Thermosensitive Hydrogel from Oligopeptide-Containing Amphiphilic

Dec 4, 2013 - We reveal that a slight change in the functional group of the oligopeptide block incorporated into the poloxamer led to drastically diff...
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Thermosensitive Hydrogel from Oligopeptide-Containing Amphiphilic Block Copolymer: Effect of Peptide Functional Group on Self-Assembly and Gelation Behavior Ping-Ray Chiang,† Tsai-Yu Lin,†,§ Hsieh-Chih Tsai,∥ Hsin-Lung Chen,*,† Shih-Yi Liu,‡ Fu-Rong Chen,‡ Yih-Shiou Hwang,⊥,# and I-Ming Chu*,† †

Department of Chemical Engineering and ‡Department of Engineering and System Science, National Tsing Hua University, Hsinchu 300, Taiwan, ROC § Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute (ITRI), Hsinchu 300 Taiwan, ROC ∥ Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei, 106 Taiwan, ROC ⊥ Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, 333 Taiwan, ROC # Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, 333 Taiwan, ROC S Supporting Information *

ABSTRACT: We reveal that a slight change in the functional group of the oligopeptide block incorporated into the poloxamer led to drastically different hierarchical assembly behavior and rheological properties in aqueous media. An oligo(L-Ala-co-L-Phe-co-β-benzyl L-Asp)-poloxamer-oligo(βbenzyl-L-Asp-co-L-Phe-co-L-Ala) block copolymer (OAF(OAsp(Bzyl))-PLX-(OAsp(Bzyl))-OAF, denoted as polymer 1), which possessed benzyl group on the aspartate moiety of the peptide block, was synthesized through ring-opening polymerization. The benzyl group on aspartate was then converted to carboxylic acid to yield oligo(L-Ala-co-L-Phe-co-LAsp)-poloxamer-oligo( L -Asp-co- L -Phe-co- L -Ala) (OAF(OAsp)-PLX-(OAsp)-OAF, denoted as polymer 2). Characterization of the peptide secondary structure in aqueous media by circular dichroism revealed that the oligopeptide block in polymer 1 exhibited mainly an α-helix conformation, whereas that in polymer 2 adopted predominantly a β-sheet conformation at room temperature. The segmental dynamics of the PEG in polymer 1 remained essentially unperturbed upon heating from 10 to 50 °C; by contrast, the PEG segmental motion in polymer 2 became more constrained above ca. 35 °C, indicating an obvious change in the chemical environment of the block chains. Meanwhile, the storage modulus of the polymer 2 solution underwent an abrupt increase across this temperature, and the solution turned into a gel. Wet-cell TEM observation revealed that polymer 1 self-organized to form microgel particles of several hundred nanometers in size. The microgel particle was retained as the characteristic morphological entity such that the PEG chains did not experience a significant change of their chemical environment upon heating. The hydrogel formed by polymer 2 was found to contain networks of nanofibrils, suggesting that the hydrogen bonding between the carboxylic acid groups led to an extensive stacking of the β sheets along the fibril axis at elevated temperature. The in vitro cytotoxicity of the polymer 2 aqueous solution was found to be low in human retinal pigment epithelial cells. The low cytotoxicity coupled with the sol−gel transition makes the corresponding hydrogel a good candidate for biomedical applications.

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INTRODUCTION

behavior may be tailored by the balance between hydrophobic and hydrophilic interactions. It is known that polypeptides with specific amino acid sequences can form well-defined secondary structures, including α helixes and β sheets. The secondary structures may

In recent years, research on thermosensitive hydrogels (THG) that exhibit sol−gel phase transition near physiological temperature has increased greatly because of THG’s potential use as an injectable carrier for the sustained release of drugs1−7 and a cell-growth matrix for local tissue engineering.8−13 Polypeptide-based materials are attractive THG systems because their sol−gel transition temperatures and gelation © 2013 American Chemical Society

Received: August 29, 2013 Revised: November 6, 2013 Published: December 4, 2013 15981

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Scheme 1. Design and Synthesis of Polymers 1 and 2

may promote intramolecular or intermolecular hydrogen bonds contributing to gelation. Poly(aspartic acid) (P(Asp)) bearing a polar carboxylic acid group has also been used as a hydrogel material because its strong polarity can promote the water absorbency and swelling ability.24 The incorporation of a small amino acid sequence may affect the properties of the conventional polymers significantly with respect to hydrogelation. Huang et al. reported that the inclusion of six tyrosine units in PEG allowed the polymer to exhibit the sol−gel transition at a polymer concentration as low as 0.25 wt %.23 Jeong et al. reported that the Pluronic F127 incorporated in the Gly-Arg-Gly-Asp (GRGD) sequence displayed a low sol−gel transition temperature and that the resultant hydrogel essentially had no loss modulus.25 The strategy of developing polypeptide-based hydrogels has shifted from the synthesis of new polypeptides to the control of desired secondary structures by chemical modification. It has been shown that the modification of the terminal groups16 or the side chains26 on the polypeptide by the incorporation of hydrophobic groups greatly affected the secondary structure. The secondary structure also depends on the sequence27,28 and length29 of the polypeptide block in the relevant block copolymers. Increasing the population of the β-sheet conformation by chemical modification was found to create a more stable hydrogel.16 It is known that the β-sheet

control the aggregation behavior of the polymer in aqueous media, which in turn determines the material properties, including the sol−gel transition temperature,14,15 gel modulus,16,17 critical gelation concentration (CGC),18 and degradability.19 Jeong et al. have developed thermosensitive polypeptide hydrogels based on block copolymers composed of hydrophobic peptide blocks such as poly(alanine) (P(Ala)),20 poly(alanine-co-phenyl alanine) (P(Ala)-co-P(Phe)),18 and poly(alanine-co-leucine) (P(Ala)-co-P(Leu))19 and hydrophilic blocks including poly(ethyl glycol) (PEG)21,22 and poloxmer (PLX).18−20 Notably, the polypeptide-based hydrogels exhibited lower a CGC than polyester-based THGs such as poly(ethylene glycol)-block-poly(lactic acid)-block-poly(ethylene glycol) (PEG-PLA-PEG), poly(ethylene glycol)block-poly(lactic acid-co-glycolic acid)-block-poly(ethylene glycol) (PEG-PLGA-PEG), and poly(2-ethyl oxazoline)-blockpoly(ε-caprolactone)-block-poly(2-ethyl oxazoline) (PEOzPCL-PEOz). Moreover, their degradation products did not produce the acidic microenvironment that may adversely affect the activity of encapsulated protein drugs or cells. Recently, Huang et al. reported THGs with very low CGC (0.25−3.0 wt %) based on oligo(tyrosine) in which the tyrosine−OH groups enhanced the molecular self-assembly via hydrogen bonding of the amide bonds in the main chain.23 Their study disclosed a phenomenon in which the polar groups 15982

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Figure 1. (a) 1H NMR spectrum of PAF−PA−PAsp (OBzyl)−PLX−PAsp(OBzyl)−PA−PAF (polymer 1). (b) 1H NMR spectrum of PAF−PA− P(Asp)−PLX−P(Asp)−PA−PAF (polymer 2) in CF3COOD.

conformation is related to the formation of amyloid β (Aβ) protein, which is the cause of Alzheimer’s disease.30 This secondary structure promotes the aggregation of Aβ peptide to form fibrils that induce neurotoxicity.31 Therefore, the β-sheet conformation, which tends to enhance the hydrophobicity of the polypeptides, is an important factor that may tailor the sol− gel transition as well as the aggregation of polypeptides in aqueous media.

In the light of the crucial role of the secondary structure and the balance between hydrophobic and hydrophilic interactions, here we reveal that a slight change in the functional group of an oligopeptide sequence in an amphiphilic block copolymer greatly modifies the secondary structure of the polypeptide block. Here we inserted a nonpolar sequence, oligo(β-benzyl-Laspartate) (OAsp(Bzyl)), and a polar sequence, oligo(Asp) (OAsp), into a peptide-containing block copolymer, oligo(Lalanine-co-L-phenyl)-poly(propylene glycol)-poly(ethylene gly15983

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Table 1. Molecular Characteristics of Polymers 1 and 2 composition ratio (mole%)a

in feed (mmole)

a

molecular weight (Mn)b

code

PLX

NCA-Asp(OBzyl)

NCA-Ala/NCA-Pha

Oligo(Asp(OBzyl))/Oligo(Asp)

Ala/Pha

1672

polymer 1 polymer 2

2.1

4.4

11.1/0.21

3.2/0 0/2.9

6.4/0.9 5.6/0.52

1958 1914

Calculated by 1H NMR. bDetermined by MALDI-TOF. precipitated in n-hexane (300 mL). NCA-Asp(OBzyl) obtained was filtered and dried under vacuum and stored at −20 °C before use.32 Synthesis of N-Carboxyl Anhydride of L-Alanine (NCA-Ala). L-Alanine (5.0 g) was dissolved in THF (130 mL) under nitrogen. Triphosgene (16.7 g) was introduced, and the mixture was reacted with gentle stirring at 45 °C. Twelve hours after the mixture turned clear, the solution was condensed to 20 mL and precipitated in nhexane (300 mL). The product was filtered and dried under vacuum. NCA-Ala was stored at −20 °C until use.33 Synthesis of N-Carboxyl Anhydride of L-Phenyl Alanine (NCA-Phe). L-Phenyl alanine (5.0 g) was dissolved in THF (56 mL) under nitrogen. Triphosgene (8.9 g) was introduced, and the mixture was reacted with gentle stirring at 45 °C. Three hours after the mixture had become transparent, the solution was condensed by reducing the pressure to remove THF and then about 20 mL of the solution was precipitated in n-hexane (300 mL). The white powder NCA-Phe thus obtained was filtered and dried under vacuum. The NCA-Phe was stored at −20 °C until use.34 Synthesis of OAF-(OAsp(Bzyl))-PLX-(OAsp(Bzyl))-OAF Block Copolymer. Poly(propylene glycol)-block-poly(ethylene glycol)block-poly(propylene glycol) bis(2-aminopropyl ether) (PLX, Mn = 1900) (4.00 g, 2.1 mmol) was dissolved in 20 mL of toluene, and residual water was removed by azeotropic distillation to a final volume of approximately 5 mL. Anhydrous chloroform/N, N-dimethylformamide (2/1 v/v, 32 mL) was added under reflux in a nitrogen atmosphere.18 NCA-Asp(OBzyl) (1.05 g), NCA-Ala (1.26 g), and NCA-Phe (0.126 g) were added to the reaction system and stirred at 40 °C for 24 h in a dry nitrogen atmosphere. The product was precipitated in cooled diethyl ether (500 mL) and dried under vacuum to yield OAF-(OAsp(Bzyl))-PLX-(OAsp(Bzyl))-OAF (polymer 1). The yield was about 69%. Synthesis of OAF-(OAsp)-PLX-(OAsp)-OAF Block Copolymer. OAF-(OAsp)-PLX-(OAsp)-OAF block copolymer was hydrolyzed with 1.5 M TFMSA-thioanisole (1/1 mol/mol) in TFA (10 mL) to remove the benzyl group on the aspartate to yield polymer 2. The reaction mixture was stirred at room temperature for 30 min and then precipitated in cooled diethyl ether (200 mL). The product was then dried overnight at 40 °C in vacuum and subsequently dissolved in DMSO. The product was purified by dialysis (MWCO 1000, Spectrum) and lyophilized.32 The yield was about 42%. The chemical structure and block composition of polymers 1 and 2 were verified by 1H NMR and ATR spectroscopy. 1H NMR spectra at 1.0−2.0 ppm {oligo-Ala: −CH(CH3)CONH−; PLX: −NHCOCH(CH3)CH2O−; −OCHH(CH3)CH2−}, 3.0−5.4 ppm {oligo-Asp(OBzyl): −NHCOCH(CH 2 COOCH 2 (C 5 H 6 ))−; oligo-Ala: −NHCOCH(CH3); oligo-Pha: −NHCOCH(CH2(C5H6))−; PLX: −NHCOCH(CH3)CH 2O-}; 7.0−7.4 ppm {oligo-(OBzyl-Asp): −NHCOCH(CH2COOCH2(C5H6))−; oligo-(Phe): −NHCOCH(CH2(C5H6))−} for polymer 1 and 1.0−2.0 ppm {oligo-Ala: −CH(CH3)CONH-; PLX: −NHCOCH(CH3)CH2O−; −OCHH(CH3)CH2−}, 3.0−5.0 ppm {oligo-Asp: −NHCOCH(CH2COOH)−; oligo-Ala: −NHCOCH(CH3); oligoPhe: −NHCOCH(CH2(C5H6))−; PLX: −NHCOCH(CH3)CH2O-}; 7.0−7.4 ppm {oligo-Phe: −NHCOCH(CH2(C5H6))−} for polymer 2, respectively (Figure 1a,b). The compositions of polymers 1 and 2 were calculated from the integral areas of 1H NMR spectra at A3.8−4.0/ A4.4−4.6 = 38.5(4/x), A3.8−4.0/A4.6−4.8 = 38.5(4/y), and A3.8−4.0/A7.0−7.2 = 38.5(4/5z) for polymer 1 and at A4.0−4.2/A4.5−4.6 = 38.5(4/x), A4.0−4.2/ A4.7−4.9 = 38.5(4/y), and A4.0−4.2/A7.0−7.4 = 38.5(4/5z) for polymer 2. On the basis of this information, Table 1 summarizes the molecular characteristics of the copolymers synthesized. The ATR absorption

col)-poly(propylene glycol)-oligo(L-alanine-co-L-phenyl alanine) (OAF-PLX-OAF), to form the OAF-(OAsp(Bzyl))PLX-(OAsp(Bzyl))-OAF multiblock copolymer (denoted as polymer 1; see Scheme 1 for its chemical structure) and the OAF-(OAsp)-PLX-(OAsp)-OAF multiblock copolymer (denoted as polymer 2; see Scheme 1 for its chemical structure), respectively. In contrast to previous studies that controlled the secondary structure of the polypeptide block by modifying the terminal groups or the side chains on the polypeptide16,26 or by changing the sequence and length of the peptide block,27,29 the new approach discloses that a slight difference in the functional group on an oligopeptide sequence drastically altered the preferred secondary structure and consequently led to different thermally induced gelation behavior. The main advantage of the present copolymer over the existing ones lies in the smaller polymer concentration required to induce gelation (and thus reduced the potential cytotoxicity caused by the incorporation of a large quantity of polymers). We will show that 2.0 wt % of the material in an aqueous medium was sufficient to induce gelation whereas the gelation of most polypeptide-based systems required a significantly higher concentration (≥5.0 wt %). As shown in Scheme 1, the two copolymers synthesized here differ only in the functional groups attached to the Asp unit, where the nonpolar benzyl and the polar carboxylic acid groups are attached to OAsp(OBzyl) and OAsp blocks in polymers 1 and 2, respectively. It will be shown that the benzyl and carboxylic acid moieties tended to induce α-helix and β-sheet conformations at low temperature, respectively. The difference in functional group further influenced the supramolecular assembly behavior of the block copolymers and the rheological properties. Although the aqueous solutions of both polymers exhibited a relatively high viscosity at room temperature, the storage modulus (G′) of polymer 2 increased drastically near physiological temperature, leading to the formation of a hydrogel. The in vitro cytotoxicity of the hydrogel that formed was also evaluated in the interest of biomedical applications.

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EXPERIMENTAL SECTION

Materials. Poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol) bis(2-aminopropyl ether) (PLX, Mn = 1900, Sigma-Aldrich) was used as received. β-Benzyl-L-aspartate (SigmaAldrich), L-alanine (Sigma-Aldrich), L-phenyl alanine (Sigma-Aldrich), triphosgene (Sigma-Aldrich), trifluoromethanesulfonic acid (TFMSA, Sigma-Aldrich), trifluoroacetic acid (TFA, Sigma-Aldrich), and thioanisole (Sigma-Aldrich) were used as received. Toluene (J.T. Baker), tetrahydrofuran (THF, TEDIA), chloroform (Mallinckrodt), and N,N-dimethylformamide (Sigma-Aldrich) were dried over calcium hydride (CaH2). Synthesis of N-Carboxyl Anhydrides of β-Benzyl L-Aspartate (NCA-Asp(OBzyl)). β-Benzyl L-aspartate (10.0 g) was dissolved in THF (100 mL) under nitrogen. Triphosgene (13.3 g) was introduced, and the mixture was reacted with gentle stirring at 45 °C. Three hours after the mixture turned clear, the solution was condensed to a final volume of 20 mL by rotary evaporation to remove THF and then 15984

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peaks associated with the functional groups of polymers 1 and 2 are presented in Figure S1 of the Supporting Information. 1 H and 13C Nuclear Magnetic Resonance Spectroscopy (NMR). The 1H NMR spectra (Varian Uniytinova-500 MHz spectrometer) in CF3COOD were used to determine the chemical structure and composition of the polymers.18,20 Moreover, the segmental dynamics of the PEG block in both copolymers was also probed by this technique at various temperatures. D2O solutions (2.0 wt %) of polymers 1 and 2 were equilibrated for 20 min at the prescribed temperatures (10, 20, 30, 40, and 50 °C), and their 1H NMR and 13C NMR spectra were collected with a Varian VNMRS-700 MHz spectrometer. Matrix-Assisted Laser Desorption/Ionization-Time of Fight (MALDI-TOF). The MALDI-TOF spectra, which showed the progress of the polymerization from PLX to polymers 1 to 2, provide quantitative information about the absolute molecular weight. The MALDI-TOF mass spectra of the polymers were obtained using a mass spectrometer (Voyager-DE STR, Applied Biosystems, Foster, CA) that was equipped with a nitrogen laser that emitted pulsed UV light at 337 nm and was operated in reflector mode at an acceleration voltage of 20 kV. A 5 μL volume of polymer matrix solution (1.0 mg/ mL) comprising an α-cyano-4-hydroxycinnamic acid solution (4.0 mg/ mL) of a 60/40 (v/v) acetonitrile/water matrix that contained 0.1% trifluoroacetic acid was deposited on the MALDI-TOF plate and dried in air at room temperature. The MALDI-TOF spectrum yielded the molecular weights of the polymers. Preparation of the Copolymer Solutions. The copolymers were dissolved in deionized (DI) water at room temperature and then stored overnight at 4 °C in a refrigerator. The aqueous solutions refer to the solutions of the copolymers in pure water. Circular Dichroism (CD) Spectroscopy. The secondary structures of polymers 1 and 2 in aqueous solution were deduced from the circular dichroism spectra. The spectra of 0.02 wt % aqueous solutions of polymers 1 and 2 were obtained at 10, 20, 30, 40, and 50 °C after the solutions had been equilibrated for 20 min at each temperature. The contents of α-helix, β-sheet, and random-coil conformations were calculated form the spectra using the selfconsistent method (SELCON 3 software) as the absolute molecular weight (Mw) ,and the concentration of the aqueous solution was known. Wet-Cell Transmission Electron Microscopy (Wet-Cell TEM). The morphologies of 2.0 wt % polymer 1 and 2 aqueous solutions were observed in situ by liquid-cell TEM. A detailed description of the method has been presented by Chen et al.35 Briefly, the samples (1.0 μL) were placed on a silicon wafer pretreated with plasma excitation and covered with a separated silicon wafer whose center consisted of a silicon nitride film. The microscopy image was obtained using a JEM2010 with an accelerating voltage of 200 kV. Rheological Property Measurement. To study the sol−gel transition behavior of the solutions of polymers 1 and 2, the changes in G′, G″, and the complex viscosity were measured in a heating cycle by dynamic rheometry (AR2000ex system, TA Instruments). Polymers 1 and 2 were separately dissolved in DI water at concentrations of 2.0, 5.0, and 7.0 wt % and subsequently stored overnight at 4 °C. Then, 0.63 mL of the polymer solution was loaded between the cone and the plate in the dynamic rheometer and equilibrated at 10 °C for 1 min. The shear rate of the rheometer was of 1.0/s, and the temperature was varied from 10 to 50 °C at a heating rate of 0.1 °C/min. In Vitro Cytotoxicity. The human retinal pigment epithelial cell line (ARPE-19) was incubated in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEA, Invitrogen, USA) and Ham’s F12 (Invitrogen, USA), supplemented with 10% FBS (Invitrogen), in a 5% CO2 and 95% humidity incubator at 37 °C for 24 h. Polymer 2 was dissolved in balanced saline solution (BSS, Alcon), which is made up to physiological pH and salt concentrations of 0.4, 2.0, and 5.0 wt %. The polymer solutions were then precooled to 4 °C, followed by sterilizing by filtration using the 0.22 μm filter. The filtered solutions were added to the ARPE-19 cell culture plate with 4.0 mL of the cultured medium for treatment for 24 h. It is noted that the solutions after filtration still formed gels upon equilibration at 37 °C, implying

that their concentrations were not perturbed significantly by the filtration. Propidium iodide (PI) staining was used to distinguish between cell apoptosis and necrosis. Cell survival was also measured by a FACS Calibur System (Becton-Dickinson, San Jose, CA), as described elsewhere.36 The experiments were performed in triplicate, and 10 000 eVents/cell were counted in each experiment. Data Analysis. Descriptive statistics are reported as the mean ± SD for continuous variables. Differences between two categorical and two normally distributed groups were evaluated by a χ2 test and a twosample student’s t test, respectively. All statistical assessments were two-sided and evaluated at the 0.05 level of significance. A p value adjusted by multiple comparison tests was calculated in each experiment.

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RESULTS AND DISCUSSION

Syntheses of OAF-(OAsp(Bzyl))-PLX-(OAsp(Bzyl))-OAF (Polymer 1) and OAF-(OAsp)-PLX-(OAsp)-OAF (Polymer 2). N-Carboxyl anhydride (NCA) polymerization is traditionally initiated using primary amines because amines are nucleophilic ring-opening chain propagators that allow the polymer chain to grow linearly with monomer conversion. Therefore, polymer 1 was synthesized by an SN2 reaction that involved N-carboxyl-β-benzyl L-aspartate anhydride (NCAAsp(OBzl)), N-carboxyl-L-alanine anhydride (NCA-Ala), and N-carboxyl-L-phenyl alanine anhydride (NCA-Phe) using PLX bis(2-aminopropyl ether) as the macroinitiator. PLX bis(2aminopropyl ether) is an electrophilic initiator that preferentially attacks the carbon atom of the anhydride on the NCA ring. NCA was continually added to the polymeric chains, causing chain propagation until NCA had been fully consumed. Then, the benzyl group of oligo-Asp(OBzyl) on polymer 1 was removed using TFMSA-thioanisole in TFA to afford the carboxylic acid group of oligp(Asp) on polymer 2 (Scheme 1). The molecular weights of polymers 1 and 2 were obtained using MALDI-TOF (Figure S2). The result showed that both polymers exhibited a narrow unimodal peak, and the molecular weight of polymer 1 was very similar to that of polymer 2, suggesting that the effect of molecular weight on the structure and properties was essentially absent. Secondary Structure of the Oligopeptide Block in the Aqueous Solutions. The secondary structures of the oligopeptide blocks of polymers 1 and 2 in aqueous solution were determined by CD. Figure 2a displays the CD spectra of polymer 1 collected at different temperatures. The spectrum showed a positive band centered at 196 nm and negative bands situating at 210 and 220 nm, suggesting that the oligopeptide block in polymer 1 formed a predominantly α-helix conformation.27 Figure 2b shows the content of α-helix, βsheet, and random-coil conformations in polymer 1 at various temperatures. It can be seen that the contents of the α helix and β sheet decreased and increased abruptly at ca. 35 °C, respectively, showing that a significant population of the α-helix conformation was disrupted and converted to the β sheet upon heating. A similar finding has also been reported by Rajesh et al., where the loss of α-helix structure in TlpA was found to occur with increasing temperature.37 Figure 2c presents the CD spectra of polymer 2, which was characterized by a positive band centered at 200 nm and a negative band centered at 218 nm.14 The spectra indicated that the oligopeptide of polymer 2 was rich in β-sheet conformation. The content of the α helix and β sheet also tended to decrease and increase, respectively, on heating (Figure 2d), but not as drastically as for polymer 1. 15985

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the stacking of the side-chain benzene rings of poly(γ-benzyl Lglutamate) was the critical factor.38 In this sense, the preferential formation of α-helix in polymer 1 may be associated with the stacking of the benzene rings on the benzyl group. Kuo et al. reported that the dipole−dipole interactions between the CO groups of poly(γ-benzyl L-glutamate) and the CO groups of poly(acetoxystyrene) also contributed to the formation of the α helix.39 Their experimental results thus implied that both the rigid benzene rings on the side chain and the dipole−dipole interaction were the key factors in enhancing the content of the α helix, as observed for polymer 1. For polymer 2, the oligopeptide block may be more flexible as a result of the removal of the bulky benzene rings. The more flexible peptide units were allowed to fold to form a β sheet to increase the amount of hydrogen bonding between the carboxyl acid groups.40 Temperature-Dependent Segmental Mobility of the PEG Block. Besides the difference in secondary structure, polymers 1 and 2 also showed different temperature dependences of the segmental dynamics. To elucidate the molecular motions at various temperatures, both polymers were dissolved in D2O, and the 2.0 wt % solutions were studied by 1H NMR spectroscopy as the temperature was increased stepwise from 10 to 50 °C. Figure 3a,b displays the peaks associated with the PEG block in the 1H NMR spectra of polymers 1 and 2, respectively. For polymer 1, the PEG peak was quite sharp, and its shape and width remained fairly constant from 10 to 50 °C, suggesting that PEG interacted freely with the water molecules over the temperature range studied (Figure 3a). A similar result was observed for PLX (Figure S3), suggesting that the chemical environment of PEG in polymer 1 was comparable to that in neat PLX. Thus, it implied that polymer 1 could form similar supramolecular structure in the aqueous solution to that of PLX (to be discussed later). However, the PEG segments of polymer 2 displayed different dynamic response to temperature change. It can be seen that the peak broadened significantly as the temperature was raised from 10 to 50 °C, indicating reduction of segmental motion in the solution upon heating (Figure 3b). The full widths at half height (ΔW1/2) of the PEG peak of polymers 1 and 2 are plotted as a function of temperature in Figure 3c. It can be seen that ΔW1/2 associated with polymer 2 increased sharply at ca. 35 °C, showing an abrupt decrease in the PEG segmental mobility across this temperature. The decrease in segmental mobility of the PEG peak of polymer 2 may stem from the significant aggregation of the copolymer molecules. Considering that the mobility of the PEG block in polymer 1 and PLX remained largely unperturbed with respect to changing temperature, such an aggregation should not be driven by the decreasing solubility of PEG at the elevated temperature. The aggregation of the β-sheet moiety of the oligopeptide blocks in polymer 2 at the elevated temperature may be responsible for the reduction of PEG mobility because this aggregation event would cause a local enrichment of the PEG concentration (where PEG and the peptide blocks are chemically linked in the copolymer molecules). The carboxylic acid group on the Asp units in polymer 2 may form hydrogen bonds with water molecules and also with other carboxylic acid groups. These two types of hydrogen bonds may compete with one another. The hydrophilicity of the copolymer was enhanced by the hydrogen bonding with water at low temperature. When the temperature was increased, a significant fraction of the hydrogen bonding was disrupted by

Figure 2. (a) CD spectra of the polymer 1 solution at various temperatures. (b) Changes in the relative contents of the secondary structures with respect to the temperature of polymer 1. (c) CD spectra of the polymer 2 solution at various temperatures. (d) Changes in the relative contents of the secondary structures with respect to the temperature of polymer 2.

The CD spectra revealed that the presence of the benzyl group on the Asp unit favored the formation of the α-helix conformation, whereas the carboxylic acid group induced the βsheet conformation. The effect of the functional group on the secondary structure may be attributed to the difference in characteristic interactions between polymers 1 and 2. Painter et al. reported that the fraction of the α-helix conformation of poly(γ-benzyl L-glutamate) increased with increasing poly(acetoxystyrene) content in their blends. It was suggested that 15986

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aggregation led to the formation of nanofibrils by polymer 2. The aggregation in turn reduced the mobility of the PEG block. The carboxylic acid group was absent in polymer 1 and PLX, so the copolymer chains did not aggregate further to perturb the chemical environment of the PEG blocks. Rheological Properties and Supramolecular Structures. Both polymers 1 and 2 were viscous liquids at room temperature, and their viscosities increased upon heating, as judged by the naked eye. For polymer 1, the more viscous solution was still able to flow at 50 °C (photograph in Figure 4a); however, the viscous solution of polymer 2 (2.0 wt %) turned into a gel that was essentially nonflowing above 35 °C (photograph in Figure 4d). To characterize the properties of both polymers more quantitatively, their storage modulus (G′), loss modulus (G″), and viscosity (η) were measured as a function of temperature in a heating cycle. Figure 4a−c displays the results for the solutions of polymer 1 with different concentrations. It can be seen that both G′ and η underwent an abrupt increase at ca. 27 °C for 2.0 and 5.0 wt % solutions and at ca. 20 °C for a 7.0 wt % solution. The transition temperatures were in close proximity to the temperatures at which the secondary structure transformed from α helix to β sheet (Figure 2b). This may imply that the significant increase in the population of the β sheet led to a more dynamically constrained supramolecular structure. The enhancement of the structure constraint, however, did not modify the segmental mobility of the PEG block significantly. Wet-cell TEM, which circumvents the problem of morphological disturbance upon solvent removal, was employed to examine the real-space morphologies of the aqueous systems. Figure 5 displays the TEM micrograph of polymer 1 quenched from 50 °C. Large particles with an average diameter of 500 nm were observed. Because the size of these particles was much larger than the radius of gyration of the block copolymer, they should not correspond to the individual micelles formed by the assembly of the copolymer molecules. Considering that the behavior of polymer 1 was analogous to that of neat PLX that formed network clusters in the aqueous media, the large nanoparticles observed were considered to be these clusters with the internal structure schematically illustrated in Figure 6a. For PLX, it has been shown that the hydrophobic PPO block may associate in the aqueous media to form microdomains. These microdomains were bridged by the hydrophilic PEG blocks, thereby yielding the network structure. In the case in which the polymer concentration was low, the network structure was unable to span the entire sample volume; therefore, microgel particles composed of networks of limiting sizes were formed. In the case of polymer 1, we proposed that a large fraction of the α-helix-forming peptide blocks aggregation to form microdomains that served as physical cross-links to generate the microgel particles (Figure 6a). Upon heating, the α helices in the microdomains transformed into β sheets and the association strength or the hydrophobic interaction became stronger so as to generate more intact microgel particles that gave rise to larger G′ (Figure 6a). The environment and local dynamics of the PEG block, however, remained largely unchanged. In the case of polymer 2, the temperature dependence on G′, G″, and η displayed in Figure 4d−f showed that these parameters also experienced abrupt increases from 25 to 35 °C upon heating. Because the solution was gel-like in appearance at 50 °C, its G′ at the higher temperature was in

Figure 3. 1H NMR spectra of 2.0 wt % solutions of (a) polymer 1 and (b) polymer 2 in D2O showing the temperature dependence of the peak associated with the PEG block. For polymer 1, the PEG peak was quite sharp, and its shape and width remained fairly constant from 10 to 50 °C. The peak broadened significantly as the temperature was raised for polymer 2, indicating the reduction of segmental motion in the solution upon heating. (c) Full widths at half height (ΔW1/2) of the PEG peak of polymers 1 and 2 as a function of temperature. It can be seen that ΔW1/2 associated with polymer 2 increased sharply at ca. 35 °C.

thermal energy, and the β-sheet moiety may then aggregate to enhance the hydrogen bonding between the carboxylic acid groups on the Asp units. It will be shown later that such an 15987

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Figure 4. Rheological properties of (a) 2.0, (b) 5.0, and (c) 7.0 wt % aqueous solutions of polymer 1. Corresponding results for polymer 2 solutions with concentrations of (d) 2.0, (e) 5.0, and (f) 7.0. (g) Concentration dependence of the maximum storage modulus (G′) of polymers 1 and 2 at 50 °C.

general much larger than that of polymer 1 at a given solution concentration, as demonstrated in Figure 4g. In fact, the G′ of polymer 2 was increased ca. 1000-fold across the transition temperature; therefore, we considered this polymer to exhibit a

thermally induced sol−gel transition such that polymer 2 can be regarded as a thermosensitive hydrogel. The dramatic enhancement of G′ attested to the fact that an obvious transformation of the supramolecular structure should 15988

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Figure 6. Schematic illustrations of the structural changes on heating the solutions of (a) polymer 1 and (b) polymer 2. Polymer 1 is proposed to form microgrel particles in which a large fraction of the αhelix-forming peptide blocks aggregated to form a microdomain that served as the physical cross-links. Upon heating, the α helices in the microdomains transformed into β sheets and the association strength became stronger. It is proposed that the morphology of polymer 2 underwent a drastic change from the microgel particle to nanofibrils across the sol−gel transition.

Figure 5. Wet-cell TEM micrographs of 2.0 wt % solutions of polymers 1 and 2 quenched from 50 °C. Polymer 1 formed microgel particles of several hundred nanometers in size (top image). By contrast, nanofibers with extensive networking were observed for polymer 2 in the gel state (bottom image).

have taken place across the sol−gel transition. Because the solutions of polymer 2 were viscous liquids at room temperature, we proposed that they also formed microgel particles in the sol state. For the gel state of polymer 2, wet-cell TEM was also employed to observe the characteristic morphology formed. The TEM micrograph in Figure 5b showed that in contrast to large microgel particles, polymer 2 in the gel state formed nanofibrils. These fibrils appeared to overlap or cross-link extensively, and the extensive networking gave rise to the strong gel property of the polymer at elevated temperature. As a consequence, we proposed that the supramolecular structure of polymer 2 underwent a drastic change from the microgel particle to the nanofibril across the sol−gel transition, as schematically illustrated in Figure 6b. Upon heating, the hydrophobicity of the β-sheet moiety was enhanced, leading to an extensive face-to-face stacking of the β sheets along the fibril axis to enhance the hydrogen bonding between the carboxylic acid groups on the sheets so as to create the long nanofibrils. The dense packing of the β sheet caused the local enrichment of the PEG block concentration such that its segmental dynamics was slowed down, as manifested by the NMR peak broadening. In Vitro Cytotoxicity of Polymer 2. Because polymer 2 can serve as a THG system, its cytotoxicity was evaluated by investigating the apoptosis of ARPE-19 cells in the polymer solutions for 24 h. Figure 7 shows the apoptosis of ARPE-19 cells in the aqueous solutions at a concentration ranging from 0.4 to 5.0 wt % as examined by flow cytometry. The wells

containing only medium were regarded as a positive control. It can be seen that the percentages of cells undergoing apoptosis in the polymer solutions were similar to that of the positive control (Figure 7b), where the differences were in general within the experimental error considering that the p values were in general greater than 0.05. This indicates that the hydrogel system has low cytotoxicity.

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CONCLUSIONS The present study reports a new biocompatible thermosensitive hydrogel system prepared by incorporating an oligopeptide block into the conventional poloxamer amphiphilic block copolymer. The sol−gel transition as mediated by the secondary-structure unit of the oligopeptide block tailored by the functional group has been investigated. The replacement of the benzyl group on the Asp unit by the carboxylic acid moiety led to the predominant formation of the β sheet in the oligopeptide block of polymer 2 in the aqueous solution. The hydrogen bonding between the −COOH groups on the β sheets might induce the stacking of these sheet entities and consequently generated nanofibrils at elevated temperatures. The extensive networking of the nanofibrils then gave rise to the gel property of polymer 2. For polymer 1, which contained a benzyl group on the Asp unit, although the secondary structure tended to transform from α helix to β sheet upon heating, such a local structure transformation was unable to drive a drastic reorganization of the supramolecular structure, 15989

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by a grant from Chang Gung Memorial Hospital (CMRPG300111). We gratefully acknowledge Prof. Ping-Chiang Lyu, Chao-Sheng Cheng, and Dr. Kuo-Chang Cheng for their kind technical assistance with circular dichroism measurements made at the Institute of Bioinformatics and Structural Biology, National Tsing Hua University. We also thank Prof. Hsiu-Fu Hsu and Mr. ChienHung Chiang in the department of chemistry of Tamkang University for their helpful comments and important advice.

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Figure 7. (a) In vitro cytotoxicity of ARPE-19 cells measured by flow cytometry at different solution concentrations. The upper left panel represents the control groups. The upper right is the result for 0.4 wt %, and the lower left and the lower right are the results for 2.0 and 5.0 wt % solutions, respectively. (b) Apoptosis percentage of ARPE-19 cells in the presence of an OAFP-PLX-OPFA aqueous solution at different concentrations (n = 3).

such that the solution remained a viscous liquid (with higher viscosity) at elevated temperature. Polymer 2 synthesized in this study may possess a great potential for encapsulating cells and drugs for extended drug release.

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ASSOCIATED CONTENT

S Supporting Information *

Molecular characteristics, design, synthesis, and characterization of polymers 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(H.-L.C.) E-mail: [email protected]. Tel: + 886-35713704. Fax: + 886-3-5715408. *(I-.M.C.) E-mail: [email protected]. Tel: + 886-35713704. Fax: + 886-3-5715408. 15990

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