Synthesis, Characterization, and Drug Delivery from pH

Research ArticleSynthesis Characterization and Drug Delivery from pH-and Thermoresponsive Poly(N-Isopropylacrylamide)Chitosan CoreShell Nanocomposites Made by SemicontinuousHeterophase Polymerization

Abraham G Alvarado1 Andres Ortega2 Lourdes A Peacuterez-Carrillo2 Israel Ceja3

Martin Arellano2 R Guillermo Loacutepez4 and Jorge E Puig2

1Departamento de IngenieriaMecanica Electrica Universidad de Guadalajara Boul M Garcıa-Barragan 1451 44430 GuadalajaraJAL Mexico2Ingenierıa Quımica Universidad de Guadalajara Boul M Garcıa-Barragan 1451 44430 Guadalajara JAL Mexico3Fısica Universidad de Guadalajara Boul M Garcıa-Barragan 1451 44430 Guadalajara JAL Mexico4Centro de Investigacion en Quımica Aplicada Av Ing E Reyna 140 25294 Saltillo COAH Mexico

Correspondence should be addressed to Jorge E Puig puigjemailudgmx

Received 31 August 2016 Revised 16 December 2016 Accepted 19 December 2016 Published 24 January 2017

Academic Editor Xuping Sun

Copyright copy 2017 Abraham G Alvarado et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Temperature- and pH-responsive coreshell nanoparticles were prepared by semicontinuous heterophase polymerization of N-isopropylacrylamide (NIPA) in the presence of chitosan micelles for drug delivery purposes Micelles of chitosan formed in anacetic acid aqueous solution at 70∘C containing potassium persulfate were fed with N-isopropylacrylamide (NIPA) at a controlledrate to produce PNIPAchitosan coreshell nanoparticles of about 350 nm Then the crosslinking agent glutaraldehyde wasadded to crosslink the nanoparticles These nanocomposites were temperature- and pH-responsive which make them suitableas controlled drug releasing agents The nanoparticles exhibit thermoreversibility to heating-and-cooling cycles and show differentresponses depending on the releasing mediumrsquos pH Drug delivery tests were performed employing as a model drug doxycyclinehyclate

1 Introduction

Environmentally sensitive nanoparticles (NPs) are receivingincreasing scientific and industrial attention as drug-deliverysystems because of their ability to release drugs to therapeutictargets at controlled and appropriate times and doses [1ndash5]Their sizes of the order of a few tenths of nanometers allowthem to go through capillary vessels avoiding being trappedby phagocytes which permits larger duration in the bloodstream

Recently biodegradable and biocompatible polymericmaterials used for drug delivery have been developedsuch as poly(lactic acid) poly(lactide-co-glycolide) poly-caprolactone polyanhydrides poly(hydroxyalkanoates)

polypeptides and polysaccharides [6ndash8] Among thempolysaccharides are the most employed polymers to prepareNPs for drug delivery purposes [9 10] Chitosan (CS) is apolysaccharide that is pH-responsive due to its amino groupsalong the chain which are able to get protonated at acid pHs[5]

Environmentally sensitive polymers have been inves-tigated in biomedical applications such as drug deliverysensors diseasesrsquo targeting and diagnostics [11ndash13] Amongthese polymers poly(N-isopropylacrylamide) (PNIPA) is themost studied thermoresponsive polymer because it has alower critical solution temperature (LCST) of ca 32-33∘C [12]below this temperature PNIPA is completely soluble in waterbut above it PNIPA and water phase separate due to the

HindawiJournal of NanomaterialsVolume 2017 Article ID 6796412 7 pageshttpsdoiorg10115520176796412

2 Journal of Nanomaterials

transition from a hydrophilic to a hydrophobic structure atthis temperature [14]

Huang et al reported in 2013 that chitosan was ableto form micelles in an acetic acid solution even thoughthis polysaccharide is insoluble in pure water these authorsdemonstrated the micelle formation and reported the crit-ical micellar concentration by fluorescence emission spec-troscopy [15] In addition Huang et al showed in the samearticle that by adding NIPA in one shot and an initiator(potassium persulfate) at 70∘C the NIPA polymerized andbecause the reaction temperature was higher than the LCSTof the PNIPA it became hydrophobic and as a consequencemigrated to the micellar core To ensure that the PNIPACSmicelles would not be disrupted in further applications theseauthors added an aqueous solution of glutaric dialdehyde tocrosslink themicellesmoreover they demonstrated the capa-bility for prolonged drug delivery of this coreshell PNIPACSmicellar solutionWang et al synthesized coreshell nanoma-terials through radical polymerization of CS and a mixtureof NIPA and acrylamide (AM) which exhibited thermallysensitive features and that allowed to tune the volume phasetransition temperature (119879VPT) up to collapsing temperaturesof ca 39∘C depending on the AMNIPA molar ratio [16]

Popat et al [17] reported a CS-coated mesoporous silicananospheres that were pH-responsive and used ibuprofenas model drug they demonstrated that under basic con-ditions the coreshell nanomaterial forms a gel structurethat is insoluble and prevents the drug release whereasbelow the CS isoelectric point (pH = 63) the drug isreleased due to the protonation of the CS amino groupsChang et al [18] produced poly[poly(ethylene glycol methylether methacrylate)-block-poly(methyl methacrylate)] blockcopolymer to encapsulate the model drug Nile Red anda chemotherapeutic drug doxorubicin (DOX) and showedthat the release of DOX from the micelles was sufficient tobe cytotoxic to human colon carcinoma cells Li et al [19]developed a potential nanocarrier for topical administrationof curcumin (CUR) a novel thiolated CS made by covalentbinding between N-acetyl-L-cysteine (NAC) and CS whichwas employed to modify the surface of the nanostructuredlipid carrier CUR Onnainty et al [20] created sustainedrelease systems of the drug chlorhexidine (CLX) based onCS and montmorillonite These hybrid carriers exhibitedsustained release ofCLXwhich allowedmaintaining theCLXantimicrobial properties

In this study we synthesized pH- and thermoresponsiveNIPACS coreshell nanoparticles by semicontinuous het-erophase polymerization (SHP) The aim was to synthesizethermo- and pH-sensitive nanoparticles with larger polymercontent than those reported by Huang et al [15] and evaluatetheir thermoreversibility and their potential application asdrug carrier and delivery systems as a function of pH andtemperature

2 Experimental Section

CS AM (99 pure) NMBA (99 pure) glutaraldehyde((GA) 50wt in water) and doxycycline (98 pure) wereobtained from Sigma-Aldrich NIPA was 99 pure from

Acros Organics KPS (99 pure) was from Fermont N2gas

was from Infra de Occidente (Mexico) and doubly distilledwater was from Productos Selectropura (Mexico)

The CS degree of deacetylation was determined poten-tiometrically with the following procedure first 04 g of CSwas dissolved in 89 g of a 01M HCl solution by constantstirring during 24 h next this solution was titrated with01393M NaOH solution with a pH-meter The volume ofthe NaOH solution added to the chitosan solution wasplotted versus the measured pH to determine two inflectionpoints that correspond to the excess HCl added volume andto the protonated CS volume respectively The differencebetween these two volumes corresponds to the amount ofHCl consumed for the protonation of the amine groups of thechitosan which allows the determination of the CS degree ofdeacetylation with the following formula

DA = 203 lowast ( 1198812minus 1198811119898 + 00042 (1198812minus 1198811)) (1)

Here119898 is themass of the sample1198811and119881

2correspond to the

volumes of NaOH solution where the inflection points werelocated 203 is the coefficient resulting from themolarmassesof the units of the CS monomer and 00042 is the differencebetween the molar mass of the units of the monomers ofquinine and CS The calculated degree of deacetylation was68

The viscosity-average molar mass (119872V) of the chi-tosan was determined in an Anton-Paar automatic Ubbe-lohde microviscosimeter For these measurements differentamounts of CS were dissolved in 50mL of a 02M NaCldissolved in 01M of acetic acid aqueous solution withagitation for 24 h Then samples were put in a constant-temperature water bath at 25∘C and their shear viscosity wasmeasured at the same temperature in the microviscometerFrom the measured viscosity of the different samples theintrinsic viscosity [120578] was determined in the usual way Theviscosity-average molar mass was then calculated using theMark-Houwkins equation given by

[120578] = 119870119872119886V (2)

with the Mark-Houwkins constants K = 18 times 10minus5 dLg anda = 093 reported by Roberts and Domszy [21] The obtainedchitosan119872V was 12 times 106 gmol

For the synthesis of the PNIPACS coreshell nanopar-ticles the following procedure was followed 2 g of CS wasmixed with 160 g of distilled water containing 07 g of aceticacid whichwas added to a 500mL three-month glass reactorthis mixture was maintained under constant stirring at roomtemperature for 12 h Then the reactor was immersed in awater bathmaintained at 70∘C next 0684 g of KPS dissolvedin 10 g of water was added to the reactor which was thenbubbled with N

2to remove the dissolved oxygen Then a

solution of 2062 g of NIPA and 0839 g of NMBA dissolvedin 349 g of water was fed at a controlled rate to the reactorduring 233min After this time the reaction was allowed tocontinue for 1 h to increase the concentration of producedPNIPA The latex was cooled down to 40∘C followed by the

Journal of Nanomaterials 3

addition of GA solution to crosslink the CS shell Sampleswere collected at given times to follow the particle sizeevolution with reaction time The final latex cooled to 25∘Cwas collected and put in a dialysis bag (119872

119899= 11200 gmol

exclusion size) which was immersed in a large amount ofdistilled water to eliminate the residual monomers initiatorandGA that had not reactedThis dialyzed sample was placedin a teflon box and put in a convection oven at 60∘C to dryuntil constant weight was achieved The sample was weighedand the final conversion was estimated with the followingformula [15]

119883 = 119882DRY minus119882CS minus119882GA119882NIPA +119882NMBA (3)

Here119882DRY119882CS119882GA119882NIPA and119882NMBA are the masses ofthe dry sample chitosan glutaraldehyde NIPA and NMBArespectively

For the drug loading in the PNIPACS nanoparticles02012 g of nanoparticles was dispersed in 80 g of watercontaining 0021 g of doxycycline (mother solution) whichwas maintained with constant agitation at pH of 45 and 25∘Cat room temperature during 24 h After incubation for 96 hthe dispersion solution was adjusted to pH of 9 and the drug-loaded nanoparticles were separated from the dispersion bycentrifugation at 13500 rpm for 5min Then the nanoparti-cles were dried by lyophilization andweighed To estimate theamount of drug loaded into the nanoparticles a calibrationcurve of absorbance versus doxycycline concentration wasmade by diluting the mother solution at pH = 9 Each of thediluted samples was put in a quartz cell which was placed in aThermoelectronUV-vis spectrophotometer (Genesis 10 UV)to measure the absorbance at the wavelength of 346 nm Theobtained calibration curve was a straight line with R-squarestandard deviation of 09995 To obtain the drug liberationprofiles 040 g of the drug-loaded nanoparticles was mixedwith 5mL of a 90wtwvNaCl aqueous solution and loadedin a dialysis bag of 1000 Dalton molecular mass cut-offThe dialysis bag was then immersed in 195mL of normal-saline solution The drug release profile was determined attwo different temperatures (25 and 37∘C) and two pHs (2 and7)

Particle size of the nanocomposites was measured with aNanosize S-90 quasielastic light scattering (QLS) apparatusfrom Malvern Instrument at 25∘C For the measurements005 g of the latices obtained at different reaction times wasdispersed in 10 g of water from this dispersion 3mL was putin a glass cell for the measurements

The morphology shape and size distribution of thenanocomposites was examined with a TESCAM Field-Emission Scanning ElectronMicroscope (FESEM) For thesetests 005 g of the nanoparticles was dispersed in 200 g ofwater 002 g of this dispersion was deposited in a Cu plaquefrozen to minus60∘C for 24 h and lyophilized at minus40∘C and 1 times10minus4 bar for 2 hThen the sample was covered with a thin goldlayer and analyzed in the FESEM

The PNIPACS nanoparticles were also examined in aJEOL 1200 EXII transmission electron microscope (TEM)For this one drop of latex diluted 20 times was deposited in

210

240

270

300

330

360

0 40 80 120 160 200 240

0 3 6 9 12 15 18 21

t (min)

DPZ

(nm

)

g NIPAm

Figure 1 Evolution of particle size as a function of time and thedosed amount of NIPA

a grid which was dried overnight before observation in theTEM

For the thermoreversibility and pH- and thermoresponsetests of the nanocomposites 05 g of the dialyzed and driednanoparticles were redispersed in 20 g of buffer solutions ofpHs = 2 4 and 7 Samples from these dispersions were placedin the glass cell for QLS measurements The temperaturerange was from 20 to 50∘C with increments of 5∘C for thetemperature-response tests the equilibration time was of15min between temperature increments to allow samplestabilization Measurements were made three times for eachsample These temperature response tests were performed atthree pHs (2 4 and 7)

The thermoreversibility of the samples was examinedusing increasing-and-decreasing temperature cycles from 20to 50∘C as a measure of the stability Measurements weremade by QLS allowing 15min of equilibration at the end ofeach temperature increase or decrease Again these tests wereperformed at pHs of 2 4 and 7

3 Results

Figure 1 depicts the evolution of particle size in the presenceof the CS micelles as a function of time and the dosedamount of NIPA Initially before the NIPA addition the CSmicelles have an average QLS size of ca 200 nm which issimilar to that reported by Huang et al [15] as expectedClearly particle size increased with increasing amount ofNIPA added indicating that PNIPA was produced and thatit was incorporated into the micellar core because at thereaction temperature (70∘C) this polymer is hydrophobic[14] and hence it enters the CS micelle hydrophobic coreFinal particle size was ca 360 nm but it increased to 380 plusmn6 nm after the crosslinking of the CS micelles with GA

Figure 2 shows a FESEM micrograph of the finalnanocomposites This micrograph shows polydispersedspheroidal particles with sizes of ca 200 to 450 nm This


Figure 2 FESEMmicrograph of the final nanocomposites

Figure 3 TEM micrograph of the obtained PNIPACS coreshellnanoparticles

size polydispersity could be due to (i) aggregation duringthe polymerization of PNIPA (ii) the treatment of thenanoparticles for FESEM examination which could haveintroduced agglomeration of the originally synthesizednanoparticles and (iii) some PNIPA nanoparticles which didnot penetrate the micelles that is unlikely moreover PNIPAnanoparticles produced by semicontinuous heterophasepolymerization (SHP) are much smaller on the order of30 nm as documented elsewhere [22]

Figure 3 depicts a TEM micrograph of the crosslinkedPNIPACS nanoparticles This micrograph reveals that thePNIPACS nanoparticles are spheroidal with average size ofca 300 nm Moreover there is an obscure contour aroundeach nanoparticle which is due to the crosslinkedCSmicellarwall surrounding the PNIPA nanoparticles that confirmedthe formation of coreshell nanostructures Moreover nosmall particles (lt50 nm) which might have formed due tothe presence of PNIPA nanoparticles are observed indicatingthat all the produced PNIPA entered into the CS micelles

100 200 300 400 500 600 800700

DPZ (nm)

0

10

20

30

[I]

Figure 4 QLS-intensity size distribution of the reacting particles atthree different reaction times that is 60min (◼) 160min (e) andat the end of the reaction (998771)

200

300

400

500

15 20 25 30 35 40 45 50

T (∘C)

DPZ

(nm

)

Figure 5 Particle size as a function of temperaturemeasured at pHsof 2 (◼) 4 (e) and 7 (998771)

Figure 4 shows QLS-intensity size distribution of thereacting particles at three different reaction times that is60min 160min and at the end of the reaction Clearly sizedistributions shift to larger sizes as the reaction proceedsin agreement with the average sizes shown in Figure 1Moreover particles are polydispersed with sizes similar tothose observed in the micrograph shown in Figure 1

Figure 5 depicts plots of particle size as a function of tem-perature measured at three different pHs The three reportedcurves exhibit an abrupt decrease in size with increasing thetemperature from 20 to ca 34∘C and then the size reductionis substantially smaller Notice that the inflection point (ca34∘C) coincides with 119879VPT of the crosslinked PNIPA at thethree examined pHs as expected since above 119879VPT PNIPAbecomes hydrophobic and then it tends to expel the watertrapped inside it Notice that pH has a smaller but detectableeffect in the swelling behavior of the nanocomposites beinglarger at pH= 2 and smallest at pH= 7 Aswewill discuss later


200

300

400

500

25 25 25 2550 50 50

T (∘C)

DPZ

(nm

)

Figure 6 Thermoreversibility tests at the pHs of 2 (◼) 4 (e) and 7(998771)

0

20

40

60

80

0 10 20 30 40 50

t (h)

F

rele

ased

Figure 7 Drug liberation profiles measured at 25 (open symbols)and 37∘C (closed symbols) at pHs of 2 (◻ ◼) and 74 (I e)

this is related to the interaction of CS and PNIPA groups withH+ andOHminus groups at the different pHs and to the isoelectricpoint (pH = 63) of CS [23]

Figure 6 reports the thermoreversibility tests at the threepH used in this paper The nanoparticles were subjectedto heating-cooling-heating cycles as a function of pH todemonstrate whether or not the particles recovered theiroriginal sizes after the conclusion of the cycles In all casessamples recovered their sizes at all the three pHs examinedbut now larger changes in size are detected at the more acidpH these changes in sizes decreased as the pHwas increased

Figure 7 shows drug liberation profiles measured at 25and 37∘C at pHs of 2 and 7 The drug liberation profilesat both pHs increased rapidly during the first ca 6 h andthen they rose more slowly but they kept raising even after50 h of testing Drug liberation is larger at pH = 2 comparedto that at pH = 74 at both temperatures However drugrelease is larger at 25∘C compared to those at 37∘C whichseems contradictory with the fact that the PNIPA volume

phase transition temperature is ca 34∘C and then it would beexpected that the drug release would be larger at 37∘CNoticehowever that drug release is fast for both temperaturesand pHs during the first 10 hrs and then it diminishessubstantially

4 Discussion and Conclusions

Semicontinuous heterophase polymerization (SHP) is a vari-ation of the semicontinuous microemulsion polymerization(SMP) which allows the synthesis of polymer nanoparticlesand nanocomposites smaller than 50 nm with narrow sizedistributions employing smaller amounts of surfactant andlarger polymer-to-surfactant ratios compared to the SMP[24ndash28] In this process and in contrast to the SMP neatmonomer is dosed at a controlled rate to an aqueous (or oleic)solution containing surfactant forming micelles initiatorand a solvent-soluble initiator An important feature of theSHP is that narrower particle size distributions with smallermolar masses compared to SMP are obtained The SHPprocess has been proved successfully to synthesize a seriesof poly(alkyl methacrylates) that have anomalously higherglass transition temperatures indicating a larger syndiotacticpolymer content [24ndash28] semiconducting polypyrrole [29]mesoporous nanoparticles [30] and polymeric nanocompos-ites [31]

Here we report the synthesis of PNIPACS coreshellnanoparticles by SHP determine their 119879VPT examine theirthermoreversibility observe their shape and size distributionby FESEM and TEM and perform drug liberation testswith a model drug doxycycline hyclate as a function oftemperature and pH First following the report of Huanget al [15] CS micelles were produced in a dilute acetic acidsolution over which NIPA was dosed at a controlled rateand polymerized at 70∘C to induce the formation of PNIPAand the subsequent entrapment in the CS micellar core sincethe reaction temperature (70∘C) was higher than 119879VPT ofPNIPA and hence the formed polymer molecules shouldmigrate inside the micellar core The growth of the initiallyformed CS micelles which have a diameter of ca 200 nmincreased with NIPA addition time as observed in Figure 1Particles grew from ca 200 nm to ca 360 nm suggesting theincorporation of the formed PNIPA into the micellar coreQLS also show that the particles are polydispersed and growwith NIPA addition time (Figure 3) which indicates thatmost or all of the PNIPA formedwas incorporated into theCSmicellar core TEMwas used to determine the size and shapeof the produced nanoparticles and it showed the presenceof spheroidal nanoparticles (Figure 3) with sizes similar tothose obtained byQLSMoreover TEMrevealed the presenceof a dark skin over the nanoparticles which might be dueto the thickness of the CS micelles around the PNIPAnanoparticles QLS also showed that the polydispersed singlepopulation coreshell nanoparticles increased with reactiontime (Figure 4)

To examine the response of temperature as a functionof pH the nanocomposites sizes were measured by QLS atdifferent pHs Results shown in Figure 5 demonstrate thatthe PNPACS coreshell nanoparticles shrink rapidly with


increasing temperature up to ca 34∘C which correspondto the hydrophilic-hydrophobic transition of PNIPA (119879VPT)at which PNIPA collapses The gradual size decrease of thenanoparticles is hence related to the expulsion of water bythe PNIPA core as the temperature is increased For temper-ature larger than 34∘C the size decrease of the nanoparticle issmaller since the remaining water in the particles continuesto be expelled but in much smaller amounts Notice that pHplays a role in the shrinking behavior of the nanocompositesbeing larger at the more acid pH and then diminishing as thepH increases Here the main role is played by the CS shellThis natural polymer has an ionization potential (IP) of 63[23] At pH values higher than this value CS forms a gel-like structure that is insoluble and it reduces the water releasewhereas at pH below its IP water can be released because ofthe protonation of the amino groups ofCS producing a softermore labile structure An important feature of the PNIPACSnanoparticles is that they exhibit thermoreversibility afterfour consecutive heating-cooling-heating cycles (Figure 6)indicating that the nanoparticles structure is maintained andthat they are thermoreversible that is they recover theiroriginal size and structure

The PNIPACS coreshell nanoparticles made here bySHP were subjected to drug delivery tests to determine theirpotential as nanocarriers As demonstrated in Figure 7 thePNIPACS nanoparticles were able to dose the model drugdoxycycline in prolonged periods of time As expected dueto the sensitivity of the CS shell to pH drug release is largerat pH= 2 than at pH = 7 regardless of the test temperatureDrug release is fast at early releasing tests and then becomesslower after ca 12 hrs for both temperatures and pHs After50 hrs approximately the 55 of the drug had been releasedat pH = 2 and at 37∘C and ca 38 at pH = 7 at 37∘C Noticehowever that drug keeps being released even after 50 hrsindicating that the PNIPACS coreshell nanoparticles exhibitlong releasing activity Moreover temperature also has animportant effect on the drug release profiles As shown inFigure 7 drug release is larger at 25∘C than at 37∘C whichis contrary to our expectation inasmuch as PNIPA collapsesat ca 34∘C However Huang et al [15] proposed that due tothe collapsing of the PNIPA at temperatures larger than 34∘Cthe remaining drug molecules get trapped into the shrinkingPNIPA core because some of the drug molecules had someinteractions with PNIPA These results suggest that somedrug molecules get trapped in the coreshell nanoparticlesinside the PNIPA nanocore even above the collapsing PNIPAtemperature and that below the119879VPT the drugmolecules areable to diffuse faster through the swollen PNIPA core and theCS shell

To determine the diffusion mechanism of the liberateddrug several models were employed mainly those of orderzero first order Higughi and Korsmeyer-Peppas [32] Ourresults (not shown) indicate that the Korsmeyer-Peppasmodel is the one that fits better the drug release at earlierreleasing times which indicate that the diffusion is non-Fickian This is to be expected inasmuch as the drug has todiffuse first from the PNIPA core to theCS shell before exitingthe nanoparticles Similarmechanisms has been proposed forthe drug release from a polymeric matrix

The comparison with Huang et al [15] clearly demon-strates that larger concentrations of drug are released insimilar times in our work 471 compared to 38 in Huanget al after 7 hrs of dosing at 37∘C which is the temperature ofinterest for mammals drug release applications Clearly thedifferences in the drug releasing times could be explained interms of the PNIPA molecular mass and internal structureevidently affected by the polymerization method [33] Huanget al added the whole amount of NIPA over the CS micellarsolutions which were at 70∘C hence one should expecta NIPA solution polymerization mechanism which shouldproduce low molecular weights and loose porous-structuredparticles [33] However the PNIPA produced by SHP haslargermolecular weight andmore compact crosslinked struc-ture as shown elsewhere [22] Hence the release of trappedsubstance within these two different structures should bequite different as is the case here

In conclusion we have reported here the synthesis by SHPof PNIPACS coreshell nanoparticles which when swollenwith water exhibit a fast size decrease at ca 34∘C due to therapid expulsion of water which is associated with 119879VPT ofPNIPA at higher temperature size also diminishes but lessrapidly with increasing temperature The nanoparticles arealso pH-responsive specially at acid pHs which is relatedto the transition from a soluble-to-insoluble gel structure ofthe CS These nanoparticles were thermoreversible that isthey recovered their size and shape after being submittedto heating-and-cooling cycles in the range of 25 to 50∘CQLS FESEM and TEM reveal that the nanoparticles arespheroidal and polydispersed Liberation studies of a modeldrug were performed that demonstrate that the PNIPACSnanoparticles exhibit a long-release behavior which makesthem suitable as drug releasing carriers

Competing Interests

The authors declare that they have no competing interests

References

[1] R Langer and D A Tirrell ldquoDesigning materials for biologyand medicinerdquo Nature vol 428 no 6982 pp 487ndash492 2004

[2] F Alexis E Pridgen L K Molnar and O C Farokhzad ldquoFac-tors affecting the clearance and biodistribution of polymericnanoparticlesrdquoMolecular Pharmaceutics vol 5 no 4 pp 505ndash515 2008

[3] Z Liu Y Jiao Y Wang C Zhou and Z Zhang ldquoPoly-saccharides-based nanoparticles as drug delivery systemsrdquoAdvanced Drug Delivery Reviews vol 60 no 15 pp 1650ndash16622008

[4] N S Rejinold P R Sreerekha K P Chennazhi S V Nairand R Jayakumar ldquoBiocompatible biodegradable and thermo-sensitive chitosan-g-poly (N-isopropylacrylamide) nanocarrierfor curcumin drug deliveryrdquo International Journal of BiologicalMacromolecules vol 49 no 2 pp 161ndash172 2011

[5] AD Sezer and E Cevher ldquoTopical drug delivery using chitosannano- and micro-particlesrdquo Expert Opinion on Drug Deliveryvol 9 no 9 pp 1129ndash1146 2012


[6] G Berth A Voigt H Dautzenberg E Donath and HMohwald ldquoPolyelectrolyte complexes and layer-by-layer cap-sules from chitosanchitosan sulfaterdquo Biomacromolecules vol 3no 3 pp 579ndash590 2002

[7] Y Hu X Jiang Y Ding H Ge Y Yuan and C YangldquoSynthesis and characterization of chitosan-poly(acrylic acid)nanoparticlesrdquo Biomaterials vol 23 no 15 pp 3193ndash3201 2002

[8] Y Hu Y Ding D Ding et al ldquoHollow chitosanpoly(acrylicacid) nanospheres as drug carriersrdquo Biomacromolecules vol 8no 4 pp 1069ndash1076 2007

[9] C Choi J-P Nam and J-W Nah ldquoApplication of chitosan andchitosan derivatives as biomaterialsrdquo Journal of Industrial andEngineering Chemistry vol 33 pp 1ndash10 2016

[10] P K Dutta Ed Chitin and Chitosan for Regenerative MedicineSpringer Series of Polymer and Composite Materials SpringerNew York NY USA 2016

[11] A Yamada Y Hiruta J Wang E Ayano and H KanazawaldquoDesign of environmentally responsive fluorescent polymerprobes for cellular imagingrdquo Biomacromolecules vol 16 no 8pp 2356ndash2362 2015

[12] Y Isikver and D Saraydin ldquoEnvironmentally sensitive hydro-gels N-isopropylacrylamideacrylamidemono- di- tricar-bixylic acid crosslinked polymersrdquo Polymer Engineering andScience vol 55 no 4 pp 843ndash851 2014

[13] F Sun Y Wang Y Wei G Cheng and G Ma ldquoThermo-triggered drug delivery from polymeric micelles ofpoly(N-isopropylacrylamide-co-acrylamide)-b-poly(n-butylmethacrylate) for tumor targetingrdquo Journal of Bioactive andCompatible Polymers vol 29 no 4 pp 301ndash317 2014

[14] H G Schild ldquoPoly(N-isopropylacrylamide) experiment the-ory and applicationrdquo Progress in Polymer Science vol 17 no 2pp 163ndash249 1992

[15] C-H Huang C-F Wang T-M Don and W-Y Chiu ldquoPrepa-ration of pH- and thermo-sensitive chitosan-PNIPAAm corendashshell nanoparticles and evaluation as drug carriersrdquo Cellulosevol 20 no 4 pp 1791ndash1805 2013

[16] Y Wang H Xu L Ge and J Zhu ldquoDevelopment of a ther-mally responsive nanogel based on chitosan-poly(N-isopropyl-co-acrylamide) for paclitaxel deliveryrdquo Pharmaceutics DrugDelivery andPharmaceutical Technology vol 103 pp 2012ndash20212014

[17] A Popat J Liu G Q Lu and S Z Qiao ldquoA pH-responsive drugdelivery system based on chitosan coated mesoporous silicananoparticlesrdquo Journal of Materials Chemistry vol 22 no 22pp 11173ndash11178 2012

[18] T Chang P Gosain M H Stenzel and M S LordldquoDrug loading of poly[(polyethylene glycol methylether methacrylate)-block-poly(methyl methacrylate)](PEGMEMA)-based micelles and mechanisms of uptake incolon carcinoma cellsrdquo Colloid and Surfaces BmdashBiointerfacesvol 144 pp 257ndash264 2016

[19] J Li D Liu G Tan Z Zhao X Yang and W Pan ldquoA com-parative study on the efficiency of chitosan-N-acetylcysteinechitosan oligosaccharides or carboxymethyl chitosan surfacemodified nanostructured lipid carrier for ophthalmic deliveryof curcuminrdquo Carbohydrate Polymers vol 146 pp 435ndash4442016

[20] R Onnainty B Onida P Paez M Longhi A Barresi and GGranero ldquoTargeted chitosan-based bionanocomposites for con-trolled oral mucosal delivery of chlorhexidinerdquo InternationalJournal of Pharmaceutics vol 509 no 1-2 pp 408ndash418 2016

[21] G A F Roberts and J G Domszy ldquoDetermination of theviscometric constants for chitosanrdquo International Journal ofBiological Macromolecules vol 4 no 6 pp 374ndash377 1982

[22] J Aguilar F Moscoso O Rios et al ldquoSwelling behavior ofpoly(N-isopropylacrylamide) nanogels with narrow size distri-butionmade by semi-continuous inverse heterophase polymer-izationrdquo Journal of Macromolecular Science Part A Pure andApplied Chemistry vol 51 no 5 pp 412ndash419 2014

[23] T Lopez-Leon E L S Carvalho B Seijo J L Ortega-Vinuesaand D Bastos-Gonzalez ldquoPhysicochemical characterization ofchitosan nanoparticles electrokinetic and stability behaviorrdquoJournal of Colloid and Interface Science vol 283 no 2 pp 344ndash351 2005

[24] M G Perez-Garcıa M Rabelero S M Nuno-Donlucaset al ldquoSemicontinuous heterophase polymerization of butylmethacrylate effect ofmonomer feeding raterdquo Journal ofMacro-molecular Science Part A Pure and Applied Chemistry vol 49no 7 pp 539ndash546 2012

[25] M G Perez-Garcıa E V Torres I Ceja et al ldquoSemicontinuousheterophase copolymerization of styrene and acrylonitrilerdquoJournal of Polymer Science A Polymer Chemistry vol 50 no 16pp 3332ndash3339 2012

[26] M G Perez-Garcıa A G Alvarado M Rabelero et al ldquoSemi-continuous heterophase polymerization of methyl and hexylmethacrylates to produce latexes with high nanoparticles con-tentrdquo Journal of Macromolecular Science A Pure and AppliedChemistry vol 51 no 2 pp 144ndash155 2014

[27] M G Perez Garcıa A G Alvarado L A Perez-Carrilloet al ldquoOn the modeling of the semicontinuous heterophasepolymerization of alkyl methacrylates with different watersolubilitiesrdquoMacromolecular Reaction Engineering vol 9 no 2pp 114ndash124 2015

[28] H Saade C Barrera G Guerrero EMendizabal J E Puig andR G Lopez ldquoPreparation and loaded with rifampicin of sub-50 nmpoly(ethyl cyanoacrylate)rdquo Journal of Nanomaterials vol2016 Article ID 384973 11 pages 2016

[29] J C Gonzalez-Iniguez V M Ovando-Medina C F Jasso-Gastinel D AGonzalez J E Puig and EMendizabal ldquoSynthe-sis of polypyrrole nanoparticles by batch and semicontinuousheterophase polymerizationsrdquo Colloid and Polymer Science vol292 no 6 pp 1269ndash1275 2014

[30] O Esquivel M E Trevino H Saade J E Puig E Mendizabaland R G Lopez ldquoMesoporous polystyrene nanoparticlessynthesized by semicontinuous heterophase polymerizationrdquoPolymer Bulletin vol 67 no 2 pp 217ndash226 2011

[31] J E Puig and M Rabelero ldquoSemicontinuous microemulsionpolymerizationrdquo Current Opinion in Colloid and Interface Sci-ence vol 25 pp 83ndash88 2016

[32] G Singhvi and M Sing ldquoReview in-vitro drug release char-acterization modelsrdquo International Journal of PharmaceuticalStudies and Research vol 2 no 1 pp 77ndash84 2011

[33] G Odian Principles of Polymerization Wiley Interscience NewYork NY USA 2004

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transition from a hydrophilic to a hydrophobic structure atthis temperature [14]

Huang et al reported in 2013 that chitosan was ableto form micelles in an acetic acid solution even thoughthis polysaccharide is insoluble in pure water these authorsdemonstrated the micelle formation and reported the crit-ical micellar concentration by fluorescence emission spec-troscopy [15] In addition Huang et al showed in the samearticle that by adding NIPA in one shot and an initiator(potassium persulfate) at 70∘C the NIPA polymerized andbecause the reaction temperature was higher than the LCSTof the PNIPA it became hydrophobic and as a consequencemigrated to the micellar core To ensure that the PNIPACSmicelles would not be disrupted in further applications theseauthors added an aqueous solution of glutaric dialdehyde tocrosslink themicellesmoreover they demonstrated the capa-bility for prolonged drug delivery of this coreshell PNIPACSmicellar solutionWang et al synthesized coreshell nanoma-terials through radical polymerization of CS and a mixtureof NIPA and acrylamide (AM) which exhibited thermallysensitive features and that allowed to tune the volume phasetransition temperature (119879VPT) up to collapsing temperaturesof ca 39∘C depending on the AMNIPA molar ratio [16]

Popat et al [17] reported a CS-coated mesoporous silicananospheres that were pH-responsive and used ibuprofenas model drug they demonstrated that under basic con-ditions the coreshell nanomaterial forms a gel structurethat is insoluble and prevents the drug release whereasbelow the CS isoelectric point (pH = 63) the drug isreleased due to the protonation of the CS amino groupsChang et al [18] produced poly[poly(ethylene glycol methylether methacrylate)-block-poly(methyl methacrylate)] blockcopolymer to encapsulate the model drug Nile Red anda chemotherapeutic drug doxorubicin (DOX) and showedthat the release of DOX from the micelles was sufficient tobe cytotoxic to human colon carcinoma cells Li et al [19]developed a potential nanocarrier for topical administrationof curcumin (CUR) a novel thiolated CS made by covalentbinding between N-acetyl-L-cysteine (NAC) and CS whichwas employed to modify the surface of the nanostructuredlipid carrier CUR Onnainty et al [20] created sustainedrelease systems of the drug chlorhexidine (CLX) based onCS and montmorillonite These hybrid carriers exhibitedsustained release ofCLXwhich allowedmaintaining theCLXantimicrobial properties

In this study we synthesized pH- and thermoresponsiveNIPACS coreshell nanoparticles by semicontinuous het-erophase polymerization (SHP) The aim was to synthesizethermo- and pH-sensitive nanoparticles with larger polymercontent than those reported by Huang et al [15] and evaluatetheir thermoreversibility and their potential application asdrug carrier and delivery systems as a function of pH andtemperature

2 Experimental Section

CS AM (99 pure) NMBA (99 pure) glutaraldehyde((GA) 50wt in water) and doxycycline (98 pure) wereobtained from Sigma-Aldrich NIPA was 99 pure from

Acros Organics KPS (99 pure) was from Fermont N2gas

was from Infra de Occidente (Mexico) and doubly distilledwater was from Productos Selectropura (Mexico)

The CS degree of deacetylation was determined poten-tiometrically with the following procedure first 04 g of CSwas dissolved in 89 g of a 01M HCl solution by constantstirring during 24 h next this solution was titrated with01393M NaOH solution with a pH-meter The volume ofthe NaOH solution added to the chitosan solution wasplotted versus the measured pH to determine two inflectionpoints that correspond to the excess HCl added volume andto the protonated CS volume respectively The differencebetween these two volumes corresponds to the amount ofHCl consumed for the protonation of the amine groups of thechitosan which allows the determination of the CS degree ofdeacetylation with the following formula

DA = 203 lowast ( 1198812minus 1198811119898 + 00042 (1198812minus 1198811)) (1)

Here119898 is themass of the sample1198811and119881

2correspond to the

volumes of NaOH solution where the inflection points werelocated 203 is the coefficient resulting from themolarmassesof the units of the CS monomer and 00042 is the differencebetween the molar mass of the units of the monomers ofquinine and CS The calculated degree of deacetylation was68

The viscosity-average molar mass (119872V) of the chi-tosan was determined in an Anton-Paar automatic Ubbe-lohde microviscosimeter For these measurements differentamounts of CS were dissolved in 50mL of a 02M NaCldissolved in 01M of acetic acid aqueous solution withagitation for 24 h Then samples were put in a constant-temperature water bath at 25∘C and their shear viscosity wasmeasured at the same temperature in the microviscometerFrom the measured viscosity of the different samples theintrinsic viscosity [120578] was determined in the usual way Theviscosity-average molar mass was then calculated using theMark-Houwkins equation given by

[120578] = 119870119872119886V (2)

with the Mark-Houwkins constants K = 18 times 10minus5 dLg anda = 093 reported by Roberts and Domszy [21] The obtainedchitosan119872V was 12 times 106 gmol

For the synthesis of the PNIPACS coreshell nanopar-ticles the following procedure was followed 2 g of CS wasmixed with 160 g of distilled water containing 07 g of aceticacid whichwas added to a 500mL three-month glass reactorthis mixture was maintained under constant stirring at roomtemperature for 12 h Then the reactor was immersed in awater bathmaintained at 70∘C next 0684 g of KPS dissolvedin 10 g of water was added to the reactor which was thenbubbled with N

2to remove the dissolved oxygen Then a

solution of 2062 g of NIPA and 0839 g of NMBA dissolvedin 349 g of water was fed at a controlled rate to the reactorduring 233min After this time the reaction was allowed tocontinue for 1 h to increase the concentration of producedPNIPA The latex was cooled down to 40∘C followed by the



119899= 11200 gmol








210

240

270

300

330

360

0 40 80 120 160 200 240

0 3 6 9 12 15 18 21

t (min)

DPZ

(nm

)

g NIPAm





3 Results








100 200 300 400 500 600 800700

DPZ (nm)

0

10

20

30

[I]


200

300

400

500

15 20 25 30 35 40 45 50

T (∘C)

DPZ

(nm

)





200

300

400

500

25 25 25 2550 50 50

T (∘C)

DPZ

(nm

)


0

20

40

60

80

0 10 20 30 40 50

t (h)

F

rele

ased
















Competing Interests


References










































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





119899= 11200 gmol








210

240

270

300

330

360

0 40 80 120 160 200 240

0 3 6 9 12 15 18 21

t (min)

DPZ

(nm

)

g NIPAm





3 Results








100 200 300 400 500 600 800700

DPZ (nm)

0

10

20

30

[I]


200

300

400

500

15 20 25 30 35 40 45 50

T (∘C)

DPZ

(nm

)





200

300

400

500

25 25 25 2550 50 50

T (∘C)

DPZ

(nm

)


0

20

40

60

80

0 10 20 30 40 50

t (h)

F

rele

ased
















Competing Interests


References










































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








100 200 300 400 500 600 800700

DPZ (nm)

0

10

20

30

[I]


200

300

400

500

15 20 25 30 35 40 45 50

T (∘C)

DPZ

(nm

)





200

300

400

500

25 25 25 2550 50 50

T (∘C)

DPZ

(nm

)


0

20

40

60

80

0 10 20 30 40 50

t (h)

F

rele

ased
















Competing Interests


References










































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




200

300

400

500

25 25 25 2550 50 50

T (∘C)

DPZ

(nm

)


0

20

40

60

80

0 10 20 30 40 50

t (h)

F

rele

ased
















Competing Interests


References










































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









Competing Interests


References










































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Documents

Synthesis, Characterization, and Drug Delivery from pH