research article synthesis, characterization, and acute...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 239838 9 pageshttpdxdoiorg1011552013239838

Research ArticleSynthesis Characterization and Acute Oral Toxicity Evaluationof pH-Sensitive Hydrogel Based on MPEG Poly(120576-caprolactone)and Itaconic Acid

Liwei Tan Xu Xu Jia Song Feng Luo and Zhiyong Qian

State Key Laboratory of Biotherapy and Cancer Center West China Hospital West China Medical School Sichuan UniversityChengdu 610041 China

Correspondence should be addressed to Zhiyong Qian anderson-qian163com

Received 21 August 2013 Revised 17 October 2013 Accepted 26 October 2013

Academic Editor Mehrdad Hamidi

Copyright copy 2013 Liwei Tan et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A kind of chemically cross-linked pH-sensitive hydrogels based on methoxyl poly(ethylene glycol)-poly(caprolactone)-acryloyl chloride (MPEG-PCL-AC PECA) poly(ethylene glycol) methyl ether methacrylate (MPEGMA MEG) NN-methylenebisacrylamide (BIS) and itaconic acid (IA) were prepared without using any organic solvent by heat-initiated free radicalmethodThe obtained macromonomers and hydrogels were characterized by 1HNMR and FT-IR respectively Morphology studyof hydrogels was also investigated in this paper and it showed that the hydrogels had good pH-sensitivityThe acute toxicity test andhistopathological study were conducted in BALBc mice The results indicated that the maximum tolerance dose of the hydrogelwas higher than 10000mgkg body weight No morality or signs of toxicity were observed during the whole 7-day observationperiod Compared to the control groups there were no important adverse effects in the variables of hematology routine testand serum chemistry analysis both in male or female treatment group Histopathological study also did not show any significantlesions including heart liver lung spleen kidney stomach intestine and testis All the results demonstrated that this hydrogelwas nontoxic after gavage Thus the hydrogel might be the biocompatible potential candidate for oral drug delivery system

1 Introduction

Hydrogels are networks which can swell in water andhold a large mount of water while maintaining theirthree-dimensional structure Over the past decades theenvironment-sensitive hydrogels which are also called ldquointel-ligentrdquo or ldquosmartrdquo hydrogels have attracted increasing atten-tion because they can show a sudden or gradual change intheir dynamic and equilibrium properties with the changesof ambient conditions such as temperature [1 2] pH [3ndash5]and ionic strength [6] They might have great potential intargeted drug delivery system on-off switches for modulateddrug delivery artificial organs and immobilization of enzymedue to their biocompatibility and resemblance to biologicaltissues [7ndash11] Among the various types pH-sensitive hydro-gels based on biocompatible copolymers had been widelyused in the area of drug delivery system [12ndash18] especiallyoral drug delivery system [19ndash21]The pH-sensitive hydrogelscould adjust their swelling behavior in different pH value In

acidic gastric environment the hydrogel is shrinking and thedrug is sustained in the hydrogel but in high-pH intestinaltract the hydrogel is swelling and the drug is released fromhydrogel So the pH-sensitive hydrogels could improve drugefficacy and reduce the side effects [22ndash24]

Poly(ethylene glycol) (PEG) and poly(120576-caprolactone)(PCL) have been widely utilized as a biomaterial for theirgreat biocompatibility Since Perret and Skoulios [25] pre-pared a series of block copolymer based onPEGandPCL thiskind of copolymer have been widely researched containingPEG PCL and poly(ethylene glycol) methyl ether (MPEG)that followed [26 27] In our previous works we suc-cessfully preparedMPEG-PCL amphiphilicmacromonomersand integrated them in the main constituent of smart pH-sensitive hydrogels [28] The carboxyl groups (ndashCOOH)make the hydrogel pH-sensitive In our previous work [29]itaconic acid (IA) was chosen to prepare a new kind ofpH-sensitive hydrogel due to its two carboxyl groups inone molecule which would achieve a better pH-responsive

2 BioMed Research International

ability Moreover the IA was water-soluble and we could usewater as the solvent This would avoid the use of organicsolvents considering the safety of hydrogel The obtainedhydrogels showed good pH-sensitivity and were employedto delivery protein dexamethasone DOX-loaded nanopar-ticles and so on

Until now we had studied the cytotoxicity of MPEG-PCL-AC (PECA) copolymer primarily byMTTmethod [29]however the toxicological study on pH-sensitive hydrogelbased on PECA copolymer has not been thoroughly exam-ined Since the safety evaluation of the smart hydrogel canhave a significant implication on its further applicationsas a biocompatible carrier for oral drug delivery systemin this paper the acute oral toxicity evaluation of pH-sensitive hydrogel based on MPEG PCL and itaconic acidwas performed

2 Materials and Methods

21 Materials Among the whole materials poly(ethyleneglycol) methyl ether (MPEG Mn = 2000) 120576-caprolactone (120576-CL) NN1015840-Methylenebisacrylamide (BIS) itaconic acid (IA)poly(ethylene glycol) methyl ether methacrylate (MPEGMAMn = 475) tin (II) 2-ethylhexanoate acryloyl chloride (AC)triethylamine (TEA) and ammoniumpersulfate (98) (APS)were purchased from Aldrich Company USA For the restof the reagents they were used as received All the reagentswere completely analytic grade Male and female BALBcmice were purchased from the Laboratory Animal Center ofSichuan University

22 Synthesis of PECA Macromonomer and P(ECA-IA-MEG)Hydrogel The MPEG-PCL copolymer was synthesized byring-opening polymerization of 120576-caprolactone initiated byMPEG using tin (II) 2-ethylhexanoate as catalyst [29 30]And the PECA macromonomer was obtained by dissolvingMPEG-PCL in dewatered methylene chloride to react withAC which has been reported before [29]

With APS as the initiator and BIS as the cross-linkingagent the P(ECA-IA-MEG) hydrogel was synthesized byheat-initiated free radical method As shown in Table 1the predetermined amounts of PECA IA MPEGMA BISand APS were dissolved in water Then the entire systemwas soaked and bathed in water at 37∘C for 4 hours Theobtained hydrogel was immersed in a plenty of distilledwater for 7 days and the water was refreshed everyday toremove the unreacted substances The purified hydrogelswere freeze-dried kept Finally the freeze dried hydrogelswere grinded into powder and suspended in 1 aqueoussodium carboxymethyl cellulose (CMC-Na) solution foracute oral toxicity test

23 Characterization of PECA Copolymer and P(ECA-IA-MEG) Hydrogel

231 1H-Nuclear Magnetic Resonance Analysis (1H-NMR)The structure of PECA copolymer was analyzed by 1H-NMRspectrum which was recorded on Varian 400 spectrometer

Table 1 The hydrogel prepared in this work

PECA MPEGMA IA BIS content (wt) IA Content (wt)3 3 4 83 166

(Varian USA) at 400MHz using deuterated chloroform(CDCl

3) as solvent and tetramethylsilane (TMS) as internal

reference standard

232 Fourier Transform Infrared (FTIR) Analysis The chem-ical structures of PECA copolymer and P(ECA-IA-MEG)hydrogel were analyzed by the FTIR (KBr) spectrum whichwere recorded on Nicolet 200SXV meter (Nicolet USA)The samples were scanned at wave number range of 4000ndash400 cmminus1

233 Scanning Electron Microscopy (SEM) Themorphologyof the P(ECA-IA-MEG) hydrogel was recorded on JEOL SEM(JSM-5900LV JEOL Japan)The hydrogels were immersed inthe aqueous solution of pH 12 and pH 68 respectively Andthe samples were lyophilized transected and sputtered goldbefore observed

234 Swelling Behavior of the Hydrogels The hydrogels wereimmersed in aqueous medium with different pH conditions(pH 12 and pH 74) at 37∘C for 1 2 4 8 24 48 and 72 hrespectivelyThen theywere taken out and the surplus surfacewater was removed by filter paper This experiment wasrepeated 3 times and the wet weights of them were recordedcarefully The swelling ratios could be calculated by thefollowing equation

Swelling ratio (SR) =119882119905

1198820

times 100 (1)

where1198820was the initial dryweight and119882

119905was thewetweight

of the hydrogel at time 119905 respectively

24 Acute Toxicity Test

241 Maximal Tolerance Dose (MTD) Owing to the greatbiocompatibility and the swelling ability of P(ECA-IA-MEG)hydrogel no lethal dose or median lethal dose (LD

50) could

be obtained according to our preliminary trials So theMaximal Tolerance Dose (MTD) method was adopted toevaluate the acute oral toxicity of the hydrogel

All BALBc mice were housed individually in a standardanimal room maintained at a temperature of 20ndash22∘C anda relative humidity of 50ndash60 The animals which had anaverage weight of 20 g were 9-10 weeks old on the day ofdosing Twenty mice of both sexes were divided into twogroups hydrogel group (119899 = 10 5 male and 5 female mice)and control group (119899 = 10 5 male and 5 female mice) Allthe mice were fasted approximately 15 h prior to dosing andwater was provided continuously Hydrogel group was givenP(ECA-IA-MEG) hydrogel suspension twice at an intervalof four hours by gavage at a total dose of 10000mgkgbody weight and a total volume of 04mL10 g body weight

BioMed Research International 3

OO O O

OO

O

OO

OOO

OH Hx x

x

y

y

OCH3 130∘C

CH3

CH3

(MPEG-PCL)

40∘C H2C C C

HCl

P (ECA-IA-MEG) hydrogel

APS (initiator)37

∘C

MPEGMA (graft)IA (graft) (PECA)

MPEG-PCLIA

MPEGMABIS

+

(MPEG)(120576-CL)

BIS (cross-linking)

Sn(Oct)2

Figure 1 Synthesis scheme of P(ECA-IA-MEG) hydrogel

Accordingly control group was treated with a same volumeof 1 CMC-Na solution The animals were given food againapproximately 4 h after dosing

After the final administration all the animals were con-tinuously observed for 7 days Observations were conductedtwice daily including morality injury abnormal behaviorand the general condition (the hair activity feces behaviorpattern and other clinical signs) Besides body weights forall mice were recorded on study days 1 3 5 and 7 Thenecropsies of dead animals were performed to observe thegross pathological changes On the study day 7 all the animalswere sacrificed

242 Hematology Routine Test Whole blood samples fromeach rat were collected by removing the eyeball for bloodroutine test which is determined by a hematology Ana-lyzer (MEK-6318 Nihon Koden) And hematology rou-tine test in our study included the following variableswhite blood cell count (WBC) red blood cell count(RBC) hemoglobin (HGB) hematocrit (HCT) platelet count(PLT) mean corpuscular volume (MCV) mean corpus-cular hemoglobin (MCH) mean corpuscular hemoglobinconcentration (MCHC) red cell distribution width (RDW)mean platelet volumes (MPV) and platelet distributionwidth(PDW)

243 Serum Chemistry Analysis Approximately 05mL ofwhole blood samples from each rat was placed into EPtubes and left to stand for 2 h to separate serum fromblood The obtained sera samples were analyzed by a clinical

chemistry analyzer (Hitachi 7020) And this analysis ofour study contained the following indexes albumin (ALB)alkaline phosphate (ALP) alanine aminotransferase (ALT)aspartate aminotransferase (AST) total protein (TP) totalbilirubin(TBIL) glucose (GLU) blood urea nitrogen (BUN)and creatinine (CREA)

244 Statistical Analyses Refering to the similar methodadopted by the previous report [31] data obtained from ourtests were analyzed by using SPSS software All quantitativedata were expressed as ldquomean value plusmn standard deviationrdquoand a value of 119875 le 005 was considered as significantlydifferent

25 Histopathologic Study Sixty mice were equally dividedinto five time groups of 2 h 4 h 24 h 3 days and 7 days anda control group (119899 = 10 5 male and 5 female mice) Afterbeing fasted over night animals of five time groupswere givenP(ECA-IA-MEG) hydrogel suspension by gavagemethod at adose of 7500mgkg bodyweight in the volume of 03mL10 gbody weight and the control group was given a same volumeof 1 CMC-Na solution

Then at time points of 2 h 4 h 1 day 3 days and 7 daysafter administration one corresponding group of mice weresacrificed and the following organs were extracted heartliver spleen lung kidney stomach intestine (duodenumjejunum ileum and colon) and testes The obtained tissuesamples were fixed via preserving in 10 buffered formalde-hyde for 48 h and then embedded in paraffin sectioned at5 120583m and visualized by hematoxylin and eosin staining [32]


37∘C

4h

(a) (b)

Figure 2 Direct observation of synthesis of the P(ECA-IA-MEG) hydrogel (a) Mixture solution of PECA IA MPEGMA BIS and APS (b)Obtained hydrogel after being heated at 37∘C for 4 hrs

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

05

(ppm)

Figure 3 1HNMR spectrum of PECAmacromonomer (in CDCl3)

3 Results and Discussion

31 Synthesis and Characterization of Macromonomer andHydrogel

311 Synthesis of P(ECA-IA-MEG) Hydrogel The MPEG-PCL copolymer was synthesized by ring-opening polymer-ization of 120576-caprolactone initiated by MPEG In our previouswork the PECA copolymer was proved to be a biocompatiblematerial with low cytotoxicity It showed the synthesis schemeof PECA macromonomer from Figure 1 As also shown inFigure 2 the sample had good solubility in water and couldform clear hydrogel after being soaked and bathed in water at37∘C for 4 hours

312 1H-Nuclear Magnetic Resonance (1H-NMR) of thePECA Figure 3 presented the 1H NMR spectrum of PECAmacromonomer The peaks at 13 16 23 and 41 ppmpertained to methane protons (ndashCH

2ndash) of PCL blocks The

signals at 33 ppm were attributed to the methyl protons(ndashCH

3) of MPEG blocks The methane protons (ndashCH

2ndash) of

MPEG segment appeared at 37 41 ppm while the peaks at58 61 and 64 ppmbelong to protons of the C=CThe resultsdemonstrated that MPEG-PCL copolymer was successfullyprepared and reacted with acryloyl chloride

4000 3500 3000 2500 2000 1500 1000 500 0

Hydrogel

C=C

PECA

Wavenumber (cmminus1)

Tran

smitt

ance

Figure 4 FT-IR spectra of PECA and P(ECA-IA-MEG) hydrogels

313 Fourier Transform Infrared (FT-IR) of MacromonomerandHydrogel P(ECA-IA-MEG) hydrogel was synthesized byheat-initiated free radical polymerization Figure 4 showedthe FT-IR spectra for PECA macromonomer and P(ECA-IA-MEG) hydrogel In Figure 4 the absorption bands at16903 cmminus1 and 11157 cmminus1 were assigned to ester andether of PCE stretching vibration modes respectivelyThe absorption bands at 1637 cmminus1 and 8356 cmminus1 wereattributed to C=C stretching of PECA macromonomerBut they decreased greatly in the FT-IR of P(ECA-IA-MEG) hydrogel which indicated that the end double bondshad been converted to carbon-carbon single bonds com-pletely during the formation of hydrogel It could be seenthat the P(ECA-IA-MEG) hydrogel was successfully pre-pared

314 Morphological Characterization of the P(ECA-IA-MEG)Hydrogel SEM was used to observe the cross section ofP(ECA-IA-MEG) hydrogel in different aqueous media with


(a) (b)

Figure 5 SEM observation of P(ECA-IA-MEG) in pH 12 (a) and pH 68 (b)

0 1 2 3 4 5 6 7 815

16

17

18

19

20

21

22

23

Male treatment groupMale control group

Female treatment groupFemale control group

Mea

n w

eigh

t of t

he m

ice (

g)

Time (day)

Figure 6 Mice body weight of each group during the observationperiod

pH 12 and 68 As clearly seen in Figure 5 there weremany pores in the hydrogels and the pores were intercon-nected with each other In pH 12 most carboxylic acidgroups were in the form of COOH and they formed thehydrogen bonds with the section of MPEG The hydro-gels were shrinking and the mesh sizes were small Con-versely when environmental pH value increased to 68 thehydrogen bond broke owing to the ionization of carboxylicacid groups meanwhile electrostatic repulsion caused thenetwork to expand The hydrogels swelled obviously andthe mesh size increased a lot These figures implied thepH-sensitive characterization of the prepared hydrogel andindicated that the hydrogels have a good pH-responsibility

315 Swelling Behavior of the Hydrogels In order to inves-tigate the influence of the external pH value on the waterabsorption behavior of obtained hydrogels the hydrogels

Table 2 Swelling behavior of hydrogels in aqueous medium withpH 12 and pH 74 at 37∘C

Time (h) Swelling Radio ()pH 12 pH 74

1 210 plusmn 3 341 plusmn 16







were immersed in different pH conditions (pH 12 and pH74) and the results were showed in Table 2 It was seenthat the equilibrium swelling ratios of hydrogels in pH 74buffer solutions were much higher than those in pH 12buffer solutions Besides the swelling ratios in pH 74 buffersolutions became higher with time while they became lowerin pH 12 buffer solutions In pH 12 the hydrogels wereshrinking because of the hydrogen bonds and the waterwas squeezed out from the hydrogels while in pH 74 thehydrogen bonds broke and the electrostatic repulsion madethe hydrogels swellingThewater entered the hydrogels easilyand the swelling ratios were increased rapidly These datashowed that the environmental pHvalue had a great influenceon the swelling behavior of hydrogels and that the hydrogelswere pH-sensitive

32 Acute Oral Toxicity Test

321 General Conditions Clinical manifestation observed inacute oral toxicity test was listed in Table 3 Neither poisoningperformance nor toxic response was demonstrated duringthe seven-day observation period and all mice had behavednormally To elaborate they were sensitive to sound lightand other stimuliTheir furs remained totally normal withoutany ulceration It was noticeable that no running nose eyesecretion salivation or vomit were observed and no mouthor nose dryness or edema were showed in this treatment


(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

(u) (v)

Figure 7 Photograph of mice cardiac muscle liver spleen lung kidney stomach duodenum jejunum ileum colon and testes after oraladministration of P(ECA-IA-MEG) hydrogel (40x) ((b) (d) (f) (h) (j) (l) (n) (p) (r) (t) (v)) and of the control group ((a) (c) (e) (g)(i) (k) (m) (o) (q) (s) (u))

group Besides the micersquos feces were in regular form withnormal color without mucus pus or blood Then Afternecropsy no macroscopic pathological alterations caused byP(ECA-IA-MEG) hydrogel were found in all mice All abovelines of evidence showed that P(ECA-IA-MEG) hydrogel wassafe for taking orally

322 Body Weight of Mice Both the control and treatmentgroups were weighted and recorded accordingly during thewhole seven-day period As illustrated by the body weightcurves in Figure 6 no significant difference was showedbetween the treatment and control group thus P(ECA-IA-MEG) hydrogel would not affect the body weight of the mice


Table 3 Toxicity signs not observed in BALBc mice treated with P(ECA-IA-MEG) hydrogel

Motor activities Restlessness hyperactivity and phonationSymptoms of nervoussystem Tail erect tremble spasm movement disorder and gesture abnormal

Symptoms of autonomicNervous system

Protrusion of eyeballs salivation weep urination diarrhea hair bristled up and discolorationdyspnea

mortality Death

Table 4 Hematology routine test of acute oral toxicity test

Femalecontrol group

Femaletreatment group

Malecontrol group

Maletreatment group

WBC (109L) 601 plusmn 110 445 plusmn 059lowast

386 plusmn 069 406 plusmn 050

RBC (1012L) 1113 plusmn 051 1136 plusmn 039 1111 plusmn 037 1133 plusmn 038

HGB (gL) 14957 plusmn 547 15263 plusmn 329 14557 plusmn 395 15150 plusmn 600

HCT () 5817 plusmn 089 5985 plusmn 153 5730 plusmn 088 6125 plusmn 177lowast

PLT (109L) 53743 plusmn 10823 66638 plusmn 12195 63586 plusmn 5657 66638 plusmn 4892

MCV (fL) 5177 plusmn 118 5233 plusmn 093 5279 plusmn 090 5384 plusmn 080

MCH (pg) 1337 plusmn 031 1336 plusmn 039 1309 plusmn 038 1331 plusmn 025

MCHC (gL) 25814 plusmn 372 25538 plusmn 612 24771 plusmn 492 24738 plusmn 437

RDW (CV) 1689 plusmn 298 1734 plusmn 092 1731 plusmn 080 1753 plusmn 070

MPV (fL) 553 plusmn 068 580 plusmn 055 539 plusmn 047 570 plusmn 025

PDW () 1354 plusmn 081 1477 plusmn 081 1405 plusmn 079 1506 plusmn 087

lowastP le 005 compared to control group

323 Maximal Tolerance Dose Due to P(ECA-IA-MEG)hydrogelrsquos excellent biocompatibility no lethal dose ormedian lethal dose could be obtained thus MTD methodwas used as an alternative to evaluate the acute toxicity ofP(ECA-IA-MEG) hydrogel Sincemortality rates remain zeroafter being treated with highest tested dose MTD of P(ECA-IA-MEG) hydrogel was higher than 10000mgkg bodyweightby oral administration for BALBc mice Higher dose couldcause side effects and possibly lead to death This is due tothe limited gastric capacity of mice rather than the toxicityof the hydrogel In order to ensure that all toxicity wascaused by P(ECA-IA-MEG) hydrogel instead of excessivevolume dose was designed at 7500mgkg for the rest of theexperiments

324Hematology Routine Test and SerumChemistryAnalysisThe purpose of hematology routine test was to investigatewhether the P(ECA-IA-MEG) hydrogel could lead to theblood systemrsquos abnormality From the results listed in Table 4we could find that there were no important adverse effects inthe hematology variables both in male or female treatmentgroup Some statistically significant differences from control(119875 le 005) were observed in white blood cell count (femaletreatment group 74 of control) and in hematocrit (maletreatment group 107 of control) However these differencewere not considered to be adverse because the differenceswere relatively minor in magnitude (26 lowerWBC and 7higher HCT resp)

The serum chemistry analysis was designed to check theliver functions (ALB ALP ALT AST TBIL and TP) renal

functions (BUN and CREA) and blood sugar level (GLU)In Table 5 compared with the control group no importantbiologically adverse effects were observed in all the listedclinical chemistry parameters in both male and treatmentgroups Additionally statistically significant increases (119875 le005) were observed in blood urea nitrogen and creatinine inthe female treatment (111 and 125 of control group resp)These differences were also relatively small inmagnitude (11higher in BUN and 25 higher in CREA)

Both of these results indicated that P(ECA-IA-MEG)hydrogel did not affect the bloody system liver function andrenal function of mice However it was difficult to eliminatethe fluctuations between treatment group and control groupbecause the individual differences existed inevitably

33 Histopathologic Study No symptoms of poisoning wereidentified after giving P(ECA-IA-MEG) hydrogel by gavageat a dose of 7500mgkg body weight In order to tracethe toxicity caused by P(ECA-IA-MEG) hydrogel accuratelyhistopathologic changes of major organs were checked ina sequential order of 2 h 4 h 1 day 3 days and 7 daysrespectively

All samples of major organs obtained were observed bylight microscope but no obvious histopathological lesionswere found Since P(ECA-IA-MEG) hydrogels should beenmetabolized completely pictures for each organ at the timepoint of 7th day were demonstrated in this paper

Figures 7(a) and 7(b) exhibited the comparison betweenthe light microscopic images of cardiac muscle of treatmentgroup and control group on the 7th day Cardiac myocytes


Table 5 Serum chemistry analysis of acute oral toxicity testFemale control group Female treatment group Malecontrol group Male treatment group

ALB 3561 plusmn 157 3500 plusmn 076 3407 plusmn 135 3434 plusmn 113

ALP 22057 plusmn 1624 23638 plusmn 2960 22500 plusmn 2930 20600 plusmn 1589

ALT 5186 plusmn 1877 4375 plusmn 623 4243 plusmn 321 4213 plusmn 458

AST 113 plusmn 1460 10938 plusmn 1264 10200 plusmn 2688 10088 plusmn 1911

BUN 901 plusmn 055 1002 plusmn 110lowast


CREA 186 plusmn 146 233 plusmn 106lowast


GLU 361 plusmn 056 398 plusmn 054 537 plusmn 134 532 plusmn 088

TBIL 007 plusmn 019 027 plusmn 024 037 plusmn 033 021 plusmn 022

TP 6254 plusmn 337 6078 plusmn 189 6031 plusmn 246 6144 plusmn 200


displayed orderly and clearly without any inflammatoryexudate necrosis or hemorrhage

Figures 7(c) and 7(d) presented difference between theoptical micrograph of livers of animals treated with andwithout P(ECA-IA-MEG) hydrogels on the 7th day Underthe microscope no obvious degeneration and necrosis werefound The dividing lines of liver lobules were clear andthe hepatic cord arranged in neat order No hypertrophyand hyperemia were observed on hepatic sinusoid andno neutrophil lymphocyte or macrophage infiltration wasfound

As shown in Figures 7(e) and 7(f) there was littledifference between the hydrogel-treated lung and the controlgroup Additionally the tissue structure of hydrogel-treatedlung did not show any bronchioles alveoli ectasia or collapsemeanwhile no inflammatory cell infiltration surrounding thebronchus was identified

The structure of spleen for both groups was representedIn Figures 7(g) and 7(h) No pathologic changes were showedthus the spleen sinus was absolutely normal without show-ing any pathologic changes

Figures 7(i) and 7(j) exhibited the cytopathologic photosof mice kidney The treated kidney was in ordinary shapecompared with the control one No degeneration bleedingnecrosis was showed within renal glomerulus and variouskidney tubes

The cytopathologic photographs of gastrointestinal tractwere also observed (Figures 7(k)ndash7(t)) The gastrointestinaltract included stomach duodenum jejunum ileum andcolon From these photographs it was easy to find that gastricglands and intestinal glands were in regular arrangementmucosa cells were clear and the basement membranes wereintact Haemorrhage hydropsy inflammatory cell infiltra-tion degeneration and necrosis were largely absent in thehydrogel-treated experimental group as well as the controlgroup

Figures 7(u) and 7(v) described the light micrograph ofmale mice testis Histopathologic examination of the sper-mary showed no significant pathologic changes

4 Conclusion

In this paper a kind of pH-sensitive P(ECA-IA-MEG) hydro-gel was successfully prepared by heat-initiated free radical

polymerization method The chemical and physical charac-terization reflected that the hydrogel was pH-sensitive Theresults of acute oral toxicity evaluation showed that therewas no toxic response or histopathological changes causedby P(ECA-IA-MEG) hydrogel in BALBc mice by gastricperfusionThus the hydrogel prepared in this papermight bea safe candidate for application in biomedical field especiallyin oral drug delivery system

Acknowledgments

This work was financially supported by the National NaturalScience Foundation (NSFC31271021 and NSFC31222023) andthe Distinguished Young Scholars of Sichuan University(2011SCU04B18)

References

[1] L E Bromberg and E S Ron ldquoTemperature-responsive gelsand thermogelling polymer matrices for protein and peptidedeliveryrdquoAdvancedDrugDelivery Reviews vol 31 no 3 pp 197ndash221 1998

[2] J Zhang L-Y Chu Y-K Li and Y M Lee ldquoDual thermo-and pH-sensitive poly(N-isopropylacrylamide-co-acrylic acid)hydrogels with rapid response behaviorsrdquo Polymer vol 48 no6 pp 1718ndash1728 2007

[3] X Qu A Wirsen and A-C Albertsson ldquoNovel pH-sensitivechitosan hydrogels swelling behavior and states of waterrdquoPolymer vol 41 no 12 pp 4589ndash4598 2000

[4] P Y Yeh P Kopeckova and J Kopecek ldquoBiodegradable andpH-sensitive hydrogels synthesis by crosslinking of N N-dimethylacrylamide copolymer precursorsrdquo Journal of PolymerScience A vol 32 no 9 pp 1627ndash1637 2003

[5] T Wang M Turhan and S Gunasekaran ldquoSelected propertiesof pH-sensitive biodegradable chitosan-poly(vinyl alcohol)hydrogelrdquoPolymer International vol 53 no 7 pp 911ndash918 2004

[6] K Zhang Y Luo and Z Li ldquoSynthesis and characterization ofa pH- and ionic strength-responsive hydrogelrdquo Soft Materialsvol 5 no 4 pp 183ndash195 2007

[7] J I Lee and H S Yoo ldquoBiodegradable microspheres con-taining poly(120576-caprolactone)-Pluronic block copolymers fortemperature-responsive release of proteinsrdquo Colloids and Sur-faces B vol 61 no 1 pp 81ndash87 2008

[8] M J Mc Gann C L Higginbotham L M Geever and MJ D Nugent ldquoThe synthesis of novel pH-sensitive poly(vinyl


alcohol) composite hydrogels using a freezethaw process forbiomedical applicationsrdquo International Journal of Pharmaceu-tics vol 372 no 1-2 pp 154ndash161 2009

[9] H He J Guan and J L Lee ldquoAn oral delivery device based onself-folding hydrogelsrdquo Journal of ControlledRelease vol 110 no2 pp 339ndash346 2006

[10] X Wei C Gong M Gou et al ldquoBiodegradable poly(120576-capro-lactone)-poly(ethylene glycol) copolymers as drug deliverysystemrdquo International Journal of Pharmaceutics vol 381 no 1pp 1ndash18 2009

[11] M Hamidi A Azadi and P Rafiei ldquoHydrogel nanoparticles indrug deliveryrdquo Advanced Drug Delivery Reviews vol 60 no 15pp 1638ndash1649 2008

[12] B Kim andN A Peppas ldquoIn vitro release behavior and stabilityof insulin in complexation hydrogels as oral drug deliverycarriersrdquo International Journal of Pharmaceutics vol 266 no1-2 pp 29ndash37 2003

[13] Y Qiu and K Park ldquoEnvironment-sensitive hydrogels for drugdeliveryrdquo Advanced Drug Delivery Reviews vol 53 no 3 pp321ndash339 2001

[14] M K Nguyen C T Huynh and D S Lee ldquopH-sensitive andbioadhesive poly(120573-amino ester)-poly(ethylene glycol)-poly(120573-amino ester) triblock copolymer hydrogels with potential fordrug delivery in oral mucosal surfacesrdquo Polymer vol 50 no 22pp 5205ndash5210 2009

[15] N M Ranjha J Mudassir and N Akhtar ldquoMethyl metha-crylate-co-itaconic acid (MMA-co-IA) hydrogels for controlleddrug deliveryrdquo Journal of Sol-Gel Science and Technology vol 47no 1 pp 23ndash30 2008

[16] P Gupta K Vermani and S Garg ldquoHydrogels from controlledrelease to pH-responsive drug deliveryrdquo Drug Discovery Todayvol 7 no 10 pp 569ndash579 2002

[17] WWu J Liu S Cao et al ldquoDrug release behaviors of a pH sen-sitive semi-interpenetrating polymer network hydrogel com-posed of poly(vinyl alcohol) and star poly[2-(dimethylamino)ethyl methacrylate]rdquo International Journal of Pharmaceuticsvol 416 no 1 pp 104ndash109 2011

[18] D Velasco C B Danoux J A Redondo et al ldquoPH-sensitivepolymer hydrogels derived from morpholine to prevent thecrystallization of ibuprofenrdquo Journal of Controlled Release vol149 no 2 pp 140ndash145 2011

[19] X Yu andM V Pishko ldquoRelease of paclitaxel from pH sensitiveand biodegradable dextran based hydrogelsrdquo Soft Matter vol 7no 19 pp 8898ndash8904 2011

[20] Z Zhang L Chen M Deng Y Bai X Chen and X JingldquoBiodegradable thermo- and pH-responsive hydrogels for oraldrug deliveryrdquo Journal of Polymer Science A vol 49 no 13 pp2941ndash2951 2011

[21] R Gong C Li S Zhu Y Zhang Y Du and J Jiang ldquoA novelpH-sensitive hydrogel based on dual crosslinked alginateN-120572-glutaric acid chitosan for oral delivery of proteinrdquoCarbohydratePolymers vol 85 no 4 pp 869ndash874 2011

[22] S Freiberg andX X Zhu ldquoPolymermicrospheres for controlleddrug releaserdquo International Journal of Pharmaceutics vol 282no 1-2 pp 1ndash18 2004

[23] F IemmaU G Spizzirri F Puoci et al ldquopH-Sensitive hydrogelsbased on bovine serum albumin for oral drug deliveryrdquo Inter-national Journal of Pharmaceutics vol 312 no 1-2 pp 151ndash1572006

[24] J-Y Yoon H-Y Park J-H Kim andW-S Kim ldquoAdsorption ofBSA on highly carboxylated microspheresmdashquantitative effects

of surface functional groups and interaction forcesrdquo Journal ofColloid and Interface Science vol 177 no 2 pp 613ndash620 1996

[25] R Perret and A Skoulios ldquoSynthese et caracterisation decopoly-mers sequences polyoxyethylenepoly(120576-caprolactone)rdquoDie Makromolekulare Chemie vol 156 no 1 pp 143ndash156 1972

[26] S Y Kim Y M Lee D J Baik and J S Kang ldquoToxic charac-teristics of methoxy poly(ethylene glycol)poly(120576-caprolactone)nanospheres In vitro and in vivo studies in the normal micerdquoBiomaterials vol 24 no 1 pp 55ndash63 2003

[27] Y Hu L Zhang Y Cao H Ge X Jiang and C Yang ldquoDegra-dation behavior of poly(120576-caprolactone)-b-poly(ethyleneglycol)-b-poly(120576-caprolactone) micelles in aqueous solutionrdquoBiomacromolecules vol 5 no 5 pp 1756ndash1762 2004

[28] KWang S Z Fu Y CGu et al ldquoSynthesis and characterizationof biodegradable pH-sensitive hydrogels based on poly(120576-caprolactone) methacrylic acid and poly(ethylene glycol)rdquoPolymer Degradation and Stability vol 94 no 4 pp 730ndash7372009

[29] J Song Y C Gu X Xu et al ldquoSynthesis and characterizationof pH-sensitive hydrogel based on methoxyl poly(ethyleneglycol) poly(120576-caprolactone) and itaconic acid for delivery ofdoxorubicinrdquo Advanced Science Letters vol 16 no 1 pp 130ndash136 2012

[30] C Shen S Guo and C Lu ldquoDegradation behaviors of mon-omethoxy poly(ethylene glycol)-b-poly(120576-caprolactone) nano-particles in aqueous solutionrdquo Polymers for Advanced Technolo-gies vol 19 no 1 pp 66ndash72 2008

[31] X Wang B Kan Y Wang et al ldquoPharmaceutical nanotechnol-ogy safety evaluation of amphiphilic three-armed star-shapedcopolymermicellesrdquo Journal of Pharmaceutical Sciences vol 99no 6 pp 2830ndash2838 2010

[32] XChen ZQianMGou et al ldquoAcute oral toxicity evaluation ofbiodegradable and pH-sensitive hydrogel based on polycapro-lactone poly(ethylene glycol) and methylacrylic acid (MAA)rdquoJournal of Biomedical Materials Research A vol 84 no 3 pp589ndash597 2008

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



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Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



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MaterialsJournal of


Nano

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Journal ofNanomaterials


ability Moreover the IA was water-soluble and we could usewater as the solvent This would avoid the use of organicsolvents considering the safety of hydrogel The obtainedhydrogels showed good pH-sensitivity and were employedto delivery protein dexamethasone DOX-loaded nanopar-ticles and so on

Until now we had studied the cytotoxicity of MPEG-PCL-AC (PECA) copolymer primarily byMTTmethod [29]however the toxicological study on pH-sensitive hydrogelbased on PECA copolymer has not been thoroughly exam-ined Since the safety evaluation of the smart hydrogel canhave a significant implication on its further applicationsas a biocompatible carrier for oral drug delivery systemin this paper the acute oral toxicity evaluation of pH-sensitive hydrogel based on MPEG PCL and itaconic acidwas performed

2 Materials and Methods

21 Materials Among the whole materials poly(ethyleneglycol) methyl ether (MPEG Mn = 2000) 120576-caprolactone (120576-CL) NN1015840-Methylenebisacrylamide (BIS) itaconic acid (IA)poly(ethylene glycol) methyl ether methacrylate (MPEGMAMn = 475) tin (II) 2-ethylhexanoate acryloyl chloride (AC)triethylamine (TEA) and ammoniumpersulfate (98) (APS)were purchased from Aldrich Company USA For the restof the reagents they were used as received All the reagentswere completely analytic grade Male and female BALBcmice were purchased from the Laboratory Animal Center ofSichuan University

22 Synthesis of PECA Macromonomer and P(ECA-IA-MEG)Hydrogel The MPEG-PCL copolymer was synthesized byring-opening polymerization of 120576-caprolactone initiated byMPEG using tin (II) 2-ethylhexanoate as catalyst [29 30]And the PECA macromonomer was obtained by dissolvingMPEG-PCL in dewatered methylene chloride to react withAC which has been reported before [29]

With APS as the initiator and BIS as the cross-linkingagent the P(ECA-IA-MEG) hydrogel was synthesized byheat-initiated free radical method As shown in Table 1the predetermined amounts of PECA IA MPEGMA BISand APS were dissolved in water Then the entire systemwas soaked and bathed in water at 37∘C for 4 hours Theobtained hydrogel was immersed in a plenty of distilledwater for 7 days and the water was refreshed everyday toremove the unreacted substances The purified hydrogelswere freeze-dried kept Finally the freeze dried hydrogelswere grinded into powder and suspended in 1 aqueoussodium carboxymethyl cellulose (CMC-Na) solution foracute oral toxicity test

23 Characterization of PECA Copolymer and P(ECA-IA-MEG) Hydrogel

231 1H-Nuclear Magnetic Resonance Analysis (1H-NMR)The structure of PECA copolymer was analyzed by 1H-NMRspectrum which was recorded on Varian 400 spectrometer

Table 1 The hydrogel prepared in this work

PECA MPEGMA IA BIS content (wt) IA Content (wt)3 3 4 83 166

(Varian USA) at 400MHz using deuterated chloroform(CDCl

3) as solvent and tetramethylsilane (TMS) as internal

reference standard

232 Fourier Transform Infrared (FTIR) Analysis The chem-ical structures of PECA copolymer and P(ECA-IA-MEG)hydrogel were analyzed by the FTIR (KBr) spectrum whichwere recorded on Nicolet 200SXV meter (Nicolet USA)The samples were scanned at wave number range of 4000ndash400 cmminus1

233 Scanning Electron Microscopy (SEM) Themorphologyof the P(ECA-IA-MEG) hydrogel was recorded on JEOL SEM(JSM-5900LV JEOL Japan)The hydrogels were immersed inthe aqueous solution of pH 12 and pH 68 respectively Andthe samples were lyophilized transected and sputtered goldbefore observed

234 Swelling Behavior of the Hydrogels The hydrogels wereimmersed in aqueous medium with different pH conditions(pH 12 and pH 74) at 37∘C for 1 2 4 8 24 48 and 72 hrespectivelyThen theywere taken out and the surplus surfacewater was removed by filter paper This experiment wasrepeated 3 times and the wet weights of them were recordedcarefully The swelling ratios could be calculated by thefollowing equation

Swelling ratio (SR) =119882119905

1198820

times 100 (1)

where1198820was the initial dryweight and119882

119905was thewetweight

of the hydrogel at time 119905 respectively

24 Acute Toxicity Test

241 Maximal Tolerance Dose (MTD) Owing to the greatbiocompatibility and the swelling ability of P(ECA-IA-MEG)hydrogel no lethal dose or median lethal dose (LD

50) could

be obtained according to our preliminary trials So theMaximal Tolerance Dose (MTD) method was adopted toevaluate the acute oral toxicity of the hydrogel

All BALBc mice were housed individually in a standardanimal room maintained at a temperature of 20ndash22∘C anda relative humidity of 50ndash60 The animals which had anaverage weight of 20 g were 9-10 weeks old on the day ofdosing Twenty mice of both sexes were divided into twogroups hydrogel group (119899 = 10 5 male and 5 female mice)and control group (119899 = 10 5 male and 5 female mice) Allthe mice were fasted approximately 15 h prior to dosing andwater was provided continuously Hydrogel group was givenP(ECA-IA-MEG) hydrogel suspension twice at an intervalof four hours by gavage at a total dose of 10000mgkgbody weight and a total volume of 04mL10 g body weight


OO O O

OO

O

OO

OOO

OH Hx x

x

y

y

OCH3 130∘C

CH3

CH3

(MPEG-PCL)

40∘C H2C C C

HCl


APS (initiator)37

∘C


MPEG-PCLIA

MPEGMABIS

+

(MPEG)(120576-CL)

BIS (cross-linking)

Sn(Oct)2











37∘C

4h

(a) (b)


80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

05

(ppm)









2ndash) of


4000 3500 3000 2500 2000 1500 1000 500 0

Hydrogel

C=C

PECA


Tran

smitt

ance





(a) (b)


0 1 2 3 4 5 6 7 815

16

17

18

19

20

21

22

23



Mea

n w

eigh

t of t

he m

ice (

g)

Time (day)

















(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

(u) (v)









mortality Death


Femalecontrol group


Malecontrol group

Maletreatment group











































4 Conclusion



Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




OO O O

OO

O

OO

OOO

OH Hx x

x

y

y

OCH3 130∘C

CH3

CH3

(MPEG-PCL)

40∘C H2C C C

HCl


APS (initiator)37

∘C


MPEG-PCLIA

MPEGMABIS

+

(MPEG)(120576-CL)

BIS (cross-linking)

Sn(Oct)2











37∘C

4h

(a) (b)


80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

05

(ppm)









2ndash) of


4000 3500 3000 2500 2000 1500 1000 500 0

Hydrogel

C=C

PECA


Tran

smitt

ance





(a) (b)


0 1 2 3 4 5 6 7 815

16

17

18

19

20

21

22

23



Mea

n w

eigh

t of t

he m

ice (

g)

Time (day)

















(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

(u) (v)









mortality Death


Femalecontrol group


Malecontrol group

Maletreatment group











































4 Conclusion



Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




37∘C

4h

(a) (b)


80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

05

(ppm)









2ndash) of


4000 3500 3000 2500 2000 1500 1000 500 0

Hydrogel

C=C

PECA


Tran

smitt

ance





(a) (b)


0 1 2 3 4 5 6 7 815

16

17

18

19

20

21

22

23



Mea

n w

eigh

t of t

he m

ice (

g)

Time (day)

















(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

(u) (v)









mortality Death


Femalecontrol group


Malecontrol group

Maletreatment group











































4 Conclusion



Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)


0 1 2 3 4 5 6 7 815

16

17

18

19

20

21

22

23



Mea

n w

eigh

t of t

he m

ice (

g)

Time (day)

















(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

(u) (v)









mortality Death


Femalecontrol group


Malecontrol group

Maletreatment group











































4 Conclusion



Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

(u) (v)









mortality Death


Femalecontrol group


Malecontrol group

Maletreatment group











































4 Conclusion



Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








mortality Death


Femalecontrol group


Malecontrol group

Maletreatment group











































4 Conclusion



Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
























4 Conclusion



Acknowledgments


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



research article synthesis, characterization, and acute...

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