Thermosensitive PEG-PCL-PEG hydrogel controlled drug delivery system: Sol-gel-sol transition and in...

Thermosensitive PEG–PCL–PEG Hydrogel ControlledDrug Delivery System: Sol–Gel–Sol Transition andIn Vitro Drug Release Study

CHANG YANG GONG, PENG WEI DONG, SHUAI SHI, SHAO ZHI FU, JIN LIANG YANG, GANG GUO,XIA ZHAO, YU QUAN WEI, ZHI YONG QIAN

State Key Laboratory of Biotherapy, West China Hospital, and School of Life Science, Sichuan University,Chengdu 610041, China

Received 24 September 2008; revised 5 December 2008; accepted 19 December 2008

Published online 2 February 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21694

Dong PW anCorresponde

85164063; Fax:E-mail: anderso

Journal of Pharm

� 2009 Wiley-Liss

ABSTRACT: In this article, biodegradable and low molecular weight poly(ethyleneglycol)–poly(e-caprolactone)–poly(ethylene glycol) (PEG–PCL–PEG, PECE) triblockcopolymers were successfully synthesized. Aqueous solution of the obtained PECEcopolymers underwent sol–gel–sol transition as temperature increased which wasflowing sol at room temperature and then turned into nonflowing gel at body tempera-ture. Sol–gel–sol phase transition behaviors of aqueous PECE solutions were studiedusing rheometry and test tube-inverting method, which were affected by many factors,including the heating/cooling procedure and different additives in copolymers aqueoussolution. In vitro drug release behavior was studied using bovine serum albumin (BSA)and Vitamin B12 (VB12) as model drugs, and the PECE hydrogel could protect BSA fromacidic degradation for 1 week at least. Therefore, PECE hydrogel is believed to bepromising for injectable in situ gel-forming controlled drug delivery system due to theirgreat thermosensitivity and biodegradability. � 2009 Wiley-Liss, Inc. and the American

Pharmacists Association J Pharm Sci 98:3707–3717, 2009

Keywords: biodegradable polymers;
thermosensitive; hydrogel; injectable; sol–gel–sol transition; drug release; protein delivery
INTRODUCTION

For the last decades, injectable hydrogels havegained considerable attention in biomedicinefields including controlled drug delivery system,implants, and tissue engineering applications.1–4

Among those hydrogels, thermosensitive hydro-gels have been extensively studied due totheir smart responsibility to the environmental

d Gong CY contributed equally to this work.nce to: Zhi Yong Qian (Telephone: 86-28-86-28-85164060;[email protected])

aceutical Sciences, Vol. 98, 3707–3717 (2009)

, Inc. and the American Pharmacists Association

JOURNAL OF PHA

stimulus and great biocompatibility.5–9 N-isopro-pylacrylamide (PNiPAAM) copolymer and Pluro-nic copolymer were the most important ones,because they were flowing liquid at or belowambient temperature and formed gels at physio-logical temperatures.10–15 However, Pluronicor PNiPAAM copolymers were not suitable forin vivo drug delivery, as they were not biodegrad-able and could be accumulated in the body.14,15

Therefore, biodegradable thermosensitive hydro-gels have been investigated as controlled drugdelivery system due to their great biodegrada-bility and nontoxicity. In recent researches, manybiodegradable thermosensitive hydrogels havebeen studied, such as poly(ethylene glycol)-poly(L-lactide) (PEG-PLLA) diblock or triblock

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3708 GONG ET AL.

copolymers,16,17 poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) (PEG-PLGA) diblock or tri-block copolymers,18–21 chitosan derivatives,22–25

methylcellulose, polyphosphazene, and etc.In our last contribution,26 we prepared biode-

gradable and injectable thermosensitive poly(ethylene glycol)–poly(e-caprolactone)–poly(ethy-lene glycol) (PEG–PCL–PEG, PECE) hydrogel thatunderwent sol–gel–sol transition, and the struc-ture-property relationship of the sol–gel–sol tran-sition was investigated, including PCL length,total molecular weight, and topological variation.Furthermore, the cytotoxicity assay and in vitrodegradation behavior of PECE hydrogel werestudied. The in vivo gel formation and degradationtest upon subcutaneous injection of PECE hydrogelin the mice model was also conducted.26

In this work, sol–gel–sol transition behavior ofthe prepared PECE hydrogels and many factorsaffecting the phase transition behavior wereinvestigated, including the heating/cooling pro-cess, aging time, and different additives incopolymers aqueous solution. Moreover, in vitrodrug release behavior and acidic degradationprotection assay were also investigated in detail.This work is the subsequent for the last work,which is very important for the further applica-tion of PECE hydrogel in clinic.26

MATERIALS AND METHODS

Materials

Poly(ethylene glycol) methyl ether (MPEG, Mn¼550 and 750, Aldrich, St. Louis, MO), Poly(ethy-lene glycol) (PEG, Mn¼ 2000, Fluka, St. Gallen,Switzerland), e-Caprolactone (e-CL, Alfa Aesar,Ward Hill, MA), Hexamethylene diisocyanate(HMDI, Aldrich), Stannous octoate (Sn(Oct)2,Sigma, St. Louis, MO), bovine serum albumin(BSA, BR, BoAo Co., Shanghai, China) and VB12

(Sigma) were used without any further purifica-tion. All the materials except BSA used in thisarticle were analytic reagent grade and used as

Table 1. The PECE Copolymers Prepared in This Work

Code CopolymerPEG/PCLa

(Theoretical)Total Mn

a

(Theoretical)

S1 E550–C2000–E550 1100/2000 3100S2 E750–C3000–E750 1500/3000 4500S3 E750–C2500–E750 1500/2500 4000

aTheoretical value, calculated according to the feed ratio.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

received. Water was ultra-purified by Milliporesystem (pH 7.0).

Synthesis and Purification of PECE Copolymers

PECE triblock copolymers were synthesized bycoupling PEG-PCL diblock copolymers usingHMDI as coupling agent, according to the protocoldescribed previously.26,27 In this article, thecopolymers were denoted as EY–C2X–EY, where2X and Y represented the number averagemolecular weight (Mn) of PCL and PEG blockrespectively. The PECE copolymers prepared herewere listed in Table 1.

The just-obtained PECE block copolymers werefirstly dissolved in dichloromethane, and repreci-pitated from the filtrate using excess cold petro-leum ether. Then the mixture was filtered andvacuum dried to constant weight at room tem-perature. The purified copolymers were kept inair-tight bags before further use.

Characterization of PECE Copolymers andPECE Hydrogels

Fourier Transform Infrared Spectroscopy (FTIR)

The copolymer samples were dissolved in chloro-form and casted on KBr plates. FTIR spectra wererecorded on a NICOLET 200SXV Infrared Spec-trophotometer (Nicolet, Boston, MA).

Nuclear Magnetic Resonance Analysis (1H-NMR)

1H-NMR spectra (in CDCl3) were recorded on aVarian 400 spectrometer (Varian, Palo Alto, CA)at 400 MHz to characterize chemical compositionand molecular weight of the copolymers.

Gel Permeation Chromatography (GPC)

GPC (Agilent 110 HPLC, Santa Clara, CA) wasused to determine the molecular weight and dis-tribution of PECE copolymers. The samples weredissolved in freshly distilled tetrahydrofuran

PEG/PCL(1H-NMR)

Total Mn(1H-NMR)

Total Mn

(GPC)PDI

(GPC)

1055/2181 3236 2992 1.261518/3116 4634 5046 1.181481/2433 3914 4373 1.24

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PEG–PCL–PEG HYDROGEL CONTROLLED DRUG DELIVERY SYSTEM 3709

(THF) at a concentration of 1–2 mg/mL. THF waseluted at a rate of 1.0 mL/min through two WatersStyragel HT columns and a linear column. Theexternal and column temperature were kept at358C. The molecular weights of samples werecalculated from polystyrene standard with narrowmolecular weight distribution.

Scanning Electron Microscopy (SEM)

SEM was employed to investigate morphology ofPECE hydrogel. The hydrogels were frozen inliquid nitrogen and lyophilized for 72 h. Then, thehydrogels were sputtered with gold before obser-vation. In this study, morphology of preparedparticles was examined on JEOL SEM (JSM-5900LV, JEOL, Tokyo, Japan).

Sol–Gel–Sol Phase Transition Behavior

Sol–gel–sol transition of PECE hydrogel wasinvestigated by rheometry (AR 2000ex rheometer,TA Instruments, New Castle, DE). The aqueousPECE copolymer solution was placed betweenparallel plates of 40 mm diameter and a gap of31 mm. The data were collected under a controlledstress (4.0 dyn/cm2) and a frequency of 1.0 rad/s.The heating rate was 18C/min.

Sol–gel–sol phase transition diagram of PECEhydrogel was also recorded using test tube-inverting method with a 10-mL tightly screw-capped vial with inner diameter of 13 mm.26,27

The sol–gel–sol transition was visually observedby inverting the vials, and conditions of sol and gelwere defined as ‘‘flow liquid sol’’ and ‘‘no flow solidgel’’ in 1 min respectively.17,20,21,28,29

In this study, two procedures were performed toinvestigate sol–gel–sol phase transition behavior,which were shown as follows:

Heating Procedure

Each sample at given concentration was preparedby dissolution in deionized water at a designatedtemperature. The volume of the solution was kept1 mL in total regardless of the concentration. Afterincubated in water bath at 08C for 20 min, thehydrous samples were slowly heated at a rate of0.58C/min, from 08C to the temperature whenprecipitation occurred.

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Cooling Procedure

The sample was first performed like in heatingprocedure. And after the precipitation occurred,the sample was then cooled to 08C at 0.58C/min.

In Vitro Drug Release Behavior

VB12 was added into aqueous PECE solution at48C to form homogenous solution. Then thefreshly prepared VB12 loaded PECE hydrogelcomplexes were used to assay in vitro releasebehavior of hydrophilic small molecular drugs. Indetail, 200 ml of VB12 loaded PECE hydrogelcomplexes containing 2 mg VB12 were placed into5 mL-Eppendorf (EP) tubes and allowed to gel inan incubator at 378C for 12 h. Then, the gels wereimmersed in 1 mL of PBS (pH 7.4) or HCl/H2O solution (pH 1.2) and then were shaken at100 rpm at 378C. At specific time intervals, all ofrelease media were removed and replaced by freshrelease media. After centrifuged at 13,000 rpm for10 min, the supernatant of the removed releasemedia were collected and stored at �208C beforeanalysis. The collected supernatants weredetected on UV spectrophotometer at 362 nm todetermine the concentration of VB12.

BSA was used as model hydrophilic proteindrug to determine the release behavior of proteinor peptide from PECE hydrogel in vitro. Thedetailed process was similar to the in vitro releasestudy of VB12, but the initial drug loading amountwere 4 and 8 mg respectively and the hydrogelconcentration was 20 wt%. The amount of BSApresent in the supernatant was determined bybicinchoninic acid (BCA) assay and BCATM

Protein Assay Kit (Pierce, Holmdel, NJ) was used.The SDS–polyacrylamide gel electrophoretic(PAGE) analysis was used to assay the stabilityof BSA in the supernatant. All the release studyexperiments were repeated three times.

Acidic Degradation Protection Assay

A series of BSA loaded PECE hydrogel sampleswere incubated in 1 mL of HCl (0.1 M) at 378C inair shaker bath (100 rpm). At each time intervals,1 mL of NaOH (0.1 M) was added to one of thesamples to neutralize HCl. Then the medium wasreplaced by 1 mL of fresh PBS (prewarmed to378C), and the hydrogel was destroyed by mildagitation. The solution was centrifuged under13,000 rpm for 10 min and the supernatant wascollected for SDS–PAGE test.

AL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

Figure 2. 1H-NMR spectra of PECE copolymer inCDCl3.

3710 GONG ET AL.

RESULTS

Synthesis and Characterization of PECE Copolymers

A series of PEG–PCL diblock copolymerswere prepared by ring-opening copolymerizationof e-CL initiated by MPEG using stannous octoateas catalyst, and biodegradable PECE triblockcopolymers were synthesized from PEG–PCLdiblock copolymers using HMDI as couplingagent. FTIR, 1H-NMR, and GPC were used tocharacterize the chemical structure of PECEcopolymers.

Figure 1 presented typical FTIR spectra ofPEG–PCL (E550–C1000) diblock copolymer (A) andPECE (E550–C2000–E550) triblock copolymer (B).The absorption bands at 1104 cm�1 (Fig. 2A)and 1243 cm�1 (Fig. 1A) were attributed tothe characteristic C–O–C stretching vibrationsof the repeated –OCH2CH2 units of PEG and the –COO– bands stretching vibrations respectively.A strong C––O stretching band appeared at1721 cm�1 (Fig. 1A), which was attributed tothe ester bond. There is no absorption in 2250–2270 cm�1(Fig. 1B), which indicates that –NCOgroups of hexamethylene diisocyanate disap-peared completely due to coupling reaction of –NCO with –OH group. The absorption bands at1528 cm�1 (Fig. 1B) were attributed to the N–Hbending vibrations, which confirmed the forma-tion of PECE triblock copolymers.

In order to further confirm the formation ofPECE triblock copolymer, 1H-NMR spectra ofPECE triblock copolymer was recorded and shown

Figure 1. FTIR spectra (KBr) of PCL–PEG (A) andPECE (B) copolymers.


in Figure 2. The characteristic absorption peakswere also designated. In Figure 2, the sharp peakat 3.60 and 3.38 ppm are attributed to methyleneprotons of –CH2CH2O– and –OCH3 end group inPEG block respectively. Peaks at 1.35, 1.62, 2.30,and 4.06 ppm are assigned to methylene protons of–(CH2)3–, –OCCH2–, and –CH2OOC– in PCLblocks, respectively. The very weak peaks at4.20 and 3.82 ppm are respectively attributed tomethylene protons of –O–CH2–CH2– in PEG endblock that linked with PCL blocks.

The number-average molecular weight of PECEblock copolymers and PEG/PCL block ratios werecalculated from 1H-NMR spectra according toEqs. (1)–(5):26,27

ID

IA¼ 2 ½4ðY � 1Þ�

4(1)

IE

IA¼ 4X

4(2)

MnðPEGÞ ¼ 2ð44Y þ 31Þ (3)

MnðPCLÞ ¼ 2ð144XÞ (4)

MnðPEG�PCL�PEGÞ ¼ MnðPEGÞ þ MnðPCLÞ

¼ ½2ð44Y þ 31� þ ½2ð144XÞ� (5)

where IA, ID, and IE were integral intensities ofpeaks at about 4.20, 3.60, and 4.06 ppm respec-tively in 1H-NMR spectrum of PECE copolymers(Fig. 2). And 2X and Y were the respective blocknumber of PCL and PEG respectively in macro-molecular structure of PECE copolymers asshown in Figure 2.

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Figure 3. GPC curve of prepared PECE copolymer.


Molecular weight and distribution of PECEcopolymers were also characterized by GPC.The typical GPC curve of E550–C2000–E550 wasshown in Figure 3. According to Figure 3,only a single peak existed, which indicated themono-distribution of molecular weight and theabsence of any homopolymer of e-CL or MPEG.The total molecular weight and polydispersity(PDI, Mw/Mn) of PECE copolymers were in therange of 3000 to 5000 and 1.18 to 1.26, respec-tively. Table 1 summarized the copolymers pre-pared in this work.

The molecular weight and PEG/PCL blockratios estimated from 1H-NMR spectrum werelisted in Table 1, which were consistent withtheoretical value calculated from feed ratio.Therefore, for simplicity, the feed ratio was usedin the following text instead of experimentalcomposition ratio calculated from 1H-NMR spec-tra. FTIR, 1H-NMR and GPC results indicatedthat the PECE triblock copolymer designed bycontrolling the feed composition were preparedsuccessfully.

Figure 4. Photograph and SEM photographhydrogel at 108C (right) and at 378C (left); (Bdetected by SEM.


Temperature-Dependent Sol–Gel–SolTransition Behavior

The PECE triblock copolymers prepared in thiswork are amphiphilic in nature due to combina-tion of hydrophobic PCL block and hydrophilicPEG block. The aqueous solution of PECEcopolymers based on central PCL blocks andend PEG blocks from S1 to S3 showed tempera-ture-dependent reversible sol–gel–sol phasetransition. The aqueous solution of PECE copoly-mers flowed freely at lower temperature, butturned into gel at body temperature about 378C(Fig. 4A).

Sol–gel–sol transition of aqueous PECE solu-tion accompanies with a significant change inmodulus. Figure 5 shows the change in storagemodulus (G0) and loss modulus (G00) of S1 aqueoussolution (30 wt%) as a function of temperature.The G0 in sol state was less than 2 Pa andincreased abruptly to greater than 100 Pa due tothe sol–gel transition as the temperatureincreased. The dramatic decrease in the G0 atabout 458C demonstrates the gel–sol transition ofthe aqueous copolymer solution.

Figures 6–8 presented the sol–gel–sol phasetransition diagram of PECE triblock copolymersaqueous solution. When the copolymer concentra-tions are above the critical gelation concentration(CGC), aqueous solutions of PECE copolymerschanged from ‘‘sol’’ phase to ‘‘gel’’ phase withincrease in temperature to the lower criticalgelation temperature (LCGT), which seems tobe driven by the micelle packing and aggregat-ing.30,31 With further increase in temperature toupper critical gelation temperature (UCGT), thesol phase occurs again, which might be due to

of PECE hydrogel (S1, 30 wt%). A: PECE) The morphology of the PECE hydrogel


Figure 5. Rheology analysis of PECE (S1) aqueoussolution (30 wt%) as a function of temperature.

Figure 7. Effect of additives on sol–gel–sol transitionphase diagram. A: Effect of NaCl concentration;B: Effect of NaSCN concentration.

3712 GONG ET AL.

the increase in molecular motion of PCL block andthe destruction of micelles structure.17,31

The sol–gel–sol transition behavior of PECEtriblock copolymers aqueous solutions wasaffected by many factors, including the heating/cooling process, aging time, and differentadditives in copolymers aqueous solution. Thedetailed results followed.

Effect of Heating/Cooling Procedure and Aging Time

Figure 6A showed the effect of heating/coolingprocedure of copolymer aqueous solution on sol–gel–sol transition behavior for S1, which wasdetermined by heating and cooling proceduresaccording to Sol–gel–sol Phase Transition Beha-vior Section, that is, the aqueous copolymersolution was heated at the rate of 0.58C, and thencooled to 08C at the same rate after the precipita-tion occurred. When the sol–gel–sol transitiondiagram was tested by cooling procedure, thehysteresis in transition temperature occurred.The UCGT decreased by about 128C, and the

Figure 6. Effect of administration process on sol–gel–sol transition phase diagram. A: Effect of heatingand cooling process; B: Effect of aging time.


LCGT decreased by about 48C, which meantthe gel region window shifted to lower tempera-ture than that tested by heating procedure.Because the decrease of UCGT is much sharperthan that of LCST, the gel phases of aqueouscopolymers solution at the concentration of 20 and25 wt% disappeared, which resulted in theincrease of CGC in cooling procedure.

Figure 6B presented the effect of aging time onsol–gel–sol transition of copolymer aqueous solu-tion. In this figure, the same samples preparedfreshly and aged at 48C for 24 h were comparedwith same treatment procedure (heating proceduredescribed in Sol–gel–sol Phase Transition Be-havior Section). Although the aging time wasdifferent, the phase transition diagrams wereapproximately the same. In other words, agingtime of PECE copolymer aqueous solution has verysmall influence on phase transition diagrams.

Figure 8. Effect of some factors on sol–gel–sol transi-tion phase diagram. A: Effect of PEG concentration; (B)effect of PBS solution; (C) effect of pH value.

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Figure 9. In vitro release behavior of VB12

from VB12/PECE (S1) hydrogel complexes in releasemedium with different pH values (n¼ 3).


Effect of NaCl and NaSCN on Hydrogel

NaCl is a kind of salting-out salt, and NaSCN is akind of salting-in salt, which would affect the sol–gel transition temperature.6 Figure 7 presentedthe influence of NaCl and NaSCN on sol–gel–soltransition of PECE hydrogel. In Figure 7A, NaClat low concentration caused the transition curveto be shifted to lower temperature withoutinfluencing CGC, whereas the NaSCN showedopposite effect according to Figure 7B. The influ-ence degree of these salts was concentration-dependent, where higher concentration led tolarger influence on transition diagram. At thesame concentration, the degree of NaCl andNaSCN influence on UCGT and LCGT wasapproximately the same, thus the transitiondiagram showed a vertical shift.

Effect of PEG Concentration, PBS Solution,and pH Value of Medium

Effect of PEG concentration on sol–gel–sol phasetransition diagram of copolymer aqueous solutionfor sample S2 is shown in Figure 8A. Sampleswith the same concentration were dissolved indeionized water containing different amount ofPEG (0, 2 wt%, and 5 wt%); and then determinedby the same treatment procedure. As was shownin Figure 8A, with increase of PEG concentrationfrom 0% to 5%, the LCGT and UCGT bothdecreased and the gelation curve shifted to thelower temperature region, while the CGC waskept constant.

Phosphate buffered saline (PBS, pH 7.4) solu-tion were widely used as drug release medium anddegradation medium. It is important to investi-gate the influence of PBS solution on the phasetransition behavior of PECE hydrogel system, andthe results were presented in Figure 8B. In PBSsolution, the LCGT and UCGT both decreasedabout 48C compared to aqueous solution, whichmeant the gelation window shifted to lowertemperature, whereas the CGC of copolymer inaqueous solution and normal saline solution waskept constant.

For investigating the effect of pH value on sol–gel–sol transition of PECE aqueous solution, HClwas added to the solution to adjust pH value to 1.2.According to Figure 8C, acidic medium have noobvious influence on the sol–gel–sol transitioncurve compared to ultrapure water.

The phase transition behavior of PECE copoly-mer hydrogel was affected by the factors mentionedabove, thus the sol–gel–sol transition temperature


range could be varied, which might be very usefulfor its application in biomedicine field.

In Vitro Drug Release Profile From PECE Hydrogel

In vitro release profiles of VB12 from VB12/PECEhydrogel complexes in PBS and HCl/H2O werestudied, and the results were shown in Figure 9.According to Figure 9, VB12 could be releasedfrom VB12/PECE hydrogel complexes in a sus-tained period and the pH value of release mediumhad great effect on VB12 release profile. In neutralmedium, VB12 was released faster, and thecumulative release (92.9%) was higher than thatin acidic medium (80.6%) in 336 h. In neutralmedium, an initial burst release of 22.7% ofloaded VB12 occurred in the first 1 h, followed byrelease of 80.9% in 1 day, whereas, in acidicmedium, the cumulative release rate of 1 h and1 day were 15.6% and 62.3% respectively.

In this study, BSA was used as a model drug toinvestigate in vitro release behavior of protein orpeptide drugs from PECE hydrogel, and the datawere summarized in Figure 10. As presented inFigure 10A, initial BSA loading amount substan-tially affected the BSA release behavior fromPECE hydrogel. The higher initial drug loadingamount resulted in slower release rate of BSAfrom PECE hydrogel. SDS–PAGE was performedto evaluate the stability of BSA in the in vitrorelease period. According to Figure 10B, the majorband for BSA appeared at about 67 kDa (lanes2–9) according to the protein marker (lane 1) andBSA standard sample (lane 10), which means thatBSA was stable in all the release period.


Figure 10. In vitro release behavior of BSA from BSA/PECE (S1) hydrogel complexeswith different initial drug loading (A) (n¼ 3) and SDS–PAGE results of BSA in vitrorelease profile (B) (Lane 1, marker; Lane 2, 1 h; Lane 3, 4 h; Lane 4, 12 h; Lane 5, 24 h;Lane 6, 72 h; Lane 7, 168 h; Lane 8, 240 h; Lane 9, 336 h; Lane 10, BSA standard).

3714 GONG ET AL.

Acidic Degradation Protection Assay

PECE hydrogel could protect BSA from acidicdegradation and the stability of the protein wasevaluated by SDS–PAGE method. According toFigure 11, BSA sample incubated with 0.1 M HClfor 2 h (lane 3) did not show the major band of BSAat about 67 kDa according to the protein marker(lane 1) and BSA standard sample (lane 2), whichindicated that BSA could be destroyed at acidmedium, whereas all the samples of BSAloaded PECE hydrogel incubated with 0.1 MHCl for 1–7 days show visible BSA major band,which means that PECE hydrogel could protectBSA from acidic degradation for 7 days at least.

Figure 11. SDS–PAGE results of acidic degradationprotection assay (lane 1, marker; lane 2, BSA standardsample; lane 3, BSA sample after incubated in 0.1 M HClfor 2 h; lane 4, sample of BSA loaded in PECE hydrogel(protection sample) after incubated in 0.1 M HCl for 2 h;lane 5, protection sample for 8 h; lane 6, protectionsample for 1 day; lane 7, protection sample for 2 days;lane 8, protection sample for 3 days; lane 9, protectionsample for 5 days; lane 10, protection sample for 7 days).


DISCUSSION

In our previous study,27 we have reported thegel–sol transition of PECE copolymer aqueoussolution and the many factors influenced thephase transition, such as chemical compositionand treatment procedure of the copolymers. Butthe transition curves of PECE triblock copolymersaqueous solution studied in this paper were quitedifferent from the results observed in our previouspaper.27 The phenomenon may be attributed tothe difference in chemical composition (PEG/PCLratio and total molecular weight) and treatmentprocedure of the copolymers in our present work.All the factors including chemical composition andtreatment procedure of the copolymers have greatinfluence on the phase transition curve of theaqueous copolymer solution. In our present paper,the chemical compositions of the copolymers(PEG/PCL< 3/5) are quite different from thosein our previous work (PEG/PCL> 1/1),27 thereforethe phase transition behaviors of the PECEcopolymers aqueous solution are also quitedifferent. Thus, by varying the chemical composi-tion of copolymers or treatment manner ofhydrous copolymers, the phase transition be-havior could be varied, which is very useful forthe application of PECE hydrogel in biomedicinefield.

In our last study,26 we have reported the sol–gel–sol phase transition of PECE triblock copoly-mers and the influence of chemical composition ofPECE copolymers and solution composition on thephase transition of PECE hydrogel. With increasein molecular weight of PCL, the gel region becamewider, and with the increase in the total molecular

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weight of the copolymer, the LCGT and UCGTincreased significantly. In this study, someother factors affected sol–gel–sol transitionbehavior of PECE hydrogel were investigated indetail, including the heating/cooling process,and different additives in copolymers aqueoussolution.

During the cooling procedure of PECE hydrogelphase transition test, hysteresis in transitiontemperature occurred. This hysteresis behavioris very common for block copolymer hydrogelincluding PEO–PPO–PEO (Pluronic1, Poloxa-mer1, Lutrol1) systems.32,33 The hysteresismight be attributed to the two primary reasons.First, the crystallization rate of melting PCL blockin water was very slow and the partial micro-crystalline structure could be formed only afterlonger time;34 In addition, the hydrophobic inter-action of the melt and swollen PCL blockhydrophobic clusters weakened on cooling, whichshould also cause the hysteresis in the phasetransition temperature than that on heatingprocedure.35,36

As shown in Figure 7, NaCl at a concentration of1 wt% caused the phase transition curve of PECEhydrogel to be shifted to lower temperaturewithout affecting CGC, whereas NaSCN dis-played an opposite effect. NaCl are known aswater structure making salt, and could cause asalt-out effect in water. Salt-out effect minimizedhydrogen bond between water and copolymer,which could obviously enhance the hydrophobicinteraction between the micelles, resulting in aphase transition at lower temperature.21,37 Theopposite effect is expected with a water structurebreaking salt. Water structure breaking salt iscapable of solubilizing the hydrophobic PCLblocks in water, inducing gelation at highertemperature.

In previous reports, free PEG could elevate thegelation temperature of Pluronic copolymers,because free PEG disturbs the Pluronic micellepacking.38 But free PEG resulted in the oppositeeffect on phase transition curve of PECE hydrogel,which could lower the gelation temperature. Thisphenomenon indicates that free PEG in watermight interact with micelles to enhance micelleaggregating rather than disturbing the micelleinteractions. This possibly caused by that freePEG in water interacts with the PEG shell whichis partly mixed with PCL core. And this interac-tion between free PEG and PECE micelleenhanced intermicellar interactions.21 In otherwords, free PEG act as bridges between the


micelles and thus lead to gelation at lowertemperature.

PBS and HCl/H2O solution were widely used todissolve various substances including some drugsor proteins. As a kind of drug delivery system, it isurgent to investigate the effect of PBS and HCl/H2O solution on the phase transition behavior ofPECE hydrogel system. The influence of PBSsolution on phase transition of PECE hydrogelseems to be contributed to the presence of NaCl inPBS solution. It was obvious that the sol–gel–soltransition behavior of PECE triblock copolymersaqueous solution depended on the differentsolutions mentioned above besides the additivesin the solution. And by altering the solutions ofcopolymers and additive in the aqueous solution,the temperature range of sol–gel–sol phasetransition could be varied, which might be veryuseful for its application in injectable drugdelivery system.

In vitro drug release behavior of PECE hydrogelwas studied and factors affected release behaviorwas also investigated. According to Figures 9–10,drugs including hydrophilic drug and protein drugcould be released from PECE hydrogel in asustained period, and drug release profiles wereaffected by initial drug loading and pH value ofrelease medium to some extent. Hydrophilicsmall molecular drug was almost completelyreleased from PECE hydrogel in a week withhigh release rate (>90% in 24 h) and high initialburst rate (about 20% in 1 h), whereas hydrophilicmacromolecular protein drug was released slowlyin a longer period with lower cumulative releaserate (45.8% for BSA in 14 days). Drug releasebehavior from hydrogel was generally driven bytwo forces: diffusion effect and erosion of thehydrogel.39 Because the hydrolytic degradationrate of poly(e-caprolactone) is very slow, drugrelease behavior of prepared PECE hydrogelmainly depended on diffusion effect. Because ofthe great solubility and small molecular size,hydrophilic small molecular drugs could diffusethrough the pores of hydrogel (Fig. 4B) in a shorttime and almost all the drug in hydrogel wasreleased. But for macromolecular protein drugs,low diffusion rate in water and strong intermo-lecular interactions with hydrogel dominated thedrug release profile, which resulted in lowerrelease rate.

It is very interesting that the pH value ofmedium did not affect sol–gel–sol transitionbehavior of PECE hydrogel significantly, but havegreat influence on drug release behavior of the


3716 GONG ET AL.

hydrogel. The phenomena might be caused bythe fact that hydronium (H3Oþ) in the medium didnot disturb the micelle packing and destruction ofmicelles (The micelle packing and destruction ofmicelles induced the lower sol–gel and highergel–sol transition of the hydrogel, respectively),but greatly affected the structure property anddiffusion property of the hydrogel (which wasrelated to the drug release behavior of thehydrogel). Bea et al.31 reported that pH value ofmedium significantly influenced the hydrogelmodulus (strength), which indicated that pHvalue had great influence on structure propertyof the hydrogel.

SUMMARY

A series of low molecular weight biodegradablePECE triblock copolymers were successfullysynthesized and then characterized by FTIR,1H-NMR, and GPC. The PECE copolymers aqu-eous solution undergoes sol–gel–sol transition asthe temperature increases. The sol–gel–sol tran-sition behavior of the copolymers was affectedsignificantly by many factors, such as the heating/cooling process and different additives in copoly-mers aqueous solution. As a result, the tempera-ture range of phase transition could be varied,which might be great useful for its application inmany fields such as controlled drug deliverysystem. Furthermore, in vitro drug releasebehavior indicated that drugs could release fromthe hydrogel in an extended period, and thehydrogel could protect protein from acidic degra-dation for at least 1 week. Owing to the greatthermosensitivity and biodegradability of thesehydrogels, a solvent-free (no organic solvent)injectable system could be designed as anin situ gel-forming controlled drug deliverysystem.

ACKNOWLEDGEMENTS

We would like to show our great thanks to Ms.Zheng Jing (Library of Chengdu University ofInformation Technology) for her great helpduring the literature retrieval and manuscriptpreparation. This work was financially support-ed by National 863 Project (2007AA021902and 2006AA03Z356), Specialized ResearchFund for the Doctoral Program of Higher Educa-tion (SRFDP 200806100065), National, Natural


Science Foundation (NSFC20704027), SichuanProminent Young Talents Program (07ZQ026-033), and Chinese Key Basic Research Program(2004CB518807).

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