Macroscopic assemblies with colloidal structures

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 dependentpK_aofchitosan[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

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).

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.

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.