Biomedical applications and colloidal properties of amphiphilically modified chitosan hybrids

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ContentslistsavailableatSciVerseScienceDirect

Progress

in

Polymer

Science

j o u r n al ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / p p o l y s c i

Biomedical

applications

and

colloidal

properties

of

amphiphilically

modiﬁed

chitosan

hybrids

Mikael

Larsson

a,b

_,

_Wei-Chen

_Huang

a,1

_,

_Meng-Hsuan

_Hsiao

a,1

_,

Yen-Jen

Wang

a,1

_,

_Magnus

_Nydén

b

_,

_Shih-Hwa

_Chiou

c

_,

_Dean-Mo

_Liu

a,∗

a_Department_of_Materials_Science_and_Engineering,_National_Chiao_Tung_University,₁₀₀₁_Ta-Hseuh_Road,_Hsinchu_City,₃₀₀_Taiwan_ROC b_Ian_Wark_Research_Institute,_University_of_South_Australia,_Mawson_Lakes_Campus,_Mawson_Lakes,_SA_5095,_Australia

c_Institute_of_{Pharmacology,}_National_Yang-Ming_University_and_Department_of_Medical_Research_and_Education,

TaipeiVeteransGeneralHospital,Taipei,Taiwan

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21December2012 Receivedinrevisedform20June2013 Accepted27June2013

Available online 12 July 2013 Keywords:

Amphiphilicmodiﬁedchitosan Colloidalproperties

Biomedicalapplications

a

b

s

t

r

a

c

t

Chitosanisamongthemostabundantbiopolymersonearthandhasbeeneitherusedor exhibitedpotentialinawidevarietyofindustrialandbiomedicalapplications.Withthe advancementofmaterialstechnologies,chitosanhasbeenchemicallymodifiedto self-assembleintonanoarchitecturesthatareusableinadvancedbiomedicalapplications,such asdrugnanocarriers,macroscopicinjectables,tissue-engineeringscaffolds,and nanoimag-ingagents.Colloidalamphiphilicallymodifiedchitosan(AMC)isarelativelyrecentmaterial receivingincreasedattentionwithnumerouspublicationsaddressingthemedical advan-tages ofspecific systems.To date,many reviews havefocusedon the synthesisand biomedicalpropertiesofchitosan-basedbiomaterials,butacomprehensivestudyfocusing onthecolloidalpropertiesofAMCinrelationtobiomedicalperformanceappearstobe lack-ing.Thisreviewprovidesasurveyofthefield,criticallyreviewingthecolloidalproperties andbiomedicalperformanceofAMCsystems,suchasnanoparticledrugdeliverysystems andmacroscopicmedicaldevices.Finally,thefuturedevelopment,marketpotential,and clinicalimplicationsofthesepromisingcolloidal-structuredbiomaterialsaresummarised. © 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction... 1308

2. Amphiphilicallymodiﬁedchitosan... 1308

2.1. Generalinformationonchitosan... 1308

2.2. Preparationofamphiphilicallymodiﬁedchitosan... 1309

2.3. Colloidalpropertiesofamphiphilicallymodiﬁedchitosan... 1309

3. Colloidalamphiphilicallymodiﬁedchitosaninbiomedicalapplications... 1311

3.1. Nanoparticles... 1311

3.1.1. Deliveryofconventionaldrugs... 1311

3.1.2. Deliveryofproteinsandpeptides... 1315

3.1.3. Genedelivery... 1315

3.1.4. Imaginganddiagnosis... 1317

∗ Correspondingauthor.Tel.:+88635712121x55391;fax:+88635724272. E-mailaddress:[email protected](D.-M.Liu).

1 _Contributed_equally_as_second_author.

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3.2. Macroscopicassemblieswithcolloidalstructures... 1318

3.2.1. Injectabledepotgelsfordrugdelivery... 1318

3.2.2. Invivocellscaffolds... 1319

3.2.3. Wounddressings... 1320

3.3. Organic–inorganichybridnanostructures... 1321

4. Clinicalimplicationsandmarketpotential ... 1322

5. Conclusions... 1322

Acknowledgments... 1323

References... 1323

1. Introduction

Recent advancements in medicine combined with improvementsinunderstandingfundamentalcellularand molecularbiologicalprocesses have ledtoan increased demand for materials that can fulfil specific functions toimprove therapeutic effects. In particular, nanoparti-cles and nano-microstructured materials can allow for functionalitiesthatarenotachievablethrougheitherthe administrationoffreedrugsortheuseofmaterialsthatlack criticalstructuralcharacteristics.Nanotechnologycanuse engineeredmoleculesand/orsupra-molecularentitiesto formassembliesonthescaleofindividualcells,organelles, or even smaller components [1]. Polymeric nanoparti-clesand structures have been heavily investigated and showngreatpotentialinnumerousmedicalapplications [2–6].Thesynthesisof“smart”moleculesandthecontrol overmolecularself-organisationintowell-defined nano-structuresarecurrentareas ofresearch,asthecolloidal properties of thesematerials can have dramatic effects onbothproperties andperformance in various applica-tions.Formedicalapplications,increasedeffortshavebeen madetopreparebiodegradable andnon-toxic polymeric amphiphilesbasedonnaturalpolymers,suchas polysac-charides.

Chitosanisapolysaccharidethathasbeenextensively investigated for biomedical applications, with many reports on modified chitosan exhibiting new function-alities [7–15]. Multiple reports on the modification of chitosanandpromiseforutilisationinpharmaceuticsand biomedicineare available in theliterature. Amphiphilic modificationsthat promote self-assemblyinto nanopar-ticles and/or provide increased interactions with lipid structures,suchascellmembranes,areofparticular inter-est.Ascolloidalpropertiesareimportantfortherapeutic effects,asoftendiscussedinindividualpapers,a compre-hensivereviewfocusingonthecolloidalbehaviourofAMC nanoparticles,theassemblyoftheseentitiesintogelsand films, andtheir effectivenessin biomedical applications is needed. This review provides a condensed sourceof newinsightsandanimprovedunderstandingontherole andprospectsofcolloidalAMCinbiomedicalapplications, critically important for advancing current promising researchintoclinicalpractices. Thisreview begins with abriefoverviewonthesynthesisofAMC followedbya criticalexaminationoftheliteratureonAMCin biomed-ical applications. Reviewing multiple applications, the examinedmaterialsrangedfromnanoparticlesto macro-scopicassemblies,includingthedeliveryofconventional drugs,proteins/peptidesandgenes,aswellastheuseof

carriers for contrastagents, andnanostructured materi-als for wound dressings and cell scaffolds.Amphiphilic chitosan-inorganic hybrids are also included, as this newly developed material class has exhibited potential for applications involving sensing, imaging, diagnosis, andmultifunctionaldrugdeliveryprocesses.Thisreview summarisesthemostimportantpapersinthefieldofthis highlyversatilepolymerclassandreflectsovertheir rele-vanceinpotentialapplicationstofacilitatethetranslation oftheseresearchinitiativesintoclinicalbenefits.

2. Amphiphilicallymodiﬁedchitosan

2.1. Generalinformationonchitosan

Chitin, the precursor of chitosan, is a biopolymer composedofN-acetylglucosamineunitslinked by␤-1,4 linkages and isamong themostabundantbiopolymers, clearlyoutrankedonlybycellulose.Chitosanisformedby extensivedeacetylation ofchitin,formingamino groups at the C2carbon (Fig.1).These groups accept protons, dramatically increasing the solubility of chitosan even at slightly acidic pH, compared to chitin. The positive chargeofchitosanhasgeneratedgreatinterestin biomed-icalresearchforthepossibleinteractionsofthispolymer withnegativelychargedbiomoleculesandtherapeutic sub-stances.

Numerousreportsonthepromisingusesofchitosanin medicalapplications,bothasmacroscopicmaterialsandas nanocarriers,areavailableintheliterature,suggestingthat chitosan hasantibacterial,biodegradable,biocompatible, andmucoadhesivepropertieswithreasonable biocompat-ibilityforbiomedicaluses[8,11,12,14,16,17].Chitosanhas alsobeenshown totransiently openthetight junctions betweencells[18,19].However,complicationshavebeen reportedforchitosanbasedmaterials.InastudybySkogh andEngstrand,aheparin-chitosanspongeusedasacarrier forrecombinanthumanBMP-2incranialrepairgenerated inﬂammatoryreactions[20].Clinicallyrelevant biodegra-dationmayvarywithchitosanpropertiesandthesiteof degradation.Thechitosanpreparedfromchitinhasvarying degreesofdeacetylation.Togetherwithmolecularweight (Mw),thisdegreeofdeacetylationdeterminesthe prop-erties,colloidalbehaviours, and biologicalperformances [11,16,17,21–23],whichinturnwillaffectbiomedical per-formance. Controlover thesetwo parametersis critical. Oneapproachtoovercomevariationsinproperties associ-atedwiththedegreeofdeacetylation(oratleastmakethe formulationsmorerobust)couldbetoattachgroupswith

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Nomenclature

AMC Amphiphilically modiﬁed chitosan (chi-tosanmodiﬁedtoenhancetheamphiphilic characteristicsofthepolymer)

BSCB Blood-spinalcordbarrier

CAC Criticalaggregationconcentration CHC Carboxymethyl-hexanoylchitosan

CPT Camptothecin

DCMC Deoxycholicacidmodiﬁed carboxymethy-latedchitosan

DOX Doxorubicin

ECPs Extracellularproducts

EPR Theenhanced permeability and retention effect

G-CS-DCA Glycidol-chitosan-deoxycholicacid Genedelivery Usedforthedeliveryofgenetic

mate-rials, including DNA, RNA and modiﬁed analogues

GP Glycerophosphate

HAp Hydroxyapatite

HGC Hydrophobicallymodiﬁedglycolchitosan hm-chitosan Hydrophobicallymodiﬁedchitosan HTCC

N-(2-hydroxy)propyl-3-trimethylammoniumchitosanchloride ICPTES 3-isocyanatopropyltriethoxysilane iPSCs Inducedpluripotentstemcells

LA Linoleicacid

LC Loadingcapacity

LCC N-lauryl-carboxymethylchitosan

LE Loadingefﬁciency

LMC Linoleic acid/poly(␤-malic acid) double graftedchitosan

LSC Lauroylsulfatedchitosan MDR Multidrugresistance MPC Methylpyrrolidonechitosan MRI Magneticresonanceimaging

Mw Molecularweight

Nanocarrier Nanoparticlesloadedwithcargo Nanoparticle Any nano-sized object (<1000nm),

irrespectiveofactualstructure

NIR Near-infrared

NMPCS N-methylenephosphonicchitosan NOSC N-octyl-O-sulfatechitosan

N/P TheratioofchitosannitrogentoDNA phos-phorus

NSC N-succinyl-chitosan

OCMCS Oleoyl-carboxymethylchitosan OcCMC N-octyl-N,O-carboxymethylchitosan OQLCS Octadecyl-quaternisedlysinemodiﬁed

chi-tosan

PDMS Polydimethylsiloxane PEG Poly(ethyleneglycol) PMLA Poly(␤-malicacid)

PTX Paclitaxel

PVP Polyvinylpyrrolidone SCI Spinalcordinjury

SOC N-succinyl-N-octylchitosan

SPIONs Superparamagneticironoxidenanocrystals

TEOS Tetraethylorthosilicate

ZrP ␣-zirconiumhydrogenphosphatehydrate

dominatingcharacteristics,suchasstronghydrophilicity and/orhydrophobicity,dependingonthedesiredresults. 2.2. Preparationofamphiphilicallymodiﬁedchitosan

ChitosancanbereadilymodiﬁedusingtheC2amino groups aswellasthe C3andC6 hydroxylgroups, with thereactivityofC6>C3forpolysaccharidereactions[24]. Extensiveworkhasbeenconductedonthemodiﬁcations ofchitosan, assummarised inseveralexcellent reviews coveringthistopic[12,14,21].

Modificationsofchitosanwithboth hydrophobicand hydrophilicgroupswerefirstreportedin1995by Yosh-ioka et al. [25]. Hydrophobic alkyl chains of various lengthswereattachedtotheaminogroupsandhydrophilic sulfate groups were attached to the hydroxyl groups, producing a modified chitosan that formed polymeric micelles.Miwaetal.synthesised N-lauryl-carboxymethyl-chitosan(LCC)byattachinghydrophobiclaurylgroupsto theaminogroupsandhydrophiliccarboxymethylgroups to the hydroxyl groups [26], also forming polymeric micelles. Based on these findings, preparations of AMC areoftenperformed witha hydrophilicmodification by carboxymethylationtoincreasewatersolubility[10]and withadditionalmodificationsusinghydrophobicgroups, such as cholesterol [27], lauryl aldehyde [26], linoleic acid (LA) [28,29], deoxycholic acid [30], and hexanoyl anhydride[31–33].Withanextensivenumberofpossible configurations, the generalmodification strategies typi-callyincludereactionswithhydrophilicgroupstoincrease watersolubilityandwithhydrophobicgroupstofacilitate theself-assemblyintonanostructuresaswellastoimprove theinteractionswithbio-relevantlipidstructures,suchas cellmembranes.Modifications of chitosan can dramati-callychangethe physiochemicalcharacteristicsand the degradationproductsofthesepolymers,suggestingthat thereisgreatneedtocharacteriseboththe physiochemi-calpropertiesandthemedicalsafetyofthesenewchitosan derivatives.

2.3. Colloidalpropertiesofamphiphilicallymodiﬁed chitosan

Self-assembled colloids have shown great potential in numerous applications, with an ease of production, regularityinboth sizeand shape,andthepresenceofa core,a bi-layer,or nano-domains withpolarproperties thatareoppositethoseofthesolvent.Particlesizecanalso beimportant, as this characteristic inﬂuencesboth cell internalisation and clearance from circulation [34–36]. Literatureguidanceoftenstatesthatnanoparticlesshould besizedsmallerthanthesinusoidinthespleenandthe fenestra of the liver (150–200nm) to avoid clearance andthatpassivetumourtargetingthroughtheenhanced permeationandretentioneffect(EPR)occursthroughgaps

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Fig.1.SchematicdrawingillustratingthestructureofAMC,withpossiblesitesforhydrophilicandhydrophobicmodifications.AMCcanself-assemble intonanoparticles.Conceptualnanoparticlestructuresarepresented,includingahydrophobiccore-hydrophilicshellparticle,abi-layercapsuleanda particlewithhydrophobicandhydrophilicnano-domains.Thenanoparticlescanbeloadedwithdrugs,whichwilldistributetominimisethechemical potential.Thenanoparticlescancarrysurfacemodificationsfortargeting.Thehydrophobicandhydrophilicmodificationscanalsobegroupsthatinduce functionality,suchastargetingorimagingcapability.Forvisualclarity,thecomponentsinthefigurearenotdepictedatscale.

of100–600nm[37].However,theauthorswanttosuggest thatpolymericnanoparticlescommonlyaredeformable, soincreasedsizesmaypassthroughporesizesthatmight nominallyexcludethenanoparticles,asreportedbyChen andWeissforredbloodcellsinthespleen[38].Thesurface ofnanoparticlesisoftencharacterisedintermsofthezeta potential,withanabsolutevalue>25mVsuggestinggood stability in aqueoussolutions. Invivo, chargedparticles interactwithbiomoleculesandcells[35,36,39],thusthe zeta-potential is a relevant property. However, these electrostaticinteractionsarecomplex,makingtheinvivo stabilitydifficulttopredictfromthezeta-potentialvalues. The nanostructure of self-assembled colloids allows for the incorporation of molecules into regions leading toreduced chemicalpotentials, surroundedbyabarrier withdifferentpolarproperties.Thenanoparticlesurface exposed to the surroundings can be modified for spe-cificneeds,includingtargetingapplications[40–42],the avoidanceofproteinadsorption,phagocyteuptake, parti-cleaggregationandbloodclottinginthebloodstream,and aprolongationofthecirculationtime[43–45](seeFig.1). Liposomes and low-molecular micelles have been widelystudied for pharmaceutical and biotechnological applications,suchasdrugdelivery,genetherapy,protein delivery,andcelltargeting[46].Thesenanostructureshave oftenbeenfoundtobestructurallyunstableunderrelevant conditions[25],and acurrent challengeis toovercome thefast elimination of these nanoparticles through the reticulo-endothelialsystem[47].

Self-assembledpolymericmicelles ofAMC havebeen proposedtobemorestablethanlowMwsurfactantbased micelles[25],producinganimprovementinadesired prop-ertyformanyapplications.Followingthereportingofthese results,colloidalpropertiesandapplicationsofAMChave generatedincreasedattentionintheresearchcommunity. Typically, new molecules are characterised withregard

tophysiochemicalproperties,suchascriticalaggregation concentration (CAC), particle sizes, zeta-potentials, and (ifpossible)nano-microstructuralcharacteristics,withthe propertiesdependingonboththenatureanddegreeof sub-stitution.Thepolaritymap andstericpropertieschange uponmodification,andelectrostaticcontributionscanalso beintroduced.Inparticular,acidicsubstituentsallowfor pH dependentinteractions with theamino groups. The rangeandcomplexityofthestructure-property relation-shipsinvariousAMCsmakesimplificationsdifficult,but severalnoteworthycommentscanbemade.

Commonly,AMCsandotherself-associatingpolymers areassumedtohaveahydrophobiccore-hydrophilicshell structure.Hydrophobicdomainsmayalsobeinterspersed through the particle, especially for short hydrophobic substituting groups. Korchagina and Philippova [48] proposed this type of structure for nanoparticles from chitosanmodifiedwithn-dodecylsidechains,suggesting a nanogel structure with hydrophobic domains at the crosslinks.Withregardstonanocapsules,Liuetal.[33,49] andLietal.[50]reportedAMCsthatself-assembledinto eithercompactcore-shellorhollowbi-layered nanopar-ticles,dependingonthespecificmolecularcharacteristics (seeschematicdrawinginFig.1).Forthesystem investi-gatedbyLiuetal.,alkylchainsconstitutedthehydrophobic groups,andthetransitionfromacompactcoretohollow capsulesoccurredwithincreasinghydrophobicity.In con-trast,Lietal.,reportedthatincreasingthegraftingofthe hydrophobicgrouppoly(L-lacticacid)producedadecrease in the fractionof thehollowcapsules. Thereshouldbe cases when particles with compact core,particles with interspersednano-domainsand hollowcapsulesbehave differentlyandofferstructurespecificopportunities,such as loading of trans-membrane structures and loading of hydrophilic substances. However, since the distinc-tion betweendifferentstructures is seldomreported in

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biomedicalstudiesusingAMC,suchdistinctionwillnotbe madeinthegeneraldiscussionsofthisreview.

Theself-assemblyofAMCisdrivenbytheorientationof hydrophobicgroupstoformhydrophobicdomainswiththe hydrophilicgroupsorientatedtowardstheaqueous envi-ronment.Generally,CACdecreaseswithincreasingdegree ofhydrophobicsubstitutionandsizeofthehydrophobic groups,asreportedintheliterature[29,33,50–53].

AbroadrangeofsizeshasbeenreportedforAMC par-ticles,rangingfromtensofnanometres[54,55]uptothe micrometrescale[50],withthemajorityofreports pre-senting values in the interval of 100–500nm. The size oftheformednanoparticlescandependonthepolymer backbonelength,thenatureof theattachedgroups and thedegreeofsubstitution.Preparationconditionscanalso greatly influence the self-aggregationresults, especially becausepolymeraggregatesystemscanformkinetically trappedstructures.ForAMCverydifferentdependencesof nanoparticlesizeonthedifferentparametershavebeen reported.Forexample,Kimetal.reportedthatfor nanopar-ticlesofdeoxycholic-acidmodifiedchitosan,anincreaseof Mwfrom5to200kDaledtoanincreaseinnanoparticle sizesfrom130nmto300nm[56].Parketal.reportedthat fornanoparticlesofglycolchitosanmodifiedwithcholanic acid,thesizeafterself-aggregationonlyincreasedslightly withMwvaluesrangingfrom20to250kDa[57].Similarly, Korchagina and Philippova reported that the aggregate sizesindilutesolutionofbothchitosanand hydrophobi-callymodifiedchitosan wereindependentof Mwinthe range55–150kDa,suggestingthatthenumberof associat-inggroupswasthedeterminingfactor[48].Theextentof broadeningoftheintervalwithoutaffectingthesizeofthe aggregateswouldbeofinterest,aswellasadetermination oftheeffectsofmorebio-relevantsolutions.

Forhydrophobicmodificationstherehavebeen multi-plereportsonhowthelengthanddegreeofsubstitution influencethenanoparticlesize.Withincreasing hydropho-bicmodifications,bothincreased[48,58,59]anddecreased [51–53,60,61] sizes, as well little effect [33] and more complicateddependences[55]havebeenreportedinthe literature.Theincorporationofdrugsubstancescanalso influencenanoparticlesize.

Forzeta-potentials,theeffectsaresomewhatless com-plicated. Typically, the characteristics of AMC can be associatedwithtwosituations:(i)thenativeaminogroups will be the dominating charge contributors, suggesting that the zeta-potential is expected to be positive and todecreasewiththeneutral oracidicsubstitutionsthat are either attached to the amino nitrogen or electro-staticallyinteractingwiththechargedaminogroups;(ii) thedominating chargecontributors arethesubstituting groups, which can be either positive or negative, with thecorrespondingzeta-potentialincreasingwith increas-ing degree of substitution. Withthe pHdependence of the amino groups and the other substituents, electro-static interactions with the loaded molecules and the structuralorientationmayincreasethecomplexityofthe zeta-potentialbehaviour.

Insummary,foragivenAMCsystem,bothparticlesize andsurfacepropertiescanbetunedbychangingthetype and degree of chemical modiﬁcation. The dependences

ofthepolymercharacteristicswithnanoparticlesizeand surfacepropertiesarecomplicated,suggestingthateach systemisunique.

3. Colloidalamphiphilicallymodiﬁedchitosanin biomedicalapplications

TheinvestigationofAMCfor biomedicalapplications hasincreasedinthelasttwodecades,withintensiﬁcation ofeffortsinrecentyears.TheliteratureonAMC nanoparti-clesfordrug,protein,genedeliveryandimaging,aswell asmacroscopicnano-microstructuredAMCfor biomedi-calapplicationsisreviewedinthiswork.Aninformative listofmultipleAMC,colloidalstructuresandbiomedical applicationsisgiveninTable1.

3.1. Nanoparticles

3.1.1. Deliveryofconventionaldrugs

Commonly,hydrophobicdomainsofamphiphilic self-assemblednanoparticlescanprovideaneffectiveloading space for hydrophobicmoieties, suchas drugs, fluores-cent probes, and contrast agents, with the hydrophilic surfaceallowingforsuspensionstabilityinaqueous solu-tions[57,62].Theloadingofhydrophilicmoietiesisalso possible(seeconceptualdrawingsinFig.1).Nanocarriers haveapplicationsinanumberofmedicalapplications,such ascancertherapy.Onecurrentchallengerelatestothelow watersolubilityofmany drugs,suchasPaclitaxel(PTX) [63],Camptothecin(CPT)[64],andDoxorubicin(DOX)[65]. In addition to improved solubility, the loadingof drug intonanocarrierscanoffera rangeofbenefits,including increasedtime in circulation, passive targetingthrough theEPR,protectionofthedrugfromdegradation,reduced drugrelatedsideeffects,andtheactivetargetingby sur-facemodifications.Thenanocarrierscanalsobedesigned torespondtoexternalstimuli,suchaspH,light,magnetic fields,etc.[4–6,21,66–71].Tosummarise,anideal poly-mericnanoparticlematrixfordrugdeliverywouldexhibit thefollowingcharacteristics:(i)allowfortheincorporation ofthedrugintothenanoparticles;(ii)provideprotection ofthedrugfromenzymedegradation;(iii)facilitate cellu-laruptakeintargetcells;(iv)releasedrugatthesiteof action(i.e.,toincreasethelocaldrugconcentrationand prolongthedurationofdrugactivity);(v)decreasedrug toxicities;and (vi)providelow manufacturingcosts. To achieve theseproperties,chitosanhasbeenmodified to producenanoparticlesandwidelyinvestigatedforuseas adrugcarrier[2,12–14,72].

Onepromisingclassofmaterialsthathasbeen devel-opedis AMC.Numerous studieshavedemonstrated the self-assembly of this material into nanoparticles with excellentdrugloadingcapacities,modiﬁedrelease prop-erties, and good colloidal stability under physiological conditions, asdiscussed below. Withexcellent colloidal stability for well-encapsulated therapeutic substances, AMCformulationsarehighlypromisingforpracticaldrug deliveryuses,especiallyformodiﬁedpolymersthatretain thebiocompatibilityofchitosan.

Miwaetal.reportedin1998thatPTXloaded nanocar-riersofchitosan derivativeswithbothhydrophobicand

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M. Larsson et al. / Progress in Polymer Science 38 (2013) 1307 – 1328 Table1

ListofselectedAMCswithcolloidalpropertiesandbiomedicalapplications.

AMC Colloidalproperties Biomedicalapplications References

Acylatedchitosan Nanoparticles,spunnanoﬁbres,microporous sponge,formingAC-cellgelnetwork.

Drugdelivery,genedelivery,cellscaffold,wound dressings

[95,131,190,191,199]

Acylatedcarboxymethylchitosan Nanoparticles Drugdelivery [79]

Alkylchain-modiﬁedsuccinylchitosan Nanoparticles Drugdelivery [96]

Benzene-n-octadecyl-chitosan Microporoussponge,forming

benzene-n-octadecyl-chitosan-cellgelnetwork

Wounddressing [200]

Carboxymethylchitosan-6-mercaptopurine pHdependentnanoparticleformation Drugdelivery [72]

Carboxymethyl-hexanoylchitosan Nanocapsules,nanoparticles,shellconstituentin core-shellnanoparticles,nano-microstructured macroscopicgels

Drugdelivery,injectabledepotgels,wound dressings,injectablecellscaffolds

[29,31–33,97,169,192,202]

Carboxymethyl-hexanoylchitosan–Silica Multilayerednanoparticles Drugdelivery [223]

Chitosan-silicahybrids Silicacore-chitosanshellnanoparticles, nanostructuredmembrane

Biomaterials,boneregenerationandseparation membranes

[219–221]

Deoxycholicacidmodiﬁedcarboxymethylated chitosan

Nanoparticles Drugdelivery [30]

Deoxycholicacidmodiﬁedchitosan Nanoparticles Genedelivery [56]

Deoxycholicacid-N,O-hydroxyethylchitosan Nanoparticles Drugdelivery [88]

DOX-conjugatedstearicacid-g-chitosan Nanoparticles Drugdelivery [89]

Folate-decoratedsuccinylchitosan Nanoparticles Drugdelivery [54]

Glycidol-chitosan-deoxycholicacid Nanoparticle Drugdelivery [58]

Hydrophobicallymodiﬁedglycolchitosan Nanoparticles Drugdelivery,genedelivery,imagingand diagnosis

[52,53,57,74–78,92,134,156]

Hydrophobicallymodiﬁedglycol chitosan-Cy5.5-RGDpeptides

Nanoparticles Proteindelivery,Imaginganddiagnosissystem [154,155]

Hydroxypropylchitosan Micro-nanostructuredorganicinorganichybrid networktogetherwithethyleneglycol functionalisednanohydroxyapatite

Cellscaffold [210]

Lauroylsulfatedchitosan Nanoparticles Drugdelivery,proteindelivery [100,101]

Linoleicacid-carboxymethylchitosan Nanoparticles Drugdelivery [28,29]

Linoleicacidgraftedchitosanoligosaccharides Nanoparticles Drugdelivery [85]

Linoleicacid/poly(␤-malicacid)doublegrafted chitosan

Nanoparticles Drugdelivery [61]

Methylpyrrolidonechitosan Gelformingfreezedriedsponge Woundhealing [201]

N-alkyl-N-trimethylchitosan Nanoparticles Drugdelivery [55]

N-alkyl-O-sulfatechitosan Nanoparticles Drugdelivery [91]

N-lauryl-carboxymethylchitosan Nanoparticles Drugdelivery [26]

N-methylenphosphonicchitosan Nanoparticles Genedelivery [136]

N-octyl-O-sulfatechitosan Nanoparticles Drugdelivery [80,81,90,98]

O-carboxymethyl-chitosan-methotrexate Nanoparticles Drugdelivery [227]

Octadecyl-quaternisedlysinemodiﬁedchitosan Nanoparticlestogetherwithcholesterol, multi-lamellarstructure,couldhavefolate-PEG coating

Drugdelivery [42]

Oleoyl-carboxymethylchitosan Nanoparticles Proteindelivery [99]

PEGylatedamphiphilicoctadecylquaternised carboxymethylchitosan

Nanoparticles Proteindelivery [42]

SPIONs-loadedwatersolublechitosan-linoleicacid nanoparticles

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hydrophilicmodification(i.e.,LCC)inhibitedhuman epi-dermoidcarcinomacellgrowthbetterthanthefreePTX [26].Thenanoparticleshadsizeslessthan100nm, zeta-potentialsbetween−25.5and−20mV,andlowhaemolytic effects. The carriers have been effective at the passive targetingoftumours.Followingthisdiscovery,numerous reports onAMCasa drugnanocarrierhavebeenmade. In2003,Kwonetal.reportedonself-assembled nanopar-ticlesofhydrophobicallymodifiedglycolchitosan(HGC) prepared bytheattachmentof 5␤-cholanicacidto gly-colchitosan[60].Thenanoparticlessizesdecreasedfrom 850 to 210nm with increasing hydrophobic modifica-tions, which wasexplainedby theformationof a more compactcore.TheHGCexhibitedsignificantlylowerCAC thanlowMwsurfactants,indicatingimprovedstability.In subsequentstudies,HGCwaseffectivelyloadedwith cis-platin[73],docetaxel[74],DOX[75,76],PTX[76],andCPT [77].Thenanocarriersprovidedasustainedreleaseofthe drugsubstance for timesexceeding oneweekand pro-tectedthedrugfromdegradation.Antitumorefficiencywas improvedinanimalmodels,withacorresponding reduc-tionintoxicity.Theimprovedpropertieswereattributed mainly to the sustained release and the passive tar-geting throughthe EPR-effect.Huo etal. compared the PTX-loadedoctyl modifiedHGCtothepharmaceutically approvedTaxol® _formulation,_which _is_based _on_PTX _in

Cremophore EL® _solubiliser. _The _HGC_nanocarriers _had

sizesof190–230nm,highpositivezeta-potentialsof+24.6 to+30.2mVandwerereportedtohavebettersafetyand toxicitypropertiesthanTaxol® _[78]_._Similar_results_have

been reported for low Mw N-octyl-N,O-carboxymethyl chitosan (OcCMC)thatwasusedasaPTX carrier.Blank nanoparticles demonstrated lower cytotoxicity towards HepG2 cells than Cremophor EL®_, _while _PTX _loaded

formulations had comparable cytotoxicities [79]. Both studiesclearlydemonstratethepotentialsafetybeneﬁtsof AMC.

Zhaoet al.developed nanoparticlesofbiodegradable LA/poly(␤-malic acid) (PMLA) double grafted chitosan (LMC), withdiametersrangingfrom190to329nm and zeta-potentials ranging from −17 to −11mV [61]. The PTXloadednanocarriersinhibitedtumourgrowthbetter thanfreePTXinS-180mice.Recently,Zhouetal. synthe-sisedglycidol-chitosan-deoxycholicacid(G-CS-DCA)and prepared nanoparticles withsizes of 160–210nm, with thesesizesincreasingwiththedegreeofhydrophobic sub-stitution,incontrastwiththereports onHGCdiscussed above.Theformednanoparticleswerereportedtobestable for up tothree months in PBS at pH 7.4, despite hav-ingazeta-potentialvaluethatwasclosetozero.Finally, the DOX loadednanocarriers couldbe efficiently inter-nalisedintoMCF-7cellsinvitro,withgoodanti-tumour efficiency(cytotoxicity),eventhoughtheefficacywasless thanfreeDOX[58].N-octyl-O-sulfatechitosan(NOSC)has alsoshownpromiseforuseincancertherapy.Zhangetal. performedsafetystudiesonNOSCinmicewithexcellent resultsandinvestigatedtheanticancerefficiencyof PTX-loadedNOSCformultipletumourtypesinmousemodels. PTX-loadedNOSChadsimilaranti-tumoureffectsasthe freedrug,withdecreasedtoxicityandincreased bioavail-ability[80,81].

Recently, succinyl chitosan modiﬁed with folic acid derivatives prepared using a simple carbodiimide reac-tion was reported for the effects of the polymer as a targeted cancer therapy [54]. The nanoparticles were 60–80nminsize,exhibitingnon-toxicinvitroeffectswith a capacity for high drug LE. The particles also exhib-itedpHdependentdrugreleasecharacteristicsandwere capable of tumour cell targeting. Similarly, folate-PEG coatednanoparticleswithmulti-lamellarstructure were preparedfromoctadecyl-quaternizedlysinemodiﬁed chi-tosan (OQLCS),folate conjugatedOQLCS,and PEGylated OQLCS [42]. The nanoparticles had small particle sizes (approximately160nm),increaseduptakeinMCF-7cells andacalceinreleaserateofapproximatelyonetenthof thatfromtraditionalphosphatidylcholine/cholesterol lipo-somes.

AhighlyrelevantresearchquestionisifAMC nanopar-ticles can help overcome multidrug resistance (MDR). For example by allowing loaded drug to escape the P-glycoprotein efflux pump in tumor cells and other multidrug-resistant proteins (MRP) or processes which cancauseexocytosis,degradation,orinactivationofdrugs [82,83]. Promising results have been reported for drug nanocarriersand,ofparticularinterestforthisreview,for nativechitosannanocarriers[84].Tothebestofour knowl-edge,AMChasnotbeenwidelyinvestigatedfortheeffects onMDR.Thesestudieswouldbeofgreatinterestandvalue, giventheurgencyofovercomingMDR.Theversatilityof modifiedchitosan shouldallowforanintelligentdesign ofapossibletherapytoovercomeMDR,while maintain-ingboththebiocompatibilityandbioactivityofchitosan. For example, hydrophobic modifications using choles-terol[27],LA[29,61,85],deoxycholicacid[30,58,86–88]or stearicacidfunctionalisation[89]couldleadtothe disrup-tionofmembranestructureandfluidity,possiblyenabling drugvehiclestobeincorporatedintothelipidbilayerof thecells.Astheintracellular-extracellularpHgradientis reversedinmanysolidtumours,amodificationwithproton donatinggroups,suchascarboxymethyl[27,29,87],sulfate [80,81,90,91]andfolicacid[87],orgroupswithstrongly pH-dependenthydrophilicity,suchasacetylhistidine[92], incombinationwiththepHsensitivityoftheaminogroups of chitosan (pKaof approximately 6.5),could allowfor improvedtargetednanoparticleaccumulationand/ordrug releaseintheextracellulartumourspaceaswellasin/from earlyendosomes.Thesepropertiescouldbehighly benefi-cialincancertherapy,asdiscussedinarecentreviewbyLee etal.[93].Onthistopic,Jinetal.preparedpH-sensitive self-aggregateddeoxycholicacidmodifiedcarboxymethylated chitosan (DCMC) nanoparticles [30]. The nanoparticles escapedthe endosomal entrapmentof MCF-7 cells and exhibitedacidicpH-inducedaggregationanddeformation, accelerating thedrugrelease at endosomal-relevant pH values. DOX-loadedDCMC was superiorto free DOXin suppressing drug resistant MCF-7/Adr cells, which was explainedbyincreasedcellularuptakeandretention.Hu et al prepared DOX-conjugated stearic acid-g-chitosan nanoparticleshavinghighlypositive zeta-potentialsand sizessmallerthan100nm[89].Theyalsoreportedbetter performanceoftheDOX-nanoparticlesthanoffreeDOX forrepressingMCF-7/Adrcells,andthatthecytotoxicity

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oftheDOX-nanoparticlesdecreasedwithincreasingDOX content.

Withregardtoadministrationroutes,themajorityof deliverysystemsonthemarketareintendedforperoral administration,asthisroutehascomparablyhighpatient complianceandisnon-invasive.AMCcanbedesignedfor peroral drugdelivery[88,90,94,95]. Throughthe proper molecularmodification,thedrugcanbeprotected from degradationbythemetabolicsystemandtheaggressive acidenvironment ofthestomach.Theintestinal attach-mentsiteasthedrugdeliverysiteshouldbetunableby usingthecorrectchemistrytoexploitthevariabilityinpH withintheintestinalsystem.Shelmaetal.investigatedthe mucoadhesivityandreleaseofhydrophobiccurcuminand hydrophilicinsulinunderstomach(pH1.2)andintestinal (pH7.4)conditionsforchitosanandhydrophobic deriva-tivespreparedbyN-acylationwithhexanoyl,lauroyl,and oleoylchlorides[95].Increased mucoadhesionwith the hydrophobicmodifications andsustainedrelease of cur-cumin were reported, with the slowest release for the oleoylsubstitutedchitosanatpH7.4.Fortheinsulin formu-lations,significantburstreleasewasobserved,followedby veryslowreleasecharacteristics.Thissystemwasreported tohave significantpotentialfor theoral administration ofdrug substances, in particularhydrophobic drugs. As theseresultswereinteresting,stabilitystudiesofthedrugs loadedinthenanocarriersshouldbeconductedatstomach pH,asdrugdegradationcouldalsopotentiallyoccurduring theretentionwithinthecarriers.

TheinvitroreleasekineticsfromAMCcanvarygreatly. Someformulations release closeto allof the drugin a fewdays,whileothertypescanprovidesustainedrelease forweeks(Fig.2).Invitroreleasecharacteristicscanvary

Fig.2. Releaseprofilesforselectedamphiphilicallymodifiedchitosanand drugs.Data-pointswereestimatedfromtheoriginalfiguresinthecited references:SOC-DOX[96],G-CS-DCA-DOX[58],HGC-PTX[76],LCC-DOX

[29],DCMC-DOX[30],HGC-CPT[77],LMC-PTX[61],NOSC-PTX[98].All releasestudieswerereportedtohavebeenconductedatpH7.4and37◦C.

greatlywithexperimentalsetups.Inparticular,the com-monly used dialysis method may generate results that suggesttooslowreleasecharacteristics,resultingfromthe diffusionbarrierofthemembrane andahighlocal con-centration of the nanocarriers in the dialysis tube that may lead tothat drugremaining within the nanocarri-ers. Nanocarriers for hydrophobic drugs commonly can beloadedthroughsimplemixingprocesses,withthelow chemical potentialof thedrug inhydrophobic domains promotingboth theloadingand retention. Nonetheless, thesecommonlyusedinvitromethodsaregood indica-tionsofformulation performance,which willbefurther evaluatedinsubsequentstagesofdevelopmentusingmore sophisticatedinvivoassays.

The relationships between the amphiphilic modifi-cations of chitosan with the drug loading and release properties have alsobeenexamined.For N-succinyl-N -octyl chitosan (SOC) [96] and G-CS-DCA [58] loaded withDOX,thereportedresultssuggestedthatincreasing hydrophobicmodificationscanleadtoimprovedLCand LEwithreductionsinthereleaserates.Similarly,Liuetal. investigatedtheself-assemblybehaviourandDOXLCof acylated carboxymethyl chitosan,reporting that LEwas enhancedbothbyanincreasedacylchain-lengthandthe degreeofacylsubstitution[49].Carboxymethyl-hexanoyl chitosan (CHC)hasbeeneffectively usedtoencapsulate drugsandmodifiedtheinvitroreleasecharacteristicsfor thesedrugs,suchasvancomycinandnaproxen[97],DOX [33],magnolol[31]anddemethoxycurcumin[32].ForLMC, increasingthedegreeofsubstitutionwithLAand increas-ingthelengthofthePMLAchainsledtodecreasedrelease ratesofPTX,asexplainedbytheincreasedLA-drug interac-tionsandtheincreasedhydrophilicshellthicknesses[61]. ForlowMwamphiphilicOcCMCloadedwithPTX,a chi-tosanprecursorwithMwof10kDaresultedinhigherLC and LE (LC=37%,LE=99%) thanthechitosan precursors withMwof5kDa(LC=32%,LE81%).Asmalldecreasein bothLEandLCwasreportedforMwvaluesincreasedto 20kDa,indicatingacomplexdependence[79].

Inadditiontomolecularcharacteristics,thedrug load-ing environment may signiﬁcantly inﬂuence the drug loadingperformanceandtheformedcolloidalstructures, asobservedinthepaperbyZhangetal.[98]investigating theNOSCself-assemblyandPTXloadinginmultiple sol-vents.Theoptimalsystemwaswater-ethanol,andthePTX insidetheNOSCcoreexistedascolloidalparticlesrather thaninamoleculardispersedstate.Theadditionalstepof dissolvingthecolloidalPTXmayhaveproducedaslower sustainedreleasecomparedtothemoleculardispersion.

Insummary,AMCnanocarriersappearverypromising forcancertherapyandotherindications.Theversatilityof modiﬁcationsandpropertiesallowsforthedesignto spe-ciﬁcneeds,suchastheprotectionofdrugfromdegradation, mucoadhesion,overcoming MDR,and targeted delivery. Important characteristics,suchasthedrugrelease rates and physiochemical properties, are often tunable for a givensystem.WorthtomentionisthatParketal.reported increasedtumouraccumulationwithincreasingMwofthe polymer chains,withoutchangesin theinvitro physio-chemicalpropertiesofthenanocarriers[28].Theincreased accumulationmayhaveresultedfromtheenhancedinvivo

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Fig.3. Serumantibodytitersincarps immunizedwith OCMCS-ECPs nanoparticlesandECPsalone.Datarepresentedthemean±SD.(n=4).

*_P_<_0.05,_as_compared_with_control_[99]_._Copyright_2012._Reproduced_with

permissionfromSpringerScienceandBusinessMedia.

stabilityandbloodcirculationtimes.Theseﬁndingssuggest thatMwmaybeanimportant,butoftenoverlookedfactor, inthecharacterisationofpolymericnanocarriers. 3.1.2. Deliveryofproteinsandpeptides

AMC hasbeenexaminedforuseintheprotectionof loadedproteinsandthecontrolleddeliveryattargetsites. Inparticular,formulationsfororaladministrations(i.e.,to protecttheloadedproteinfromdegradationinthestomach andpromoteintestinaltransmucosaldelivery)have gen-eratedsignificantinterest.Recently,oleoyl-carboxymethyl chitosan(OCMCS)waspreparedbychemicalmodification witholeicacidandmonochloroaceticacid.Nanoparticles preparedbyionic-gelationusingsodiumtripolyphosphate wereeffectivelyloadedwithextracellularproducts(ECPs). Thenanoparticleshadtunablesizesrangingfrom160to 400nmwithoutECPs,andslightlylargerwhenloadedwith ECP.Thenanoparticleshaduniformsizes,highLEvalues, abilities for thesustainedrelease oftheECPs, enhance-mentstomucosaldelivery,andgreatlyimprovedantibody responses compared withthefree ECPs,as observed in Fig. 3 [99]. Consistent with these results, Shelma and Chandrapreparedlauroylsulfatedchitosan(LSC)by mod-ificationwithhydrophilicsulfogroups andhydrophobic lauroylgroups,formingnanoparticlesusing tripolyphos-phate [100]. A thorough characterisation of theformed LSCwasperformed,andtheresultingproductshad excel-lentbloodcompatibilitycharacteristics.Subsequently,LSC wasinvestigatedforuseasdrugandpeptidenanocarriers, withtheresultssuggestingthatLSChadhigher mucoadhe-sionthanchitosanandnocytotoxicity.Furthermore,LSC increasedthepermeabilityofcaco-2celllayers,inhibited theenzymatic degradationofinsulinand displayed pH-dependentreleaseofinsulinwithdramaticallyincreased releaseratesathighpHvalues.Insummary,LSCseemsvery promisingasanoralpeptide-proteindeliverysystem[101]. Loadedpeptidesorproteinsshouldberetainedinthe parti-clesandprotectedfromdegradationduringthemigration throughthestomach,withthesubsequentreleaseofthe drugproductoccurringintheintestinewiththeincreased

pHvalues.Althoughconﬁrmationalstudiesarestillneeded, thisdesignapproachappearstoholdpotentialfortheoral deliveryofinsulinandotherproteins.

Incontrastwiththegenerallyapplicableoraldelivery route, a speciﬁc and challenging indication for peptide orproteindeliveriesisspinalcordinjury(SCI)repair.In clinicalpractices,effectivetreatmentsofSCIinvolvelocal injections of high-dose drugs [102,103]. Unfortunately, high-dose drugs are commonly associated with multi-plesideeffects.Improvedtreatmentscouldpotentiallybe achievedbythetargeteddeliveryofproteinsthat stim-ulatehealing at thesite ofinjury. Nanocarriers maybe usefulin achieving this goal byusing a suitable design todelivertherapeuticmacromolecularagentsacrossthe blood-spinalcordbarrier(BSCB)[104].

One approach to direct nanoparticles to a target area is through the inclusion of magnetic components and the useof an external magnetic field for guidance [105,106].Functionalisationwithtat-peptides,which con-taintransmembraneandnuclearlocalisationsignals,can increasethecellularinternalisationofparticlesand pep-tides[106,107].Usingthisapproach,Wangetal.elegantly developedtat-modified,ironoxidecontaining nanoparti-clesfromPEGylatedoctadecylquaternisedcarboxymethyl chitosan and cholesterol [108].The nanoparticles effec-tivelycrossedtheBSCBandpenetratedintonervecellsina SCI-ratmodelusinganexternalmagneticfieldforguidance. Thesedatasuggestthatthenanoparticlesarepromisingfor thedeliveryofmacromolecules,suchasproteins,acrossthe BSCBtoSCIrepairsites.

3.1.3. Genedelivery

Genetherapyhasgeneratedextensiveinterestinthe researchcommunityandholdsgreattherapeuticpromise. Intheeffectivedeliveryofgenes,polymeric nanocarrier-baseddeliverysystemsmaybeanattractivealternativeto virus-basedsystemsfor whichthereareproductionand safetyconcerns.Generally,nanocarriersforefﬁcientgene transfectionmustmeetanumberofrequirements, includ-ingtheformationofcomplexescondensingtheDNA,the protectionofgeneticmaterialfromdegradation,the pro-motionof efﬁcient cellular uptake, an ability to enable endo-lysosomal escape and the localisation of genetic material into the nucleus as well as the release from thecarriers.Cationicpolymersandliposomeshavebeen extensivelyinvestigatedasgenecarriers,whereespecially polycationsenableeffectiveDNAcondensation[109,110]. Complexationwithpolymericcationshavebeenreported toprotectgeneticmaterialagainstnucleasedegradation, whilefacilitatingbothcellularuptakeandendo-lysosomal escape[111].

Withcationicchargecharacteristics,chitosanhasbeen widely examined as a non-viral vector for transfection [3,112–118]. The effects of specific colloidal properties, including size, colloid stability, surface charge, and pH stability,ontransfectionefficiencyhavebeenwidely con-sidered. Several studies have been conducted on the structural optimisation of chitosan/gene complexes to enhance delivery by controlling the chitosan Mw, the degree of chitosan deacetylation, and theplasmid con-centration [9,112,119,120]. The transfection efficiency

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using chitosan is still low compared with the current “golden standard” polyethylenimine (PEI), most likely resultingfromthepoorsolubilitycharacteristicsof chi-tosan at physiological pH and the strong interactions within the chitosan-DNA/RNA polyelectrolyte-complex preventingdissociationinthenucleus.Toaddressthese dif-ﬁculties,modiﬁedchitosanhasbeeninvestigatedforgene delivery.

A popular choice for the hydrophilic modification of gene-nanocarriers has been PEG, allowing for mul-tifunctionality. PEG can reduce the cytotoxicity of the carriers,increase circulationtimes, improvewater solu-bility/colloidal stabilities, shield excess charges, and be usedasaspacerbetweenthecarriers andtargeting lig-ands,allowingforanimprovedaccesstotargetedreceptors [45,121].WiththepositiveresultsusingPEIasa nanocar-rierin genedelivery[122,123],chitosansmodified with PEIhavealsobeen investigated[124,125].In 2008, chi-tosanbasedgenenanocarriersmodifiedwithbothPEGand PEIwereexaminedbybothJiangetal.[40]andWuetal. [126].

Anothernotablemodifiedchitosaninthegene deliv-ery field, galactosylated chitosan was able to improve the galactose induced hepatocyte-targeted gene deliv-ery when hydrophilically modified with dextran, PEG, polyvinylpyrrolidone (PVP), and PEI.The polymer-DNA self-aggregatesrangedfromsizeslessthanfifty nanome-trestoseveralhundredsofnanometres[40,127–129].The colloidalstabilityin PBSofthehydrophilicallymodified chitosan-DNAcomplexes wassuperior to non-modified galactosylated chitosan-DNA complexes [127,128], Fur-thermore,aPVP modificationincreasedstabilityagainst albumin-induced aggregation compared with conven-tionalPEInanocarriers[129].TheDNA-nanocarriershad improvedtransfectionincellspresenting asialoglycopro-tein receptors that allowed for interactions with the galactose groups [40,127,128], demonstrating that the hydrophilic modification did not significantly interfere withtheligand-receptorinteractions.Inamurinemodel, galacosylated chitosan nanocarriers modified withboth PEGand PEI had increased circulation times and accu-mulationinthelivercomparedwithPEIandPEI-chitosan nanocarriers [40]. These results demonstrate that the nanocarrierdesigncanaffectthebiodistribution charac-teristics,boththroughactivetargetingandphysiochemical properties.

Hydrophobic modifications of nanocarriers for gene deliverycan allow for improvedloading of the genetic material,reducedseruminhibitions,increasedattachment toandtranslocationacrosscellmembranes,anda weak-eningof the ionicinteractions betweenthecarrier and thegeneticmaterial[56,130,131],promotingtransfection efficiencies. Liu et al. investigated N-alkylated chitosan as a nanocarrier for gene delivery, observing that the transfection efficiency increased with increasing chain length up toeight alkyl carbons [131]. Kim et al. pre-pareddeoxycholic-acidmodified chitosanthatproduced transfectionefficienciesthatwerestronglydependenton theMwoftheprecursorchitosan [56].Theoptimal for-mulationdependedonthetransfectionconditions(with or without serum). The transfection efficiencies were

discussedprimarilyintermsoftheDNA-polymer interac-tionstrengthandtheDNAcondensation,withapossible inﬂuencefrombothsizeandstructurealsomentioned.

An interesting approach for the enhanced delivery usingthiolatedchitosan-DNAcomplexeshasbeenreported by Lee et al. [132]. The complexes were between 75-120nmwithazetapotentialof+2.3to+19.7mVandhad improvedstabilitythatpreventeddegradationwithDNase Idigestion,leadingtohigherDNAexpressioninHEK293, MDCK,and Hep-2celllinescompared withthe unmod-iﬁed chitosan.In addition,invivoexperimentsin Balb/c micedemonstratedthattheintranasaladministrationof thecomplexesproducedgene expressionforatleast14 days.

The combination of both hydrophilic and hydropho-bicmodificationsofcationicpolymersforthesynergetic effects on gene delivery has led to AMC receiving increasedresearchattention[87,133–136].Yooetal. pre-paredHGCnanocarriersfromglycolchitosan,5␤-cholanic acid-modified-glycol chitosan and hydrophobised DNA. In addition to electrostatic forces, hydrophobic inter-actions were a driving force used to encapsulate the modified DNA [134]. In vitro transfection efficiencies using HGCwere increasedup toeight-foldhigher than theexperiments usingchitosan. Inseveralanimal stud-ies,HGChadsignificantlyhighertransfectionefficiencies than both naked DNA and commercially available PEI (Superfect®_). _Both _the _in _vitro _and _in _vivo _results

sug-gested increased transfectionefficiency withincreasing HGCcontent,withincreasedHGCcontentcorresponding withdecreasednanocarriersizes.Theimprovedefficiency mayhave resultedfromthedecreased nanocarriersize, the improved stability of thecomplexes, theimproved resistance toserum proteins, or a combination thereof. Similarly, Opanasopit et al. [135] prepared methylated N-(4-pyridinlymethyl) chitosan, and suggested that the permanentpositivechargeofthetrimethylatedchitosan wasakeyfactorincondensingandprotectingtheDNA, whilethehydrophobicN-4-pyridinylmethylgroupplayed an important role in the transfection efficiency. Germ-ershaus et al. [133] reported that PEG-graft-trimethyl chitosan/DNA had improved transfection efficiencies in various cell lines, including NIH/3T3, L929, and MeWo. A549 cells were alsoobserved tobe resistantto trans-fection, indicating the presence of cell line specific characteristicsthatcaninfluencetransfection.These char-acteristics couldinclude themembrane charge density, differentinternalisationroutes,thesurfacereceptors,and otherattributes.

In summary, multiple reports have been generated ontheinﬂuenceof both propertiesand formulationsof amphiphilicpolymersontheperformanceofnanocarriers for the delivery of genetic material. The delivery per-formance is inﬂuenced bytheinteractions betweenthe polymerandthegeneticmaterial,aswellasthesizeand thezeta-potentialofthenanocarriers.Anotheressential parameteristhepolymernitrogentoDNAphosphorous ratio (N/P), with a clear but complicated dependence [133,136,137]. Non-electrostatic interactions appear to allow for improved dissociation of the loaded genetic material with a corresponding increase in transfection

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efficiency. Establishing clear dependences between the transfectionefficiencyandthevariousparameterscanbe difficult asthey arecommonly not independent, which confoundsthecorrelations.Tothebestofourknowledge, nosystematicstudyorevendiscussionhasbeenreported on theoptimalratio of the hydrophobicto hydrophilic substitutions. As a high degree of hydrophobic substi-tution might induce strong cytotoxicity and extensive hydrophilic substitution could lessen cellular uptake, a modelsystemexploringinvitroandinvivocharacteristics, including colloidal properties, stability, cell internalisa-tion andtransfectionefficiency forthevarious ratiosof hydrophilic/hydrophobicsubstitution,wouldbean impor-tantcontributiontothefield.

3.1.4. Imaginganddiagnosis

In addition to use as nanocarriers for therapeutic substances, self-assembled nanoparticles can provide a potentialplatformfor effectivediagnosticimaging, pos-sibly in combination with the delivery of substances [92,138–140].Thesenanodevicesmayallowforselective imaging andinvivomonitoringofbiological conditions. Nanoparticle-based optical imaging agents require sev-eral crucial properties: (i) stability; (ii) resistance to metabolic disintegration withnon-toxicities; (iii) excel-lent absorbance and quantumyields; and (iv)adequate dispersibility in physiological environments [141]. For effective diagnostic imaging, additional requirements include excellent spatialresolution in three dimensions andanaccumulationofthenanoparticlesatthetargetsites (ifdesired).

Superparamagnetic iron oxide nanocrystals (SPIONs) areimagingagentsthathavebeenwidelystudiedfor mag-neticresonanceimaging(MRI).SPIONbasednanoparticles canbeusedbothasdrugnanocarriersandforinvivo imag-ing,allowingforsimultaneoustreatmentandmonitoring ofeffects[142].Fortheseapplications,SPIONsshouldhave a narrowsizedistributionandbestabilised inan aque-ous media[143]. Toachieve theseproperties, Leeet al. developedhepatocytetargetingself-assembled nanoparti-clescomposedofchitosan–LAconjugates,carryingSPIONs ascontrastagents [144].LA accumulatesin hepatocytes andplaysacentralroleincholesterolandtriglyceride syn-thesis[145,146],suggestingthattheconjugationofLAto chitosanmayenhancetheaccumulationoftheconjugate intheliver.Theamphiphilicchitosan–LAcarrierimproved thecolloidalsuspensionstabilityoftheSPIONs,reduced cytotoxicityandallowedfortargetedMRIofthe hepato-cyteswithoutimagingtheKupffercells.Inanotherstudy, Shietal.reportedonsurfacedmodiﬁedSPIONsprepared byanattachmentwithcarboxymethylchitosan.The parti-cleshadexcellentimagingpropertiesincombinationwith several advantages, such as improved biocompatibility, effectiveuptakeintocells,andthepossibilityofstemcell tracking[147].

Recently,nanotechnologyandmolecularimaginghave beenusedtoproducemultifunctionalnanoparticlesthat couldfacilitatesimultaneousearly-stagecancerdiagnosis, targeteddrugdelivery,andrealtimemonitoringofcancer treatment[148–150].ThecombinationofMRIand near-infraredﬂuorescenceopticalimagingforcancerdiagnosis

hasseveralattractivebenefitsthathavedriventhe devel-opmentofmolecularimagingusingthesefunctionalities [151–153].Onthistopic,Bhattacharyaetal.prepared SPI-ONssurfacemodifiedwithcarboxymethylchitosan,adding rhodamine isothiocyanate for optical imaging and folic acidfortargeting.Theparticleshadexcellentdispersion propertiesunder physiological conditions, optical imag-ingcapabilities,magneticactivityunderexternalmagnetic fieldsandincreaseduptakeintoHeLacells,which overex-pressfolatereceptors[41].

Therehasbeenconsiderableworktowardstherapeutic imagingusingHGCnanoparticles,whichisespecially inter-estinggiventheirpotentialforcancertherapy[53,74–77]. In onestudy, thenearinfrared (NIR)ﬂuorophore Cy5.5 wasconjugatedtoanti-angiogenicRGDpeptidesthatwere loadedintotheHGCnanocarriers [154].The HGC-RGD-Cy5.5nanoparticlesallowedfortheinvivoNIRimagingof theRGD-Cy5.5biodistribution,whichatearlytimesshould correspondtoparticledistribution.Inanother investiga-tion,theHGCwasconjugatedwithCy5.5[74],allowing the imaging of the actual polymer chains constituting the particles. The HGC nanoparticleswere loaded with docetaxel and had good imaging properties as well as therapeuticpotential. Recently, HGCnanoparticleshave been developed for dual NIR/MR imaging by modiﬁca-tionsoftheglycolchitosan-5␤-cholanicacidwithCy5.5for NIRimagingandagadolinium(Gd(III))chelatingagentto incorporateGd(III)forMRI.Thepolymer-Gd(III)complexes self-assembled into nanoparticles having average sizes of approximately 350nm and accumulated in tumours afterintravenousadministrationinamurinemodel.The nanoparticleswerethenusedforsimultaneousNIRandMR imaging[155].Bycombininganimprovedtumour accu-mulation,themolecularsensitivityofNIRimagingandthe threedimensionalcapabilityandspatialresolutionofMRI, theCy5.5-HGC-Gd(III)nanoparticleshavethepotentialto beusedasoptical/MRdualimagingagentsincancer ther-apeuticsandmonitoring.

HGCnanoparticlescanalsobesurfacefunctionalisedto improvetargetingefficiencies.InanelegantworkbyPark etal.,peptide-conjugatedHGCnanoparticleslabelledwith Cy5.5selectivelyboundtomembranesofactivatedbovine aorticendothelialcellsandexhibitedhigherbinding affin-ityfortheatheroscleroticaortaofanLdlr−/−mousethanto theaortaofanormalmouse,asdeterminedbyNIR imag-ing[156].Theseresultsclearlydemonstratethepotential ofHGCbasednanodevicesinatheroscleroticlesion imag-ingtomonitorpathophysiologicalchanges.Insubsequent work,Naetal.investigatedtheinvivoimagingcapability of Cy5.5labelled HGCnanoparticlesin various tumour-bearingmousemodelswithexcellentresults[157].HGC nanodevicesappeartohavegreatflexibilitywithregard toimagingandtargetingmodifications,whichcouldallow for thedevelopmentof multifunctional nanodevicesfor a range of indications. Incorporating both imaging and thesimultaneousreleaseofdrug(possiblywith imaging-designthatcouldallowfordetailedinformationonthedrug statetoconsiderboththeconcentrationandlocalisation withinoroutsideofnanoparticles)wouldconstitutea fur-thertherapeuticandimagingdevelopmentthatmaybeof significantinterest.

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3.2. Macroscopicassemblieswithcolloidalstructures 3.2.1. Injectabledepotgelsfordrugdelivery

Withmultipledesirablebiomedicalproperties,chitosan hasbeeninvestigatedforuseininjectabledepotdrug deliv-ery systems. In 2000, Chenite et al. developed a novel thermo-gelling hydrogelbased on solutions of chitosan withasmallamountof␤-glycerophosphate(␤-GP)[158]. The mechanism of gelation has beenascribed to chain aggregationupontheproton transferfromthechitosan tothe␤-GP.Thistransferresultsfromthetemperature dependentpKaofchitosan[159].Anothercommon

expla-nationisthatatlowtemperaturesthe␤-GPassumesan entropicallydisfavouredarrangementsurroundingthe chi-tosan,while athigher temperaturesthearrangementis broken-up,whichallowthechitosanchainstoaggregate. Thisformulationwasshowntobeaviabledelivery sys-temfor growthfactorsandfor livingchondrocytes,and clearlyhaspotentialforthedeliveryofothertherapeutic substances,as discussedbelow. Kashyapet al.used the chitosan-␤-GPsystemasaninsitugellingdepotfor pul-satiledeliveryofinsulin[160].LoadedwithPTX,CPTor DOX,thisdrugdeliverysystemhasalsobeeninvestigated forthetreatmentsofbreastcancer,RIF-1ﬁbrosarcoma,and cervicalcancer[161–163].Evenwiththeelegantdesignof thechitosan-␤-GPsystem,improvementscanbemadeto reduceseveralcommonlyobserveddisadvantages,suchas theburstreleaseofdrugs,poorwater-solubilityandlow thermo-sensitivity.Tothisendchitosanandformulations havebeenmodiﬁed toincrease thedrugdeliverydepot functionsforarangeofapplications.

Bhattarai et al. developed an injectable thermo-sensitivehydrogelbased ona solutionof poly(ethylene glycol)-graft chitosan (PEG-g-chitosan) using genipin for crosslinking in situ under physiological conditions [164,165].The hydrogel provided close to a linear sus-tained release of bovine serum albumin for up to 40 days, withonly a small amountof burstrelease. Simi-larly,Luetal.usedN-succinyl-chitosan(NSC)andoxidised carboxymethylcelluloseasthematrixinaninjectable pro-teindeliverysystem[166].ThesystemwasbothpH-and thermo-sensitive,formingsolid gelsatphysiological pH andtemperature.Thegelationtime,equilibriumswelling anddegradationcouldbetunedbyadjustingthe oxida-tion degree of the carboxymethylcellulose. The porous structureof thehydrogelscouldbetuned, providingan attractivemethodforcontrollingthediffusionrate,and thusthereleaseoftheloadedmacromolecules.

Wu et al. prepared injectable hydrogels by prepar-ingN-(2-hydroxy)propyl-3-trimethylammoniumchitosan chloride(HTCC)with␣-␤-GP[167].DOXreleasefromthe hydrogelshadhigherpHsensitivitythanchitosan-␣-␤-GP hydrogels.Gelsfromboth HTCC and chitosandisplayed increased release rates at low pH. An additional nasal formulationintendedforthedeliveryofmacromolecular drugswasdevelopedbycombiningHTCC withPEGand ␣-␤-GP[168].Theformulationunderwentasol-gel tran-sitionat37◦C.Loadedwithinsulin,thegelreleaseproﬁles andinitialburstrelease weredeterminedtobe depend-enton both the gelcomposition and thedrug loading, withdecreasedreleaseratesascribedtotheformationofa

tightgelnetwork.Forgelsloadedwithinsulinand admin-isterednasallyintherat,theHTCC-PEG-_␣-␤-GPreduced bloodglucoselevelswithvaluesthatweresimilarto sub-cutaneouslyinjectedinsulinsolution,butwithadelayed response. Incontrast, nasallyadministered insulin solu-tionsdidnot have a signiﬁcanteffect onblood glucose levels.

Thenano-microstructureofinjectablehydrogelsisan important parameterfor controllingthereleaserates of theloadedsubstances.Onemethodtoachieve extended releasewithminimalburstreleasefrominjectableinsitu gellingsystemsistoloadthetherapeuticsubstanceinto nano-ormicro-carriersthataredispersedinamacroscopic gel.Hsiaoetal.recentlydevelopedaninjectablecolloidal hydrogelcomposedofself-assemblednanocapsulesofCHC and␤-GP[169].TheCHCprovidedboth the encapsulat-ing functionality andthe coherentgelnetwork, making the preparation simple. The gels had extended release characteristicswithoutburst releasefor thehydrophilic drugethosuximide.Conventionalinjectablehydrogeldrug deliverysystemscommonlyexhibitburstrelease charac-teristics[170],withthereleaseofsmallhydrophilicdrugs often completed in less than 24h[171]. Currently, the authorsareinvestigatingthereleaseratesofcolloidalCHC systemscontrolledbythegelmicrostructure,asachieved with variations in the gelation conditions and kinetic parameters.

Similarly,Dingetal.reportedaninjectableamphiphilic pH sensitive hydrogel composed of glycol chitosan and benzaldehyde-capped poly(ethyleneglycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (OHC-PEO-PPO-PEO-CHO)[172].The gelsformedinsitu through theformation ofcovalentbenzoic-imine bonds hadthecapacitytoloadbothhydrophilicandhydrophobic drugs. Subsequently, a glycol chitosan/OHC-PEO-PPO-PEO-CHOhydrogelwaspreparedwithhydrophilicDOX, mixedintheglycolchitosansolutionandhydrophobicPTX, blended and solubilised in the OHC-PEO-PPO-PEO-CHO solution, before combining the two [173]. The authors describedthattheDOXwasloadedintheaqueousregions of thegels, withthePTX locatedin OHC-PEO-PPO-PEO-CHO micelles. The gels had pH dependent release of bothDOXandPTX,withasmalldecreaseinpHfrom7.4 resultingin anaccelerated release.Thegelformulations withbothDOXand PTXwereusedfor thetreatmentof B16F10murinemelanomacancerinmicebyintratumoural administration,resultinginimprovedtumourtreatments thatwerenotobservedwiththesingledrugformulations. In conclusion, conventional injectable hydrogel sys-temsthatarecomposedofdrugandasimplepolymeric matrix areusuallynotideallysuited asinjectabledepot drugdeliverysystems;themedicalapplicationsofthese hydrogels are limited bybiodegradation, biocompatibil-ity issues, thermo/pH sensitivity problems, cytotoxicity, andnon-idealreleaseproﬁles.Multipleinvestigationshave focusedonthesynthesisofnewpolymersorthemixingof functionalmolecules/polymerstoproducenovelinjectable hydrogels thatareeasiertousewiththeimproved per-formanceproﬁlesthatarenecessaryforsuccessfulclinical applications.Oneapproachtoaddressingthese improve-mentsistodesignacomplexgelstructure,suggestingthat

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Fig.4.Schematicdrawingillustratingthesimpliﬁedstructuresofconventionalhydrogels,interpenetratingnetworkhydrogelsandpolymerbased nanopar-ticlehydrogels.

thedevelopmentofthesehydrogelshasproceededfrom singlepolymersystemstointerpenetratingnetwork struc-turesandself-assemblednanoparticlegels(seeschematic drawingsinFig.4).FromthediscussedreportsonAMC, thismaterialclasshasalsodevelopedtoexhibitthese char-acteristicstobecomeasuccessfulcandidateforinjectable hydrogelswithdesignedstructure-functionalityproﬁles. 3.2.2. Invivocellscaffolds

In recent years, several significant developments in cell therapies have been made, as reported in multi-ple recent reviews [174–176]. Promising results using cell therapy have been reported for the treatments of metastaticmelanoma[177],post-acutemyocardial infarc-tion [178],and muscular dystrophy in a murine model [179].In addition,significantadvanceshave beenmade in replacing the extensively discussed embryonic stem cellsanddonorderivedculturedstemcellswithinduced pluripotentstemcells(iPSCs)thatarereprogrammedfrom selectedcellsderivedfromthepatient[180,181].Asnovel therapeuticalternativeshaveemerged,asubsequentneed forsuitabledeliveryplatformshasalsoevolved.For effec-tiveorimprovedcelltherapies,adeliveryplatformneeds tobebiocompatibleandprovideatemporaryporous well-defined3D-environmenttoallowforcellproliferationand differentiationtothedesiredcelltype[182].In particu-lar,cell-scaffoldinteractionsandnano-microarchitecture needstobefavourable,asdemonstratedinrecent publi-cations[183–185].Tosustainviabletherapeuticcellular alternatives,scaffoldsalsoneedtoallowforeasyandrobust implantationinpatientsinaclinicalsetting.Injectablecell scaffoldsystemswould behighlydesirable,allowingfor minimalinvasivesurgeryaswellastheabilitiestoform complexshapes,providegoodcontactwiththe surround-ing tissue, and enable thehomogeneousdistribution of cellsandsignallingsubstances[182,186,187].

Asobservedintheliterature,anumberofpromising chi-tosancontaining[182,186,188,189]andmodiﬁedchitosan based[7,188,189]injectablecellscaffoldsfortissue engi-neeringhavebeenreported.TheuseofAMCincellscaffolds

isstillnew.SeveralstudieshavesuggestedthatAMCmay beusefulasacellscaffoldmatrix.Xuetal.demonstrated inratsthatN,O-hexanoylchitosandidnotaffectthe pro-liferationandviabilityofdermalfibroblasts,hadexcellent biocompatibilityandwasdegradedinvivo,withthe degra-dationratetailoredbyalteringthedegreeofsubstitution [190].Comparedwiththecommonlyusedbiodegradable polymerPLGA,amilderandlowerinflammatoryresponse wasobserved.Inanotherstudy,Neamnarketal.usedfibres ofelectrospunhexanoylchitosan asscaffoldsforhuman keratinocytesandfibroblasts[191].Basedonproliferation andcellintegrationresults,thecreatedfibrousmatswere reportedtohavepotentialasascaffoldingmaterialin tis-sueengineering.The resultsof Neamnarketal. and Xu etal.providesomeindicationthatAMCmaybeusefulin tissueengineering.Inarecentstudy,Chienetal. demon-stratedthepre-clinicaluseofamphiphilicCHCasathermo gellinginjectablecellscaffold carryingiPSCs forcorneal repair[192],concludingthattheinjectableCHCgelmay provideasafeinjectablescaffolddeliveringstemcellsfor thetreatmentofcorneal injury.TheCHCgelswerefirst reportedbyHsiaoetal.asapotentialdepotdrugdelivery system[169].CHCnanocapsulesincombinationwith_␤-GP hadformedamacroscopicgeluponheatingto37◦C,with theformedgelhavingamicrometrescalenetworkinwhich thenanocapsulesremainedintact,producinggelswitha porousstructureonthemicrometrescaleanda nanostruc-turedcell-scaffoldinterfacethatmightallowforanideal scaffoldingperformance.

In conclusion, there are very few studies on AMC as in vivo cell scaffolds. The available reports clearly demonstrate significant potential for this new class of materialinthefieldofcell-therapyandtissue engineer-ing.Inparticular, theself-assembly intonanostructures, thecapacitytoloaddrugsandgrowthfactorsinto nanopar-ticles,theabilitytotunethemicroscalenetwork-structure, andtheeasewithwhich chitosancanbemodified may allowfor thesimple preparation of injectable cell scaf-folds to meet new needs (see Fig. 5 for conceptual drawing).

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Fig.5.Conceptualschemeillustratingthepreparationofinjectablenanoparticlebasedcellscaffoldshavingthecontrolledreleaseofdrugsandgrowth factorsaswellasadesignednano-microstructure.

3.2.3. Wounddressings

Manyinjuriesandmedicalconditionsinvolvedamage totheskinbarrier(i.e.,wounds).Thesewoundscanbedeep orlocatedonthesurfaceoftheskin.Forsmallwoundsin healthyareas,thebody canmanagethehealingprocess unlesscomplications,suchasinfections,occur.Formore seriousinjuries,therapeuticinterventionsareneeded.For extensivewoundswithbleeding,awounddressingis usu-allyusedtostoporreducethebleeding.Forlargewound areas,asmayoftenbethecaseforburninjuries,or underly-ingmedicalconditionsthatpreventwoundhealing,suchas chroniculcers,therapeuticinterventionsarenecessary.To preventtheinfectionofthewound,wounddressingsand antibacterialagents are commonlyused.A combination ofantibacterialeffectswithotherfeatures thatcan pro-motehealingwouldbedesirablefeaturesfortheintegral functionsofthewounddressing.Inadditiontoproviding amicrobialbarrier,thedressingalsoneedstohaveproper liquidandvapourmanagement.Excesswoundliquidneeds tobeabsorbed and/orevaporatedthrough thedressing, maintainingasuitablymoistenvironmentforwound heal-ing[193].Inappropriateliquidmanagementmayleadto environmentsthataretoodryortoowet,impairingthe healing process and damaging theskin. These require-mentsareespeciallytruefordressingsthataretoremain inplaceforlongperiodsoftime.

Withexcellent biocompatibility,biodegradabilityand antibacterialfeatures,chitosan hasbeeninvestigatedfor use in wound healing and several possibly beneficial effectshavebeenreported[194].Thepositive chargeof protonated amino groups may attract the anionic gly-cosaminoglycansthatarelinkedwitha largenumberof cytokines and growth factors. Chitosan can also act as a chemo-attractant for neutrophils [195]. Chitosan has beenconfirmedtoimproveboththefunctionandspeed of migration to theareaof interest for cells associated withinflammatoryresponses,leadingtoimproved heal-ingthroughareducedriskforinfections[8].Thesefeatures combined with the haemostatic properties of chitosan

[196]andtheabilitytoincreasecollagenaseactivitywould suggestthatchitosanmaybesuitedforuseinwound dress-ings.Indeed,multiplereportsontheuseofchitosan-based wound dressings, in particular on the HemCon dress-ing, have demonstrated efficacy. These types of wound dressingswereusedinOperationIraqiFreedomand Oper-ationEnduringFreedom[197]withexcellentresults,and appearsafetousewithinjuredU.S.soldiers[198]. Clini-calfailureshavealsobeenreportedandascribedtopoor adhesiveness aswell aslargebatch tobatch variability forthechitosanwounddressings[199],indicatinganeed todevelop improvedchitosan based dressings.Dowling et al. reported that chitosan modified with hydropho-bic benzene-n-octadecyl side-chains (2.5mole% relative to amine groups) couldbind non-covalently tocells in the blood and self-assemble to form a hydrogel with thecells in theblood [200](see Fig.6).The hydropho-bicchainsmayhaveintegratedintothecellmembranes sothat thepolymerchainsand thecells formeda self-supportingnetwork.Subsequently,asimilarAMCformed by modifyingchitosan withn-dodecyl side-chains(at a different mole%) was used as a wound dressing for a lethal arterialinjury in swine [199]. The bandage from the hydrophobically modified chitosan was superior to boththebandagefromthepristinechitosanandthe stan-dard gauze for reducing bleeding. In fact, none of the eight test animals died within the180-min test period using themodified chitosan, whilealltest animalsdied inapproximately90minusingthepristinechitosan and in less than 10min using standard gauze. The success ofthemodifiedchitosanwasattributedtotheincreased tissue adhesiveness, as previously described (i.e., the hydrophobicside-chainsanchortothecellmembranes). Inotherrelevantwork,Muzzarellietal.usedfreeze-dried methylpyrrolidone chitosan (MPC) to augment wound healing after dental surgery [201]. MPC induced osteo-conduction and angiogenesis,which combinedwiththe biocompatibilityofthematerialmaketheseresultsvery interesting.

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Fig.6. Mechanismforgelationofbloodbyhm-chitosan.Ontheleftthepolymerisshownschematicallywithitshydrophilicbackboneinblueandthe graftedbenzyloctadecylhydrophobesinpurple.Whenaddedtoliquidblood,thecomponentsassembleintoathree-dimensionalnetwork(gel),asshown ontheright.Thisisdrivenbyinsertionofhydrophobesintobloodcellmembranes(asdepictedinthetopinset);therebythepolymerchainsconnect (bridge)thecellsintoaself-supportingnetwork.[200],Copyright2011.ReprintedwithpermissionfromElsevierLtd.

In additionto improvedhaemostaticfunction, liquid management and healing augmentation, the controlled deliveryofdrugsorothertherapeuticagentscould rep-resent additional wound care applications for AMC. As discussedinSection3.1,AMCcanself-assembleinto nano-structures, offering extensive control over the release proﬁlesoftherapeuticsubstances.Recently,Linetal. pre-pared all-trans retinoic acid loaded CHC nanocapsules embeddedinanalginatehydrogelmatrix[202].Alginate is commonly used in wound dressings [203], and the alginate-CHC systemhad low cytotoxicity,as evaluated usingboth theMTT assayand thelackof skinirritation onrabbits.Thealginate-CHCsystemseemstobea promis-ingdressingmaterialwithadditionalcontroloverthedrug releaseproﬁle.Thissystemcouldbeusefulasalong-term therapeuticdressingwithimprovedhealing characteris-tics.

Basedonthelimitedliteratureavailable,AMCappears suitableforwoundhealingapplications.Theanchoringof hydrophobicside-chainsintocellmembranesandthe col-loidalstructurecouldprovidewounddressingswithboth verygoodtissueattachmentandimprovedcontrolofthe drug release profiles tomeet various clinical demands. With both technical interest and medical importance, hydrogel dressings could be produced via a controlled self-assemblyofAMC,leadingtodesignednanostructures andmicrostructureswithcontrolleddrugreleaseprofiles, evaporationratesandfluidaccessibility,allowing exten-sive structural design for therapeutic improvements in woundhealingapplications.

3.3. Organic–inorganichybridnanostructures

Recently, organic–inorganic hybrid materials and moleculeshavereceivedincreasedinterestintheresearch community. The aim for creating these materials is to

combinesynergisticallythepropertiesofboththeorganic and the inorganic components, to achieve improved properties and/or functions, as compared to the indi-vidual components. Severalexamples of improvements couldincludeimprovedmechanicalproperties,improved biocompatibility, introduction of responses to external stimuli,imagingcapabilities,enhancedcontrolover diffu-sion,etc.Aschitosanhasmultipledesirablepropertiesfor biomedicalapplications,chitosan-inorganichybridshave alsobeeninvestigated,producinginterestingresults.

One widely investigated use of chitosan is for the preparation of composites with hydroxyapatite (HAp) nanoparticles for use in biomedical applications, typi-cally bone replacement and/or hard tissue engineering [204–206].Thebeneﬁtsofthesecompositescanincludea porousstructurewithtunabledegradationrates,improved injectability characteristics, reduced damage from HAp nanoparticles migrating into surrounding tissues, bio-functionality improvements, improved control over the HAp nanoparticle structure, and control of the nano-microstructurescaffold[204,205,207–210].

Anotherhybridmaterialclassincludeschitosancoated metal/metal-oxides,suchasAg/chitosan[211,212], Iron-oxides/chitosan [41], TiO2/chitosan [213], Au/chitosan

[214,215],and␣-zirconiumhydrogenphosphatehydrate (ZrP)/chitosan[115,216].Thehybridscanexhibitboththe propertiesofmetals/metal-oxidesandchitosan-exclusive characteristics,whichallowsforgraftingwithadditional functionalandimprovedbiocompatibility.

Inorganicnanoparticlescanbemanufacturedinto mul-tipletypesofshapes.Surprisingly,thisstructuraldesign approach appears to be lacking in the literature on chitosan-inorganichybridmaterials.Aschitosancoatings commonly are templated onto inorganic particles, the shape andsize oftheinorganic corecouldbe variedto allowfordesiredstructuralcharacteristicsofthehybrids.