novel applications of nanotechnology in medicine

Introduction

Nanomedicine involves utilization ofnanotechnology for the benefit of human health and well being. The use of nanotechnology in varioussectors of therapeutics has revolutionized the field of medicine where nanoparticles of dimensionsranging between 1 - 100 nm are designed and usedfordiagnostics,therapeuticsandasbiomedicaltoolsfor research1. It is now possible to provide therapyat a molecular level with the help of these tools,thus treating the disease and assisting in study ofthe pathogenesis of disease. Conventional drugssuffer from major limitations of adverse effectsoccurring as a result of non specificity of drug action

Novelapplicationsofnanotechnologyinmedicine

A.Surendiran,S.Sandhiya,S.C.Pradhan&C.Adithan

Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education & Research Puducherry, India

ReceivedOctober29,2008

Current modalities of diagnosis and treatment of various diseases, especially cancer have major limitations such as poor sensitivity or specificity and drug toxicities respectively. Newer and improved methods of cancer detection based on nanoparticles are being developed. They are used as contrast agents, fluorescent materials, molecular research tools and drugs with targeting antibodies. Paramagnetic nanoparticles, quantum dots, nanoshells and nanosomes are few of the nanoparticles used for diagnostic purposes. Drugs with high toxic potential like cancer chemotherapeutic drugs can be given with a better safety profile with the utility of nanotechnology. These can be made to act specifically at the target tissue by active as well as passive means. Other modalities of therapy such as heat induced ablation of cancer cells by nanoshells and gene therapy are also being developed. This review discusses the various platforms of nanotechnology being used in different aspects of medicine like diagnostics and therapeutics. The potential toxicities of the nanoparticles are also described in addition to hypothetical designs such as respirocytes and microbivores. The safety of nanomedicine is not yet fully defined. However, it is possible that nanomedicine in future would play a crucial role in the treatment of human diseases and also in enhancement of normal human physiology.

Key wordsDiagnostics-drugs-nanomedicine-nanotechnology-therapeutics

and lack of efficacy due to improper or ineffective dosage formulation (e.g., cancer chemotherapy andantidiabeticagents).Designingofdrugswithgreaterdegree of cell specificity improves efficacy and minimizesadverseeffects.Diagnosticmethodswithgreaterdegreeofsensitivityaidinearlydetectionofthediseaseandprovidebetterprognosis.Nanotechnologyisbeingappliedextensivelytoprovidetargeteddrugtherapy,diagnostics,tissueregeneration,cellculture,biosensors and other tools in the field of molecular biology. Various nanotechnology platforms likefullerenes, nanotubes, quantum dots, nanopores,dendrimers, liposomes, magnetic nanoprobes andradiocontrollednanoparticlesarebeingdeveloped.

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IndianJMedRes130,December2009,pp689-701

Review Article

Current status of therapeutics

The major factors influencing the treatment outcome in a patient are the efficacy and safety profile of the drug more so when used for cancer chemotherapy. Thesedrugs have poor cell specificity and high toxicity like bone marrow suppression, gastric erosion, hair loss,renaltoxicity,cardiomyopathy,andseveraleffectsonother systems. Similarly treatment for diabetes faceschallenges with the route of delivery and inadequateglycaemic control. Availability of non-parenteraldosageformsofinsulinwouldbeabreakthroughanddevelopment of a suitable drug delivery device canaidinthisapproach.Inmanycases,thesensitivityandspecificity of various diagnostic methods as in radio imagingandvariousassaysfordetectionofmalignancyare not sufficient enough for early detection and treatment.

Nanotechnology and medical applications

Development of newer drug delivery systemsbased on nanotechnology methods is being tried forconditionslikecancer,diabetes,fungalinfections,viralinfections and in gene therapy. The main advantagesof thismodalityof treatmentare targetingof thedrugand enhanced safety profile. Nanotechnology has also founditsuseindiagnosticmedicineascontrastagents,fluorescent dyes and magnetic nanoparticles (Table 1).

Liposomes

Liposomes discovered in mid 1960s were theoriginalmodelsofnanoscaleddrugdeliverydevices.They are spherical nanoparticles made of lipidbilayer membranes with an aqueous interior but canbe unilamellar with a single lamella of membraneor multilamellar with multiple membranes. They

Table. Somenanoparticlesusedformedicalapplications*

Studyphase Product Description Use Manufacturer

Preclinical MRX952 Nanoparticlepreparation–toencapsulatecamptothecinanalogues

Tumours IMARxTherapeutics

Preclinical TargetedNanoTherapeutics(TNT)™system

TNTwithpolymercoatedironoxidemagneticparticle

Solidtumours TritonBiosystems

Preclinical AuroLase™ Goldnanoshell Headandneckcancer

NanospectraBiosciencesInc

Preclinical Dendrimer-Magnevist# PAMAMdendrimer MRIimagingagent DendriticNanotechnologiesInc

Phase1 VivaGel® Dendrimerbasedmicrobicidegel

HIVprevention StarpharmaPtyLtd

Phase1 INGN401 NanoparticleformulationoftumoursuppressiongeneFUS1

Lungcancer IntrogenTherapeuticsInc

Phase1&2 Cyclosert-Camptothecin–IT101

β-Cylcodextrin polymer drugdeliverysystem

Solidtumours CalandoPharmaceuticals

Phase2 VivaGel® Dendrimerbasedmicrobicidegel

HSVprevention StarpharmaPtyLtd

Phase2 MRX815 Nanobubbletechnology Treatmentofintravascularclot

IMARxTherapeutics

Phase3 Combidex®/Ferumoxtran10

Ironoxidenanoparticle MRIcontrastagent AMAGPharmaceuticals

Marketed Abraxane® Albuminboundtaxaneparticles

Nonsmallcelllungcancer AbraxisOncology

Marketed AmBisome® LiposomalpreparationofamphotericinB

Fungalinfection AstellasPharmaUS

Marketed Doxil® Liposomaldoxorubicin Ovariantumour OrthoBiotech

* Informationobtainedfromrespectivecompanywebpageoninternet#AvailableatNanotechnologyCharacterizationLaboratoryWebpageat http://ncl.cancer.gov/

690 INDIANJMEDRES,DECEMBER2009

can be used as effective drug delivery systems.Cancer chemotherapeutic drugs and other toxicdrugs like amphotericin and hamycin, when used asliposomal drugs produce much better efficacy and safety as compared to conventional preparations.These liposomes can be loaded with drugs either inthe aqueous compartment or in the lipid membrane.Usually water soluble drugs are loaded in aqueouscompartmentandlipidsolubledrugsareincorporatedin the liposomalmembrane2.Themajor limitationofliposome is its rapid degradation and clearance bythelivermacrophages3, thusreducingthedurationofaction of the drug it carries. This can be reduced toa certain extent with the advent of stealth liposomeswhere the liposomes are coated with materials likepolyoxyethylene4 which prevents opsonisation of theliposome and their uptake by macrophages5. Otherwaysofprolongingthecirculationtimeof liposomesare incorporation of substances like cholesterol6,polyvinylpyrollidone polyacrylamide lipids7 andhigh transition temperature phospholipids distearoylphosphatidylcholine8.

Targeting of liposomal drugs: Liposomes can betargeted to specific organ or tissue by passive as well as active methods.As the liposomal drug actsminimally on other tissues, the safety profile is better thannon-liposomaldrug.Thevascularity in tumourtissue is poorly organized and significant leak occurs frombloodvesselinthetumourtissue.Theliposomaldrugsgetaccumulatedinthetumourtissuepassivelyandproduceenhancedeffects.Activetargetingofthedrugcanbeachievedbyusingimmunoliposomesandliganddirectedliposomes.

Immunoliposomesareliposomesconjugatedwithanantibodydirectedtowardsthetumourantigen.Theantibodycanbeconjugatedtothesurfaceofastealthliposome, the polyoxyethylene coating of a stealthliposomeoronthesurfaceofanonstealthliposome.Theseimmunoliposomeswheninjectedintothebody,reaches the target tissue and gets accumulated in itssiteofaction.Thisreducesunwantedeffectsandalsoincreases the drug delivery to the target tissue, thusenhancing its safety and efficacy9.

Antibody directed enzyme prodrug therapy(ADEPT) consists of liposomes conjugated with anenzymetoactivateaprodrugandanantibodydirectedtoatumourantigen(enzymelinkedimmunoliposomes).These are administered prior to administration of aprodrug.Theantibodydirectstheenzymetothetarget

tissue where it activates the prodrug selectively andconverts it to itsactiveform.Thisway,actionof thedrugisavoidedinothernormaltissues,thusminimizingthe toxicity of drug10-12. Such studies are being triedwithepirubicinanddoxorubicin11,13.

Ligand bearing liposomes are conjugated withspecific ligands which are directed towards target structures.Inovariancancer,overexpressionoffolatereceptorsby the tumour tissueoccurs.The liposomaldrugcanbeconjugatedwithfolatesoastodirectthemolecule to the tumour14. This method is also beingtriedinthetreatmentofleishmaniasiswhereliposomalhamycin conjugated with mannosyl human serumalbumin are targeted towards human macrophages15.Asialofeutin conjugation is being tried to targetliver cells forgene therapy16.The targeted liposomalpreparations are found to have a better efficacy than nontargetedliposomes.

Nanopores

Nanopores designed in 1997 by Desai andFerrari17,consistofwaferswithhighdensityofpores(20nmindiameter).Theporesallowentryofoxygen,glucoseandotherproductslikeinsulintopassthrough.However, it does not allow immunoglobulin andcells to pass through them. Nanopores can be usedas devices to protect transplanted tissues from thehost immune system, at the same time, utilizing thebenefit of transplantation. β cells of pancreas can be enclosedwithinthenanoporedeviceandimplantedinthe recipient’s body. This tissue sample receives thenutrientsfromthesurroundingtissuesandatthesametime remains undetected by the immune system andhencedonotgetrejected.Thiscouldserveasanewermodality of treatment for insulin dependent diabetesmellitus18.

Nanopores can also be employed in DNAsequencing. Branton’s team at Harvard University19,has been working on modified nanopores that have the ability to differentiate DNA strands based ondifferencesinbasepairsequences.Nanoporesarealsobeing developed with ability to differentiate purinesfrompyrimidines.Further,incorporationofelectricityconducting electrodes is being designed to improvelongitudinal resolution for base pair identification19.Suchamethodcouldpossibly reada thousandbasesper secondperpore.Thesecanbeused for lowcosthighthroughputgenomesequencing20whichwouldbeof great benefit for application of pharmacogenomics indrugdevelopmentprocess.

SURENDIRANet al:NANOTECHNOLOGyINMEDICINE 691

Fullerenes

Fullerenes,acarbonallotrope,alsocalledas“buckyballs” were discovered in 198521. The buckminsterfullerene is the most common form of fullerenemeasuringabout7Åindiameterwith60carbonatomsarrangedinashapeknownastruncatedicosahedrons22.It resembles a soccer ball with 20 hexagons and 12pentagonsandishighlysymmetrical23.

Types of fullerenes: Alkali doped fullerenes arestructureswithalkalimetalatomsinbetweenfullerenescontributing valence electrons to neighbouringfullerenes24.Theyoccurbecauseoftheelectronegativenatureofthefullerenes.

Endohedralfullereneshaveanotheratomenclosedinside the buckyball. If a metallic atom is enclosed,these are called as metallofullerenes25,26. Due to thesmallsizeofC60 fullerene, it is difficult to synthesize endohedralC60 fullerenes.However, larger fullerenessuchasC82orC84fullerenesareusedforsynthesizingendohedral fullerenes. Endohedral metallofullerenescanbeusedfordiagnosticpurposesasradiocontrastmedia in magnetic resonance imaging and otherimaging procedures. Since the radioactive metal isenclosedwithinthebuckyball,thesearelesstoxicandsafer.Thismethodcanalsobeemployedforimagingorgansasradioactivetracers26.

Exohedral fullerenes also called as fullerenederivatives, are synthesized by chemical reactionbetween the fullerene and other chemical groups.These are also called as functionalized fullerenes.Such fullerenes can be used as photosensitizers inphotodynamictherapyformalignancies.Thesegeneratereactiveoxygenspecieswhenstimulatedbylightandkills the target cells. This method is now also beinginvestigatedforantimicrobialpropertyasthesecausecellmembranedisruptionespeciallyinGrampositivebacteriaandmycobacterium27-29.

Heterofullerenes are fullerene compounds whereoneormorecarbonatomsarereplacedbyotheratomslikenitrogenorboron21.

Fullerenes are being investigated for drugtransportofantiviraldrugs,antibioticsandanticanceragents27-30. Fullerenes can also be used as freeradical scavengers due to presence of high numberof conjugated double bonds in the core structure.Thesearefoundtohaveaprotectiveactivityagainstmitochondrial injury induced by free radicals31.However,fullerenescanalsogeneratereactiveoxygen

speciesduringphotosensitization.Thispropertycanbeusedincancertherapy32.

Fullerenes have the potential to stimulate hostimmune response and production of fullerene specific antibodies. Animal studies with C60 fullereneconjugated with thyroglobulin have produced aC60 specific immunological response which can be detected by ELISA with IgG specific antibodies. Thiscanbeused todesignmethodsofestimationoffullerenelevelsinthebodywhenusedfortherapeuticor diagnostic purposes33. On intravenous injection,thesegetdistributedtovariouspartsof thebodyandget excreted unchanged through the kidney. Solublederivates of fullerenes are more biocompatiblecompared to insoluble forms of fullerenes and havelowtoxicpotentialevenathigherdoses33.Further,thedegree of purification of fullerene determines its cost and highly purified fullerenes are expensive, restricting its application in medical field21.

Nanotubes

Carbonnanotubesdiscoveredin199134aretubularstructureslikeasheetofgraphiterolledintoacylindercappedatoneorbothendsbyabuckyball.Nanotubescan be single walled carbon nanotube (SWCNT) ormultiwalledcarbonnanotube(MWCNT)inconcentricfashion.Singlewallednanotubehasaninternaldiameterof 1-2 nm and multiwalled nanotube has a diameterof2-25nmwith0.36nmdistancebetween layersofMWCNT. These vary in their length ranging from1µmtoafewmicrometers35.Thesearecharacterizedbygreaterstrengthandstabilityhencecanbeusedasstable drug carriers. Cell specificity can be achieved by conjugating antibodies to carbon nanotubes withfluorescent or radiolabelling36.Entryofnanotubesintothecellmaybemediatedbyendocytosisorbyinsertionthroughthecellmembrane.Carbonnanotubescanbemademoresolubleby incorporationofcarboxylicorammoniumgroups to theirstructureandcanbeusedfor the transport of peptides, nucleic acids and otherdrug molecules. Indium-111 radionuclide labelledcarbon nanotubes are being investigated for killingcancercellsselectively35.

Amphotericin B nanotubes has shown increaseddrug delivery to the interior of cells compared toamphotericin B administration without nanotubes37.The efficacy of amphotericin B nanotubes was greater as an antifungal agent compared to amphotericin Balone and it was effective on strains of fungi whichareusuallyresistanttoamphotericinBalone.Further,


there was reduced toxicity to mammalian cells withamphotericinBnanotubes37.Theabilityofnanotubesto transport DNA across cell membrane is used instudiesinvolvinggenetherapy.DNAcanbeattachedtothetipsofnanotubesorcanbeincorporatedwithinthe tubes. Prato et al37 showed greater expression ofthe β galactosidase marker gene through nanotubes comparedtotransferofnakedDNA.Thisconferstheadvantageofnonimmunogenicityincontrasttoviralvectorsusedforgenetransfer.Genesilencingstudieswithsmall interferingRNA(siRNA)havebeendoneasamodalityofcancertherapywheretumourcellswillbeselectivelymodulated.FunctionalizedsinglewalledcarbonnanotubescanbeusedwithsiRNAtosilencetargetedgeneexpression38.

It was observed that carbon nanotubes, exceptacetylatedones,whenbondedwithapeptideproducea higher immunological response compared tofree peptides. This property can be used in vaccineproduction to enhance the efficacy of vaccines. Further, it was also found that compounds bound tonanotubes increase the efficacy of diagnostic methods likeELISA.Thesecanalsobeused fordesigningofbiosensorsowingtopropertyoffunctionalizationandhighlengthtodiameteraspectratiowhichprovidesahighsurfacetovolumeratio39,40.

Water insoluble forms of nanotubes like pristinecarbonnanotubeshavehighin vitrotoxicitycomparedto modified water dispersible forms of nanotubes. It was also seen that the toxic potential decreases withfunctionalization. Further, functionalization alsoaffects the elimination of the nanotube. SWCNTswithout conjugation to monoclonal antibody have ahighrenaluptakeandmodestliveruptakeascomparedtoSWCNTswithconjugationtomonoclonalantibodyhavinghigherliveruptakeandlowerrenaluptake41.

Quantum dots

Quantumdotsarenanocrystalsmeasuringaround2-10 nm which can be made to fluorescence when stimulated by light. Their structure consists of aninorganiccore,thesizeofwhichdeterminesthecolouremitted, an inorganic shell and an aqueous organiccoating to which biomolecules are conjugated. Thebiomolecule conjugationof thequantumdots canbemodulatedtotargetvariousbiomarkers42.

Quantumdotscanbeusedforbiomedicalpurposesasadiagnosticaswellastherapeutictool.Thesecanbetaggedwithbiomoleculesandusedashighlysensitive

probes. A study done on prostate cancer developedin nude mice has shown accumulation of quantumdotsprobebyenhancedpermeabilityandretentionaswellasbyantibodydirectedtargeting43.Thequantumdots conjugated with polyethylene glycol (PEG)and antibody to prostate specific membrane antigen (PSMA)wereaccumulatedandretainedinthegraftedtumourtissueinthemouse43.

Quantum dots can also be used for imaging ofsentinelnodeincancerpatientsfortumourstagingandplanningof therapy.Thismethodcanbeadopted forvariousmalignancieslikemelanoma,breast,lungandgastrointestinaltumours42.QuantumdotprobesproviderealtimeimagingofthesentinelnodewithNearInfraRed (NIR) fluorescence system. The NIR region of the electromagnetic spectrum produces reducedbackgroundnoiseanddeeperpenetrationofrays,ofupto2to5cmintothebiologicalsample.However,thetraditional fluorescence dyes yield low signal intensity whenusedinNIRregion.Thislimitationisovercome,by using NIR fluorescence system with quantum dot probes. The fluorescence produced by quantum dots ismuchbrighterthanthoseproducedbyconventionaldyes when used with NIR fluorescence system44.However,theapplicationofquantumdotsinaclinicalsettinghaslimitationsowingtoitseliminationfactors.Functionalizationofthequantumdotswhichprotectsfrom the toxic core, leads to increase in size of thenanoparticlegreaterthantheporesizeofendotheliumandrenalcapillaries,thusreducingitseliminationandresultingintoxicity.Also,in vivostudiesarelackingonthemetabolismandexcretionofquantumdots42.

Nanoshells

NanoshellsweredevelopedbyWestandHalas45atRiceUniversityasanewmodalityoftargetedtherapy.Nanoshells consist of nanoparticles with a core ofsilica and a coatingof thinmetallic shell.These canbe targeted todesired tissuebyusing immunologicalmethods.Thistechnologyisbeingevaluatedforcancertherapy.Hirshet al46usednanoshellswhicharetunedtoabsorb infraredrayswhenexposedfromasourceoutside the body to demonstrate the thermo ablativepropertyofnanoshells.Thenanoshellswhenexposedto NIR region of the electromagnetic spectrum getheatedandcausedestructionofthetissue.Thishasbeenstudied inboth in vitro and in vivo experimentswithHER2expressingSK-BR-3humanbreastcarcinomacells.Thecontrolcellsdidnotlosetheirviabilityevenafter treatment with nanoshells with non specific anti IgGorPEGandNIRablation47.


Nanoshells can also be embedded in a hydrogelpolymer containing the drug. After directing thenanoshells to the tumour tissue by immunologicalmethods,withaninfraredlaser,thesecanbemadetogetheatedup,meltingthepolymerandreleasingthedrugatthetumourtissue.Targetingthedrugreleaseavoidsthetoxicityofcancerchemotherapydrugs.Nanoshellsarecurrentlybeinginvestigatedformicrometastasisoftumoursandalsofortreatmentofdiabetes19,48.

Nanoshellsarealsousefulfordiagnosticpurposesinwholebloodimmunoassays.Goldnanoshellscanbecoupled to antibodies and the size canbemodulatedsothatitrespondstoNIRwavelength,whichhastheabilitytopenetratewholebloodspecimens.Withthismethod it ispossible todetect immunoglobulins at aconcentration range of nanograms per millilitre inplasmaandwholeblood19.

Nanobubbles

Cancertherapeuticdrugscanbeincorporatedintonanoscaledbubblelikestructurescalledasnanobubbles.Thesenanobubblesremainstableatroomtemperatureandwhenheated tophysiological temperaturewithinthebodycoalescetoformmicrobubbles.Thesehavetheadvantagesoftargetingthetumourtissueanddeliveringthe drug selectively under the influence of ultrasound exposure.Thisresultsinincreasedintracellularuptakeof the drug by the tumour cells. It also provides anadditional advantage of enabling visualisation of thetumourbymeansofultrasoundmethods49,50.Rapaportet al51 havedemonstratedtheutilityofnanobubblesindelivery of drugs like doxorubicin based on in vitroand in vivo experiments using breast cancer cellsMDAMB231andmicewithbreastcancerxenograftrespectively.Onadministrationofnanobubbleloadeddoxorubicin, these reach the tumour tissue throughleaky vasculature and get accumulated at the site oftumour.Thisisfollowedbyformationofmicrobubblesbycoalescingofnanobubbleswhichcanbevisualisedbyultrasoundtechniques.Whenthesiteisfocusedwithhigh intensity focused ultrasound (HIFU), it causesdisruptionof themicrobubblesresultinginreleaseofthedrug.ThemicrobubblesretainedthedruginastablestateuntilstimulatedbyHIFU.Thisresultsinattainmentofhigher levelsofdrug in the targetcellsandhencereduced toxicity and increased efficacy. This method needs further exploration for its utility in treatmentofvariousmalignancies.Liposomalnanobubblesandmicrobubbles are also being investigated for theirrole as effective non viral vectors for gene therapy.

Nanobubbles combined with ultrasound exposurehasshownimprovedtransferofgeneinboth in vitroand in vivo studies52,53. Nanobubbles are also beingtried as a therapeuticmeasure for removal of clot invascular system in combination with ultrasound, aprocesscalledas sonothrombolysis.Thismethodhasadvantages of being non invasive and causing lessdamagetoendothelium54.

Paramagnetic nanoparticles

Paramagnetic nanoparticles are being triedfor both diagnostic and therapeutic purposes.Diagnostically,paramagneticironoxidenanoparticlesare used as contrast agents in magnetic resonanceimaging.Thesehaveagreatermagneticsusceptibilitythanconventionalcontrastagents.Targetingofthesenanoparticles enables identification of specific organs andtissues55.TheuseofironoxideinMRIimagingfaces limitations like specificity and internalization by macrophages56. Paramagnetic nanoparticlesconjugatedwithantibodies toHER-2/neuwhichareexpressedonbreastcancercellshavebeenusedwithMRI to detect breast cancer cells in vitro57. StudydonebyLeuschneret al58hasdemonstratedthein vivodetection of breast cancer cells using paramagneticnanoparticles conjugated with luteinizing hormonereleasing hormone as breast cancer cells expressLHRH receptors. Thus, use of antibodies to directthenanoparticletothetargetsitehelpedtoovercomeproblems with specificity of action. Internalization ofthenanoparticlesbymacrophagescanbereducedbytreatmentwithdrugslikelovastatinwhichreducemacrophagereceptorexpressionforthenanoparticleby reducing the recycling of receptors56. Further,injection of decoys of nanoparticle can be used toeliminate plasma opsonins and reduce uptake ofthenanoparticles.Also,changeofsurfacechargeofthe nanoparticle to neutral by covalent coupling tochemicalsleadstoanincreaseincirculationtime56.

Monocrystalline iron oxide nanoparticles(MIONs) have been studied by Knauth et al59 inmagnetic resonance imaging of brain. MIONs helpinovercomingthedisadvantageofsurgicallyinducedcontrastenhancementwith traditionalcontrastagentsresulting in misinterpretation during intra-operativeMR imaging of brain. Surgically induced contrastenhancement occurs in brain due to leak of contrastmaterial from the cut end and oozing blood vesselsin brain when MR imaging is done post-operatively.ThisisavoidedwhenMIONsareusedpre-operatively.


These are rapidly taken up by the tumour cells60,producinglonglastingcontrastenhancementoftumourandtheremainingnanoparticlesareremovedfromthecirculationbyreticuloendothelialsystem61.

Magnetic microparticle probes with nanoparticleprobes have been used for identification of proteins like prostate specific antigen. Here magnetic microparticles coatedwithantibodiestogetherwithnanoprobeswithsimilarcoatingandauniquehybridizedDNAbarcodeare used. The microparticle coated with antibodydirected against prostate specific antigen combines withittoformacomplexandcanbeseparatedbyusingmagneticseparation.Thepresenceof theseseparatedcomplexes is determined by dehybridization of thecomplexed DNA barcode sequence and polymerasechain reaction for the oligonucleotides. This allowsprostate specific antigen detection at 30 attomolar concentration62.This sensitivity is much greater thanconventional assays for prostate specific antigen.

Magneticnanoprobesareusedforcancertherapy.Ironnanoparticlescoatedwithmonoclonalantibodiesdirectedtotumourcellscanbemadetogeneratehighlevels of heat after these accumulate in their targetsite by means of an alternating magnetic field applied externally.Thisheatkills thecancercellsselectively.ThismethoddesignedbyTritonBiosystems,isabouttoenterclinicaltrialsforsolidtumoursin200963.

Nanosomes

Raoul Kopelman’s group at the University ofMichigan, USA, has been working on nanosomesalso called as PEBBLEs (Probes Encapsulated byBiologically Localized Embedding) which integratevarious aspects of medical applications such astargeting, diagnosis and therapy. These nanosomesarebeingdevelopedfortreatmentofvarioustumours,in particular CNS tumours. Silica coated iron oxidenanoparticles coated with polyethylene glycol64 andaffixed with targeting antibody and contrast elements like gadolinium are used to access specific areas of braininvolvedwithtumour.Targetingaidsinbindingthe nanoparticle specifically to the tumour cells and the contrastelementshelpsinbetterdetectionwithmagneticresonance imaging. Subsequent treatment with lasercandestroythecellsloadedwiththesenanoparticlesbytheheatgeneratedbyironoxideparticlesbyabsorbingtheinfraredlight.Nanosomescanalsobe integratedwithaphotocatalystwhichproducesreactiveoxygenspecieswhenstimulatedbylightanddestroythetargettissue.Thismethodhas advantageover conventional

drugsinbeingmuchsaferwithouttheadverseeffectsofcancerchemotherapydrugsandalsotheabsenceofdevelopmentofdrugresistance.Nanosomesarebeingdevelopedtointegratemoreandmorecomponentsinitfor flexibility of its applications19,65.

Dendrimers

Dendrimers are nanomolecules with regularbranching structures. The number of branchingdetermines the size of the dendrimer which can becontrolled.Thebranchesarisefromthecoreinshapeofasphericalstructurebymeansofpolymerisation19.Thisresults in formation of cavities within the dendrimermolecule which can be used for drug transport. Theendsof thedendrimermoleculecanbeattachedwithother molecules for transport. These molecules givethe dendrimers various functional applications66.Tectodendrimers are complexes of dendrimers, witheach dendrimer module of the complex performingdifferent functions such as targeting, diagnosis ofdisease state, delivery of drug and imaging. Thisextended nanodevice has potential applications incancer chemotherapy as a mode of targeted drugtherapy67.

Dendrimers can be used for gene therapy wherethesecanreplaceconventionalviralvectors.TheyenterthecellsbyendocytosisandtheDNAgetstransportedintonucleusfortranscriptionoftheappliedgene.Theadvantage of dendrimer based therapy is absence ofstimulationofimmunereaction.Dendrimershavebeentestedinmammaliancelltypesandinanimalmodels.Huanget al68havedemonstrated thepotentialuseoftransferring conjugated PEG modified polyamidoamine (PAMAM) dendrimers for targeted gene delivery tothebrain.Panet al69 have demonstrated the efficacy of magnetic nanoparticle modified PAMAM dendrimers in transfer of antisense survivin oligonucleotides intumourcelllines.Thesemethodsprovideaneffectivealternativetoviralvectorsofgenetransferfortreatmentof various tumours69. NanojuiceTM Transfection Kitproduced by EMD Chemicals Inc. and Superfect®TransfectionReagentofQiagenaredendrimerbasedDNAtransfectionkitsusedfordeliveringDNAintothecell.Theseareclaimedtohaveimprovedtransfectionefficacy and low toxicity to cells70,71.

Dendrimer based drugs are being tried for anti-retroviral therapy70and it is in stagesofclinical trialafter getting clearance from US-FDA on July 2003.ThismoleculewasfoundtosuccessfullypreventsimianHIVinfection19.PAMAMdendrimerscanalsobeused


intreatmentofcancerbyconjugatingwithanti-cancerdrugs like cisplatin, adriamycin or methotrexate72.Calabrettaet al73havedemonstrated theantibacterialproperty of amino-terminated polyamidoamine(PAMAM)dendrimersandtheirpartiallypolyethyleneglycol (PEG)-coated derivatives. The antibacterialproperty was observed against both Gram negativePseudomonas and Gram positive Staphylococcus aureus.However, thePEGcoatingof thedendrimer,which reduces the cytotoxicity of unmodified PAMAM dendrimers, reduces the efficacy against Gram positive bacteria without change in efficacy against Gram negativebacterialikepseudomonas73.

Dendrimers are also used as contrast agentsfor imaging. The 1,4-diaminobutane (DAB) coredendrimer and the PAMAM dendrimer are wellstudied commercially available dendrimers forimaging studies. Renal excretion is the main routeof clearance and is dependent on the size of theparticleandmorethan60percentofinjectedDABorPAMAMdendrimerisclearedfromcirculationwithin15min74.Smallersizeddendrimersundergorapidrenalclearance whereas dendrimers with charged surfaceor hydrophobic surfaces are rapidly cleared by theliver. Those dendrimers with a hydrophilic surfaceescape renal clearanceandhaveagreater circulationtime75.

Cationic dendrimers have a greater potential tocause cytotoxicity compared to anionic dendrimeror PAMAM dendrimers. It is proposed to cause cellmembrane instability and cell lysis. The toxicity ofdendrimerisdependentonthesizeoftheparticleandincreasewithsize.Itcanbereducedbymeansofsurfacemodification of the dendrimers with incorporation of PEGorfattyacids76.

Nanotechnology in gene therapy

Genetherapyisanewermodalityofapproachfortreatmentofmanygeneticdisordersincludingdiabetesmellitus77, cystic fibrosis78, and alpha 1 antitrypsindeficiency79.Viralvectorsusedforgenetransferhavethe limitations of safety concerns and stimulation ofimmunesystemwithproductionofantibodiesagainstthe viral vectors. Further, naked DNA cannot crossthe negatively charged cell membrane as these arealsonegativelycharged79,80.Hence,thereisaneedforother modes of transfer of genetic material such asnanoparticlebasedgenetherapy.Liposomesmeasuringless than100nmcanbeusedfordeliveryofgeneticmaterial into cells. Liposomes incorporated with

polyethylene glycol and galactose target liver cellseffectivelydue to their rapiduptakeby liverKupffercells. Thus gene therapy may be tried with suchliposomalnanoparticlesforvariousliverdisorderssuchasWilson’sdiseaseandhereditaryhemochromatosis79.

Niu et al77 used human insulin gene in chitosannanoparticles to transfect STZ diabetic rat throughgastrointestinal tract. They found a significant decrease in fasting blood glucose level, increase in plasmainsulin levels and expression of human insulin genemRNA in the study rats.This study may lead to thedevelopmentofanewermodalityoftherapyfortype1diabetesmellitus77.

A “hybrid nanodevice” designed by workersof Northwestern University and Argonne NationalLaboratory consists of a titanium dioxide (TiO2)semiconductor nanoparticle of dimension 4.5 nmlinked covalently to oligonucleotide DNA19. Theoligonucleotide sequence can direct the hybridnanodevicetothecorrespondingDNAsegmentwithinthecellnucleus.WhenstimulatedwithlightorXrays,TiO2inducesnucleicacidendonucleasewhichcleavesoff the specific DNA fragment. This device is yet to be testedinalaboratorymodel.However,itmayhaveapotentialutilityintreatmentofvariousmalignanciesinthefuture.

Respirocytes

Respirocytes are hypothetical artificial red blood cells51 are nanodevices which can function as redblood cells but with greater efficacy. These have higher capacity to deliver oxygen to tissues, supplying 236times more oxygen per unit volume than natural redbloodcells.Thesedeviceshavesensorsonthesurfacewhich can detect changes in the environment and theonboard nanocomputer will regulate the intake andoutput of the oxygen and carbon dioxide molecules.Aninfusionofonelitredoseof50percentrespirocytessalinesuspensioninahumancantheoreticallykeepthepatientoxygenatedup to fourhours followingcardiacarrest19,81.Respirocytes,consideredasadevicebyFDAareregulatedundertheprovisionsoftheMedicalDeviceAmendmentsof1976,theSafeMedicalDevicesActof1990,andtheMedicalDeviceAmendmentsof199282.

Microbivores

Microbivores83 are hypothetical structures whichfunction as white blood cells in the blood streamdesignedtotrapcirculatingmicrobes.Theyareexpectedto have greater efficacy than cellular blood cells in


phagocytosis. The microbivores surface is arrangedwithprocesseswhichcanextendinlengthandsecurethemicrobewhichgetsincontactwithit.Themicrobewillbegraduallymanoeuvredtotheingestionportandundergoestheprocessofmorcellizationandenzymaticdegradation.The endproducts are released as aminoacids,fattyacids,nucleotidesandsugars.Applicationof the microbivores in human circulation couldtheoreticallyclearthebloodstreaminsepticaemiaatamuchgreaterratethanthenaturaldefencemechanismwithantibiotics19,83.

Regulatory challenges with nanomedicines

Regulatory issues play a major role in thedevelopment of nanoformulation drugs. Theseinclude, the type of nanodrug produced and thevariousregulatoryrequirementsthatthemanufacturersmust follow during the manufacturing of nanodrugs.A nanoformulation of a drug which is based on apreviously approved drug in microformulation canundergo a shorter approval pathway by means ofabbreviated new drug application if bioequivalencecan be demonstrated to its microformulation drug.However, if bioequivalence cannot be demonstrated,itwouldnecessitateapprovalofallthestagesofnewdrugapplication.Further,whenananodrugisdesignedas a new chemical entity, the evaluation procedurebecomesmorestringent84.

NanodrugmanufacturersmustcomplywithFDA’sCurrent Good Manufacturing Practices (CGMP) andQualitySystemRegulations(QSR)85.Noncompliancewith these regulations would warrant enforcementactionsbytheFDA.Forexample,21CFR211.25ofFDA’s CGMP requires that the personnel involvedin the manufacturing process, in providing trainingfor the workers and the personnel supervising themanufacturing process have adequate training,education and experience. Further, the training mustbe conducted at adequate frequency and there mustbe adequate number of qualified personnel for the assignedduties86.Ananodrugmanufacturermustinvestconsiderable amount of financial resources to have such qualified personnel in the working unit. Maintenance of equipment formanufactureofnanodrugsandcontrolofcontaminationarealsoregulatoryrequirementsformanufacturers. The drug products are purified by the use of filters and CGMP demands that the filters do not release fibres87. However, when liquid filtration is used, nanodrug manufacturers will not be able to complywith CGMP, since the smallest filtration level available

isapproximately15nmandnanodrugscouldbeattherangeof5to6nmlong84.

The FDA centres namely, the Center for DrugEvaluation and Research (CDER), the Center forDevices and Radiological Health (CDRH) and theCenterforBiologicsEvaluationandResearch(CBER)regulatedrugs,devices,andbiologicsrespectivelyandareresponsibleforregulatingnanomedicalproducts.FDA classifies medicinal products as drug, device or biologicsaccordingtotheirprimarymodeofactiontoassignacentrefortheirprimaryjurisdictionduringtheevaluation process. In case of a nanodrug it is difficult to classify it as a drug, device or biologics since ittendstohaveacombinationoftheabove.Hence,theassignment of the Centre becomes difficult. Further, thedrughastopassthroughall theCentresofFDAowingto itscomplexity.Thisresults ingreater timeperiod for approval of the drug. The staff of FDAmust also be sufficiently educated and trained in nanotechnology in the field of medicine to evaluate nanodrugproducts.88

Potential hazards of nanoparticles

Nanoparticles, as a result of their extrememicroscopic dimension, which gives uniqueadvantage,havepotentialhazardssimilartoparticulatematter89. These particles have the potential to causevaried pathologies of respiratory, cardiovascular andgastrointestinal system1. Intratracheal instillation ofcarbon nanotube particles in mice, has shown thatcarbon nanotubes have the potential to cause variedlungpathologieslikeepitheloidgranuloma,interstitialinflammation, peribronchial inflammation and necrosis oflung.Thetoxicityproducedbycarbonnanotubewasfoundtobegreaterthanthatproducedbycarbonblackandquartz90.

Nanoparticlescanenterthecentralnervoussystemeitherdirectlythroughaxonsofolfactorypathwayorthrough systemic circulation that C60 fullerene cancauseoxidativestressanddepletionofGSHinbrainin fishes by entering through the olfactory bulb91.Involvementofolfactorybulbinhumansispossibleincaseofinhalationalexposure.Studiesdoneonmonkeysand rats have shown accumulation of carbon andmanganesenanoparticlesintheolfactorybulbthroughtheolfactorypathway92,93.Thisshowsthatnanoparticlemediated delivery can in future provide a means ofalternateroute,circumventingthebloodbrainbarrier.However, this can also result in the inflammatory reactionsinthebrainwhichneedstobeevaluated.


Radomskiet al94haveobservedtheproaggregatoryeffectsofnanotubesonplateletsinin vitrostudiesandaccelerationofvascularthrombosisinrat.Itwasalsoobserved that fullerenes do not have the property ofinducing platelet aggregation. Thus, for designingnanoparticle based drug delivery systems, fullerenesmaybeasaferapproachascomparedtonanotubes1,94.

Thetoxicityofnanoparticlescanalsobeextrapolatedto gastrointestinal system, resulting in inflammatory bowel diseases. The toxicity of nanoparticles may berelated to its ability to induce release of pro-inflammatory mediators resulting in inflammatory response and organ damage. If ingested, the nanoparticles can reach thecirculation and reach different organs and systems andpossibly result in toxicity95. These have been studiedin vitro and inanimalmodelsand theeffectonhumansystem is difficult to extrapolate from such studies. Their useinhumansrequirefurtherresearchandmuchneededcaution.

Conclusion

Although the expectations from nanotechnologyin medicine are high and the potential benefits are endlessly enlisted, the safety of nanomedicine is notyet fully defined. Use of nanotechnology in medical therapeuticsneedsadequateevaluationofitsriskandsafetyfactors.However,itispossiblethatnanomedicinein future would play a crucial role in treatment ofhuman diseases and also in enhancement of normalhuman physiology. With concurrent application ofnanotechnology in other fields, its utility is likely to extend further into diagnostics, molecular researchtechniquesandtools.

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Reprint requests:DrC.Adithan,DirectorProfessor&Head,DepartmentofPharmacology,JawaharlalInstituteofPostgraduate MedicalEducation&Research(JIPMER),DhanvantariNagar,Puducherry605006,India e-mail:[email protected]


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