Biocompatible and Antibacterial Nitric Oxide-Releasing Pluronic F-127/Chitosan Hydrogel for ...

19 Pages·2017·3.31 MB·English

polymers
Article
Biocompatible and Antibacterial Nitric
Oxide-Releasing Pluronic F-127/Chitosan Hydrogel
for Topical Applications
MilenaT.Pelegrino1,2,BrunadeAraujoLima3,MônicaH.M.doNascimento2,3,
ChristianeB.Lombello1,4,MarceloBrocchi3andAmedeaB.Seabra1,2,* ID
1 CenterforNaturalandHumanSciences,UniversidadeFederaldoABC,Av.dosEstados5001,SantoAndré,
SP,CEP09210-580,Brazil;[email protected]
2 NanomedicineResearchUnit(NANOMED),UniversidadeFederaldoABC,Av.dosEstados5001,
SantoAndré,SP09210-580,Brazil;[email protected]
3 TropicalDiseaseLaboratory,DepartmentofGenetics,Evolution,MicrobiologyandImmunology,Instituteof
Biology,UniversityofCampinas,Campinas,SP13083-862,Brazil;[email protected](B.d.A.L.);
[email protected](M.B.)
4 CenterforEngineering,ModelingandAppliedSocialScience,UniversidadeFederaldoABC,
AlamedadaUniversidadesemnumero,SãoBernardodoCampo,SP,CEP09606-045,Brazil;
[email protected]
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:16March2018;Accepted:16April2018;Published:18April2018
Abstract: Nitricoxide(NO)isinvolvedinphysiologicalprocesses,includingvasodilatation,wound
healingandantibacterialactivities. AsNOisafreeradical,designingdrugstogeneratetherapeutic
amounts of NO in controlled spatial and time manners is still a challenge. In this study, the
NO donor S-nitrosoglutathione (GSNO) was incorporated into the thermoresponsive Pluronic
F-127 (PL)-chitosan (CS) hydrogel, with an easy and economically feasible methodology. CS is
a polysaccharide with known antimicrobial properties. Scanning electron microscopy, rheology
and differential scanning calorimetry techniques were used for hydrogel characterization. The
resultsdemonstratedthatthehydrogelhasasmoothsurface,thermoresponsivebehaviorandgood
mechanicalstability. ThekineticsofNOreleaseandGSNOdiffusionfromGSNO-containingPL/CS
hydrogeldemonstratedasustainedNO/GSNOrelease,inconcentrationssuitableforbiomedical
applications. TheGSNO-PL/CShydrogeldemonstratedaconcentration-dependenttoxicitytoVero
cells,andantimicrobialactivitytoPseudomonasaeruginosa(minimuminhibitoryconcentrationand
minimum bactericidal concentration values of 0.5 µg·mL−1 of hydrogel, which corresponds to
1mmol·L−1 ofGSNO).Interestingly,theconcentrationrangeinwhichtheNO-releasinghydrogel
demonstratedanantibacterialeffectwasnotfoundtobetoxictotheVeromammaliancell. Thus,the
GSNO-PL/CShydrogelisasuitablebiomaterialfortopicalNOdeliveryapplications.
Keywords: chitosan; thermoresponsive hydrogel; nitric oxide; S-nitrosothiols; biocompatibility;
antimicrobial;Pseudomonasaeruginosa;PluronicF127
1. Introduction
Nitricoxide(NO)isanendogenousmoleculeinvolvedinseveralbiologicalsignalingpathways.
Atlowconcentrations(pico-tonanomolar),NOhasregulatoryfunctions,suchasthepromotionof
vasodilation,woundhealing,cellproliferation,inhibitionofplateletadhesionandaggregation[1–3].
At higher concentrations (micro- to millimolar), NO has antitumor and antimicrobial effects [2–4].
NOisconsideredapromisingalternativetoconventionalantimicrobials. However,itisachallengeto
Polymers2018,10,452;doi:10.3390/polym10040452 www.mdpi.com/journal/polymers
Polymers2018,10,452 2of19
Polymers 2018, 10, x FOR PEER REVIEW 2 of 19
deliverapreciseNOconcentrationdirectlytothetargetsiteofapplication,insustainedandconvenient
matnon deerslivaenrd aw pitrhecmisein NimOa lcsoindceeneftfreactitosn[ 1d].irectly to the target site of application, in sustained and
conNvOeniisenatf mreaenrnaedrisc aanld(h walift-hl imfeinoinmeatlo sifidvee esffeeccotsn [d1s]). ,andcanbeinactivatedinbiologicalsystems
uponreNacOti oisn aw firtehe briaodmicoalle (chuallef-sli(fseu ochnea stoh feimveo sgelcoobnind)s,),t haunsd lcimani tbineg initascetifvfeactetsd. iTno boivoelorcgoicmale styhsitseimsssu e,
sevueproanl NreOac-rtieolnea wsiinthg bbiioommoalteecruialelss h(sauvceh baese hnepmroegplaorbeidn)t, othcuasr rlyimaintidngd eitlsiv eefrfetchtes.r aTpoe ouvtiecrcaommoeu tnhtiss of
issue, several NO-releasing biomaterials have been prepared to carry and deliver therapeutic
NOfordifferentbiomedicalapplications[4]. Intopicalapplications,NO-releasingbiomaterialscan
amounts of NO for different biomedical applications [4]. In topical applications, NO-releasing
beusedforcoatingmedicaldevices[5]. Indermatologicalapplications, itcanbeusedtopromote
biomaterials can be used for coating medical devices [5]. In dermatological applications, it can be
dermalbloodflow[6–9],forantimicrobialeffects[2,10,11],cellproliferation[4],woundrepair[12–15],
used to promote dermal blood flow [6–9], for antimicrobial effects [2,10,11], cell proliferation [4],
controllinginflammatorypain[16]andlocalwarming[17].Inaddition,administrationofNO-releasing
wound repair [12–15], controlling inflammatory pain [16] and local warming [17]. In addition,
materialsonhumanskinmighthaveatherapeuticeffectagainstendothelialdysfunctions,suchas
administration of NO-releasing materials on human skin might have a therapeutic effect against
vasoconstriction,vasospasmandthrombosis[5,16,18].
endothelial dysfunctions, such as vasoconstriction, vasospasm and thrombosis [5,16,18].
Inthiscontext,thereisgreatinterestinthedevelopmentofNO-releasingbiomaterialscapable
In this context, there is great interest in the development of NO-releasing biomaterials capable
ofreleasingNO/NOdonorsinacontrollablemannerfortopicalapplications. Inthisstudy,theNO
of releasing NO/NO donors in a controllable manner for topical applications. In this study, the NO
donorS-nitrosoglutathione(GSNO)wasincorporatedintoathermoresponsivehydrogelcomprisedof
donor S-nitrosoglutathione (GSNO) was incorporated into a thermoresponsive hydrogel comprised
poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide),PEO-PPO-PEO(PluronicF127)and
of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEO-PPO-PEO (Pluronic F127)
chiatonsda cnh(iCtoSs)an(F (iCgSu)r e(F1ig).ure 1).

Figure 1. Schematic representation of the formation of three-dimensional polymeric network of
Figure 1. Schematic representation of the formation of three-dimensional polymeric network of
Pluronic F-127 (PL) hydrogel structure containing chitosan (CS) and S-nitrosoglutathione (GSNO) to
PluronicF-127(PL)hydrogelstructurecontainingchitosan(CS)andS-nitrosoglutathione(GSNO)to
delivery NO.
deliveryNO.
GSNO is an endogenous molecule that spontaneously releases free NO [1,2]. Hydrogels are soft
GSNO is an endogenous molecule that spontaneously releases free NO [1,2]. Hydrogels are
materials with a three-dimensional network and viscoelastic properties. Thermoresponsive
sofhtymdraotgereilas lcsanw cithhanageth trheeeir- drihmeoelnosgiiocanla plrnoeptewrtoiersk wainthd cvhiasncgoeesl ainst itcempproerpaetrutriee s[.19T]. hPelurmroonriec s(pPoLn) sisiv e
hydapropgroevlsedca fnorc hhaunmgaent huesiersr bhye otlhoeg Ficoaoldp raonpde Drtrieusg wAidthmcihniasntrgaetisoinn (tFeDmAp)e, raantudr hea[s1 9b]e.ePnl uerxotennicsi(vPeLly) is
appemropvleodyefdo rash ua mdraung ucaserrsiebry atnhde fFoor otidssaune denDgrinuegerAindgm [2in0i]s. tPrlautrioonnic(Fs D(SAig)m,aan) dorh Paoslobxeaemneerx (tBeanyseivr)e ly
emaprleo cyoe-dpoalsymaderrsu lgarcgaerlryi eursaedn dfofro hrytidsrsougeele pnrgeipnaereartiinogn.[ T20h]e.yP alurer omnaidcse (oSfi gpmolya)(eothryPloenloex oaxmideer)-(pBoalyye r)
are(pcoro-ppyolleynme eorxsidlaer)g-peolylyu(estehdylfeonreh oyxdidroe)g (ePlEpOre-PpPaOra-tPioEnO.) Tuhneitys.a PrPeOm saedgemoefnptso alrye( emthoyrele hnyedoroxpidheo)b-pico, ly
(prwophyelreenase PoxEiOd ei)s- pmoolyre(e hthyydlreonpehoilxici d[2e1)–(P23E]O. T-PhPerOe -aPrEeO s)evuenriatls .tyPpPeOs osef gPmluernotnsicasr,e dmepoernedhiyndg roonp hthoeb ic,
whneuremabsePr EoOf PiEsOm aonrde PhPyOdr uonpihtsi l[ic23[]2. 1I–n2 t3h]i.s Tsthuedrye, awree usesvede rtahlet Pypluersonoifc PFl-u1r2o7n (iPcLs,) dweitphe 1n0d6i nugniotsn otfh e
nuPmEbOe raonfdP 7E0O unaintsd oPfP POPOu n[i2t3s].[ 2P3l]u.rIonnitch ihsasst ualdsoy ,bweeenu fsoeudntdh etoP dluecrroenaisce Ft-h1e2 7vi(sPcoLs)itwy iothf t1h0e6 bulonoitds, of
prevent aggregation of erythrocytes and adhesion to the vascular endothelium layer [21]. In aqueous
PEOand70unitsofPPO[23]. Pluronichasalsobeenfoundtodecreasetheviscosityoftheblood,
environments, PL chains undergo a spontaneous self-organization due to the differences is solubility
preventaggregationoferythrocytesandadhesiontothevascularendotheliumlayer[21]. Inaqueous
between PEO and PPO units, leading to the formation of micelles (micellization process) [22–24]. The
Polymers2018,10,452 3of19
environments,PLchainsundergoaspontaneousself-organizationduetothedifferencesissolubility
betweenPEOandPPOunits,leadingtotheformationofmicelles(micellizationprocess)[22–24]. The
PPOsegmentofPLiscomprisedofthehydrophobiccoreofthemicelles,whereasthePEOsegmentof
PLiscomprisedofahydrophilicshell[21]. Thegelationprocessdeterminesthespaceorganizationof
themicelles(hexagonal,cubicorlamellarphases)[23]. Uponanincreaseinthetemperatureand/orPL
concentration,micellesautoassemble,leadingtoathree-dimensionalpolymericnetwork,asillustrated
inFigure1[23]. Thegelationprocessleadstoanincreaseintheviscosityofthepolymersolution,
leadingtoagelstate[23]. ThePluronicsystemsarethermosensitive,whichmeanstheyexistasliquids
atlowtemperaturesandbecomesemi-solidathighertemperatures. Thisphenomeisreversible[21,23].
This feature is suitable for pharmacological formulations, since it is possible to incorporate active
drugsatlowtemperature,andtheincreaseinthetemperatureleadstotheformationofahydrogel
containingthedrug. ThemainadvantageofPLhydrogelsistheeaseofpreparation,thatdoesnot
requiretheuseorganicsolvents[21].
The polysaccharide chitosan (CS) was incorporated into PL hydrogel. CS is biodegradable,
biocompatible, non-allergenic and an inexpensive biological polymer extensively used for tissue
engineeringanddrugdelivery[25–27].Chitosanisobtainedfromchitin,whichisthemajorcomponent
incrustaceanshells,suchascrabsandinsects[26],soitsuseintechnologicalproductsisofecological
and sustainable importance [28]. In addition, CS has important effects, such as antimicrobial,
anticholesterolemic,antitumor,hemostaticandantioxidantactivities[25–27].
Inthispresentwork,anNO-releasinghydrogelwaspreparedbyincorporatingtheNOdonor
(GSNO)intoaPL/CShydrogel. BothPLandCSarebiocompatiblepolymersextensivelyemployedin
medicalapplications. PLisresponsivetoformingthehydrogelnetwork. Thismatrixpermitsaneasy
topicalapplicationallowingasite-specificreleaseoftheNOdonor/NO.ThepresenceofCSintothe
hydrogelmatrixprovidesadvantagesduetoitsmuco-adhesivenesscharacteristicovercommercial
formulations [25–27]. In biological systems, at low concentrations, GSNO has regulatory effects
(promotionoftissueregeneration,woundhealingandvasodilation),whereasathighconcentrationsit
hasantimicrobialeffects[3]. Therefore,allofthesecombinedpropertiescancomposeaversatileand
suitablematerialfordrugdeliveryofNOintopicalapplications. Tothebestofourknowledge,this
isthefirstpapertodescribethepreparationandcharacterizationofNO-releasingPL/CShydrogel
withbiocompatibilityandantibacterialeffect. OurresultsdemonstratedasustainedNO/NOdonor
releasefromthehydrogelmatrix,attherapeuticlevels. ThecombinationofNOandCSdemonstrated
asynergisticeffectagainstPseudomonasaeruginosa. Interestingly,theconcentrationofNO-releasing
hydrogelrequiredforanantibacterialeffectwasnottoxictowardsmammaliancells,indicatingthe
promisingandsafeusesofthisbiomaterialintopicalapplications.
2. MaterialsandMethods
2.1. Materials
Chitosan (CS, low molecular weight, 75% deacetylation), glutathione (GSH), sodium nitrite
(NaNO ), Pluronic F-127 (PL) ([poly(ethylene oxide)] [poly(propylene oxide)] [poly(ethylene
2 106 70
oxide)] , molar weight of 12,600 kDa), phosphate buffer saline (PBS, pH 7.4), MTT
106
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and phenol were obtained from
Sigma-Aldrich (St. Louis, MO, USA). Acetone, hydrochloric acid and lactic acid were obtained
fromLabsynth(Diadema,SP,Brazil). Allexperimentswerecarriedoutusinganalyticalgradewater
fromaMilliporeMilli-QGradientfiltrationsystem(resistivitybelow18.2MΩ·cm−1at25.0◦C).The
VerocelllinewasobtainedfromInstitutoAdolfoLutz(SãoPaulo,SP,Brazil). Theculturemedium,
supplements,antibioticsandbovinefetalserumwereobtainedfromCultilab(Campinas,SP,Brazil).
TheMueller-Hintonmedia(brothandagar)werepurchasedfromDifco(FranklinLakes,NJ,USA),
andthesodiumchloride(NaCl,P.A.grade),usedtoobtainsalinesolution0.85%w/v,waspurchased
fromJ.T.Baker(CiudaddelMexico,Mexico).
Polymers2018,10,452 4of19
2.2. PreparationofPL/CSHydrogel
PL/CShydrogelwaspreparedbasedonthecoldmethodfirstdescribedbySchmolkaetal.[29].
ThismethodhasbeenreportedextensivelyforthepreparationofPL-basedhydrogelscontainingdrugs,
particlesorotherpolymers[27,30–32]. First,0.1gofCSwasdissolvedin10mLoflacticacid(2%)
undermagneticstirringatroomtemperature. Next,2.0gofPLwasgraduallyaddedtotheCSsolution
undergentlemagneticstirring. Thetemperaturewasmaintainedat4◦Cuntilcompletedissolutionof
PLpowder[20]. ThefinalconcentrationsofPLandCSinthehydrogelwere20%w/vand1%w/v,
respectively. ThismaterialisreferredasPL/CShydrogel.
2.3. SynthesisofGSNO
GSNOwassynthesizedaspreviouslydescribed[33–35]. GSH(1228mol·L−1)wasdissolvedinto
HCl(1mol·L−1), followedbytheadditionofanequimolaramountofNaNO , correlatedtoGSH.
2
Thenitrosationreactionwascarriedoutfor20–30min,inanicebath,undermagneticstirringand
inthedark,leadingtotheformationofapinkprecipitateofGSNO.Thismaterialwaswashedwith
coldwaterandfilteredundervacuumusingaMilliporecellulosemembrane. TheobtainedGSNO
was freeze-dried for 24 h. Solid GSNO was maintained in a recipient with humidity controlled at
−20◦C[22].
2.4. IncorporationofGSHorGSNOintoPL/CSHydrogel
GSHorGSNOwereincorporatedintothePL/CShydrogelmatrixfollowingtheprocessdescribed
byPelegrinoetal. withmodifications[33]. TherequiredamountofpowderedGSHorGSNOwas
addedtothePL/CSsolution,undergentlemagneticstirringandlightprotection,at5◦C.Thismixture
wasmaintainedat5◦CuntilthecompletedissolutionoftheGSHorGSNO.Thisprocessledtothe
formationofaPL/CShydrogel,atroomtemperature(finalconcentrationsofPLandCSwere20%and
1%ofCSw/v,respectively)containing50mmol·L−1ofGSHorGSNO(finalconcentration). Thisis
henceforthreferredtoasGSH-PL/CShydrogelorGSNO-PL/CShydrogel,respectively.
2.5. MorphologicalCharacterizationofPL/CSandGSH-PL/CSHydrogels
ThemorphologyofthePL/CSandGSH-PL/CShydrogelswasinvestigatedbyscanningelectron
microscopy(SEM).Priortheanalysis,thesampleswerefrozenat−80◦Cfor12handfreeze-driedfor
36h(Labconco,84C,KansasCity,MO,USA),aspreviouslydescribed[24,36–38]. Thedriedmaterials
werespreadonadouble-sidedconductionadhesivetape,pastedonametallicstubandanalyzedusing
abenchtopSEM(JCM-6000PLUSNeoScope,JEOLTechnics,Tokyo,Japan). Theanalysiswasdone
underhighvacuumandatanoperatingvoltageof5kV.
2.6. RheologicalMeasurementsofPL/CSandGSH-PL/CSHydrogels
RheologicalmeasurementsofPL/CSandGSH-PL/CShydrogelswereperformedonarotational
rheometeratoscillationmodus(KinexusLab,MalvernInstrumentsLtd.,Worcestershire,UK)witha
parallelplate(PP)geometry(diameter70mm,angle2.576radandgapbetweentheplatesof1.02mm)
andaPeltiertemperature-controlledholder. Fordeterminationofelastic(G(cid:48))andloss(G”)moduli,
avolumeof1mLofeachhydrogelsampleweretransferredtotherheometer. Atemperatureramp
wasappliedfrom10to50◦Cataheatingrateof5.0◦C·min−1,withafixedfrequencyof1.0Hzand
ashearstressof2.0pA.ThetemperaturewhenG(cid:48) >G”,andthesampledemonstratedasolid-like
gelformation, wasrelatedtogellingtemperature(T ). Eachexperimentwasrepeatedtwiceand
gel
analyzed using rSpace software (software version 2.00, Worcestershire, UK). In a further step, the
temperaturewasfixedat32.5◦Candthefrequencywasvariedfrom0.1to10Hzinordertoevaluate
thestabilityofthehydrogel. EachexperimentwasrepeatedtwiceandanalyzedusingrSpacesoftware.
Polymers2018,10,452 5of19
2.7. ThermalAnalysesofPL/CSandGSH-PL/CSHydrogels
ThermalcharacterizationofthePL/CSandGSH-PL/CShydrogelswasperformedbyDifferential
ScanningCalorimetry(DSC)employingtheDSCQ200equipment(TAInstruments,NewCastle,DE,
USA).Thereferenceinallcaseswasastandardaluminumsampleholder. Samplesof0.2mLofPL/CS
orGSH-PL/CShydrogelwereanalyzedusingheating–coolingcyclesintherangeof0–65◦C,witha
heatingrateof5◦C·min−1,employingnitrogentopurgethegaschamber(50mL·min−1). Thecycles
wereasfollows: cycle1cooledfrom25to−10◦C,cycle2heatedfrom−15to65◦C,cycle3cooled
from65to−15◦Candcycle4heatedfrom−15◦Cto65◦C.ThedatawereanalyzedusingUniversal
Analysis2000software(TAInstrumentsversion4.5A,NewCastle,DE,USA).Thecriticalmicellization
temperature(CMT)wasdeterminedfromtheendothermicpeakintherecordedDSCthermographs,
andtheenthalpychange(∆H)oftheendothermicpeakwascalculated.
2.8. KineticsofNORelease
KineticsofNOreleasefromaqueousGSNOandfromGSNO-containinghydrogel(GSNO-PL/CS)
weremonitoredbymeasuringchangesinabsorbanceintensityat545nm(nN→π*transition),byusing
theUV–visiblespectrophotometer(Agilent8454,PaloAlto,CA,USA),withatemperature-controlled
sample holder. Changes at 545 nm are solely associated with S–N bond cleavage and free NO
release [33,34,39]. Kinetic data were acquired at temperatures of 25, 32.5 and 37 ◦C. The initial
concentration of GSNO in all groups was 50 mmol·L−1, as confirmed by the detection of the
characteristic GSNO absorption band at 545 nm (nN→π*) [33,39]. The quantity of NO released
overtimewascalculatedfollowingEquations(1)and(2)[22,40]. Theresultswerereportedasthe
mean ± standard deviation (SD) of three independent experiments and expressed in terms of the
NOconcentration:
[NO]t=[GSNO] −[GSNO], (1)
0 t
A b A b
[NO]t = 0 − t . (2)
εGSNO εGSNO
Equations(1)and(2)relateNOconcentrationattime,t,([NO]t)totheGSNOabsorptionband,
wherein[GSNO] and[GSNO] aretheconcentrationsofGSNOatthebeginningofthereactionandat
0 t
time,t,respectively. VariablesA andA aretheGSNOabsorbances,at545nm,atthebeginningofthe
0 t
monitoringandattime,t,respectively. VariableεGSNOisthemolarabsorptioncoefficientofGSNOat
545nm(εequalsto18.4mol−1·L·cm−1),andbistheopticalpathofcuvette,whichcorrespondsto1cm.
2.9. InVitroGSNODiffusion
TheinvitrokineticsofintactGSNOdiffusionfromaqueousGSNOandfromGSNO-containing
hydrogel(GSNO-PL/CS)werecarriedoutbyusingaverticaldiffusioncell(standardcell,15mmof
diameter,7mL,HansonResearchCorporation)[41].Thecellconsistedofdonorandreceptorchambers,
separatedbypolysulfonemembranediscfilterswitha450nmporesize(Tuffryn,PallCorporation,
PortWashington,NY,USA).Thedonorcompartmentwasfilledwith2.5mLaqueousGSNO(GSNOin
PBS)orGSNO-PL/CShydrogel,inwhichtheinitialGSNOconcentrationwas50mmol·L−1,inboth
cases. ThereceptorcompartmentwasfilledwithPBS,keptundermagneticstirringat32.5±0.5◦C
andtheentirecellwasprotectedfromambientlight. Avolumeof500µLwaswithdrawnfromthe
receptorcompartment,in30minintervals,andreplacedbyanequivalentvolumeoffreshPBSsolution.
ThewithdrawnsampleswereimmediatelyanalyzedbyUV–visiblespectrophotometry. Theamount
ofintactGSNOdiffusedthroughthemembranewasdeterminedbymonitoringthespectralchanges
at336nm(ε=980mol−1·L·cm−1),andassignedtothepresenceofintactGSNO[33,34,40]. Theresults
werereportedasthemean±standarddeviation(SD)ofthreeindependentexperimentsandexpressed
intermsoftheGSNOpercentage.
Polymers2018,10,452 6of19
2.10. MathematicalModels
InordertoinvestigatethemechanismofGSNOdiffusionfromthePL/CShydrogel,theHiguchi
mathematicalmodelwasappliedtothekineticcurveobtainedinitem2.9(Equation(3))[42]:
Q =K t0.5, (3)
t H
whereQ isthecumulativeamountofdrugreleasedattime,t,K isthereleaseconstant,andtisthe
t H
time[33,43,44].
2.11. CytotoxicityofCS/PLandGSNO-PL/CSHydrogels
The cytotoxicity effects of CS/PL and GSNO-CS/PL hydrogels were evaluated towards Vero
cellsbyMTT-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)assay[45,46]. Verocells
passage173(CCIAL-057)wereseededin24-wellplates. After24h,ataconfluenceof80%andafter
formingamonolayer,theculturemediumwasremovedandsubstitutedbyelutionmedium,prepared
usingcellculturemediumandthehydrogels. ThehydrogelswereaddedtoeachwellcontainingVero
cellsatconcentrationsof0.2,1.2,2.4,6.0and9.5µg·mL−1ofPL/CShydrogel. Theseconcentrations
correspondto0.2,1.2,2.4,6.0and9.5mmol·L−1 ofGSNO,respectively(finalconcentrations. After
addingeachformulationtoVerocells,theywereincubatedfor24hat37◦Cina5%CO atmosphere.
2
Thecellsintheculturemediumwithoutanytreatmentwereemployedasthenegativecontrol,anda
solutionofphenol(0.25%)wasusedasapositivecontrol(cytotoxic)[47–49]. AfterVerocellincubation
for 24 h with the PL/CS hydrogel and GSNO-PL/CS hydrogel, a volume of 100 µL of 10% MTT
solution (5 mg·mL−1) in PBS was added to each of the wells, and the cells were incubated for an
additional4h. AfterremovingMTTsolution,50µLofdimethylsulfoxidewasaddedtoeachwell
for 10 min, and the absorbance at 570 nm was measured with an automated spectrophotometric
microtiterplatereader(SpectraMaxM5,MolecularDevices,Sunnyvale,CA,USA).Theexperiment
wasperformedintriplicate. Theresultswerepresentedasthemean±standarddeviation(SD).The
ANOVAtest(one-way)wasdone,followedbytheposthocTukeytestforcomparisonbetweenthe
twogroups. Statisticalsignificancewassetatap-valueof≤0.001.
2.12. MorphologicalEvaluationofVeroCellsafterTreatmentwiththeHydrogels
ThemorphologyofVerocellsafter24hincubationwithpurePL/CSorGSNO-PL/CShydrogels
(at final hydrogel concentration of 0.2–9.5 µg·mL−1) was observed with an inverted microscope
(AxioVertA1,Zeiss,Jena,Germany)andphasecontrast[49,50].
2.13. AntibacterialActivityofPL/CSandGSNO-PL/CSHydrogels
Theminimalinhibitoryconcentration(MIC)andminimalbactericidalconcentration(MBC)of
thePL/CSandGSNO-PL/CShydrogelswerecarriedoutbymicrodilutionassay,asdescribedbythe
ClinicalandLaboratoryStandardsInstitute[51]. Thebacterialstrainevaluatedwasthegram-negative
(standard CLSI) Pseudomonas aeruginosa ATCC 27853 (PAR). Initially, the bacteria were grown in
Mueller–Hintonsolidmediumat37 ◦Ctoobtaintheisolatedcolonies. Subsequently, thecolonies
weresolubilizedinasalinesolution0.85%(w/v)andadjustedtothe0.5indexoftheMacFarlandscale
(1.5 × 108colony-formingunits(cfu)permL).ThissolutionwasdilutedinMueller–Hintonbroth
anddistributedina96-wellplateatadensityof106cfu/well. Eachwellwastreatedwithdifferent
concentrationsofPL/CSandGSNO-PL/CShydrogelsof0.3,0.5,1.0,2.1,3.15and4.2µg·mL−1,which
correspondtoNOfinalconcentrationsof0.5, 1.0, 2.0, 4.0, 6.0and8.0mmol·L−1, respectively. The
plateswereincubatedfor18handthebacterialgrowthwasmeasuredafterthisperiod. TheMBC
testswereperformedaftertheMICtests. After24hofincubation,dropsofbacterialbrothfromthe
cavitywithnovisualgrowthwereaddedtoaPetridishwiththeMueller–Hintonsolidmedium,and
bacterialgrowthwasobservedafter48h. Thesetestswerecarriedoutintriplicate.
Polymers2018,10,452 7of19
3. ResultsandDiscussion
Biocompatiblehydrogelsmightsimulatelivingtissuesmorethananyotherclassofsynthetic
biomaterial,duetotheirsimilarities[31,52,53]. Theyareeasytoapplyintoseveralshapesandsurfaces
becauseoftheirsoftness.Thus,theyarepredominantlyusedinbiomedicalandtopicalapplications[31].
Polymers 2018, 10, x FOR PEER REVIEW 7 of 19
PLhydrogelsareversatilematerialssinceitispossibletoincorporateeitherhydrophilic[22,40,49,54]
oprrohpyedrrtoiepsh foobri cdrdurgu gdseilnivtoerPyL anhdy dtirsosgueel sen[5g5i]n.TeehreinCgS -[b38a,s5e6d–5b8io].m Tahtee riinatlesrhaacvtieong aoifn pedosaitliovte opfraotttoennatitoend
dCuSe atomtihneoi rgbriooucpoms wpaittihb itlhitey ,nbeiogdaetigvrealdya cbhilaitrygeadn dmhuecmino sltaaytiecr parnodp etritgiehst fjourndcrtuiognds eclaivne rfyacainlidtatties stuhee
etrnagnisnpeoerritn ogf h[3y8d,5ro6–p5h8il]i.cT mheacirnotmeroalcetciounleos fthproosuitgivhe thper omtouncaotseadl bCaSrraimeri [n5o9]g. rTohuips ssewctiitohnt hdeesncergibaetsiv aenlyd
cdhisacrugsesdesm tuhcei nchlaayraecrtaernidzattiigohnt ojuf nPcLti/oCnSs acsa nbifoaccoilmitaptaetitbhlee thrayndsrpoogretl ofofrh ytodpriocpahl ialpicpmlicaactrioomnso ilnec NulOes-
tthhreoruapgyh. themucosalbarrier[59]. ThissectiondescribesanddiscussesthecharacterizationofPL/CS
asbiocompatiblehydrogelfortopicalapplicationsinNO-therapy.
3.1. Characterization of PL/CS and GSH-PL/CS Hydrogels
3.1. CharacterizationofPL/CSandGSH-PL/CSHydrogels
In this study, GSH is the precursor of the NO donor molecule, GSNO [60]. Due to the intrinsic
thermInalt hainsdst updhyo,toGcShHemisictahle ipnrsetacubirlsiotyr ooff tGheSNNOO d[7o,2n2o,r40m],o tlheceu cleh,aGraScNteOriz[a6t0i]o.nD oufe mtootrhpehionltorginicsaicl,
trhheeromloaglicaanld anpdh othtoecrhmeaml ipcraolpienrsttiaebs iloift ythoef hGySdNroOge[l7 w,22a,s4 0p]e,rtfhoremcehda rwacitther itzhaet iGoSnHo-fPmL/oCrSp hhoyldorgoigcaell.,
rThheeo lmogoircpahloalnodgyt hoefr PmLa/lCpSr aonpde rGtiSesHo-PfLth/CeSh yhdyrdorgoeglewls awsapse orfbosremrveedd wbyit hSEthMe aGnSaHly-sPeLs /oCf Slyhoypdhirloizgeedl.
Tfohremmuolartpiohnolso [g2y4,o3f7P].L F/iCguSraen 2dAG eSxHh-iPbLit/s CaS rheypdrersoegnetlastwivaes iombasgerev oedf tbhye SPELM/CaSn ahlyydserosgoefll. yAop shmiloizoetdh
fsourrmfauclea twioinths [d24e,f3in7]e.dF iegdugrees 2Aanedx haimbiotsrpahroeupsr essternutcattuivrees imcaang eboef othbesePrLve/dC. SFhigyudrreo g2eBl. Aexhsmiboitos tha
srueprfraecseenwtaittihved iemfiangeed oef dthgee sGaSnHd-PaLm/CoSr phhyodurosgsetlr.u Tchtue rGesSHca inncboerpoobrasetirovne din.to Fthigeu hreyd2rBogeexl hleibaditss tao
rae pmroersee nirtraetigvuelaimr saugrefaocfet. hIte sGhSoHul-dP Lbe/ CnSothedyd trhoagt edl.ynTahmeiGc SsHurfinacceosr,p aosr aotbiosenrvinetdo itnh eFihgyudrero 2gBe,l mleiagdhst
tioncaremasoer eceirlrl egaudlhaersisounr,f amcei.grIattsiohno ualdndb edneotatcehdmthenatt, dwynhaicmhi ciss udrfeascierasb,laes ofobrs etrivsseude inenFgiginuereeri2nBg,
mapigphlitcainticornesa s[e6c1e].l lTahdeh essuiornfa,cme iogfr ahtiyodnraongdel diest atchhem iennitti,awl haincdh ipsrdimesairrayb lseitfeo rotfi sisnuteereancgtiionne ewrinitgh
aspuprrloicuantidoinnsg[ 6c1e]l.lsT ahneds utirsfsauceeso. fInh ybdioromgeedliicsatlh aepinpiltiicaaltaionndsp, irnimclaurdyinsigte ino ftiisnstuerea ecntigoinnweeirtihnsgu, rhryodurnodginelgs
cweliltsha rnodugtihssnueesss. sIunrbfaiocmese adriec acloanpspidliecraetdio snus,itianbclleu dfoinr gceinll taitstsaucehemnegnint,e ceerliln pg,rohlyifderroagtieolsnw, tiitshsureo uggrhownetshs,
spuarsfsaacgees oafr neuctorinesnitdse, rderdugs uloitaadbilnegf aonrdc delelliavtetarych. Imn ethnits, sceenllsep,r tohleif seurarftaiocne ,mtiosrspuheoglorgoyw othf t,hpea hsysadgroegoefl
n(purteriseenntcse, dorf upgorloeas dainndg raonudgdhenleivsse)r yc.anIn atfhfeiscts ethnes eb,itohmeastuerrfiaacl ebmioocorpmhpoalotigbyiliotyf tthherohuygdhro mgeold(ipfirceasteinocnes
oinf pceolrle spraonlidferroautigohnns easns)d caatntaacfhfemcetntht etob iao msoalitder isaulbbsitoractoem. Apast isbhiloiwtynt hinro Fuigghurme o2d, itfihcea tGioSnHs-iPnLc/CelSl
phryodlirfoegraetli ohnass aan rdoautgtahcnhemsse nsutrtofaaces,o wlidhislue bPsLtr/aCteS. hAysdsrhoogweln hiansF aig sumreo2o,thth seuGrfSaHce-P. RL/oCugShhnyedsrso sguerlfhaacse
amroorupghhonloegssy smurifgahcte ,inwchreilaeseP Lth/eC cSehlly adtrtaocghemlheanst aansmd othoeth cseullr fparcoel.ifReroautigohnn, ewsshsicuhr faarcee dmeosrirpahbolelo fgoyr
mtisisguhet ienncgreinaeseertihnegc aepllpaltitcaacthiomnes n[t61a]n. dTthhee hcyedllrporgoellisfe srtautidoined,w hheirceh haarveed seismirialabrle mfoorrptihssouloegeinegs itnoe tehrionsge
adpepsclircibateido nbsy [L61e]y.vaT-hGeomhyeds reotg aell.s, wsthuod icehdarhaecrteerhizaevde PsLim ainladr PmL/oCrpS hhoyldogroiegseltso ptrheopsaeredde sbcyr igbaemdmbya
Lireryavdaia-Gtioonm [e3s7e].t al.,whocharacterizedPLandPL/CShydrogelspreparedbygammairradiation[37].

Figure 2. Scanning electron microscopy (SEM) micrographs of lyophilized (A) PL/CS hydrogel and
Figure2.Scanningelectronmicroscopy(SEM)micrographsoflyophilized(A)PL/CShydrogeland
(B) GSH-PL/CS hydrogel, using a 400× of magnification. The scale bar represents 50 µm.
(B)GSH-PL/CShydrogel,usinga400×ofmagnification.Thescalebarrepresents50µm.
Due to the presence of PL into the hydrogels, PL/CS and GSH-PL/CS hydrogels became
thermoresponsive hydrogels [62]. At low temperatures (0–5 °C), PL/CS and GSH-PL/CS hydrogels
had viscous behavior, whereas at higher temperatures (20–40 °C) the hydrogel had a solid-like
behavior [62]. The gelling temperature (Tgel) indicated the temperature required to change the
rheological property of the material, and is understood as when elastic modulus (G′) becomes higher
than loss modulus (G″) during a temperature ramp [63]. This feature can be related to the presence
of PL into the hydrogel matrix, and has attracted significant interest involving drug release
applications and tissue engineering [64].
Polymers2018,10,452 8of19
Due to the presence of PL into the hydrogels, PL/CS and GSH-PL/CS hydrogels became
thermoresponsivehydrogels[62]. Atlowtemperatures(0–5◦C),PL/CSandGSH-PL/CShydrogels
had viscous behavior, whereas at higher temperatures (20–40 ◦C) the hydrogel had a solid-like
behavior [62]. The gelling temperature (T ) indicated the temperature required to change the
gel
rheologicalpropertyofthematerial,andisunderstoodaswhenelasticmodulus(G(cid:48))becomeshigher
thanlossmodulus(G”)duringatemperatureramp[63]. Thisfeaturecanberelatedtothepresenceof
PLintothehydrogelmatrix,andhasattractedsignificantinterestinvolvingdrugreleaseapplications
andtissueengineering[64].
T values of PL/CS and GSH-PL/CS hydrogels were found to be 28.72 ± 0.98 ◦C and
gel
29.95±0.85◦C, respectively (Table 1). The GSH incorporation on the PL/CS hydrogel slightly
increased T by 1 ◦C. Kkatheb et al. showed a similar result upon the addition of laxacin into
gel
PLhydrogel,wheretheirresultsshowedanincreaseofT approximatelyof1◦C[65].
gel
Table1.Tonset(◦C),criticalmicellartemperature(CMT)(◦C),Tendset(◦C)and∆H(J·g−1)acquiredusing
DSCanalysesandT (◦C)acquiredusingrheologyanalysesofPL/CSandGSH-PL/CShydrogels.
Polymers 2018, 10, x FOR PEEsoRl/ RgeElVIEW 8 of 19
MTgaetle vriaallues of PLT/oCnSse ta(n◦dC )GSH-PLC/CMST h(y◦Cd)rogels wTeenreds feotu(◦nCd) to be 2∆8H.7(2J ·±g −0.19)8 °C andT 2ge9l.9(◦5C ±) 0.85
°C, rePsLp/eCctSively (Table 112)..7 1The GSH inco1r6p.1o5ration on the1 9P.4L8/CS hydrogel4 .s0l1ig3htly incre2a8s.7e2d± Tg0e.l9 b8y 1
GSH-PL/CS 6.57 10.69 14.75 4.237 29.95±0.85
°C. Kkatheb et al. showed a similar result upon the addition of laxacin into PL hydrogel, where their
results showed an increase of Tgel approximately of 1 °C [65].
OOsscciillllaattiioonn sswweeeepp ffrreeqquueennccyy tetesststs wwereer epperefrofromrmede dono nPLP/LC/SC aSnda nGdSHG-SPHL-/PCLS/ hCySdrhoygderlos gaet l3s2a.5t
3°C2. 5(t◦hCe (stkhiens tkeimntpeemrapteurraet)u troe )etvoaelvuaaltuea ttheet heeffeefcfte cotf oGfSGHSH inicnocroproproartaitoinon uunnddeer rtthhee lliinneeaarr vviissccooeellaassttiicc
rreeggiioonn [[6644]].. FFiigguurree 33AA sshhoowwss tthhaatt tthhee GG(cid:48)′ aanndd GG”″ mmoodduullii ddiidd nnoott ssiiggnniifificcaannttllyy cchhaannggee iinn tthhee aapppplliieedd
ffrreeqquueennccyy rraannggee ((00..11––1100 HHzz)).. IInn aaddddiittiioonn,, tthheerree wwaass aa lliinneeaarr rreeggiioonn wwiitthh aa ssiimmiillaarr pprroofifilleef foorrP PLL//CCSS
aanndd GGSSHH--PPLL//CCSS hhyyddrrooggeellss ffoorr aallll tteesstteedd ffrreeqquueenncciieess ((FFiigguurree 33AA)).. TThhuuss,, tthhee GGSSHH iinnccoorrppoorraattiioonn iinnttoo
PPLL//CCSS hhyyddrrooggeell ddiidd nnoott ssiiggnniiffiiccaannttllyy cchhaannggee tthhee hhyyddrrooggeell mmeecchhaanniiccaall aanndd ggeelllliinngg pprrooppeerrttiieess..

FFiigguurree 33.. ((AA)) EEllaassttiicc ((GG(cid:48)′)) aanndd lloossss ((GG”″)) mmoodduullii ooff PPLL//CCSS hhyyddrrooggeell ((bbllaacckk lliinnee)) aanndd GGSSHH--PPLL//CCSS hhyyddrrooggeell
((bblluuee lliinnee)) dduurriinngg aann oosscciillllaattiioonn sswweeeepp ffrreeqquueennccyy tteesstt aatt 3322..55 ◦°CC;; ((BB)) DDiiffffeerreennttiiaall ssccaannnniinngg ccaalloorriimmeettrriicc
(DSC) thermographs of PL/CS hydrogel (black line) and GSH-PL/CS hydrogel (blue line).
(DSC)thermographsofPL/CShydrogel(blackline)andGSH-PL/CShydrogel(blueline).
Table 1. Tonset (°C), critical micellar temperature (CMT) (°C), Tendset (°C) and ∆H (J·g−1) acquired using
DSCisanimportanttechniquetoevaluatethetemperaturethatinducesthermaltransitionsin
DSC analyses and Tsol/gel (°C) acquired using rheology analyses of PL/CS and GSH-PL/CS hydrogels.
solutions of thermoresponsive polymers [65]. Figure 3B exhibits the thermographs of PL/CS and
GSH-PL/CShyMdraotgeerilas,l withTtohnseet a(i°mC)t oiCnvMesTt i(g°aCte) thTeernmdsaetl (t°rCan) sit∆ioHn (cJh·gan−1g) esafTtegerl G(°SCH) incorporation.
ThePL/CShydrPoLge/ClsSh oweda1n2.e7n1d otherm16p.1e5a kwitha1n9.o4n8s ettemp4e.0r1a3tu re(T28.72) ±a 0t.1928. 71◦C,apeak
onset
at16.15 ◦CanGdSHan-PeLn/dCSs ettem6.p5e7r ature(T10.69 )of191.448.7◦5C (Tabl4e.213).7 The2e9n.d9o5 t±h e0r.8m5 peakcanbe
endset
DSC is an important technique to evaluate the temperature that induces thermal transitions in
solutions of thermoresponsive polymers [65]. Figure 3B exhibits the thermographs of PL/CS and
GSH-PL/CS hydrogels, with the aim to investigate thermal transition changes after GSH
incorporation. The PL/CS hydrogel showed an endotherm peak with an onset temperature (Tonset) at
12.71 °C, a peak at 16.15 °C and an end set temperature (Tendset) of 19.48 °C (Table 1). The endotherm
peak can be related to the critical micellization temperature (CMT) process. In addition, Tonset is
mainly related to the beginning of micelle formation, whereas Tendset shows the completion of the
micelle formation process [65]. The incorporation of GSH into the PL/CS hydrogel decreased the Tonset
by 6.14 °C, CMC by 5.46 °C and Tendset by 4.73 °C (Table 1 and Figure 3B). Thus, the GSH incorporation
decreased the CMT by 40%. The micelle formation process is related to the dehydration of PPO block
units in PL structure. The incorporation of GSH (hydrophilic molecule) is expected to form into a
hydrophilic layer of the micelle, so the presence of GSH caused the dehydration of PPO block and,
consequently, decreased the CMT value. The same trend was observed by Nie et al. by adding
paclitaxel into PL hydrogel [22,42].
Enthalpy variation (∆H) is the area under the endothermic peak related to CMC. The ∆H values of
PL/CS hydrogel and GSH-PL/CS hydrogel were found to be 4.013 and 4.237 J·g−1, respectively (Table 1).
All formulations showed a positive ∆H. This result is related to the dehydration of the PPO block, which
Polymers2018,10,452 9of19
relatedtothecriticalmicellizationtemperature(CMT)process. Inaddition,T ismainlyrelatedto
onset
thebeginningofmicelleformation,whereasT showsthecompletionofthemicelleformation
endset
process[65]. TheincorporationofGSHintothePL/CShydrogeldecreasedtheT by6.14◦C,CMC
onset
by5.46◦CandT by4.73◦C(Table1andFigure3B).Thus,theGSHincorporationdecreasedthe
endset
CMTby40%. ThemicelleformationprocessisrelatedtothedehydrationofPPOblockunitsinPL
structure. TheincorporationofGSH(hydrophilicmolecule)isexpectedtoformintoahydrophilic
layerofthemicelle,sothepresenceofGSHcausedthedehydrationofPPOblockand,consequently,
decreasedtheCMTvalue. ThesametrendwasobservedbyNieetal. byaddingpaclitaxelintoPL
hydrogel[22,42].
Enthalpyvariation(∆H)istheareaundertheendothermicpeakrelatedtoCMC.The∆Hvalues
ofPL/CShydrogelandGSH-PL/CShydrogelwerefoundtobe4.013and4.237J·g−1,respectively
(Table1). Allformulationsshowedapositive∆H.ThisresultisrelatedtothedehydrationofthePPO
block,whichisanendothermicprocess. Thus,GSHincorporationintoPL/CShydrogelincreased
the ∆H value by 10%. Akkari et al. have shown a similar behavior by adding budesonide to PL
hydrogels[66].
Taking together, the morphological, rheological and thermal properties of the GSH-PL/CS
hydrogelwerefoundtobesuitablefordrugdelivery,sincethermoresponsivebehavioranddesirable
stabilityagainstoscillationwereobserved. Thesecombinedfeaturesmakethishydrogelapotential
candidatefortopicalapplicationsofactivemolecules,suchasNO/NOdonors[31].
3.2. NOReleasefromGSNO-PL/CSHydrogel
GSHistheprecursormoleculeoftheNOdonor,GSNO.Inthiswork,GSHwasnitrosatedby
reactingwithanequimolaramountofNaNO ,inacidmedium,formingtheGSNO(Equation(4)).
2
Inthiscase, thenitrosatingagentwasnitrousacid(HNO ), whichisformedbythedissolutionof
2
NO − inaqueousacidifiedsolution. Oncesynthesized,GSNOwasincorporatedintoPL/CShydrogel:
2
GSH+HNO →GSNO+H O. (4)
2 2
In this work, the kinetics of NO release from GSNO (either dissolved in aqueous solution or
incorporatedintoGSNO-PL/CShydrogel)wasmonitoredfor24h,atdifferenttemperatures. GSNO
undergoes to a spontaneous decomposition releasing free NO and yielding oxidized glutathione
(GSSG)(Equation(5))[1,9,22,33]:
2GSNO→2NO+GSSG. (5)
ThisreactionoccursthroughthehomolyticcleavageoftheS–Nbond,whichcanbecatalyzedby
thepresenceofmetalions(e.g.,copper),lightandheat[1,9,22,33].
Figure 4 shows the NO release profile from free GSNO and from GSNO-PL/CS hydrogel at
differenttemperatures(25,32.5and37◦C)for24h. AsthestabilityofGSNOisknowntobedependent
on the temperature [39], the NO release profile was monitored at room, skin and physiological
temperatures(25,32.5and37◦C,respectively). Figure4showsthatbothGSNOinaqueoussolution,
and GSNO incorporated into the hydrogel matrix, were able to release NO for at least 24 h at the
differenttestedtemperatures. Inaddition,theincreaseofthetemperatureincreasedtheratesofNO
releasefrombothaqueousGSNOandhydrogelcontainingGSNO.Thisresultisinaccordancewith
previouspublications[7,9,22,30].
Furthermore,theincorporationofGSNOintoPL/CShydrogeldecreasedtheratesofNOrelease,
comparedtoGSNOdissolvedinaqueoussolution(Figure4),aspreviouslyreported,whencomplexed
to other materials [9,20,54]. This decrease was assigned to the cage effect promoted by the higher
viscosityofthehydrogelmatrix,incomparisontotheaqueousenvironment. Thesuperiorviscosity
ofthehydrogelmatrixfavorstheradicalpairrecombinationofNO• andGS•,restoringtheGSNO
Polymers 2018, 10, x FOR PEER REVIEW 9 of 19
is an endothermic process. Thus, GSH incorporation into PL/CS hydrogel increased the ∆H value by
10%. Akkari et al. have shown a similar behavior by adding budesonide to PL hydrogels [66].
Taking together, the morphological, rheological and thermal properties of the GSH-PL/CS
hydrogel were found to be suitable for drug delivery, since thermoresponsive behavior and desirable
stability against oscillation were observed. These combined features make this hydrogel a potential
candidate for topical applications of active molecules, such as NO/NO donors [31].
3.2. NO Release from GSNO-PL/CS Hydrogel
GSH is the precursor molecule of the NO donor, GSNO. In this work, GSH was nitrosated by
reacting with an equimolar amount of NaNO2, in acid medium, forming the GSNO (Equation (4)). In
this case, the nitrosating agent was nitrous acid (HNO2), which is formed by the dissolution of NO2−
in aqueous acidified solution. Once synthesized, GSNO was incorporated into PL/CS hydrogel:
GSH + HNO2 → GSNO + H2O. (4)
In this work, the kinetics of NO release from GSNO (either dissolved in aqueous solution or
incorporated into GSNO-PL/CS hydrogel) was monitored for 24 h, at different temperatures. GSNO
undergoes to a spontaneous decomposition releasing free NO and yielding oxidized glutathione
(GSSG) (Equation (5)) [1,9,22,33]:
2 GSNO → 2 NO + GSSG. (5)
This reaction occurs through the homolytic cleavage of the S–N bond, which can be catalyzed
by the presence of metal ions (e.g., copper), light and heat [1,9,22,33].
Figure 4 shows the NO release profile from free GSNO and from GSNO-PL/CS hydrogel at
different temperatures (25, 32.5 and 37 °C) for 24 h. As the stability of GSNO is known to be
dependent on the temperature [39], the NO release profile was monitored at room, skin and
physiological temperatures (25, 32.5 and 37 °C, respectively). Figure 4 shows that both GSNO in
Polymers2018,10,452 10of19
aqueous solution, and GSNO incorporated into the hydrogel matrix, were able to release NO for at
least 24 h at the different tested temperatures. In addition, the increase of the temperature increased
tmheo lreactuelse ,oafn NdOin rcereleaassineg frtohmer abtoetsho afqNuOeoruesle GasSeN. IOn tahnids sheyndser,otgheel vciosncotasiintyinogf tGhSeNmOed. Tiuhmis craensublet uiss eind
atoccmoroddaunlcaete wthiteh rparteevsioofuGs SpNubOlidcaetcioomnsp [o7s,9it,i2o2n,3,0a]n. dconsequentlytheratesofNOrelease[31].

FFiigguurree 44.. KKiinneettiiccss ooff NNOO rreelleeaassee ffrroomm GGSSNNOO ((iinniittiiaall ccoonncceennttrraattiioonno off5 500m mmmooll··LL−−11)) iinn aaqquueeoouuss ssoolluuttiioonn
((bbllaacckk lliinneess)) aanndd iinnccoorrppoorraatteedd iinnttoo PPLL//CCSS hhyyddrrooggeell ((bblluuee lliinneess)),, aatt 2255,, 3322..55 aanndd 3377 °◦CC.. TThhee rreessuullttss aarree
rreeppoorrtteedd aass tthhee mmeeaann ±± sstatannddaarrdd ddeevviaiatitoionn ((SSDD)) ooff tthhrreeee ininddeeppeennddeenntt eexxppeerriimmeennttss..
Furthermore, the incorporation of GSNO into PL/CS hydrogel decreased the rates of NO release,
Figure4showsthattheconcentrationofNOreleasefromGSNOwasinthemillimolarrange.
compared to GSNO dissolved in aqueous solution (Figure 4), as previously reported, when
Atthisrange,NOisexpectedtohavetherapeuticeffects[60],suchasantibacterialactivity. After24h
complexed to other materials [9,20,54]. This decrease was assigned to the cage effect promoted by the
at37◦C,thetotalconcentrationofNOreleasefromaqueousGSNOwasca. 36mmol·L−1,whilethe
concentrationofNOreleasefromthehydrogelwas30mmol·L−1 (Figure4). Theincorporationof
GSNOintothePL/CShydrogeldecreasedtheamountoftotalNOreleasedby20%,atthesameperiod
oftime.
It should be noted that for therapeutic purposes, a sustained and localized release of NO is
desirable. Figure4showsthattheincorporationofGSNOintoPL/CShydrogelpromotesasustained
NOrelease,inconcentrationssuitableforbiomedicalapplications,foratleast24h,atphysiological
andskintemperatures. Inaddition,thehydrogelallowsanincreaseinresidencetimeoftheNOdonor
directlyonthetargetsiteofapplication,makingthisformationagoodcandidateforatopicalNO
deliverysystem[1,7,67–69].
3.3. GSNOReleasefromGSNO-PL/CSHydrogel
TheverticalFranzdiffusioncellwasusedtoevaluatethediffusionofintactGSNOthrougha
membraneversusGSNOdissolvedinaqueoussolutionandGSNO-PL/CShydrogel,at32.5◦C(skin
temperature). Figure5showsthatinbothcases,atwo-phasereleaseprofileofGSNOwasobserved,
whichincluded: (1)aninitialburstofGSNOdiffusion(withinthefirst1.5hofmonitoring);and(2)the
establishmentofaplateau,whichcorrespondedtoasteady-stateofGSNOdiffusionrelease[20,70].
This diffusion profile is similar to other reports published by the authors [30,32,33]. Additionally,
Wuetal.showedasimilarGSNOreleaseprofilefromfreeGSNOandGSNOencapsulatedintopolymer
nanocompositeparticles[71].
ThediffusionofGSNOfromaqueousGSNOreachedavalueof50%ofthetotalGSNOcontent
after4.5hofmonitoring,whereasinthecaseofGSNO-PL/CShydrogeltheamountofGSNOthat
diffused was 20%, at the same time frame (Figure 5). Indeed, the incorporation of GSNO into the
PL/CShydrogeldecreasedtheamountthatGSNOdiffusedby60%. Thisresultisinaccordancewith
Figure4(i.e., kineticsofNOreleasefromaqueousandGSNO-containinghydrogel). Thesuperior