has a single stranded loop with a double stranded neck which can hybridise with a complementary hairpin to release free energy. However, the process is hindered due to the neck-like structure of the hairpins. At least one neck needs to be opened to allow hybridisation to occur. For this purpose, a short strand is introduced as a catalyst which opens one of the loops. This opened loop can then hybridise with another loop releasing the introduced short strand catalyst. The reaction continues and a cascade of hairpin-hairpin reactions are followed551. Although there are currently no motors that successfully use this as an energy source, a model of one has been proposed552.

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5.4 Additional Demand for Research

Although there have been significant advances in the development of synthetic cells and molecular motors, the field is still in its infancy. The current level of research deals with the development of fundamental units of molecular motors and switches. However, research into molecular motors is important as they are central to biological functions. Understanding how these motors functions and the creation of controlled molecular motors will improve our knowledge of biological functions and aid in the design of functional materials accordingly. The following list provides an overview of some of the research requirements for this sector.

DNA machines have shown promise in detecting biomolecules with potential applications in drug delivery. Further research is required to develop this potential for use in cells.

Research on sources of energy for synthetic molecular machines to function is required, particularly natural molecular motors utilising energy from e.g. ATP.

Additional research is required to make multidirectional movement molecular motors.

Multidisciplinary research teams are required to design and assemble molecular devices for electronic applications.

Molecular devices are currently proof of principle. Further research is required to go beyond this level to create integrated molecular devices which can function in the real world.

Additional research is required to create suitable interfaces between molecules and electrodes in molecular electronic circuits.

Another key issue is the development of suitable models for scale up and manufacturing of integrated molecular devices.

Further study on nanomaterials in the design of synthetic membranes is required e.g. to control their self assembling properties and to enhance their integration with natural biomolecules.

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6. Cosmetics Keywords: nano-emulsions, liposomes, niosomes, solid lipid nanoparticles, metal oxide nanoparticles, fullerenes, dendrimers, nanocapsules, cosmetics

6.1 Definition

For the purpose of this report, cosmetics covers different products applied to skin or hair.

6.2 Short Description

The applications of nanotechnology and nanomaterials can be found in many cosmetic products including moisturisers, hair care products, make up and sunscreen.

Almost all the major cosmetic manufacturers use nanomaterials in their products. L’Oréal has a number of nanotechnology-related products in the market and ranks 6th in US in the number of nanotech related patents in US553. The European Commission estimated in 2006, that 5 % of cosmetic products contained nanoparticles.

The application of nanomaterials in cosmetic products has been the subject of continuous discussion in the media, scientific circles and among policy makers for the past few years. Toxicity issues have been raised due to conflicting research papers about the safety of nanomaterials and lack of agreement between researchers on whether the nanomaterials are safe for dermal use. There are a number of classes of nanoparticles used, or proposed for use, in cosmetic applications.

In cosmetics there are currently two main uses for nanotechnology. The first of these is the use of nanoparticles as UV filters. Titanium dioxide (TiO2) and zinc oxide (ZnO) are the main compounds used in these applications. Organic alternatives to these have also been developed.

The second use is nanotechnology for delivery. Liposomes and niosomes are used in the cosmetic industry as delivery vehicles. Newer structures such as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been found to be better performers than liposomes. In particular, NLCs have been identified as a potential next generation cosmetic delivery agent that can provide enhanced skin hydration, bioavailability, stability of the agent and controlled occlusion. Encapsulation techniques have been proposed for carrying cosmetic actives. Nanocrystals and nanoemulsions are also being investigated for cosmetic applications. Patents have been filed for the application of dendrimers in the cosmetics industry.

Other novel materials, such as fullerene, have also appeared in a small number of beauty products.

This report looks into some of the nanotechnologies used in the cosmetic industry and provides an overview of current activity in this area.

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6.3 State of R&D

6.3.1 Vesicular Delivery Systems

Liposomes are vesicular structures with an aqueous core surrounded by a hydrophobic lipid bilayer, created by the extrusion of phospholipids. Phospholipids are GRAS (generally recognised as safe) ingredients, therefore minimising the potential for adverse effects. Solutes, such as drugs, in the core cannot pass through the hydrophobic bilayer however hydrophobic molecules can be absorbed into the bilayer, enabling the liposome to carry both hydrophilic and hydrophobic molecules. The lipid bilayer of liposomes can fuse with other bilayers such as the cell membrane, which promotes release of its contents, making them useful for drug delivery and cosmetic delivery applications. Liposomes can vary in size, from 15 nm up to several µm and can have either a single layer (unilamellar) or multilayer (multilamellar) structure. Liposomes that have vesicles in the range of nanometres are also called nanoliposomes. A new type of liposomes called transferosomes, which are more elastic than liposomes and have improved efficiency, have been developed554. Transferosomes with sizes in the range of 200-300 nm can penetrate the skin with improved efficiency than liposomes555. These self assembled lipid droplets with elastic bilayers are capable of spontaneous penetration of the stratum corneum through intracellular or transcellular routes and have potential applications in cosmetics and drug delivery556.

The first liposomal cosmetic product to appear on the market was the anti-ageing cream ‘Capture’ launched by Dior in 1986. Since then several hundreds of products which utilise liposomal delivery capabilities have been introduced into the market, however only some contain liposomes in the nanoscale. Liposomes are unstable due to their susceptibility to oxidation and the breakdown of liposomal structure. However, formulations have been developed that are more stable by optimising the storage conditions and adding chelators and anti-oxidants557. It is also possible to add cryoprotectants (substances to protect biological tissue from freezing damage)v to liposomes to store them in frozen or lyophilized form.

One of the reasons for the widespread use of liposomes in the cosmetic industry is their ease of preparation and the ability to improve the absorption of active ingredients by skin. The ease of scale up made the widespread use of liposomes in cosmetic applications a reality.

Liposomes have been formed that facilitate the continuous supply of agents into the cells over a sustained period of time, making them an ideal candidate for the delivery of vitamins and other molecules to regenerate the epidermis558. Several active ingredients, biomolecules (e.g. vitamins A and E) and antioxidants (e.g. CoQ10, lycopene and carotenoids) have been incorporated into liposomal membranes to increase their delivery559. Efforts to improve the encapsulation capability of liposomes by adding emulsifiers have been proposed. However, this compromises the barrier integrity and the lipids are easily removed by washing.

Phosphatidylcholine, one of the main ingredients of liposomes, has been widely used in skin care products and shampoos due to its softening and conditioning properties. Liposomes have proved to be a convenient way to deliver phosphatidylcholine.

Liposomes have also been used in the treatment of hair loss. Minoxidil, a vasodilator, is in the active ingredient in products like Regaine (www.regaine.co.uk) that claim to prevent or slow hair loss. It is formulated in liposomes to improve the flux of contents through the skin560. Minoxidil sulphate in propylene glycol (PG)-coated liposomes is also marketed as Nanominox-MS (http://www.sinere.com/nanominox-ms_en.html).

v http://en.wikipedia.org/wiki/Cryoprotectant

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Niosomes are non-ionic surfactant based vesicles that have a similar structure to that of phospholipid vesicles like liposomes. They can be used to encapsulate aqueous solutes and act as drug and cosmetic carriers. They are formed by the self-assembly of non-ionic surfactants in aqueous media. The application of heat or physical agitation helps niosomes to attain a closed bilayer structure561. The hydrophobic parts are shielded from the aqueous solvent while the hydrophilic head groups are in contact with it. They have been used for the delivery of anti-inflammatory agents562 and anti-infective agents563. They have also been used to enhance transdermal drug delivery.

Niosomes were developed and patented by L’Oréal (www.loreal.com) in the 1970s and 80s564,565. The first product ‘Niosome’ was introduced in 1987 by Lancôme (www.lancome.com; a L’Oréal company). The advantages of using niosomes in cosmetic and skin care applications include their ability to increase the stability of entrapped drugs, improved bioavailability of poorly absorbed ingredients and enhanced skin penetration. However, niosomes do not contain GRAS components and are known to be more irritating than liposomes.

Van Hal et al.566 reported that niosome encapsulated estradiol can be delivered through the stratum corneum, which is known to be a highly impermeable protective barrier. Niosomes made from a novel surfactant (Bola surfactant), have been found highly effective for percutaneous drug delivery applications567. Studies have shown that they improve percutaneous passage of drugs through human stratum corneum and epidermis and are non-toxic.

There are hundreds of products on the market using these technologies, particularly liposomal based ones, so interest in new cosmetic applications has deteriorated. However, they are still of great interest for pharmaceutical applications (see Therapeutics subsector report).

6.3.2 Nanoemulsions

Nanoemulsions are dispersions of nanoscale droplets of one liquid within another568. These emulsions are metastable systems whose structure can be manipulated based on the method of preparation to give different types of product e.g. water-like fluids or gels569.

Nanoemulsions have a number of advantages over larger scale emulsions. They can be stabilised to increase the time before creaming occurs, therefore increasing the shelf life of products containing them570. They are transparent or translucent, and have a larger surface area due to the small particle size. It has been found that the smaller the size of the emulsion, the higher the stability and better suitability to carry active ingredients571. The components of nanoemulsions are usually GRAS compounds, therefore they are considered relatively safe systems which can break down to their safe components.

Several cosmetic products are available that use nanoemulsions, including Korres’ Red Vine Hair sunscreen (www.korres.com).

Several companies supply ready to use emulsifiers for creating stable nanoemulsions for cosmetic applications, including Nanocream® from Sinerga (www.sinerga.it) and NanoGel from Kemira (www.kemira.com)572. They produce a product called Nanogel- UV for sun care applications. L’Oreal own several patents on nanoemulsion based technologies573.

6.3.3 Solid Lipid Nanoparticles

Solid lipid nanoparticles (SLNs) are nanometre sized particles with a solid lipid matrix. They are oily droplets of lipids which are solid at body temperature and stabilised by surfactants. Their production is a relatively simple process where the liquid lipid (oil) in a nanoemulsion is exchanged by solid lipids574. This process does not require organic solvents.

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SLNs offer a number of advantages for cosmetic products. They can protect the encapsulated ingredients from degradation. Compounds, including coenzyme Q10575 and retinol576 can remain stable in SLNs over a long time period. They can be used for the controlled delivery of cosmetic agents over a prolonged period of time and have been found to improve the penetration of active compounds into the stratum corneum.

SLNs have occlusive properties making them ideal for potential use in day creams. In-vivo studies have shown that an SLN-containing formulation is more efficient in skin hydration than a placebo577. They have also been found to show UV resistant properties which were enhanced when a molecular sunscreen was incorporated and tested578. Enhanced UV blocking by 3,4,5-trimethoxybenzoylchitin (a good UV absorber) was seen when incorporated into SLNs579.

SLNs have also been tested in perfume formulations. Chanel’s Allure perfume was incorportated into SLNs and nanoemulsions580. SLN formulations delayed the release of perfume over a longer period of time. This slow release profile is also desirable for insect repellents.

Although SLNs are promising for cosmetic purposes they suffer some drawbacks. The production process needs improvement to increase loading capability and stop expulsion of the contents during storage. These problems are caused by the tendency for the particle matrix to form a perfect crystal lattice when solid lipids are used. The high water content of SLN dispersions can also be problematic.

6.3.4 Nanostructured Lipid Carriers

In order to overcome issues associated with SLNs, a second generation of lipid particles have been developed by mixing solid lipids with liquid lipids. These are known as nanostructured lipid carriers (NLCs). Compared to SLNs, NLCs have a distorted structure which makes the matrix structure imperfect and creates spaces to accommodate active compounds. The high loading capacity and long term stability offered by the NLCs make them superior to SLNs in many cosmetic applications. However, Müller et al.581 suggest that SLNs are better for applications such as UV protection where a high level of crystallinity is required for the carrier.

Similar to SLNs, NLCs are also capable of preventing the active compounds from chemical degradation582. They also possess a high occlusion factor and high level of skin adherence properties. When the particles adhere to the skin a thin film layer is created which prevents dehydration. As the size of the particles decreases the occlusion factor increases583. Due to this, NLCs offer the possibility of controlling occlusion without altering the properties e.g. increasing the occlusion of day creams without the glossiness of night creams. It has also been found that the release profile of the active compounds can be manipulated by changing the matrix structure of nanoparticle. Lipid nanoparticles have been found to increase the penetration capabilities of active compounds compared to microparticles584. The lubricating effect and mechanical barrier of lipid nanoparticles are also desired in skin care applications for reducing irritation and allergic reactions.

Lipid nanoparticles can make products appear white, rather than yellowish, which is more desirable for consumers585.

The first products containing lipid nanoparticles appeared on the market in 2005 (Nanorepair cream and lotion, Dr. Rimpler GmbH, Germany), offering increased skin penetration. More than 30 cosmetic products containing NLCs are currently available worldwide (e.g. in South Korea, Supervital products in the ‘IOPE’ line from AmorePacific). Recent reviews of these products and their ingredients have been written by Müller et al.586,587.

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6.3.5 Dendrimers and hyperbranched polymers

Dendrimers and hyperbranched polymers have also been considered for use in the cosmetic industry. Dendrimers are unimolecular, monodisperse, micellar nanostructures, around 20 nm in size, with a well-defined, regularly branched symmetrical structure and a high density of functional end groups at their periphery. They are known to be robust, covalently fixed, three-dimensional structures possessing both a solvent-filled interior core (nanoscale container) as well as a homogenous, mathematically defined, exterior surface functionality (nano-scaffold)588. They are prepared in a step-wise fashion, with an architecture like a tree branching out from a central point. Hyperbranched polymers are effectively disorganised, unsymmetrical dendrimers that are prepared in a single synthetic polymerisation step, making them much more cost-effective than dendrimers. The large number of external groups suitable for multifunctionalisation which is a requirement for its use as a cosmetic agent carrier.

L’Oréal have a patent for a formulation containing hyperbranched polymers or dendrimers which form a thin film when deposited on a substrate589. This formulation could be used for a wide variety of cosmetics e.g. mascara or nail polish. A problem of current polymers is that films are formed too soon after deposition. The new formulations will form highly adherent, water washable films only upon oxidation, usually by exposure to air. It is also possible to incorporate cosmetic agents into the medium to help form films for different applications. They have also developed a formulation comprising of a tanning agent and dendrimers for artificial skin tanning590. Unilever have a patent for hydroxyl-functionalised dendrimers from polyester units to create formulations for use in sprays, gels or lotions591. Several patents have been filed for the application of dendrimers in hair care, skin care and nail care products592,593.

6.3.6 Nanocrystals

Nanocrystals have been used in the pharmaceutical industry for the delivery of poorly soluble actives (Elan; see Therapeutics subsector report). They are aggregates comprising several hundred to tens of thousands of atoms that combine into a "cluster". Typical sizes of these aggregates are between 10-400 nm and they exhibit physical and chemical properties somewhere between that of bulk solids and molecules. By controlling the size and surface area, other properties such as bandgap, charge conductivity, crystalline structure and melting temperature can be altered. The crystals must be stabilised to prevent larger aggregates from forming.

The first cosmetic products appeared on the market recently; Juvena in 2007 (rutin) and La Prairie in 2008 (hesperidin). Rutin and hesperidin are two, poorly soluble, plant glycoside antioxidants that could not previously be used dermally. Once formulated as nanocrystals, they became dermally available as measured by antioxidant effect. This dermal use of nanocrystals is protected by patents594. Other examples are resveratrol and ascorbylpalmitate nanocrystals. Incorporation of nanocrystals into cosmetic products is a straight forward process. Nanocrystals dispersed in water (i.e. a nanosuspension) is admixed with a cosmetic product (typical dilution factor: 50). Nanocrystals for cosmetic applications are commercially available via the contract manufacturer PharmaSol/Berlin.

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6.3.7 Nanoencapsulation and controlled release

Encapsulation technologies have been widely used for a long time in the pharmaceutical industry for drug delivery applications. The emergence of nanotechnology and the availability of novel tools have paved the way for new type of particles which can be used for targeted delivery and that can carry drug payloads for localised action. Such nanosized particles which have a shell and an interior space that can be used to load drugs are called nanocapsules. Different types of nanocapsules are required depending on the nature of the material (hydrophobic or hydrophilic) to be incorporated. It is also possible to functionalise these particles to target specific molecules. Polymers, proteins and different types of biomolecules have been proposed as suitable materials to coat nanocapsules. Polymers have been widely used to create nanocapsules which are then functionalised for various applications. The release of the payload could be organised by an external trigger (ultrasound or magnetic field etc.) or the materials can be designed to release the payload depending on the environment (pH, temperature, light exposure etc.). Such polymers that can respond to the changes and react accordingly are called adaptive polymers.

Hydrophobically modified polyvinylalcohol 10 000 (PVA) with fatty acids (FAs) have been used to create polymeric nanoparticles for cosmetic applications595. PVA was substituted with saturated FAs to give the polymer sufficient lipophilicity and used to test the percutaneous absorption of benzophenone-3 (BZP), a widely used UV filter. As sunscreen filters are meant to work on the periphery of the skin, percutaneous absorption is highly discouraged. Studies showed that PVC nanoparticles can limit the adsorption of BZP, with nanoparticles with a high degree of substitution preventing absorption more efficiently.

The trend in the skincare applications of such polymers is moving from self repair (those that repair the skin damage according to changes in the environment using nanoencapsulation, controlled release etc.) to self predicting polymers that can predict future changes and change their property accordingly to prevent the damage. An example of this is a hydrogel developed by Hu and co-workers596 which can respond to temperature and be used as a facial mask. The patented technology called Facial Switch™ shrinks the hydrogel if the temperature is increased and releases nutrients597. They have also created smart fabrics with antibacterial properties that can respond to external stimuli and fabrics that turn from semi transparent to opaque in response to stimuli.

UK-based Vivamer (http://www.vivamer.com/) have developed a range of responsive polymers that can be attached to or encapsulate nanoparticles, films or gels that incorporate various ingredients. The polymer can be designed to release contents on response to external environment. The polymer capsules could be incorporated into perfumes to release the contents on exposure to sunlight or hot weather. The materials are stable in aqueous solution, non-toxic and biodegradable.

Hollow silica nanoshells, which have already found applications in drug delivery, have been proposed as suitable cosmetic agent carriers. Exilica Ltd (www.exilica.co.uk) have developed a method for making spherical polymer micro-beads and silica nano-shells to be used for molecular containment and slow release. Another company, MiCap (http://www.micap.biz/) is exploring the possibilities of using microbial cells and cell walls for the controlled delivery of perfumes.

6.3.8 Cubosomes

Cubosomes are discrete, sub-micron, nanostructured particles of bicontinuous cubic liquid crystalline phase598. Bicontinuous cubic liquid crystalline phase is an optically clear, very viscous material that has a unique structure at the nanometer scale599. It is formed by the self assembly of liquid crystalline particles of certain surfactants when mixed with water and a microstructure at a certain ratio.

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Cubosomes offer a large surface area, low viscosity and can exist at almost any dilution level. They have high heat stability and are capable of carrying hydrophilic and hydrophobic molecules600. Combined with the low cost of the raw materials and the potential for controlled release through functionalisation, they are an attractive choice for cosmetic applications as well as for drug delivery. However, at present cubosomes do not offer controlled release on their own601. They have also been modified using proteins602,603.

A number of companies including L’Oréal604, Nivia605,606,607,608 and Procter and Gamble are investigating cubosomes for cosmetic applications. Despite this interest, cubosomes have not yet led to products609. The methods of formation must be efficient and cost-effective for scale up before this type of technology can be applied. The presence of large amounts of water during cubosome formation makes it difficult to load water soluble actives.

6.3.9 Nanotechnology for UV protection

Zinc oxide (ZnO) and titanium dioxide (TiO2) particles have been widely used for many years as UV filters in sunscreens. Recently, nanoparticles of these oxides have become popular as they retain the UV filtration and absorption properties while eliminating the white chalky appearance of traditional sunscreens. Products using nanoparticles of ZnO or TiO2 are transparent so have increased aesthetic appeal, are less smelly, less greasy and more absorbable by the skin. Many sunscreens and moisturisers available now use these nanoparticles, including products from Boots, Avon, The Body Shop, L’Oréal, Nivea and Unilever.

A number of modifications to the standard ZnO or TiO2 UV protection system have been reported. Oxonica have developed Optisol, a UV absorption system which contains TiO2 and 1 % manganese610. Dispersing carnauba wax nanoparticles with TiO2 nanoparticles was found to increase the sun protection factor (SPF)611. Nanphase Technologies, who supply nanoparticles to companies including BASF, produce controllable polymeric nanocrystals of ZnO with a size less than 35 nm for personal care applications612.

Other nanoparticles have been developed for UV protection. Rohm and Haas (www.rohmhaas.com) produce hollow styrene acrylate copolymer nanoparticles, ~300 nm in size, that are reported to increase SPF by about 70 % 613. Silica nanoshells have been used by Sol-Gel Technologies (http://www.sol-gel.com) to encapsulate cosmetic ingredients. Their first product ‘UV Pearls TM’ contains UV filters encapsulated in silica shells kept on the top layers of the skin to block UV rays. The product provides improved photostability and reduces the filter uptake by the skin. Another product, ‘Cool PearlsTM BPO’, encapsulates benzoyl peroxide crystals in the silica shells for acne treatment.

Ciba Specialty Chemicals have developed TINOSORB®UV, a 50 % dispersion of a novel broad spectrum organic UV filter614. The particles are less than 200 nm in size, soluble in oils and can be used in sunscreens as an alternative to TiO2 and ZnO.

6.3.10 Nanomechanical and Nanotribological study of hair

Nanotechnology has been used to study the mechanical characteristics of hair. Understanding the differences between hair types allows cosmetic companies to create products to suit individual hair types (e.g. ethnic differences between Caucasian, Asian and African hair) as these can respond differently to activities like shampooing, styling or colouring. The hair care industry is also interested in the effect of water on the nanomechanical properties of hair.

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Bhusan et al. have conducted nanoscratch tests, using Nano Intender II (MTS Nanosystems), to understand properties of different types of hairs at the nanoscale615. The studies demonstrated the difference in scratch resistance of single hair fibres of different ethnic regions as well as the coefficient of friction of hairs. They found that the first 200nm of the hair surface, irrespective of origin, is softer than the underlying layer. A further study into the hair fatigue due to stress and tension using AFM has shown that the ethnic hairs vary in mechanical properties616.

A good review of the nanotribological and nanomechanical properties of different types of skin and hair based on ethnicity, damage, conditioning treatment and various environments was written in 2008617.

6.4 Additional Demand for Research

Although there are many products available in the market using nanomaterials, there are still opportunities to exploit the benefits of nanotechnology in the cosmetic industry.

TiO2 and ZnO are widely used in cosmetic formulations. There is a need for an in-depth study into the toxicity effects of these materials as the studies so far have brought mixed results.

Encapsulation techniques and trigger-release mechanisms have been developed for the active delivery of cosmetic molecules. However, there is a need for reliable, cost effective triggers for controlled release.

Improvements in the drug loading efficiency of lipid based nanoparticles (SLNs and NLCs) and nanocapsules are required.

Better understanding of how lipid nanoparticles modify drug penetration into the skin, how they affect the drug penetration and how they interact with lipids of the stratum corneum is required618.

Fundamental conditions for the formation of SLNs and NLCs and the effect of surfactants used for modifications need to be studied further.

Further in vivo studies on the effect of cosmetics that contain nanomaterials.

6.5 Applications and Perspectives

Although nanotechnology has featured in cosmetic formulations for many years, there are only a handful of technologies used, mainly liposomes, nanoemulsions and metal oxide nanoparticles. These offer advantages and improved characteristics compared with traditional formulations. Many of the newer technologies being investigated for drug delivery may also have applications in cosmetics. Along with the formulations discussed in this report, nanoparticles of silver, copper, silicone and silica have been reported as ingredients for cosmetics. A number of companies also claim to use fullerenes in their products due to the radical scavenging properties619. However, there are concerns over their toxicity.

It should be assumed that research in this area is being affected by calls for bans and a moratorium on nanotechnology based cosmetic products by many organisations. This has led to reluctance to talk about nanotechnology in cosmetics by a number of organisations.

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A list of products claiming to use nanomaterials has been compiled in the report ‘Nanomaterials, sunscreens and cosmetics: Small ingredients, big risks’620. (A response to this report was published in Cosmetics & Toiletries Magazine in January 2009621). The Nanotechnology Consumer Products Inventory, run by the Project on Emerging Nanotechnologies at the Woodrow Wilson International Center for Scholars, aims to list all nanotechnology-based consumer products available. However, current regulation of cosmetics means that these products may or may not actually be using nanomaterials, and there may be many more that do.

Which?, the UK-based consumer group, published a report titled “Small Wonder? Nanotechnology and Cosmetics” in November 2008622. In this they contacted a number of cosmetics companies about their use of nanotechnology. Only eight companies agreed to give details of their products. This could indicate a worrying silence and concealment of the use of nanomaterials in cosmetic products. The report highlighted a lack of information available on nanoscale ingredients in cosmetics and the safety concerns about this. They proposed the development of new regulation for reporting and assessing the use of nanomaterials in cosmetics.

Updated regulation and increased information sharing has also been called for by the SCCP (EU Scientific Committee on Consumer Products) and the European Commission. Recently, MEPs approved updates on cosmetic legislation623. The changes apply across all 27 member states and will come into effect from 2012. The new regulations introduce a safety assessment procedure for all products containing nanomaterials, which could lead to a ban on a substance if there is a risk to human health. Also, any nanomaterials present in cosmetics must be mentioned in the list of ingredients on the packaging. The establishment of an official EU-wide register for cosmetics was also proposed.

Toxicity of nanomaterials is currently the subject of an increasing amount of research. Further investigation by academia and industry is required before materials can be deemed as safe. This research, along with better regulation and reporting, will enable consumers to choose products with confidence. This in turn will allow companies to benefit from these novel technologies in the long term while retaining customer confidence.

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