chapter-1 review of literature -...

Chapter-1 Review of Literature


Outline

Introduction 3 Historical Perspective 3 A Survey of Synthesis, Properties and Biological Applications of

Important Nanoparticles 6 (i) Gold Nanoparticles (ii) Supramagnetic Iron Oxide Nanoparticles (iii) Silica Nanoparticles (iv) Quantum Dots (v) Carbon Nanotubes (vi) Other Inorganic Nanoparticles (vii) Dendritic Polymers (viii) Liposomes

Biocompatibility of Nanoparticles 23 References 26

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Introduction

The term nanoparticle is used to describe a wide variety of materials of

submicron size. An internationally accepted definition for nanoparticles does not

exist. It is used differently, according the context of material being described.

According to a recent definition suggested by British Standards Institution

“Nanoparticles are the particles with one or more dimensions at the nanoscale.” They

have defined the nanoscale as dimensions of the order of 100 nm or less. It is critical

length scale at which certain novel size related properties develop and the materials

start behaving differently than the molecules or bulk material (PAS71 2005). Here

novel properties refer to optical magnetic and electrical properties which typically

appear in materials below 100 nm size. However, many a times, besdes “strictly

nano” (1-100 nm) all submicron colloidal particles i.e. particles with at least one

dimension in the scale of 1-1000 nm, also called mesoscale, are referred as

nanoparticles, to include organic polymers and vesicles widely used in the area of

drug delivery (Leary et al. 2005, Gelperina et al. 2005). At this scale the properties of

the matter are different from atomic or molecular properties which are governed by

laws of quantum mechanics, or the properties of bulk materials determined by laws of

classical physics. So the dimension of 1-100 nm may be considered as an intermediate

state between atomic or molecular state and bulk state where materials exhibit some

unexpected and unusual new properties which can not be defined by classical laws of

physics (Hochella, Jr. et al. 2008). It is these unusual properties which have attracted

immense attention of researchers from almost every field science including biology

and medicine. Investigation of novel properties of nanoparticles and their application

has become very active area of research.

Historical Perspective

Nanoparticles have been in use in pottery and medicine since ancient times.

Historical evidences suggest that Gold nanoparticles were used as drug by Chinese

during 2500 BC. Red colloidal gold is still in use under the name of Swarna Bhasma

and Makaradhwaja” in traditional medicine system of India called Ayurveda, which

dates back to 1st millennium BC (Bhattacharya & Mukherjee 2008). Recent scientific

study of the a vessel of Roman period (4th century AD) called “Lycurgus Cup,” kept

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in British Museum, London shows the use of Nanoparticles of Gold-Silver alloy for

its decoration (Freestone et al. 2007). Similarly, churches of Middle Ages used gold in

colloidal state trapped within the matrix of glass to make aesthetically pleasant ruby

coloured glasses of different hues and colours (due to the formation of nanoparticle of

different sizes). In 16th Century Europe an aqueous form of colloidal gold called

“Aurum Potabile (drinkable gold)” was thought to have curative properties for many

diseases (Caseri 2000). In 1857 Michael Faraday described methods for synthesis of

stable aqueous dispersions and optical properties of gold nanoparticles (Faraday

1857). In 1915, in his famous book “The World of Neglected Dimensions”, Wolfgang

Ostwald recognized colloidal particles as unique state of matter, whose particles “are

so small that they can no longer be recognized microscopically, while they are still

too large to be called molecules.” However the credit of realising the enormous

potential of nanoparticles and their possible implications

in different fields is given to Richard P. Feynman. In his

classical lecture in 1959 at California Institute of

Technology (Caltech) during Annual meeting of the

American Physical Society Feynman has stated:

“............I would like to describe a field, in which

little has been done, but in which an enormous amount

can be done in principle. This field is not quite the same

as the others in that it will not tell us much of

fundamental physics (in the sense of, What are the

strange particles?)..........

Richard P. Feynman (1918-1988)

........... In the year 2000, when they look back at this age, they will wonder

why it was not until the year 1960 that anybody began seriously to move in this

direction.....

When we get to the very, very small world---say circuits of seven atoms---we

have a lot of new things that would happen that represent completely new

opportunities for design. Atoms on a small scale behave like nothing on a large scale,

for they satisfy the laws of quantum mechanics. So, as we go down and fiddle around

with the atoms down there, we are working with different laws, and we can expect to

do different things…………

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………The principles of physics, as far as I can see, do not speak against the

possibility of maneuvering things atom by atom. It is not an attempt to violate any

laws; it is something, in principle, that can be done; but in practice, it has not been

done because we are too big ………

………The problems of chemistry and biology can be greatly helped if our

ability to see what we are doing, and to do things on an atomic level, is ultimately

developed---a development which I think cannot be avoided.”

There is Plenty of Room at Bottom (Feynman 1959).

Because of such brilliant foresight and visionary thinking about the properties

of nanoparticle and their future implications, Feynman is often referred as Father of

the field of nanoparticle research (Erren 2007). Later Eric Drexler Published His book

“Engines of Creation: The Coming Era of Nanotechnology” which brought

Feynman’s vision to a broader audience (Wilsdon 2004). It gave a detailed view of

concepts, its potentials and even dangers if it is misused.

However the, availability of suitable methods for synthesis of nanoparticles

of uniform size and techniques for their characterization was a limiting step in

realization of Feynman’s dream till early nineties. During this period work in

groups of Turkovich, Frens, Stöber, Iijima, Bawendi and others has resulted into

development of synthetic methods to produce uniform nanostructures of gold,

silica, carbon, cadmium etc. with sizes in the range of 1 to 100 nm (Jaiswal &

Simon 2004). During the same period development of technique like Atomic Force

Microscopy, Scanning Tunneling Electron Microscopy, Dynamic light scattering

have enabled detailed study and manipulation of materials at nanoscale. This has

set the stage for a burst of research activities involving study, manipulation and

application of nanoparticles. Initially nanomaterials research was mainly focussed

in the area of materials science, mainly in development of microelectronic and

optoelectronic devices. However, in last 10 years, the dramatic size dependent

optical, electronic, magnetic, and mechanical properties of nanoparticles have

attracted attention of researchers from almost every science including biology and

medicine (Tan et al. 2004).

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A Survey of Synthesis, Properties and Biological Applications of

Important Nanoparticles

The high aspect ratio and resultant special properties exhibited by matter at

nanoscale has been a great attraction for development and study of nanoparticles from

every possible material. In order to study and exploit the enormous potential provided

by “nanoscale”, every possible building block for development of nanoparticles is

being explored. The design and synthesis and surface modification of nanomaterials

with novel properties has become an exciting area of research. Considering that

primary limit is only the size range of 1-1000 nm, there is virtually no limit of the

possible ways, for fabrication of nanoparticles. Indeed, almost limitless types of

nanoparticles of different shape, size and surface properties are being developed from

a range of materials of inorganic, organic, biological or hybrid nature. Availability of

new methods of fabrication and tools for characterization and manipulation has

resulted in a variety of innovative application of nanoparticles (Rao & Cheetham

2001). In last ten years, the nanomaterials research which was initially restricted to

materials science has seen cross-disciplinary expansions to almost every field of

science including biology and medicine.

The novel properties of nanoparticles hold enormous potential for applications

in both basic and applied area of research in Biology. Addressing different problems

in biology using the nanoparticles has been an active area of research. This merger of

nanomaterials research with Biotechnology has given birth to a new discipline called

Nanobiotechnology, in which innumerable types of nanoparticles are being constantly

developed and investigated for better understanding of biological system as well as

development of new products and technologies (Niemeyer 2001, Roco 2003).

Nanobiotechnology is now a burgeoning field, having influence in almost

every aspect in biomedical research. Because of the diversity of the field it is almost

impossible to review different aspects of every type of nanoparticle. So let us examine

few most important nanoparticles which are being studied for applications in biology

and medicine.

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i) Gold Nanoparticles

Amongst an array of nanomaterials being investigated for applications in

biology, Gold nanoparticles (GNP) are probably most extensively studied

nanoparticles. In fact, as described in previous section, gold nanoparticles has been in

use since ancient times in one or other form, be it in traditional Chinese, Indian or

European medicine or for decorative purpose in “Lycurgus Cup.” The work of

Faraday has pointed towards specific optical properties of colloidal gold, long before

the recognition of changing properties of matter at nanoscale. Development of

synthetic protocols for reproducible synthesis of monodisperse gold particles, initially

by Turkevich and then by Frens gave a further boost to gold nanoparticle research

Currently a number of techniques for preparation of gold nanoparticles of

various shape and size in both aqueous and organic medium are available (Daniel &

Astruc 2004). Most of these protocols employ reduction of gold ions from a gold salt in

presence of a capping agent which prevents the aggregation of particles. The most

common method of synthesis of gold nanoparticles for application in biology is the one,

first described by Turkevitch in 1951 and further developed by Frens in 1973

(Turkevich et al. 1951, Frens 1973). In this method, GNP are obtained by heating a

solution of HAuCl4 in presence of Sodium citrate. In this reaction citrate initially acts

as reducing agent to convert Au(III) to Au(0) to form particles and then it acts as a capping

agent by adsorbing to nanoparticle surface and repelling particles from each other,

thereby preventing aggregation. This protocol can be used to prepare spherical sizes

from a range of sizes from 16-147 nm (Daniel & Astruc 2004). The temperature, ratio

of gold to citrate, and the order of addition of the reagents control the size distribution

of gold nanospheres generated by this method (Eustis & El-Sayed 2006). In a slightly

modified method, smaller size (Approx. 3.5 nm) of GNP can be prepared by reducing

Chloroaurate ions with cold solution of Sodium Borohydrate (NaBH4) instead of

heating in presence citrate ions. In later case, citrate acts as the capping agent only, as it

cannot reduce gold salt at low temperature (Jana et al. 2001). The small nanoparticles

prepared in this way can be used as seed for preparing particles of higher size or

different geometry. Very small nanoparticles (1.5-5.2 nm) with narrow dispersity can be

prepared using two phase Burst-Sciffrin method. In these methods the particles are first

synthesized by reducing chloroaurate ion in organic medium, in presence of thiols,

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xanthates, disulphides, phosphines, amines or carboxylates as capping agent. Then

depending upon capping agent the particles can be dried and re-dispersed in other

organic solvents or water. This method has been method of choice for synthesis of so

called monolayer protected clusters (MPCs) (Daniel & Astruc 2004).

A variety of methods are available for conjugation of ligands and

biomolecules with GNP for different biological applications. The gold particles has

been directly obtained as biomolecule protected GNP using some peptides, lipids and

other ligands as capping agent (Guo & Wang 2007). Passive adsorption of antibodies

and proteins with colloidal gold has been in practice since long time. However, most

commonly used current methods for specific immobilization of molecules at GNP

surface exploit strong affinity of thiols with GNP. A –SH modified biomolecule eg.

mercaptoalkylolgonucleotides can be directly chemisorbed to GNP surface by

formation of strong Au-S bond. Alternatively GNP can be functionalized using a

bifunctional linker having –SH group at one end. Then the desired ligand can be

conjugated at the surface of functionalized nanoparticle using a suitable conjugation

chemistry for the functional group at other end (e.g. EDC coupling for –COOH group

at functionalized GNP and –NH2 group at proteins) (Sperling et al. 2008).

Figure 1.1: A typical UV/Visible spectrum of Gold nanoparticles. Inset: Different sizes of Gold nanoparticles.

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Because of Au, being a heavy element gold nanoparticles form sharp electron

micrographs with excellent contrast. In fact, because of its electron density, availability in

a range of size, ease in synthesis, versatility in adsorption to macromolecules and its

ability to be measured morphometrically GNP conjugated to antibodies or other specific

ligands serves an excellent cytochemical marker in Electron Microscopy (EM). Actually

colloidal gold has been in use in EM long before term nanoparticles came in fashion

(Bendayan 2001). However, most popular property of the GNP for biomedical

applications is its unusual optical properties which arise due to localized Surface Plasmon

resonance (SPR). The SPR in GNP arises due to resonance of collective oscillation of

electron cloud of 6S electrons of conduction band at surface (surface plasmon) of

particles and electromagnetic field of incoming light. Because of SPR, GNP show heavy

absorption of visible light at 520 nm. This gives brilliant red colour to GNP which varies

according to their size (Figure 1.1). The resonance frequency (absorption maxima)

depends upon the shape size and the dielectric constant of the particle and medium. The

absorption maxima of GNP show characteristic shifts upon changes in environment at

particles surface and change in inter-particle distance. A change in local environment due

to binding or conformational changes at particle surface and thereby change in electron

density results into resonance frequency. The resonance frequency also shifts dramatically

with change in inter-particle distance. These two characteristics form the basis for

probably most popular applications of GNP in detection of biomolecules (Sperling et al.

2008). Measurable shifts in SPR spectra of GNP has been observed upon pH induced

conformational changes in yeast iso-1-cytochrome c adsorbed at its surface (Chah et al.

2005). A GNP based protease assay with nanomolar sensitivity using C- and N-terminal

cysteinyl derivative of peptide substrate specific to target protease has been reported

(Guarise et al. 2006). Lactose conjugated GNP havs been shown to undergo visible colour

changes upon binding with lectins. The colour change was reversible and dependent upon

lectin concentration (Otsuka et al. 2001). Using same principle competitive colorimetric

assay for detection of protein protein interaction has been poposed. Visible colorimetric

changes have been observed for interaction of Thyroglobulin with Concanavalin-A and

other lectins using mannose modified GNP. Even binding constants could be calculated

based on the wave length shifts (Tsai et al. 2005). Pioneering work from Mirkin’s group

has resulted into a number of applications of SPR properties of GNP conjugated with

oligo-nucleotides for detection of DNA, RNA and proteins (Nam et al. 2003, Han et al.

2006, Sperling et al. 2008).

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Owing to its high absorbance spectra gold nanoparticles can quench many

fluorophores and act like an acceptor in a Förster Resonance Energy Transfer (FRET)

pair. GNPs have been proven to be better than organic quenchers with higher quenching

efficiency and over greater distances. Using a Oligo conjugated GNP and a Cy5 labeled

oligo probes Mirkin’s group has reported a “Nano-flare” which could be used detect

desired RNA species inside cell (Seferos et al. 2007). Molecular beacons having GNP

as quencher has been shown to be 8 fold more efficient in detecting a single base DNA

mismatch than other fluorophore labelled molecular beacons (Dubertret et al. 2001).

Because of very high curvature, Raman Scattering of molecules closed to GNP surface

is greatly enhanced. This is called Surface Enhanced Raman Scattering (SERS). This

property can be used to detect the biomolecules potentially at single molecule level.

Recently SERS effect produced by GNP through Raman active reporters has been

demonstrated for both in vivo and ex-vivo detection of different analytes and proteins,

even beyond pico-molar sensitivity (Kneipp et al. 2008).

Gold nanoparticles are naturally taken up by many cell types and do not cause

any detectable cytotoxicity. This fact has been extensively exploited for delivery of

DNA inside cells. Antisense oligonucleotide conjugated GNP has been shown to be

spontaneously taken up by cells and bring down the expression level of target genes

(Rosi et al. 2006). Cationic polymer agents show improved transfection efficiency

when conjugated to GNP (Thomas & Klibanov 2003). Because of the versatility in

surface modification of GNP with specific ligands GNP based transfection agents also

have potential of targeted gene delivery. After excitation with a suitable light source

GNP get heated up and dissipate heat to its immediate environment. This property is

called hyperthermia. With careful design and excitation with a suitable light source

heat produced by hyperthemia in GNP is sufficient enough to produce localized

heating and killing a cell or manipulating tissue environment. Together with a variety

of available method for selective targeting, hyperthermia has been used for selective

killing of cancerous cells both in vitro and in vivo, either directly by heating or by

localized release of anti cancerous molecules trapped in hydrogels (Everts 2007).

ii) Iron oxide nanoparticles

Nanoparticles from iron oxide have gained attention of researchers because of

their extra ordinary magnetic and optical properties. Initially research efforts for

applications of iron nanoparticles (INP) were limited to development of high density

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magnetic storage media primarily because of lack of sufficient surface chemistry.

However, recent development at synthetic front has enabled numerous application of

INP in biosensing, protein separation and purification, imaging, drug delivery,

diagnostics etc (Gu et al. 2006).

There are three most common approaches for synthesis of magnetic iron oxide

(Fe3O4 and γFe2O3) nanoparticles: (1). The classical co-precipitation of aqueous

Fe2+/Fe3+ ions in presence of an alkali hydroxide or ammonia solution; (2) thermal

decomposition of an iron complex under high pressure and (3) Sonolysis of organo

metallic complexes (Cornell & Schwertmann 1996). The particles synthesized with

these methods tend to aggregate through non-covalent interactions. This can be

avoided by using stabilizers. Many surfactants and other organic compounds with

specific functional groups have been developed for this purpose (Lu et al. 2007).

Among these, water soluble stabilizers like PEG, polyvinyl alcohol, polyamines etc.

are especially useful for synthesis of INP for biomedical applications. The stabilizers

can be incorporated at the time of synthesis itself or during a coating step after the

synthesis of particles. The stabilizers present in medium at the time of synthesis of

INP prevent particle coalescence during formation. The particles size can also be

controlled by varying stabilizer concentration (Si et al. 2004). Specific biomolecular

ligands can be conjugated to INP surface using specific functionality of stabilizers,

which in turn can be used for selective targeting to specific cells, tissues or organs.

INP serve excellent contrast agent for magnetic resonance imaging (MRI). The

ability of INP as MRI contrast agent, together with potential for selective targeting

has resulted into wide range of studies for potential applications in MRI based

imaging and diagnostics. Several antibodies and other ligands has been conjugated to

INP and tested for MRI imaging of tumours (Laurent et al. 2008). Recently,

simultaneous MRI imaging and destruction of breast cancer cells was demonstrated

using targeted delivery of INP, entrapped into PLGA (Poly-D, L-lactide-co-glycolide)

nanoparticles containing anticancer drug Doxurubicin and antibody Herceptin1.

Fluorophore conjugated INP called magnetofluorescent nanoparticles have also been

developed for multimodal optical and MRI imaging (Mrinmoy De et al. 2008). Using

INP conjugated with cell surface receptor specific ligands, a modified cellular

Enzyme Linked Immunosorbent Assay (ELISA) called Cellular Magnetic Linked

Immunosorbent Assay (C-MALISA) has also been developed (Burtea et al. 2005).

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Small INP are magnetized in presence of an external magnetic field. This

property has been used for magnetic separation of proteins. Xu et. al. have

successfully demonstrated affinity purification of recombinant His-Tag proteins using

Ni-NTA terminated INP (Xu et al. 2004). They have incubated Ni-NTA terminated

INP with lysate of recombinant His-tag protein (Figure 1.2). The protein binds with

INP because high affinity of His-Tag with Ni-NTA present at its surface and can be

pulled out easily using a magnet. Use of magnet also makes washing and elution of

recombinant proteins easier than traditional Ni-NTA resin columns. Moreover,

because of its high surface to volume ratio, fast movement and good dispersability

this system should perform better than traditional micron sized particles and should

show better binding rate and binding capacity. High binding rate should also reduce

non-specificity, because due to quick binding of target molecule, availability of

unoccupied area for nonspecific adsorption will be reduced (Gu et al. 2006).

Figure 1.2: Purification of His-tag proteins using Ni-NTA terminated INP (a) Optical images of a magnet attracting 6c and 6c-6xHis-GFP; (b) surface-modified magnetic nanoparticles selectively binding to histidine-tagged proteins in a cell lysate; (c) SDS/PAGE analysis of the cell lysate (lane 1), the fraction (lane 2) washed off a commercial Ni2+–NTA column; the fraction washed with the freshly made 6c using imidazole solution (10 mM, lane 3; 80 mM, lane 4; 500 mM, lane 5); fractions washed off the reused 6c using imidazole solution (10 mM, lane 6; 20 mM, lane 7; 500 mM, lane 8); and (d) the cell lysate (lane 1), the molecular weight marker (lane 8), the fractions washed from the freshly made 9b (lanes 2 and 3), boiled 9b (lanes 4 and 5), and the commercial HiTrap affinity column (lanes 6 and 9), and the concentrations of imidazole are 10 mM (lanes 2, 4, and 6) and 500 mM (lanes 3, 5, 9). 6c: A NTA terminated FePt magnetic nanoparticle; 9b: NTA terminated Core Shell magnetic nanoparticle. (Reproduced with permission from Gu et. al. 2006; © Royal Society of Chemistry, UK)

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The magnetic property of INP also holds potential for applications in drug and

gene delivery. A number of studies have focused on development of biocompatible

INP with positively charged surfaces for binding to and delivery of DNA inside cell.

Often suitably modified INP itself is able to transfect DNA in many cell types.

Application of strong magnetic pulses has been shown to promote transfection levels

(Dobson 2006). Magnetic INP conjugated with anticancer drug Methotrexate, which

can kill many cancerous cells overexpressing folate receptor has shown higher

accumulation of INP and release of methotryxate and killing of cultured breast and

cervical cancer cells (Kohler et al. 2005). Alexiou et al. has shown that accumulation

of INP upon application of magnetic field at tumour location (Alexiou et al. 2006).

Like GNP and other metal nanoparticles INP also show hyperthermia. Iron

nanoparticles targeted to tumours can be used for simultaneous MRI imaging and

destruction by heating. Ability of INPs to be targeted to tumours, MRI contrast agent,

delivery of anticancer drugs into tumour cells and hyperthermia makes them ideal

candidate for cancer management. An INP conjugated to a tumour specific ligand and

a anticancer drug will be a wonderful chemotherapy strategy having ability for

simultaneous MRI detection and multimodal destruction of tumour by

chemotherapeutic agent as well as a heating by a LASER.

iii) Semiconductor

Quantum Dots

Semiconductor quantum dots

are probably first nanomaterials to be

adopted for biological applications.

Quantum Dots (QD) are small

nanocrystals of semiconductor compounds (eg. CdSe, CdTe, CdS, ZnSe, InP and InS)

which have got importance because

of their excellent fluorescence

properties. Owing to their small size, semiconductor QD can exhibit a rare quantum

mechanical phenomenon called quantum confinement. This gives rise to many

important features of the excitation and emission spectra of QD, distinct from

conventional organic fluorophores (Chan & Nie 1998). Notably, QD exhibit high

quantum yield, are resistant to phtobleaching, broad excitation spectrum and narrow

Figure 1.3: Schematic representation of a typical Core Shell quantum dot.

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emission spectrum. The QD can be excited for hours without any significant loss in

photostability making them fluorophore of choice for experiments involving

recording of fluorescence for longer periods. The narrow emission spectrum of QD

depends upon the size and constituent elements. Depending upon requirement, QD

with different sizes and composition having emission spectra ranging from UV to far

red region are available. Moreover, the broad excitation range allows excitation of

multiple QDs using same wavelength which will emit light at different wavelength

depending upon their size and composition. These unique optical properties of QD

make them excellent candidates for multiplex assays, enabling simultaneous probing

of different molecules or cells labelled QDs of with different colour, using single

excitation source. Because of their unique features QD are considered next generation

fluorescence marker with potential of revolutionalizing the fields of diagnostics and

imaging (Bruchez, Jr. et al. 1998, Cai et al. 2007).

A number of synthetic approaches for QD have been explored. Most common

and successful methods involve synthesis in organic solvents. QD of CdTe, CdSe, and

CdS can be obtained by injecting liquid precursors (eg Dimethyl cadmium or

Cadmium oxide and Selinium for CdTe) into hot (300°C) co-ordinating organic

solvents. The size of crystals depends of temperature, time of reaction and amount of

limiting reagent. Usually the resultant crystal are coated with a shell of higher band

gap material like ZnS and CdS (Michalet et al. 2005). This gives the so called name

“Core Shell” nanoparticles to QDs (Figure 1.3). The shell coating improves the

fluorescence yield as well as provides protection against oxidation to core. The QD

synthesized through these methods are usually not soluble into water, a prerequisite

for most biological applications. This limitation has been overcome by

functionalization of QD surface by a hydrophilic ligand or encapsulating into a thick

organic or inorganic coating. The coat improves colloidal stability, insulated

deterioration of QD surface in aqueous media. It also as provides points for

attachment of specific ligands for different biological applications.(Medintz et al.

2005) The biomolecules can be attached to QD surface by a partial ligand exchange.

Alternatively they can be covalently linked by taking advantage of functional groups

provided in surface coating.

The development of surface chemistry for conjugation of biomolecules has

dramatically increased the utility of unique optical properties of QD for applications

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in biology. Streptavidin coated QD have been used to label biotinylated protein

present at cell surface (Howarth et al. 2005). The extraordinary photostability, unique

optical properties together with ability to conjugate antibodies have led extensive

application of QD in fluorescence immuno labelling and imaging applications

(Sukhanova et al. 2004, Gao et al. 2004, Howarth et al. 2005, Pinaud et al. 2006).

Jaiswal et al. have demonstrated long term multi colour imaging of live cells upto 12

days, using multi colour quantum dots. The cells were labelled by endocytic uptake of

QD or using QD conjugated to antibodies against cell surface markers (Jaiswal et al.

2003). Because of high quantum yield, QDs along with organic donors has been used

as FRET pair in a number of applications. An inhibition assay for biomolecules using

GNP and QD as FRET pair has been demonstrated for determining avidin

concentration in sample solution (Oh et al. 2005). Because of the size dependent

emission spectra QD has been used to determine the optimal size of nanoparticles for

renal clearance (Choi et al. 2007). So et al. have used energy of luciferase catalysis to

create a QD based self illuminating in vivo imaging system. As this system does not

require any external energy source for excitation, the problems of opacity and

autofluorescence are greatly reduced (So et al. 2006). Using streptavidin coated QD

and biotin tagged phage detection of up to 10 bacterial cells in artificial samples has

been reported (Edgar et al. 2006). Surface modified QD has been used as a

multipurpose vector delivery of siRNA, real time tracking and ultrastructural

localization (Yezhelyev et al. 2008).

Clearly, QD have appeared to be a boon for many fluorescence based

applications in biology. However, semiconductors are made from toxic heavy metals

like cadmium which can be toxic potential. Recent research has shown that QD are

toxic for cells because of release of Cd++ ions inside cells. This poses a big limitation

for their in vivo imaging applications. Photoblinking is another limitation which

reduces their applications requiring single molecule detection (Alivisatos et al. 2005).

iv) Silica Nanoparticles

Among an array of inorganic nanomaterials, silica nanoparticles (SiNP) have

made their own niche because of easy synthesis and availability of rich surface

chemistry. SiNP are generally synthesized by hydrolysis followed by condensation

polymerisation reaction of Tetraethyl orthosilicate (TEOS) catalyzed by liquid

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ammonia in following two ways. The first method called sol-gel method or Stöber

synthesis involves ammonia induced hydrolysis of TEOS in presence of water into

ethanol solution under constant stirring or sonication. The particle size can be

controlled by changing ammonia and water content (Rossi et al. 2005). In second

method the polymerisation reaction takes place in confined environment, into “reverse

micelles” formed by emulsification of a surfactant into a hydrophobic solvent (Figure

1.4). The hydrophilic core of reverse micelle acts as nanoreactor for the

polymerisation reaction. The size of nanoreactor and thereby the size of the particles

can be changed by changing the water to surfactant ratio (Bagwe et al. 2004). Usually

the particles prepared using these methods are functionalized by coating with a shell

of silica using functionalized alkoxysilanes. Depending upon the silane used, the

compact shell provides desired functional groups which can be used for conjugation

of biomolecules.

Figure: 1.4: Synthesis of dye doped SiNP by Reverse micelle method. Upper Panel: Polymerization reaction of TEOS in presence of NH4OH. Lower Panel: Formation of DDS in reverse micelle. Dye and TEOS get confined inside the hydrophilic core of reverse micelle which acts as a “nanoreactor” for polymerisation of TEOS to form spherical silica matrix in which dye molecules get trapped. (Adopted with permission from Tan et. al 2004 © Wiley-Liss, Inc. a subsidiary of John Wiley & Sons, Inc.)

Silica is a chemically inert and optically transparent material which makes it

an excellent matrix for encapsulation of molecules sensitive to change in

environment. The transparent nature of SiNP provides one of the most widely used

application as a matrix for entrapment of fluorescent organic dyes. The fluorescent

dyes can be encapsulated into silica matrix by doping the TEOS with dye during

polymerisation reaction. Different dye doped SiNP (DDS) has been synthesized from

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both Stöber and Reverse Micelle methods described above (Santra et al. 2001, Rossi

et al. 2005). However because of lack of covalent bonding, dye molecules tend to

leach out over a period of time. This problem has been overcome in a recently

reported core shell SiNP in which a core is synthesized by an alkoxysilane covalently

attached to dye molecule and then coated with a tight silica shell for protection from

solvent interaction and to provide functional group for conjugation of ligands and

biomolecules (Ow et al. 2005).

Encapsulation of fluorescent dye into silica matrix provides a pseudo solid

environment, sequestered from solvent which prevents its self quenching, solvochromic

shifts and phtobleaching. As many dye molecules are entrapped in one SiNP, quantum

yield is also greatly enhanced. A wide number of dyes can be incorporated to cover

whole range of spectrum which is highly advantageous for multiplex assays. Even a dye

which is not optimal for a particular solvent can also be used because the optical

properties of dye do not depend upon the solvent. Moreover, unlike quantum dots DDS

do not suffer the problem of stochastic blinking. Finally availability of various

oraganosilicates ensures possibility of creating various functional groups at the surface

with ease, which provides flexibility in choosing protocols for conjugation of different

biomolecules (Burns et al. 2006a). These features have resulted into wide number of

applications of DDS in biology. The streptavidin conjugated DDS has been used for

various fluorescence based techniques like fluorescence-linked immunosorbent assay,

immunocytochemistry, immunohistochemistry, DNA microarray, and protein

microarray. DDS were shown to perform better than organic dye Texas red and even

QD (Lian et al. 2004). Organically modified SiNP with a positively charged surface and

doped with fluorescent dyes has been used for delivery and real time tracking of nucleic

acids and drug molecules inside cells (Roy et al. 2005, Fuller et al. 2008). In an

intresting application of fluorescent Core shell SiNP, Weisner’s group developed a

ratiometric pH sensor. They synthesized a core shell SiNP with core containing a pH

insensitive dye TRITC which served as standard. A pH sensitive dye FITC was

conjugated at the surface of shell. The changes in pH could be quantitatively measured

by comparing the change in fluorescence of pH sensitive FITC to that of TRITC which

is present at core and remains unaffected to pH variations. They have demonstrated the

utility of this system in vivo by measuring the pH in various cellular compartments of

RBL mast cells (Burns et al. 2006b). Because of being optically transparent, availability

17


of rich surface chemistry and inert nature, silica has been used for coating other

nanoparticles like QD and INP for easy surface modification (Yi et al. 2005). Silica

coated INP and DDS conjugated with aptamer has been used for selective collection

and detection of cancerous cells (Herr et al. 2006). Application of DDS has also been

demonstrated for detection of bacteria up to single cell level (Zhao et al. 2004).

v) Carbon Nanotubes

Carbon nanotubes (CNT) are a form of fullerenes, one of the three allotropic

forms of carbon along with graphite and diamond. First described by Iijima in 1991

CNT consist of graphitic sheets arranged into a tubular structure. The sheets can bee

arranged into a single layer called single walled CNT (SWNT) or multiple concentric

layers called multi-walled CNT (MWNT). The diameter of the CNT is typically

below 100 nm. However, the length can vary from few nanometres up to several

micrometres. The CNT are materials with high surface to volume ratio, ultra light

weight, good electrical and thermal conductivity, metallic or semi-metallic behaviour

and mechanically tough ordered structure (Sun et al. 2002). Several methods e.g. arc

discharge, laser ablation, chemical vapour deposition, etc. have been developed for

SWNT and CWNT (Dai 2002).

The CNT had limited biological applications because pristine CNT

synthesized by above method are insoluble. However, in last few years several

strategies have been developed for making CNT compatible with physiological

conditions. For example, surface oxidization of CNT using strong acids can be used to

generate carboxylic groups, which increase their dispersibility in aqueous solutions.

Alternatively Addition reactions to the CNT external walls can also employed to

make them soluble in water. Functionalised CNT can be conjugated to a wide variety

of molecules including peptides, proteins, nucleic acids and other therapeutic agents

according to the requirement of application (Tasis et al. 2006). The CNT have

intrinsic property of absorbing low energy Near Infra-Red (NIR) and radiofrequency

radiation. Like metal nanoparticles this induces an increase in local temperature of

CNT. This has been used to selectively kill malignant cells without damaging normal

cells both in vitro and in vivo (Bianco et al. 2008) CNT conjugated with a folate

moiety has been used to selectively kill cancerous cell over expressing folate receptor

(Kam et al. 2005). Using Radiofrequency, CNT containing cells residing deeper in

18


side body into liver has been shown to be killed in rabbit models (Gannon et al. 2007).

Polyethylene oxide functionalized CNT conjugated to specific receptors has been

used for sensitive and specific electronic detection of biomolecule (Chen et al. 2003).

Suitably functionalized CNT can cross cell membrane to enter into cytoplasm and

even nucleus. This has been exploited for CNT mediated delivery of physically

adsorbed or covalently attached peptides, nucleic acids and drugs in a number of

reports (Bianco et al. 2005).

vi) Other Inorganic Nanoparticles

Apart from the nanoparticles discussed above, Inorganic nanoparticles from

many other elements have been synthesized either in native form or as composites.

According to a recent estimate different forms of nanoparticles of at least 44 elements

from periodic table, each with its own novel nanoscale features, are already available

commercially and the list is growing (www.etcgroup.org 2008). However,

applications of many of these nanomaterials have been limited to materials science.

Besides the GNP, INP, QD, SiNP and CNT other nanomaterials worth mentioning for

biological applications include nanoparticles of Ag, Ti, Zn, Pt, Pd, Gd, Cu etc.

The metal nanoparticles are commonly synthesized by controlled reduction

of their respective salts in aqueous medium. For example, a common method for the

synthesis of silver nanoparticles is the reduction of AgNO3 with NaBH4. It is

called, Creighton method and produces approx. 10 nm particles of narrow size

distribution. Variations of this method can also be adapted for synthesis of

nanoparticles from other metals such as Pt, Pd, Cu, Ni, etc. The specific protocols

depend on the reduction potential of the source ion (Evanoff & Chumanov 2005).

The nanoparticles synthesized using these methods generally tend to aggregate and

require a capping agent for stabilization. The capping agent can be directly added

into the solution at the time of reduction. The shape and size of nanoparticles

depend upon the reaction condition and capping agent used. Alternatively,

nanoparticles can be capped with desired molecules after the synthesis to tailor their

surface chemistry.

19


vii) Dendritic Polymers

Dendritic polymers represent two architecturally similar classes of polymers

called dendrimeric polymers or simply dendrimers and hyperbranched polymers

(Paleos et al. 2007). They are distinguished by highly branched tree like scaffold from

which they derive their name (Greek dendron, meaning “tree”). Dendrimers are highly

branched, structurally well defined and monodisperse, globular macromolecules with

symmetrical, nanometer-sized architecture which consists of a central core, branching

units, and terminal functional groups. Hyperbranched polymers are also similar to

dendrimers, having branched scaffold, but in contrast to dendrimers, they are

asymmetrical and polydispersed (Figure 1.5). Due to their hyperbranched scaffold

dendritic polymers offer several adavantages over linear polymers in terms of

solubility, lower cytotoxicity, loading capacity, avalibity of large number of terminal

functional groups for desired medication for biomedical applications. They are being

extensively investigated to be used as drug delivery systems, as gene delivery vectors,

or as drugs on their own (Stiriba et al. 2002). Biologically active molecules can be

encapsulated within dendrimers or physically adsorbed or chemically attached at their

surface for delivery inside cells (Jain & Asthana 2007). Dendritic polymers have also

shown promise in diagnostics and imaging, tissue repair and sensor applications (Lee

et al. 2005).

Figure: 1.5: Schematic representation of dendritic polymers. (Left): A multifunctional dendrimers (Right): A hyperbranched polymer

Dendrimer molecules are composed of multiple perfectly branched monomers

which originate radially from a central core. Generally, they can be synthesized by

20


either of following two different multistep polymerization approaches called divergent

and convergent approach. In the divergent approach the dendrimer is synthesised from

the core as the starting point and built up step by step according the required degree of

branching. In the second, convergent approach synthesis starts from the surface and

ends up at the core, where the dendrimer segments (dendrons) are coupled together

(Boas & Heegaard 2004). Hyperbranched polymers can be prepared in a one step self

polymerisation reaction. The polymerization reactions to prepare hyperbranched

polymers can be classified into three categories: A: Polycodensation B: Self

condensing viny polymerization and C; Ring opening opening polymerisation (Jikei

& Kakimoto 2001). All three approaches can give rise to hyperbranched polymers

which are rather imperfectly branched and having higher polydispersity but posses

many dendrimers like properties. Moreover, since they can be synthesized in single

step, their cost of production is considerably lower. Therefore, hyperbranched

polymers are being considered as an alternative to dendrimers in many applications

which can tolerate polydispersity to some extent and do not require a very well

defined branched architecture (Lee et al. 2005).

viii) Liposomes

Liposomes are spherical soft-matter particles composed of one or more

phospholipid bilayers membrane(s), encapsulating a volume of aqueous medium.

Liposomes resemble cell membranes in their structure and composition and can be

used for encapsulating bioactive molecules for a variety of biological applications

(Lasic 1998). Depending upon the desired application a number of procedures are

available for preparation of liposomes. In its simplest form liposomes can be prepared

by mixing phospholipid in aquous environment under non shearing condition. Due to

their amphiphilic nature, in aqueous environment phospholipids align themselves into

bilayers with their hydrophobic tails facing each other and their hydrophilic

headgroups facing toward the aqueous medium to minimize energy (Figure 1.6)

(Jesorka & Orwar 2008). To further minimize the energy, edges of the lipid bilayer

join to form a spherical vesicle called liposome. The charge stability and other surface

properties of the liposomes depend upon the phospholipid used for their preparation.

The most commonly utilized lipids for preparation of liposomes are natural

phospholipids like phosphatidylcholine phosphatidic acid, phosphatidylglycerol,

21


phosphatidylserine, and phosphatidylethanolamine, Stearylamine etc. (Fahy et al.

2005). However, synthetic lipid with desired functional groups for different purposes

like coupling reactions, chelating metal ions, or even multifunctional lipids may be

used (Roy et al. 2000, Chikh et al. 2002, Hassane et al. 2006). Liposomes may be

prepared in a range of sizes starting from 20 nm to few µm. More over depending

upon method of preparation and the desired application they may be unilamellar

having only one lipid bilayer, multilamellar with many lipid bilayers or even

multivescicular having more than one small liposomes encapsulated in a large

liposome (Jesorka & Orwar 2008).

Figure 1.6: Schematic representation of self-assembly process of a liposome from phospholipid molecules. (a) Amphiphilic lipid molecules with hydrophilic head and hydrophobic tails (b), Phospholipid molecules arrange into lipid bilayer with their hydrophobic tails facing each other and their hydrophilic headgroups facing toward the aqueous medium (c). The edges of the lipid bilayer join to form a spherical liposome. (d ). Apart from the structural characteristics of the lipid molecules themselves, the properties and functionality of liposomes are largely defined by their size and the composition of the four distinct regions highlighted in panel c. (Adopted with permission from Jesorka & Orwar 2008 © Annual Reviews, USA)

The liposomal shell can be used to enclose or bind many different classes of

substances for both in vitro as well as in vivo delivery. Liposomes has been

successfully utilized for the delivery of therapeutic agents like antibacterial, antiviral,

and anticancer drugs, as well as hormones, enzymes, and nucleic acids (Lasic et al.

1999, Weissig et al. 2006, Eckstein 2007). In fact, many liposome based commercial

formulations are already available for delivery of nucleic acids and drugs (Barenholz

2001). The encapsulation of drug molecules inside liposomes offers several

advantages like: increases in the circulation time, protection from degradation by

22


enzymes, slow and sustained release etc., which are essential prerequisite for good

delivery agents. Moreover, with the encapsulation inside liposomes it is possible to

deliver even hydrophobic toxic drugs which can not be directly injected (Torchilin

2005). These properties together with ability to tailor the lipids used for liposome has

been used to deliver nucleic acids and drugs for targeted delivery to desired cellular

compartments, cells, tissues or organs (Torchilin 2006). Liposomes labelled with

radio isotopes or MRI contrast agents has also been used for in vivo imaging

(Torchilin 1996, Martina et al. 2005, Goins 2008).

Biocompatibility of Nanoparticles

Owing to their unique properties nanoparticles are being considered to address

many of the issues facing society in the areas of health, agriculture, environment,

energy, information technology, education, manufacturing, sustainability,

transportation homeland security etc. (Helmus 2006). Keeping enormous potential of

nanomaterials in view, their commercial application is still in its infancy (Mazzola

2003). Nevertheless, nanoparticle based products are becoming increasingly common

(www.nanotechproject.org/ 2008). While unique properties of nanoparticles offer

enormous avenues for potential applications in different fields, their xenobiotic nature

raise worries regarding their safety. Concerns have been raised that the very properties

of nanomaterials which make them attractive, could cause unforeseen health or

environmental hazards (Maynard et al. 2006). The commercial applications of

nanoparticles require large scale production, use, and disposal of products containing

nanomaterials. This may lead to their appearance in air, water, soil, organisms or the

human body posing risk to human health and environment. Moreover, problem to

human health could be more serious in biomedical applications of nanoparticles as

drug-delivery agents, biosensors, or imaging contrast agents which require deliberate,

direct administration of nanoparticles into the body.

There are several reports of nanoparticles being toxic. Large variations in toxic

potential and underlying mechanism behind toxicity of different nanomaterials are

observed. Because of very small size nanoparticles delivered into body fluids can

bypass from circulation accumulate into organs and tissues and even enter inside cell.

Whole body studies show that inhalation of nanoparticles and entry via the lungs is

followed by rapid translocation to vital organs like the kidney and liver. Once inside the

23


organs they can cause injury by various mechanisms (Borm et al. 2006, Nel et al. 2006).

Inhalation of titanium oxide nanoparticles have been shown to induce lung injury by

affecting expression of genes involved in pathways involved in cell cycle, apoptosis,

chemokines, and complement cascades (Chen et al. 2006). Redox-active, lipophilic

fullerenes cause significant lipid peroxidation in brain and glutathione depletion in gills

in an aquatic species largemouth bass. Nanoparticles derived from toxic metals like

CdSe and CdSe/ZnS may eventually release toxic ions into the living system (Kirchner

et al. 2005). Though nanoparticles of CuO have been shown to be highly toxic, the

toxicity could not be explained by release of Cu ions alone (Karlsson et al. 2008). Apart

from chemical composition, surface properties of the nanoparticles differ considerably

with their shape and size. This may influence the uptake of different nanoparticles.

While functionalized nanoparticles may interact specifically with certain biomolecules

like proteins or DNA, non-specific interaction with sub-cellular structures may depend

upon more general features like charge and shape. For instance, because of higher

penetrance facilitated by their shape, carbon nanotubes seem to be more prominent in

causing epithelioid granulomas and interstitial inflammation than 14 nm carbon black

particles in mice (Lam et al. 2004). Recently, upon comparison of cytotoxicity data on

carbon, metal, and semiconductor based nanoparticles Lewinski et al. found that while

the causes for the increase in cell death observed at higher concentrations and longer

exposure times may be material specific, the generation of reactive oxygen species and

the influence of cell internalization of nanoparticles are two common causes which

mainly govern nanoparticle toxicity (Lewinski et al. 2008).

Due to extraordinarily large surface-to-size ratios and unique surface properties

nanoparticles can specifically adsorb to proteins and biomolecules. In fact, size and

surface properties of nanoparticles has been found to play a very significant role in

determining the adsorption of proteins at nanoparticle surface (Lundqvist et al. 2008).

Due to their ability to adsorb biomolecules, nanoparticles can catalyze biomolecular

reactions. While this might be useful for some therapeutic applications, products of

these catalyzed reactions may also be toxic themselves or produce damaging free

radicals. Moreover, adsorption of endogenous proteins to nanoparticles surface could

lead change in the protein confirmation and elicit immune response (Daughton 2004).

In this way nanoparticles made up of even chemically inert materials might prove toxic

24


because of their surface properties. Moreover, in such cases toxic potential will vary

with the variation of surface properties of particles with size.

Therefore, extensive characterization of interactions nanoparticles between

biological surfaces, cells and nanoparticles is an essential pre-requisite for

nanoparticle based clinical applications. Secondly, environmental effects due to aerial

emissions, prolonged presence and accumulation in soil, water and food sources need

to be evaluated from an environmental standpoint. Lastly, exposure levels in

manufacturing sites need to be standardized for occupational safety. Widespread

applications of nanoparticles requires that their potential toxic effects need to be

evaluated in much the same way as clinical testing of other chemical or

pharmaceutical is undertaken. Moreover, keeping in view, the variation in the

properties of nanoparticles with their shape and size, each engineered nanomaterials,

even chemically identical but of different in shape and sizes should be evaluated

separately and the information should be catalogued like e.g. Chemical Abstract

Service (CAS) Registry used for other manufactured chemicals.

25


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