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International Journal of Biological Macromolecules 96 (2017) 589–599

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

reparation and characterization of oxidizedtarch/poly(N,N-dimethylaminoethyl methacrylate) semi-IPNryogels and in vitro controlled release evaluation of indomethacin

iana Felicia Apopei Loghin, Gabriela Biliuta, Sergiu Coseri, Ecaterina Stela Dragan ∗

Petru Poni” Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley 41A, Iasi 700487, Romania

r t i c l e i n f o

rticle history:eceived 15 November 2016eceived in revised form 6 December 2016ccepted 28 December 2016vailable online 29 December 2016

eywords:ryogels

a b s t r a c t

Fabrication of novel semi-interpenetrating polymer network (semi-IPN) cryogels by cross-linking poly-merization of N,N-dimethylaminoethyl methacrylate (DMAEM) in the presence of either oxidized potatostarch (OPS) or oxidized wheat starch (OWS) and their characterization are presented in the paper. Theinfluence of the nature of entrapped polymer on the properties of the composite cryogels was evaluatedby the swelling kinetics, FT-IR spectroscopy, scanning electron microscopy, and response at external stim-uli such as temperature, pH, and ionic strength. Indomethacin (IDM), taken as a model anti-inflammatorydrug, was easily loaded into the composite cryogels by the solvent sorption-evaporation strategy. The

ndomethacinolysaccharidestimuli-sensitive polymersrug delivery systems

in vitro release of IDM from the semi-IPN cryogels was low in simulated gastric fluid at pH 1.3, irrespectiveof the nature of the entrapped oxidized starch, and consistent in simulated intestinal fluid (SIF) at pH7.4, the influence of the entrapped polysaccharide being evident. The release mechanism of IDM fromthe composite cryogels was discussed based on two kinetic models, finding that the drug release at 37 ◦Cwas pseudo-Fickian diffusion, regardless the cryogel composition.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

The hydrogels obtained from hydrophilic synthetic and/oratural polymers [1,2] that swell and shrink in response to environ-ental stimuli such as temperature [3], pH [4], and ionic strength

5] have attracted much of attention during the past decades,ue to their potential applications in numerous fields including:ood additives, pharmaceuticals, cell culture, biomedical implants,nd wastewaters treatment [6–8]. Polysaccharide based compositeydrogels are especially attractive for their applications in con-rolled drug release [9–12]. The low-cost, abundant supply, goodrocessability, biodegradability, and ease of chemical modifica-ions make starch one of the most promising raw materials forhe synthesis of novel composite hydrogels [4,6,13–16]. Stimuli-esponsive composite hydrogels, having native [17,18] or modifiedtarch [4,9] entrapped in a matrix of a synthetic polymer, haveeen successfully used in the preparation of drug delivery sys-

ems (DDSs). Among the composite hydrogels, interpenetratingolymer networks (IPN), endowed with controlled morphologynd increased levels of functionalities, have been reported last

∗ Corresponding author.E-mail address: sdragan@icmpp.ro (E.S. Dragan).

ttp://dx.doi.org/10.1016/j.ijbiomac.2016.12.071141-8130/© 2016 Elsevier B.V. All rights reserved.

decade [19–23]. The morphology of hydrogels, as well as the drugproperties such as solubility, size and the interactions betweenthe drug and the hydrogel chains should be taken under con-sideration when DDSs are of interest. However, in the case ofconventional hydrogels, because of the high swelling ratio, slowresponse at swelling/reswelling and poor mechanical strength,a burst release of drugs is usually reported. To overcome thisdrawback, macroporous composite hydrogels based on polysac-charides entrapped in stimuli responsive synthetic polymers asmatrix have been designed [21,23,24]. Macroporous semi-IPNhydrogels have been recently prepared in our group by entrappingnative potato starch (PS) or anionically modified PS, synthe-sized by the saponification of polyacrylonitrile grafted on PS, in amatrix of poly(N,N-dimethylaminoethyl methacrylate) (PDMAEM)[25]. PDMAEM has been used as a matrix because it undergoescontrollable volume changes in response to small variation ofpH, temperature and ionic strength [26,27], property which rec-ommends it for the preparation of novel DDSs. Among varioustechniques available for the generation of pores in hydrogels, cryo-gelation has been used because is a facile and green technique,

where the ice crystals act as porogen [28–30].

It is well established that the hydroxyl groups of starchcan be subjected to various chemical reactions, such as oxida-tion, esterification, etherification, and grafting [31,32]. A way of

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s(wcwohpocar(ewisptpoi

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90 D.F. Apopei Loghin et al. / International Journ

hemical modification of hydroxyl groups into carboxyl and/orarbonyl functional groups is oxidation, the water-soluble sta-le nitroxyl radical 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO)eing widely used for selective oxidation of the primary OH groupsf polysaccharides [33–37].

To extend the application range of the macroporous compositeemi-IPNs based on starch as raw material, oxidized potato starchOPS) and oxidized wheat starch (OWS) were synthesized in thisork and entrapped in a matrix of PDMAEM, the synthesis of the

omposite gels being conducted under the freezing temperature ofater (−18 ◦C). It is hypothesized that the nature and the content

f oxidized starch obtained by TEMPO oxidation, which ensures aighly selective and uniform distribution of the –COOH groups atrimary C6 in the starch backbone, will influence the propertiesf the composite gels such as the pore size, the response of theomposite cryogels at the external stimuli including pH, temper-ture, and ionic strength, as well as the profile of the controlledelease of drugs as a function of external stimuli. IndomethacinIDM) was chosen as a model anti-inflammatory drug with well-stablished biological action in reducing pain and inflammations,hich is almost insoluble in acidic solution (pKa 4.5) but dissolves in

ntestinal fluid. Therefore, the sorption and release upon/from theemi-IPN cryogels of IDM as model drug triggered by pH and tem-erature were evaluated in this work. To the best of our knowledge,his is the first study on the synthesis of multiresponsive macro-orous composite cryogels based on PDMAEM as a matrix andxidized starch as entrapped polysaccharide, and on their potentialn controlled release of IDM.

. Experimental

.1. Materials

Potato starch (PS), humidity content <10%, ash <0.5% andheat starch (WS), humidity content <15%, ash <0.5%, purchased

rom Fluka, were used as received. N,N-dimethylaminoethylethacrylate (DMAEM) was distilled at 47 ◦C, under

educed pressure (about 4 mmHg), and kept at 4 ◦C. N,N′-ethylenebisacrylamide (BAAm), ammonium persulfate (APS),,N,N′,N′-tetramethylethylenediamine (TEMED), IDM, TEMPO 99%,ll purchased from Sigma-Aldrich, were used as received. Otherhemicals, e.g. sodium hypochlorite (NaOCl, 8–15% chlorine),odium bromide (99% Alfa Aesar), ethanol and methanol, pur-hased from Chemical Company Romania, were used as received;aCl from Fluka, CaCl2 from Lach-ner and Na2SO4 from Reactivulucuresti, were used as received.

.2. Synthesis and purification of the oxidized starch

The introduction of negatively charged groups in starch was car-ied out as follows: 5 g of starch was dispersed in distilled water150 mL) under vigorous stirring. Separately, TEMPO (0.14 g) andaBr (0.84 g) were mixed, in 50 mL distilled water. After the com-lete dissolution of NaBr, this mixture was added drop wise to thetarch dispersion, and the flask was placed into a bath ice. Sub-equently, a volume of 100 mL solution NaOCl was added slowly,y carefully checking the pH value, which was kept as close as 10alue by adding 4 M aqueous HCl. Once the reaction started, plentyf carboxylic groups are introduced in the anhydroglucose units oftarch, leading to a sharp decrease of the pH value. The reactionH was maintained at 10 by adding 0.5 M NaOH solution. After 4 h,

he reaction was stopped by quenching the unreacted NaOCl with0 mL methanol, and then acidified to a pH value around 6.8. Theeaction products were precipitated with ethanol, the precipitateeing collected by centrifugation. The polymer was redissolved in

iological Macromolecules 96 (2017) 589–599

water, the solution was desalted and the oligomers were removedby diafiltration through a Millipore polyethersulfone ultrafiltrationmembrane (cut-off: 10,000 g cm−1) in Amicon cell equipped with atank filled with pure water (conductivity lower than 3 �S m−1). Thediafiltration was stopped when the filtrate conductivity was lowerthan 10 �S m−1 and the polymer was recovered by freeze-drying.

2.3. Preparation of the semi-IPN cryogels

Free radical cross-linking copolymerization of DMAEM andBAAm in the presence of oxidized starch (OPS and OWS) asentrapped polymer in an aqueous medium, at −18 ◦C was usedto prepare semi-IPN cryogels. The initial monomer concentrationof (DMAEM + BAAm), Co, was 15 wt.%, whereas the oxidized starchwas added as powder in a content of 3.35 wt.% and 5 wt.%. Stocksolutions (of 25 mL Milli pore water, each) APS and TEMED wereprepared by dissolving 0.5 g of APS and 1 mL of TEMED, respectively.Stock solutions of BAAm were prepared dissolving 0.5035 g BAAmin 25 mL Milli pore water, at 30 ◦C, under magnetic stirring, andused for hydrogels synthesis after 24 h. Briefly, for the synthesis ofsemi-IPN cryogels having Co = 15 wt.%, and 5 wt.% of entrapped oxi-dized starch, 1.535 mL of DMAEM, 0.074 g OPS (or OWS) as powder,3.032 mL of aqueous solution of BAAm and 0.75 mL of TEMED stocksolutions were first homogenized in a 10 mL graduated flask. Thereaction mixture was cooled at 0 ◦C in an ice-water bath and purgedwith nitrogen gas for 20 min, and then 1 mL of APS stock aque-ous solution was added, the whole mixture being further stirredfor about 20 s. The synthesis of cryogels was carried out at −18 ◦C,as previously shown [25]. After polymerization, the gels were cutinto small pieces (about 10 mm), and immersed in distilled waterto wash out the unreacted monomers, initiator and soluble poly-mers, changing water at 6 h, for one week. Thereafter, the swollengel samples were dried by lyophilization as previously shown [25].The feed composition and the samples code of the composite gelsare presented in Table 1. The general code of semi-IPN cryogels isPDMAEM followed by OPS and OWS used as entrapped polymers.

2.4. Characterization methods

2.4.1. FT-IR analysisA Bruker Vertex 70 FTIR spectrometer was used to record

all the infrared spectra, including potato starch, TEMPO-oxidizedpotato starch, PDMAEM, semi-IPN cryogels having OPS and OWSentrapped in PDMAEM as matrix. The samples were prepared byKBr pellet technique (about 5 mg of sample used in each pellet)and all spectra were acquired by accumulation of 32 scans with aresolution of 2 cm−1, recorded in the range of 400–4000 cm−1.

2.4.2. 13C NMR analysisFor 13C NMR analyses of the potato starch and TEMPO-oxidized

potato starch, an amount of 50 mg sample was dissolved in 0.7 mLdeuterium oxide. The resulted solution was then added to a stan-dard 5 mm NMR tube, using a pipette containing glass wool toretain any undissolved fraction. The NMR spectra were recordedon a Bruker Avance DRX 400 MHz Spectrometer, equipped with a5 mm QNP direct detection probe and z-gradients.

2.4.3. Morphological characterizationAny change on the surface morphology and/or internal struc-

ture of the dried gels were analyzed by using a Quanta 200 ScanningElectron Microscope (FEI) operating at 20 kV in Low Vacuum mode,equipped with a secondary electron detector LFD. The samples

preparation for SEM analyses, suppose a fragmentation of the cryo-gels into small pieces. For this purpose, the samples were cut andthe obtained fragments were mounted on aluminum stubs usingcarbon double-sided tapes. The ImageJ 1.48 v software was used

D.F. Apopei Loghin et al. / International Journal of Biological Macromolecules 96 (2017) 589–599 591

Table 1Feed composition and sample code for the synthesis of semi-IPN cryogels.

Semi-IPN code Cross-linker inPDMAEM matrixa

Cib, Entrapped polysaccharide

% w/v mol/L Nature wt.%

PDMAEM5.15 5 15 0.955 – –PDMAEM5.15OPS3 5 15 0.955 OPS 3.35PDMAEM5.15OPS5 5 15 0.955 OPS 5

15

twa

2

edir

S

S

wts

2

sepwc1aT

D

wtitoc

2

ifliScI2o

PDMAEM5.15OWS5 5

a Mols BAAm/100 mols DMAEM.b Initial concentration of monomers.

o calculate the average diameter of pores (a number of 15 poresere analyzed for each studied photography) utilizing the previous

cquired SEM pictures [38].

.4.4. Swelling experimentsSwelling properties of semi-IPN cryogels were gravimetrically

valuated by immersion the completely dried hydrogel samples inouble distilled water at 21 ◦C [25,39]. Based on the swelling exper-

mental results, the swelling ratio, SR, and equilibrium swellingatio, SReq, were calculated by Eqs. (1) and (2), respectively:

R = Wt

Wd(1)

Req = We

Wd(2)

here: Wt represents the mass of gel, g, in swollen state at time, Wd is the mass of gel, g, in dry state, and We is the mass of gelwollen at equilibrium, g.

.5. Drug loading into cryogels

IDM loading into composite cryogels was assessed by using theolvent sorption/evaporation strategy. A 3 mg IDM/mL solution inthanol:acetone (1:1, v/v) was prepared, and then disk shaped sam-les of semi-IPN cryogels of about 25 mg were allowed to interactith this solution, the complete adsorption of the solution by the

ryogels occurring within 1–2 h. The samples were kept in air for h to evaporate the most part of the solvent, being then addition-lly dried in vacuum for 48 h, until the total removal of the solvent.he drug loading efficiency (DLE) was calculated with Eq. (3):

LE (wt%) = qe

qmax× 100 (3)

here: qe is the adsorbed amount of IDM (mg g−1) calculated ashe difference between the mass of gel loaded with IDM and thenitial mass of gel divided by the initial mass of gel; qmax representhe maximum theoretical adsorbed amount (mg g−1), which wasf 120 mg for a disk of 25 mg contacted with 1 mL solution with aoncentration of 3 mg/mL.

.6. In vitro release study

The in vitro release of IDM was done using 2 pH values: pH 1.3,n simulated gastric fluid (SGF) and pH 7.4 in simulated intestinaluid (SIF) (phosphate buffer saline, PBS), respectivelly, by immers-

ng cryogel samples loaded with IDM in a 10 mL volume of SGF orIF in PBS. Samples of supernatant (2 mL each) were withdrawn at

ertain time intervals, and immediately analyzed to determine theDM concentration using a UV–vis Spectrophotometer (SPECORD00 Analytik Jena), at �max of 317 nm. To ensure a constant volumef the analyzed medium, the withdrawn volume was each time

0.955 OWS 5

replaced with an identical volume of fresh releasing solution. Thecumulative release of IDM was calculated using Eq. (4) [24,39]:

%IDM (released) =(

10Cn + 2∑

Cn−1)

m0× 100 (4)

where: 10 represent the total volume of the releasing medium used,mL; Cn and Cn-1 represent the drug concentrations (mg L−1) in thereleasing sample corresponding to n and n-1 withdrawing steps,respectively; 2 means the volume of supernatant taken for analy-sis, mL; n indicates the number of withdrawing steps of releasingmedia; m0 represents the drug (mg) amount loaded in the cryogelsample.

2.7. Drug release mechanism

The drug release of IDM molecules from the semi-IPN cryogelswere kinetically analyzed by the semi-empirical equation proposedby Korsmeyer and Peppas (K-P) (Eq. (5)) [40] and Higuchi equation(Eq. (6)) [41]:

Mt

M∞= ktnr (5)

where: Mt ⁄M∞ represents the fraction of the drug released, Mt andM∞ are the cumulative amounts of IDM released at time t and atinfinite time (the maximum released amount found at the plateauof the release curves), respectively, k is a constant related to thematrix and the drug, and the exponent nr gives information aboutthe release mechanism.

Mt

M∞= kt

1⁄2 (6)

where: Mt ⁄M∞ represents the fraction of the drug released, Mt andM∞ are the cumulative amounts of IDM released at time t and atinfinite time (the maximum released amount found at the plateauof the release curves), respectively, k is a constant related to thematrix and the drug.

2.8. Statistical analysis

All statistical analyses were carried out using the SigmaPlot 12software. To determine how dissolution of cryogel samples (influ-enced by time and temperature), the diffusional parameters werestudied by two-way ANOVA (with time as repeated factor). The uti-lized variables in this study were cumulative percentage values ofIDM dissolved in a certain time interval. For multiple comparisonspost hoc was applied Holm-Sidak method. Statistical significancewas set at p < 0.05.

2.9. Erosion experiment

The weighed dried sample of semi-IPN cryogels was placed inbaskets which were then placed in PBS (pH 7.4) at 37 ◦C, in a ther-mostated shaker. At well-defined time intervals, the samples werewashed with distilled water to remove the buffer ions and dried

592 D.F. Apopei Loghin et al. / International Journal of Biological Macromolecules 96 (2017) 589–599

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

potato starch

oxidized potato starch

1159

-11

52 -

C-O-C

1609

- 1419

-

- COO -

200 18 0 16 0 14 0 12 0 10 0 80 60 40 20 0Chemical Shift (pp m)

102.

27

79.5

374

.17

73.8

371

.99

63.0

9

200 180 160 140 120 100 80 60 40 20 0Chemical Shift (pp m)

178.

81 99.8

9

78.8

4 75.7

374

.56

63.2

7

A

B

dent [

up[erc

%

wd

3

3

ba

t

Fig. 1. FT-IR spectra of the native PS and its TEMPO-oxidized correspon

nder vacuum at 50 ◦C, till adsorbed water was removed. The sam-les were weighed again and placed in the PBS for another interval42]. The experiments were continued until the results support therosion of the matrix. Each experiment was done in triplicate, theesults being given as percentage of weight loss (mean ± SD; n = 3),alculated with Eq. (7):

Wtloss = W0 − Wt

W0x100 (7)

here, W0 is the initial weight of cryogel, Wt is the weight of theried cryogel after being kept in PBS a certain interval.

. Results and discussion

.1. Synthesis of oxidized starch with TEMPO

Starch, as a mixture of linear and helical amylose and the

ranched amylopectin, possesses several primary OH groupsvailable for oxidation [43–45]. Nonetheless, there are primaryOH groups which are inaccessible for oxidation, originating from

he branched amylopectin. The primary OH groups available for

A] and 13C NMR spectra of unoxidized (left) and oxidized PS (right) [B].

oxidation are converted to carboxyl moieties in the presence of thenitrosonium ion generated in situ, the oxidized form of TEMPO.Due to the presence of NaOH, the carboxyl groups are formed as

COONa. The TEMPO-mediated oxidation of starch is depicted inScheme 1.

During oxidation process, the nitrosonium ions are reduced tothe hydroxyl amine form of TEMPO, which in turn is oxidized byhypobromide ions, to regenerate the catalyst, TEMPO, thus the oxi-dation cycle being reactivated.

3.2. Characterization of oxidized starch

Fig. 1A shows the FTIR spectra of the native and oxidized PS.The main differences consist of the appearance in the FTIR spectraof oxidized samples of the new double sharp peaks at 1609 cm−1

and 1419 cm−1 indicating the presence of the asymmetrical andsymmetrical stretching vibrations of COO− groups, as a result of

the primary OH group oxidation.

Also, the bell shaped peak centered at 3420 cm−1, assigned tothe OH groups in starch, are still present in the oxidized samples,suggesting that the secondary alcohol groups are unaffected by the

D.F. Apopei Loghin et al. / International Journal of Biological Macromolecules 96 (2017) 589–599 593

OOHO

OOH

OH

O

HO

OOH

OH

O

HO

OOH

OH

OOHO

OOH

OH

O

HO

OOH O

HO

OOH

OH

O

HO

OOH

OH

Unaccesible OH groups

N

O

N

OH

N

O

TEMPO

Nitrosoniumion

Hydroxylamine

NaBrO

NaBr

NaClO

NaCl

OO

OOH

O

O

HO

OOH

O

O

HO

OOH

O

OOHO

OOH

NaO

O

HO

OOH O

HO

OOH

O

O

HOOH

O

+

ONa

ONa

ONa

ONa

ONa

O

HO

EMPO

Tasa

sttdpCateiw

Scheme 1. General scheme of T

EMPO-mediated oxidation. On the contrary, the peak located atround 2930 cm−1 in native starch, assigned to CH2 stretching,hifted to 2937 cm−1 in oxidized starch, and became much smallers a consequence of the conversion of the CH2OH groups intoCOO− groups. The C O C skeletal vibrations at 1159 cm−1 are

till present in the oxidized samples (at 1152 cm−1) confirminghe preservation of the polymer structure of the oxidized products,herefore the depolymerization processes were negligible. Fig. 1Bisplays the 13C NMR spectra of the native and oxidized PS sam-le. The native PS shows a typical spectrum of starch, with peaks of6 at 63 ppm, C1 at 102 ppm, and those originating from C2, C3, C4nd C5 between 72 and 79 ppm. After TEMPO-oxidation, the almostotal disappearance of the peak from 63 ppm is visible, the pres-nce of a new and strong peak being detectable at 178 ppm, which

s ascribed to the newly formed COOH group. No chemical shifts

ere observed in the range 185–205 ppm, indicating the absence of

O

-mediated oxidation of starch.

any ketone groups from the oxidation of secondary alcohol groups[46].

3.3. Synthesis of semi-IPN cryogels

The anionic semi-IPN composite cryogels were prepared byfree radical cross-linking copolymerization between DMAEM andBAAm, in the presence of oxidized starch as entrapped polymer,below the freezing point of the reaction solution (Scheme 2).

The ice crystals act as porogen during gelation, leading to asuperporous structure by melting at the end of the gel preparation[47–51].

3.4. Structural characterization of cryogels by FTIR analysis

The FTIR spectra of the two composite cryogels, OPS andPDMAEM matrix are presented in Fig. 2. The spectrum of OWS

594 D.F. Apopei Loghin et al. / International Journal of Biological Macromolecules 96 (2017) 589–599

Scheme 2. Schematic representation of t

3000 2000 1000Wavenu mbe r (cm-1)

3433

29512824

2772

2038

17301627

14621396

1273

1150110010421016

962852778749

532455

PD M AEM3430

29502824

2773

17291667

14611393

1272

115011001039

1017964

881853 779 749

723620

534

P DM AE M 5.15 O P S5

3429

29492823

2773

17301671

14611392

1272

11501100

10411016

964853779749

533456

PD M AE M 5.15O W S 5

W avenu m b er (cm -1)

3409

2929

1610

14201311

12421152 1072

1028951

785 725 635573

O P S

4000

FP

ii

dtasia

ig. 2. FT-IR spectra of semi-IPN cryogels having OPS and OWS entrapped inDMAEM as matrix compared with the spectra of OPS and PDMAEM.

s almost identical with that of OPS, and therefore has been notncluded in Fig. 2.

In the spectrum of PDMAEM matrix can be seen the followingistinct bands: 2951 cm−1, corresponding to the stretching vibra-ion of C H from CH3 and CH2 groups; 2824 and 2773 cm−1

ssigned to the stretching vibration of CH from N(CH3). Theharp peak at around 1730 cm−1, was assigned to the C O stretch-ng vibration in the ester groups, while the band at 1462 cm−1 wasttributed to the C H stretching in PDMAEM and in the cross-

he synthesis of semi-IPN cryogels.

links. The intense peak at 1150 cm−1 was attributed to the C Nstretching in tertiary amine. The most representative peaks in thespectrum of OPS are located as follows: 1610 cm−1 and 1420 cm−1,assigned to the asymmetrical and symmetrical stretching vibra-tions of COO−, respectively, and the three peaks located at 1152,1072, and 1028 cm−1 assigned to C O stretching vibrations in theanhydroglucose units of starch [52].

The presence of PDMAEM in the PDMAEM5.15OWS5 andPDMAEM5.15OPS5 is supported by the sharp peak at 1730 cm−1

and 1729 cm−1, respectively, and by the strong band located at1461 cm−1; as can be seen, the characteristic peaks of PDMAEMlocated at 1042, and 1016, are present in the spectra of semi-IPNcryogels. The broad absorption peaks at 3429–3430 cm−1 can beascribed to the O H stretching vibration, while the asymmetric andsymmetric stretching vibration of C H from aliphatic ( CH2 and

CH3) groups in PDMAEM matrix were evidenced by the appear-ance of the absorption peaks at 2950, 2823 and 2773 cm−1. Thepresence of OPS and OWS in the composite cryogels is supportedby the small shoulder located at 1609 cm−1, corresponding to asym-metrical stretching vibrations of COO− groups of oxidized starch.The peaks at 1072, and 1028 cm−1, assigned to C O stretchingvibrations of polysaccharide [52], are screened by the intense bandsof the PDMAEM matrix.

3.5. Morphological analysis of semi-IPN cryogels

SEM was used to investigate the surface morphology andinternal structure of the dried cryogels. The SEM images of thefreeze-dried cryogels show a strong influence of the starch natureand concentration, and provide relevant information regarding thepore size and geometry of the cryogel network (Fig. 3).

All semi-IPN cryogels, appear as homogeneous structures, withclearly defined pores of different sizes. The values of the averagediameter of pores, calculated by the ImageJ 1.48v program areas follows: 46.28 ± 2.93 �m, 57.65 ± 3.12 �m, 52.78 ± 4.39 �mand 43.28 ± 2.19 �m for PDMAEM5.15, PDMAEM5.15OPS3,PDMAEM5.15OPS5 and PDMAEM5.15OWS5. Thus, the pore sizedecreased with the increase of polysaccharide concentration,being smaller in the case of OWS as entrapped polymer comparedwith OPS, at the same concentration.

This difference could be attributed to a stronger electrostaticinteraction between the positive charges of the matrix and COO−

groups when OPS was entrapped in PDMAEM compared with OWS.

3.6. Stimuli response of semi-IPN cryogels

The temperature-sensitive hydrogels are able to swell andde-swell as a result of changes in the temperature of the surround-

ing fluid [27,53]. The swelling kinetics of two semi-IPN cryogelsdifferent only by the nature of the entrapped polymer, at four tem-peratures: 21 ◦C, 30 ◦C, 42 ◦C and 47 ◦C (Fig. 4a), and the SReq as afunction of temperature (Fig. 4b) are given below.

D.F. Apopei Loghin et al. / International Journal of Biological Macromolecules 96 (2017) 589–599 595

Fig. 3. SEM images of semi-IPN cryogels: mag 150× (inset 500×); the scaling bar 500 �m (inset 200 �m).

0 10 20 30 40 50 60 70 80 90 1000

5

10

15 PDMAEM5.15OPS 5 T = 21 oC T = 30 oC T = 42 oC T = 47 oC

PDMAEM5.15OWS5 T = 21 oC T = 30 oC T = 42 oC T = 47 oC

SR

Time, min

0 10 20 30 40 50 60 700

5

10

15

20

25

30 PDMAEM5.15 PDMAEM5.15OPS3 PDMAEM5.15OPS5 PDMAEM5.15OWS5

SReq

Temp., oC

a b

F four te( tion (n

bbsti

pctfbc

ig. 4. Swelling kinetics of PDMAEM5.15OPS5 and PDMAEM5.15OWS5 cryogels at

b) for all semi-IPN cryogels; the results are represented as means ± standard devia

The influence of the origin of starch on the swelling kinetics cane observed in Fig. 4a, the SR values of cryogel with OWS entrappedeing higher than those with OPS, at all temperatures. As Fig. 4ahows, the equilibrium of swelling was reached in about 10 min forhe composite cryogels and the swelling values decreased with thencrease of temperature.

Therefore, to determine the values of the SReq at different tem-eratures (Fig. 4b) the time was fixed at 30 min. In the case ofryogels having OPS as entrapped polymer, when the concentra-

ion of the OPS was 3 wt%, the SReq values were higher than thoseound for the cryogel having 5 wt% of OPS. This difference coulde attributed to the electrostatic interaction between the positiveharges of the PDMAEM matrix and the COO− groups of the OPS,

mperatures (a) and equilibrium swelling ratios (SReq) as a function of temperature = 3).

which are stronger at a higher concentration of OPS. The influenceof the origin of starch on the swelling kinetics can be observedin Fig. 4b, the SReq values of cryogels with OWS entrapped beinghigher than those with OPS, at all temperatures, and this could beattributed to the higher hydrophilicity of the gel prepared withOWS.

The values of SReq of the semi-IPN cryogels under study wereplotted as a function of temperature (Fig. 4b) to evaluate the VPTT.The values of VPTT evaluated as the inflection point of the curves,

◦ ◦ ◦ ◦

were as follows: 32 C, 35 C, 35 C and 35 C for PDMAEM5.15,PDMAEM5.15OPS3, PDMAEM5.15OPS5 and PDMAEM5.15OWS5.Even if the values of VPTT were almost the same for all compos-

596 D.F. Apopei Loghin et al. / International Journal of B

0 2 4 6 8 10 120

10

20

30

40

50

60 PDMAEM5.15 PDMAEM5.15 OPS 3 PDMAEM5.15OPS5 PDMAEM5.15 OWS5SR

eq

pH

Ft

iv

tcrabm

cwtw

Osbiowisa

aeNibctNaat

ttsipitstaw

The difference between PDMAEM5.15OPS5 and

ig. 5. Equilibrium swelling ratio (SReq) as a function of pH for semi-IPN cryogels;he results are represented as means ± standard deviation (n = 3).

te cryogels, differences between them will be seen later at the initro release of IDM.

The presence of PDMAEM, which is a weak polycation, makeshese gels pH responsive. The swelling behavior of the semi-IPNryogels was studied at various pH values in the range 2.0–11.0, atoom temperature (21 ◦C). As can be seen in Fig. 5, the SReq valuesbruptly decrease when pH increases from 2 to 3 for all cryogels,ecause the protonation degree of the tertiary amine groups in theatrix decreased.In the range of pH 5–11, the values of SReq remained almost

onstant, because almost all tertiary amine groups in the matrixere deprotonated. The presence of oxidized starch is supported by

he lower values of the SReq found for the composite gels comparedith that of the PDMAEM5.15 matrix.

The polyelectrolyte nature of the PDMAEM matrix and of thePS and OWS as entrapped polymer could influence the behavior of

emi-IPN cryogels in aqueous solution of salts, because the swellingehavior of ionic hydrogels is sensitive to salt concentration and

on valence [54]. In order to determine the effect of salt naturen the swelling of cryogels, four neutral salts as aqueous solutionsere used: NaCl, NaBr, Na2SO4 and CaCl2, the salt concentrations

ncreasing from 10−3 M to 1.0 M. The SReq plotted as a function ofalt concentration for the PDMAEM5.15 and the semi-IPN cryogelsre given in Fig. 6.

As can be seen, the SReq values of both the PDMAEM5.15 (Fig. 6a)nd the composite cryogels with oxidized starch as polysaccharidentrapped (Fig. 6b) monotonously decreased with the increase ofaCl concentration. The increase of NaCl concentration resulted

n diminishing of electrostatic repulsion between polymer chainsearing tertiary amine groups due to the screening of cationicharges by the excess of Cl− ions, which limited the relaxation ofhe chains. The influence of the ionic strength was stronger whenaCl was replaced with NaBr. It is well known that the solubility of

linear polyelectrolyte and the swelling of a cross-linked polyionre decreasing all the more as the counterions are stronger boundo the ionized groups.

The values of SReq in Na2SO4 aqueous solutions were even lowerhan those found in NaCl and NaBr, for all cryogels, because whenhe hydrogel was immersed in multivalent salt solution, the expan-ion of hydrogel network accelerates first the diffusion of all ionsnto the gel network, and then the multivalent ions could com-lex with the hydrophilic groups such as tertiary amine groups. The

onic complexing interaction increases the cross-linking points inhe interior or at the surface of hydrogel network, and caused thehrinkage of the swollen hydrogel and an appreciable decrease in

he swelling capacity of the gels at equilibrium. Fig. 6 illustrateslso the influence of salt concentration when NaCl was replacedith CaCl2, the swelling capacity decreases in the case of CaCl2 due

iological Macromolecules 96 (2017) 589–599

to the change of counterion species. Also can be seen, the valuesof SReq in CaCl2 were even higher than those found in Na2SO4. Byincreasing the charge of anion, the degree of crosslinking increasedand the swelling capacity decreased. Thus, the increase of multi-valent anions causes a decrease in swelling capacity. The swellingcapacity of semi-IPN cryogels with oxidized starch decreases morein the presence of solution of salts compared with PDMAEM5.15,probably due to the complexation of the carboxylate groups fromoxidized starch inducing the formation of intramolecular and inter-molecular complexes, resulting in further increase of the apparentcross-linking density of the network, and finally in the decrease ofwater absorbancy. In the case of semi-IPN cryogels with oxidizedstarch, the values of SReq of cryogels with OPS were lower thanthose of the cryogels with OWS; this behavior could be attributedto the lower hydrophilicity of the gels with OPS (see Fig. 4a). Itwas found that the SReq values decreased with the increase of saltconcentration and with the increase of the counterion valence, theorder of SReq values being: Na2SO4 < CaCl2 < NaBr < NaCl.

3.7. Loading and in vitro release of IDM

[1-(p-Chlorobenzoyl)-5-methoxy-2-methyl-3-indole aceticacid] (IDM) is a nonsteroidal anti-inflammatory drug used to treatacute and chronic rheumatoid arthritis and osteoarthritis [55].Because of its low solubility in water, the evaluation of the optimalconditions for the controlled release of IDM has attracted muchof attention, the composites based on polysaccharides having acentral place [56–58]. Two composite cryogels, PDMAEM5.15OPS5and PDMAEM5.15OWS5, were selected for the in vitro releaseexperiments in this work, based on their response at temperature(Fig. 4) and pH (Fig. 5). The average DLE, calculated with Eq. (3),was: 69.03 ± 1.1% and 67.49 ± 0.34% for PDMAEM5.15OPS5 andPDMAEM5.15OWS5, respectively. The driven force during theloading of IDM into the cryogel disks was the fast sorption of IDMsolution by convection, this being a characteristic of porous gelssuch as cryogels. Inside the cryogel, the IDM molecules have beenstabilized by electrostatic interactions with the positively chargedmatrix, as well as by hydrogen bonds.

To evaluate in vitro release profiles of IDM from these two cryo-gels, the experiments were performed in SGF (pH 1.3) and in SIF(pH 7.4), at two temperatures, 37 ◦C, and at 42 ◦C (Fig. 7).

The release of IDM from the composite cryogel should bestrongly dependent on the medium pH, because, in acidic range,IDM molecules are very hydrophobic and it is expected to remainentrapped in the positively charged matrix, while in basic range(pH 6.8–7.4), both IDM molecules and oxidized starch are nega-tively charged and hydrophilic, and therefore, the drug moleculesare forced to leave the environment of the composite cryogel by theelectrostatic repulsion. The release kinetics would be depended onthe composite morphology, the cross-linking degree, the contentof oxidized starch and by the temperature of the environment. AsFig. 7 shows, the release rate of IDM from the semi-IPNs cryogelsin SGF was very low (about 10% in 2 h), mainly because of the lowsolubility of IDM [56,59]. Changing the release environment withSIF (pH = 7.4), an initial fast release up to about 60–67%, followedby a slow release, up to ∼ 94% in the case of PDMAEM5.15OPS5, and∼ 97% in the case of PDMAEM5.15OWS5 can be observed, at 37 ◦C(Fig. 7A). Thus, the loaded IDM was slowly and steadily releasedinto the SIF over a period of about 8 h, when OPS was entrapped inPDMAEM network, and of about 6 h, when OWS was the entrappedpolysaccharide, which represents sufficient time for the night treat-ment.

PDMAEM5.15OWS5 composites was higher when IDM releasewas conducted at 42 ◦C, a “burst” release being observed mainlyfor PDMAEM5.15OWS5 composite cryogel. This behavior could be

D.F. Apopei Loghin et al. / International Journal of Biological Macromolecules 96 (2017) 589–599 597

10-3 10-2 10-1 1005

10

15

20

25 PDMAEM5.15 NaCl NaBr Na2SO4 CaCl2

SReq

Ion ic strength, M10-3 10-2 10-1 100

5

10

15

20

25PDMAEM5.15OPS5

NaCl NaBr Na2SO4 CaCl2

PDMAEM5.15OWS5 NaCl NaBr Na2SO4 CaCl2

SReq

Ion ic strength, M

a b

Fig. 6. Equilibrium swelling ratio (SReq) as a function of ionic strength; the results are represented as means ± standard deviation (n = 3).

0 100 20 0 30 0 40 0 500 600 70 00

102030405060708090

100

T = 37 oC PDMAEM5.15OPS5 PDMAEM5.15OWS5C

umul

ativ

e re

leas

e, %

Time, min

pH =

1,3

pH =

7,4

0 50 10 0 15 0 20 0 25 0 3000

102030405060708090

100110

T= 42 oC PDMAEM5.15 OPS 5 PDMAEM5.15 OWS5

Cum

ulat

ive

rele

ase,

%

Time, min

pH = 1,3 pH = 7,4A B

Fig. 7. Kinetics of IDM release from two semi-IPN cryogels, in buffer at pH 1.3, and in PBS at pH 7.4: 37 ◦C (A); 42 ◦C, (B); the results are represented as means ± standarddeviation (n = 3).

dels in

aOspbtct

3

e

Fig. 8. Linear forms of Korsmeyer-Peppas (A) and Higuchi (B) mo

ttributed to the higher hydrophilicity of the gel prepared withWS compared with OPS (see Fig. 4b). Thus, the origin of oxidized

tarch can be considered a useful parameter to modulate theerformances of DDSs based on such composites. As Fig. 7 shows,oth PDMAEM5.15OPS5 and PDMAEM5.15OWS5 composites seemo be promising for the pulsatile release of IDM, because higherumulative release values were found in a shorter time when theemperature increased from 37 ◦C up to 42 ◦C.

.8. Drug release mechanism

Based on the release data presented in Fig. 7, the K-P and Higuchiquations were plotted in Fig. 8(A and B).

PBS (pH 7.4) at 37 ◦C (open symbols) and 42 ◦C (filled symbols).

The constants nr and k of K-P equation were calculated fromthe slopes and intercepts of the plots of log (Mt /M∞) versus logt, their values being presented in Table 2. As can be seen, the nr

values for both composite cryogels were in the range 0.39–0.44,which would indicate a Fickian diffusion, except the IDM releasefrom PDMAEM5.15OPS5 at 42 ◦C when nr was 0.62, indicating anon-Fickian diffusion. Higuchi model describes also very well therelease of IDM from the semi-IPN cryogels. Therefore, based on thevalues of the correlation coefficient (R2 = 0.991–0.998, Table 2), we

assume that both models are suitable to describe the delivery ofIDM from these composite cryogels. The description of the IDMrelease from the composite cryogel PDMAEM5.15OWS5 at 42 ◦C by

598 D.F. Apopei Loghin et al. / International Journal of Biological Macromolecules 96 (2017) 589–599

Table 2IDM release evaluated by Korsmeyer-Peppas and Higuchi equations at pH 7.4.

Sample Temp., ◦C Korsmeyer-Peppas model Higuchi model

nr k, min−nr R2 k, min−1/2 R2

9.65 −2 −2

9.667.33

KI

3

msrtttfmd

3

lovce4aoscap

4

PeemsctmcossuacctdgAre

[

[

[

[

[

[

[

PDMAEM5.15OPS5 37 0.3964

PDMAEM5.15OWS5 37 0.4471

PDMAEM5.15OPS5 42 0.6279

-P and Higuchi models was not possible because the release ofDM was about 80% in the first 30 min.

.9. Statistical analysis

When the cryogel pieces (group) were exposed to dissolutionedium (Fig. 7), the drug loaded in cryogel pieces diffuses very

lowly into the acidic medium (pH 1.3), but diffusion occurs moreapidly into the alkaline dissolution medium (pH 7.4). For dissolu-ion test in pH 7.4, the RM-ANOVA revealed significant effects forhe factor time (F1,22 = 76.825, p < 0.001) and for time x tempera-ure interaction (F = 64657.480, p = 0.003), but no significant effectsor factor temperature (F2,22 = 2.682, p = 0.151; not significant). For

ultiple comparisons the post hoc tests indicate that the effect ofifferent levels of time depends on what level of temperature.

.10. Erosion test

The erosion of cryogels is based on the removal of the surfaceayers due to the polymer-solvent interactions and, in turn dependsn the chemical composition of the surface. Erosion test suppliesaluable information about the biodegradability of the matrix andorrelated with this on the kinetics of drug release. The results ofrosion test of two semi-IPN cryogels at pH 7.4 and 37 ◦C, after

h was 78.44 ± 1.56% and 87.87 ± 0.13% for PDMAEM5.15OPS5nd PDMAEM5.15OWS5, respectively. The higher level of erosionbserved for the semi-IPN after 4 h could be explained by the higherusceptibility at hydrolysis of the ester group in PDMAEM. In thease of the PDMAEM5.15OWS5, the level of erosion was highernd this could be attributed to the higher hydrophilicity of the gelrepared with OWS.

. Conclusions

The synthesis of multiresponsive semi-IPN cryogels withDMAEM as a matrix and oxidized starch (OPS and OWS) asntrapped polysaccharide was investigated in this work. The prop-rties of the semi-IPN cryogels, such as swelling ratio, internalorphology, and pores size greatly depended on the origin of

tarch. Thus, the pore size decreased by the increasing the polysac-haride concentration; at the same concentration of used starch,he pore size was smaller when OWS was used as entrapped poly-

er as compared with OPS. The SReq values of the compositeryogel with OWS as entrapped polymer were higher than thosef the composite with OPS, at the same content, which support atronger interaction between OPS and PDMAEM. The effect of ionictrength on the swelling of semi-IPN cryogels was investigatedsing four neutral salts as aqueous solutions: NaCl, NaBr, Na2SO4nd CaCl2. The SReq values decreased with the increase of the saltoncentration, in the order: Na2SO4 < CaCl2 < NaBr < NaCl. The IDMumulative release in SGF (pH 1.3) could be rather neglected, whilehe cumulative release in SIF (pH 7.4) was significant. The releaseata of IDM were analyzed by K-P and Higuchi models for both cryo-

els at 37 ◦C, and supplementary for PDMAEM5.15OPS5 at 42 ◦C.ccording to the experimental data, both models describe well theelease process. Based on the values of the exponent nr in the K-Pquation, it was found that the release of IDM occurred by Fickian

[

× 10 0.9984 4.894 × 10 0.9984 × 10−2 0.9951 5.699 × 10−2 0.9945 × 10−2 0.9921 6.149 × 10−2 0.9977

diffusion at 37 ◦C, and by anomalous diffusion at 42 ◦C. The abilityof these composite cryogels to adsorb and release IDM in a con-trolled manner, changing the pH and temperature, support theirpotential as DDSs for IDM, reducing thus the frequency of drugadministration and the risk of gastrointestinal side effects.

Acknowledgement

The results presented in this manuscript have been financed bya grant of the Romanian National Authority for Scientific Research,CNCS—UEFISCDI, project number PN-II-ID-PCE-2011-3-0300.

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