ORIGINAL PAPER

Photo-crosslinked polyaspartamide hybrid gel containingthermo-responsive Pluronic triblock copolymer

Jong Hyun Park & Jong Rok Moon &

Kwang Hyun Hong & Ji-Heung Kim

Received: 11 September 2009 /Accepted: 22 February 2010 /Published online: 17 March 2010# Springer Science+Business Media B.V. 2010

Abstract Thermo-responsive hybrid hydrogels based onbiodegradable poly(N-2-hydroxyethyl-DL-aspartamide)(PHEA) and Pluronic were prepared by photo-crosslinkingof their methacryloyl (MA) derivatives in aqueous solution.Pluronic component is expected to exhibit, so called,reverse thermal gelation behavior within the swollen gelmatrix. Swelling properties of hybrid gels and theirdependence on the different MA content and the mediumtemperature were investigated. In addition the thermo-responsive change in the morphology of porous gels wasobserved by SEM.

Keywords Hydrogel . Thermo-responsive . Pluronic .

Photo-crosslinking . Swelling behavior . Polyaspartamide

Introduction

The importance of biodegradable and biocompatible hydro-gels is being increasingly recognized, and extensive studieshave been conducted on their uses in various biomedicalfields including drug delivery carriers, artificial muscles,biosensors, scaffolds for tissue engineering [1–5]. Amongthem, hydrogel systems that undergo sol-gel and volumetransition in response to environmental stimuli such astemperature and pH have been widely investigated for usein immobilization of cells, chemical actuators and drugdelivery systems [3–5].

Various synthetic polymers such as poly(N-isopropylacrylamide), poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers (Pluronic®),poly[(lactic acid)-co-(glycolic acid)]-b-poly(ethylene oxide)-b-poly[(lactic acid)-co-(glycolic acid)] triblock copolymersare known to exhibit thermo-responsive phase transitionbehavior in aqueous solution. At high concentrations, theyself-assemble to form a highly water-absorbing three-dimensional gel network in response to temperature. Thesehydrogls have been widely investigated in controlled drugdelivery and injectable gel systems [6–11].

Polypeptides and their related synthetic poly(amino acid)shave become important on account of their biocompatibilityand biodegradability, which are useful in various bio-relatedapplications [12]. Poly(N-2-hydroxyethyl-DL-aspartamide),PHEA, is one of these synthetic polymers, which is water-soluble, biodegradable, and biocompatible. The attachmentand chemical modification of the pendent groups eitherthrough an aminolysis reaction to polysuccinimide (PSI) orvia a secondary reaction with the hydroxyl groups onPHEA can provide a variety of biodegradable functionalpolymers with specific properties. Giammona and cow-orkers developed PHEA derivatives with methacrylatependent groups (using methacrylic anhydride or glycidylmethacylate) and studied their photo-crosslinked hydrogelsin drug delivery system [13, 14].

Kim et al. investigated on the preparation of variousfunctional polyaspartamide derivatives and the crosslinkedhydrogels, which contain basic alkyl amine pendants toidentify new stimuli-responsive polymers for potentialbiomedical applications [15–17]. These polymers and gelsexhibited pH and/or temperature sensitive phase or volumetransitions in aqueous environment.

In our previous studies, we investigated PHEMA hydro-gels containing the Pluronic, which showed thermo-

J. H. Park : J. R. Moon :K. H. Hong : J.-H. Kim (*)Department of Chemical Engineering,Polymer Technology Institute, Sungkyunkwan University,300 Chunchun, Jangan,Suwon, Kyunggi 440–746, Koreae-mail: kimjh@skku.edu

J Polym Res (2011) 18:273–278DOI 10.1007/s10965-010-9415-3

responsive swelling-deswelling behavior caused by thetemperature-induced sol-gel transition of Pluronic compo-nent within the hybrid gel matrix [18]. In this work, it wasinteresting to introduce Pluronic triblock copolymer into asynthetic PHEA to prepare the corresponding gel withtemperature-sensitive properties. Biodegradable homoPHEA and hybrid hydrogels with Pluronic F127 wereprepared by photo-crosslinking in aqueous solution at313 nm. The thermo-responsive swelling behavior andmorphology of the hybrid hydrogels were discussed.

Experimental

Materials

L-aspartic acid (98+%), o-phosphoric acid (98%), N,N-dimethylformamide (DMF, anhydrous 99.8%), ethanolamine(EA, 99+%), methacrylic anhydride (94%), triethylamine(TEA, >99.5%), N,N-dimethylacetamide (DMA, 99%),Pluronic F127 ((PEO)99(PPO)67(PEO)99), chloroform(99.5%), 1-methyl-2-pyrrolidinone (NMP, 99+%), 2,2-dime-thoxy-2-phenyl acetophenone (DMPAP, 99%), phosphatebuffered saline (PBS pH 7.4) were purchased from AldrichChemical Co. and used as received. Acetic acid glacial,methanol (MeOH, 99.5%), isopropyl alcohol (IPA, 99%),acetone (99.5%), diethylether (99%) were obtained fromSamchun Chemical Co. (Korea). All other chemicalspurchased were of the highest quality and used withoutpurification.

Synthesis of Pluronic-dimethacrylate (Pluronic-DMA)

Preparation of Pluronic F127 containing methacrylategroup at both ends was carried out according to a literaturemethod [18, 19]. 10 g (1.0 eq) of Pluronic F127 wasdissolved in 20 mL chloroform in a three-neck round-bottomed flask equipped with a nitrogen inlet and outlet.1,140 μL (8.0 eq) methacrylic anhydride and 880 μL(8.0 eq) TEA, as a catalyst, were added dropwise at 0°C.The reaction flask was placed in a water bath and stirred at50°C for 1 day. The final solution was then poured into a500 mL of cold ethyl ether under vigorous stirring. Theprecipitate was filtered, washed with cold ethyl etherseveral times, and then dried in a vacuum for 2 day.

Synthesis of poly(N-2-hydroxyethyl-DL-aspartamide)with methacrylate pendants (PHEA-MA)

Polysuccinimide (PSI) was prepared using a previouslyreported method [15]. PHEA was derived from PSI viaaminolysis reaction with EA as the procedure reportedelsewhere [20]. The weight-average molecular weight of

PHEA was determined to be 127,000 g/mol using gelpermeation chromatography (GPC). PHEA partially deriv-atized with methacrylate pendants, PHEA-MA, wasobtained by reacting PHEA with methacrylic anhydride inDMF according to a literature method [14]. A typicalprocedure used is as follows: 0.5 g of PHEA was dissolvedin 10 mL DMA in a three-neck round-bottomed flaskequipped with a nitrogen inlet and outlet. 40 mol% (basedon hydroxyl group on the structure) methacrylic anhydrideand TEA, as a catalyst, were added dropwise at 0°C. Thereaction flask was placed in a water bath and stirred at 40°Cfor 2 day. The final solution was precipitated in 400 mL ofcold 2-propanol and centrifuged for 20 min, at 11000 rpmand 5°C. The final product was washed with cold2-propanol and acetone several times, and then dried invacuum for 1 day. PHEA-MA copolymer was dissolved in40 mL pure water and dialyzed using the membrane(MWCO 12000–14000) to remove the unreacted monomerand residual solvent. Finally, the dialysis product wasfreeze-dried.

1H-NMR (500 MHz, DMSO): δ 1.8–1.9 (s, 3H, -CO-C(CH3) = CH2), δ 2.3–2.8 (br, 2H, CH-CH2-CO-NH), δ 3.0–3.2 (m, 2H, NH-CH2-CH2-OH), δ 3.2–3.3 (br, 2H,NH-CH2-CH2-CO-C(CH3) = CH2), δ 3.3–3.5 (m, 2H,NH-CH2-CH2-OH), δ 4.0–4.15 (m, 2H, NH-CH2-CH2-CO-C(CH3) = CH2), δ 4.3–4.7 (br, 1H, CH-CH2-CO-NH), δ5.55–5.7 and δ 5.95–6.1 (m, 2H, -CO-C(CH3) = CH2).

Preparation of PHEA/Pluronic hybrid gel

PHEA-MA with different MA contents or PHEA-MA withPluronic-DMA was dissolved in water to give 10%(w/v)aqueous solutions. Then, DMPAP of 2% (w/v) was addedto the polymer solution as the photo-initiator. The reactionmixture was then transferred to a mold (3 cm × 3 cm) andirradiated at 313 nm for 2 h. The resulting gel product wasplaced in a steel mesh and washed with a large amount ofdistilled water for 1 day to completely remove the solublecomponents. Finally, the washed gel product was freeze-dried in a vacuum (see Scheme 1).

Instruments

The 1H-NMR spectra were recorded on a Unity Inova-500(Varian, USA) spectrometer using DMSO-d6 as the solvent.The FT-IR spectra were obtained on a Perkin-Elmer FT-IRspectrometer (SPECTRUM 2000). The average molecularweights of the synthesized copolymers were measured by agel permeation chromatography (GPC) system equippedwith KF-803 L and KF-802.5 (Shodex) columns in series,using N,N-dimethylformamide (DMF) containing lithiumbromide (50 mM) as the eluent at a flow rate of 1 ml/min at50°C. Polystyrene standards (Waters, molecular weight of

274 J.H. Park et al.

Scheme 1 Preparation ofthermo-responsive PHEA/Plur-onic hybrid gel

6

NH

NH

O

O

OH

m

NH

NH

O

O

O

n

C C

O CH3

CH2

b

cd

ef

g

h

a b

a

d cb

a

g

bcedfh

ppm

(A)

(B)a

D2O H2O DMSO

5 4 3 2

Fig. 1 1H-NMR spectra ofPHEA (a) and PHEA-MA (b)derivatives

Photo-crosslinked polyaspartamide hybrid gel containing Pluronic 275

185 K, 80 K, 10 K and 3 K) were used for calibration todetermine the molecular weight.

Measurement of swelling capacity

The swelling ratios of prepared hydrogels were measuredby gravimetric analysis. The dried samples were placedin aqueous solution and then the swelled gel wasremoved from the solution at regular time intervals. Theweight of the hydrogel was measured after wiping off thesolution on the surface of the hydrogels with moistenedfilter paper. The temperature dependence of the swellingratio of hydrogels was also measured by gravimetricanalysis. The swelling ratio (SR) was defined as follows:

Swelling ratio ðSRÞ ¼ Ws=Wd

Where, Wd and Ws are the weights of the dry hydrogel andswollen hydrogel at equilibrium, respectively.

Scaffold morphology

The dried hydrogel was equilibrated in aqueous solution for24 h at different temperature. The swollen gel sample was

quickly quenched and then cross-sectioned in liquid nitrogen.The morphology of the freeze-dried hydrogel was observedby field emission scanning electron microscopy (SEM, JEOLJSM-6390, Japan). Porous gel sample was mounted onto ametal stub with double-sided carbon tape and coated with Aufor 2 min under vacuum (10−3 Torr) using a plasma sputteringmethod (Ion sputter coater HC-21).

Results and discussion

Preparation and characterization of PHEA/Pluronichybrid gels

Both PHEA and Pluronic containing methacrylate groupswere prepared by the derivatization reaction with meth-

4000 3500 3000 2500 2000 1500 1000 500

A

CTra

nsm

itta

nce

Wavenumber (cm-1)

B

Fig. 2 FT-IR spectra of PHEA (a), PHEA-MA (b), PHEA/Pluronichybrid gel (c)

Table 1 Synthesis of PHEA-MA derivatives

PHEA-MA Content of MAa/mol%

A 8

B 12

C 17

D 20

aMA content was determined by 1 H-NMR

0 5 10 15 20 250

3

6

9

12

15

5 o

C

10 oC

25 oC

40 oC

Swel

ling

rati

o (S

R)

Time (hr)

Fig. 4 Temperature dependence of hydrogel (B) swelling ratio in PBS(pH7.4)

0 5 10 15 20 250

3

6

9

12

15

A B C

Swel

ling

rati

o (S

R)

Time (hr)

Fig. 3 Swelling ratios of PHEA (three different MA content)/Pluronic, 50/50, gels in PBS (pH7.4)

276 J.H. Park et al.

acrylic anhydride as the procedures were described in theexperimental part. These two polymers have been used asstarting materials to produce thermo-responsive hybrid gelsby crosslinking with UV irradiation (313 nm) in a mouldunder argon atmosphere. The pendent methacryloyl groupson PHEA and the terminal methacrylate groups on Pluronicshould provide reactivity toward radical crosslinkingpolymerization activated by UV rays, where ester groupsconfer a potential biodegradability to these hydrogels. Thereaction is shown in Scheme 1. Figure 1 shows the 1H-NMR spectra of PHEA (A) and PHEA-MA (B). Themethylene proton peaks c and d were assigned to thehydroxyethyl pendant, and the e & f, g and h were assignedto the methylene, methyl and vinyl protons of themethacrylate group, respectively. Table 1 show the DS(degree of substitution) of MA in the PHEA derivatives.The DS of MA was determined by comparing the peak

intensity of the methine proton of the PHEA backbone at b(δ 2.5–2.7) with that of the MA moiety at h (δ 5.5–6.1). TheDS was represented by the following ratio:

DS¼ vinyl proton; h=methylene proton of PHEAbackbone; bð Þ�100

PHEA-MAs with lower MA content (A, B, and C) werefreely soluble in water. However, PHEA-MAwith 20% MAwas only soluble at low temperature and exhibited LCSTbehavior (i.e. the clear aqueous solution become turbid ataround 23–24°C and this phenomena was reversible)induced by phase separation due to the increased hydro-phobic character of vinyl moieties.

Figure 2 shows the FT-IR spectra of Pluronic-DMA (a),PHEA-MA (b), and PHEA/Pluronic hybrid gel (c). Spec-trum (a) shows characteristic strong absorption bands at1110 cm−1 from C-O-C and at 2890 cm−1 from CH2 of thePluronic polymer backbone. Spectrum (b) shows charac-teristic strong bands at 1650 cm−1 and 1545 cm−1 (amide I& II) and 3500–3200 cm−1 (hydroxyl group) correspondingto the aspartamide backbone along with the band at1740 cm−1 corresponding to the carbonyl vibration of theester group. The spectrum (c) of hybrid gel shows bothPluronic and PHEA components in their characteristicbands.

0

3

6

9

12

15

Swel

ling

rati

o (S

R)

Time (hr)

282217116

40

5

0

ToC

Fig. 5 Reversible swelling-deswelling curve of hybrid hydrogel(B)upon a cyclic temperature change

Fig. 6 SEM images offreeze-dried hydrogel swollenat 5°C (a) and 40°C (b)

Fig. 7 Photographs of swelled hydrogel at 5°C (a) and 40°C (b),representing thermo-responsive volume change

Photo-crosslinked polyaspartamide hybrid gel containing Pluronic 277

Swelling behavior of photo-crosslinked homo PHEAand hybrid gels with pluronic

The homo gel from PHEA-MA (A, B, and C, respectively)and PHEA/Pluronic, 50/50, hybrid gels were prepared inthe mold by photo-crosslinking method. The homo PHEAgels showed swelling degrees in the range of 8–9 g/g inwater, but the hydrogels were soft and weak. Figure 3shows the swelling curves of 50/50 PHEA/Pluronic hybridgels from PHEAs of different MA content (A, B, and C inTable 1) measured in PBS (pH 7.4) at 25°C. The initial fastswelling appeared to level off in 3∼5 h. The swellingdegrees were changed within the range of ca 7.5∼12 g/g,where the degree of swelling decreased in parallel with theincreasing MA content of PHEA. The higher MA moietyshould provide denser network structure simply due to theincreased crosslinking density.

In this study we combined a thermo-responsive Pluroniccopolymer with biodegradable PHEA. Figure 4 shows theswelling curves of the PHEA/Pluronic hybrid gels, preparedfrom 50/50 mixture of PHEA-MA (B) and Pluronic-DMA(10 wt/v %), which are measured in PBS (pH 7.4) atvarious temperatures. A significant difference in thedegrees of swelling was observed depending on thetemperature, showing thermo-responsive swelling behaviorof this hybrid gel system. The swelling ratios were in therange of ca 7.5∼13 g/g and they showed the reversedependence of swelling as function of temperature. Thedecreasing swellability of the hybrid gel upon a temperatureincrease are already expected from the inherent reversethermal gelation (RTG) behavior of Pluronic copolymer. Atlow temperatures, the hydrogen-bonds between watermolecules and hydrophilic EA groups and PEO segmentresult in higher swelling. When the external temperature isincreased, the hydrogen bonding interaction decrease and atthe same time the hydrophobic interaction of PPO,polypropylene oxide, segment in the gel matrix increaseto induce gel shrinkage. To investigate the dynamics ofequilibrium swelling ratio of hybrid hydrogel as a functionof temperature, the swelling-deswelling of hydrogel mea-sured at the temperature of 5 and 40°C in a cyclic function.As the results shown in Fig. 5, the PHEA/Pluronic hybridgel responded to temperature cycle repeatedly with similarmanner.

Figure 6 shows SEM morphology at two differenttemperature from the cross-section of freeze-dried hybridhydrogel. Distinct changes in porous structure wereobserved by varying temperature. At low temperature (a),the microporous structure with 25∼50 μm pore sizes wasobserved. When the temperature was raised to 40°C (b), thegel shrank and the gel matrix became dense withsignificantly reduced pores. The thermo-responsive behav-

ior in the volume-phase transition from this hydrogel wasdemonstrated as the typical photograph is shown in Fig. 7.A detailed study on the different Pluronic content gel anddrug release behavior of these hybrid gels are currentlyunderway. These thermo-responsive hydrogels can beutilized beneficially in controlled drug delivery andregenerative scaffold applications.

Conclusion

Photo-crosslinkable hybrid hydrogels based on poly(N-2-hydroxyethyl-DL-aspartamide) and thermo-responsivePluronic were investigated. The swelling degrees of gelscould be modulated by changing methacrylate contents.The PHEA/Pluronic hybrid hydrogels showed thermo-responsive swelling behavior with the reverse dependenceof swelling degrees on the temperature. The reversibleswelling/deswelling and micron-sized pore structure of thehybrid gel were demonstrated as a function of temperature.

Acknowledgement This work was supported by the Korea ResearchFoundation Grant (KRF-2006-005-J04602) and BK21 program.

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