Reverse Thermal Gelation of PAF-PLX-PAF Block Copolymer

Jul 28, 2009 - Young Mi Lee,‡ and Byeongmoon Jeong*,†. Department of Chemistry and Nano Science, Department of Bioinspired Science, Ewha Womans...
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Biomacromolecules 2009, 10, 2476–2481

Reverse Thermal Gelation of PAF-PLX-PAF Block Copolymer Aqueous Solution Eun Hae Kim,† Min Kyung Joo,† Kyung Hyun Bahk,† Min Hee Park,† Bo Chi,† Young Mi Lee,‡ and Byeongmoon Jeong*,† Department of Chemistry and Nano Science, Department of Bioinspired Science, Ewha Womans University, Daehyun-Dong, Seodaemun-Ku, Seoul, 120-750, Korea, and Department of Life Science, Ewha Womans University, Daehyun-Dong, Seodaemun-Ku, Seoul, 120-750, Korea Received April 18, 2009; Revised Manuscript Received July 2, 2009

The aqueous solution of poly(L-Ala-co-L-Phe)-poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol)poly(L-Ala-co-L-Phe) block copolymers (PAF-PLX-PAF) in a concentration range of 6.0-10.0 wt % underwent sol-to-gel transition as the temperature increased from 10 to 50 °C. Circular dichroism spectra, hydrophobic dye solubilization, dynamic light scattering, and transmission electron microscopy image of the polymer suggest that the polymers form micelles in water, where the hydrophilic (PLX) blocks form a shell and the hydrophobic (PAF) blocks form a core of the micelle. Circular dichroism, FTIR, and 13C NMR spectra suggest that sol-to-gel transition accompanies partial strengthening of the β-sheet structure of PAF and a decrease in molecular motion of the PLX. The sol-to-gel transition temperature could be controlled by varying the molecular weight of PAF and PLX blocks, the ratio of Ala to Phe, and the corresponding secondary structure of the polypeptide.

Introduction As minimally invasive depot forming materials, reverse thermal gels have been extensively searched during the past decade.1-4 They can form a low viscous aqueous solution at low temperature, such as 20 °C, and a gel depot at the body temperature of warm-blooded animals by the thermal energy induced sol-to-gel transition. The gel depot containing pharmaceutical agents or cells can act as a sustained release system of a drug or cell-growing matrix. The reverse thermal gelation comes from the delicate balance between hydrophilicity and hydrophobicity of a polymer. A drastic change in solubility in a physiologically important temperature range of 10-50 °C is a key property in designing the polymer. In addition, the polymer should hold an appropriate amount of water in a gel state to keep the hydrogel property. Typically, polyester, polysaccharide, polyphosphazene, polycarbonate, polycyanoacrylate, and so on have been used as a biodegradable hydrophobic block, whereas poly(ethylene glycol) has been used as a hydrophilic block.5-13 As polypeptide thermogelling materials, β-lactoglobulin, elastin-like polypeptide (ELP) based polymer, and silk-elastinlike polymer were reported.14-16 Changes in secondary structure or denaturation of the polypeptides accompany the sol-to-gel transition. For example, de novo synthesized reverse thermogelling polypeptide (MAX) underwent a transition from random coils to β-hairpin-like structures as the temperature increased.17 In designing a reverse thermal gelling material, enzyme directed hydrolysis can be an efficient method due to the fact that the polymer can be designed to be degraded only after the in vivo applications. Recently, we reported a polyalanine-based thermogelling biomaterial.18-20 In particular, a poly(ethylene glycol)-poly(L-Ala-co-L-Phe) (PEG-PAF) diblock copolymer was proven to be degraded by proteases such as cathepsin B, * To whom correspondence should be addressed. E-mail: bjeong@ ewha.ac.kr. † Department of Chemistry and Nano Science, Department of Bioinspired Science, Ewha Womans University. ‡ Department of Life Science, Ewha Womans University.

cathepsin C, or elastase that are present in the subcutaneous layer of mammals.20 In this paper, a series of poly(L-Ala-co-L-Phe)-poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol)-poly(LAla-co-L-Phe) block copolymers (PAF-PLX-PAF) showing reverse thermal gelation were synthesized. Molecular aggregation behavior was investigated by fluorescence spectroscopy, UV-vis spectroscopy, dynamic light scattering, transmission electron microscopy, and scanning electron microscopy. The conformational changes of PAF and PEG as a function of temperature were investigated using FTIR, circular dichroism (CD) spectroscopy, and 13C NMR spectroscopy. In addition, the structure-property relationship of the sol-to-gel transition of the PAF-PLX-PAF aqueous solutions was investigated by varying PAF length, PLX length, and L-Ala/L-Phe ratio.

Experimental Section Materials. Poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol) bis(2-aminopropyl ether) (PLX; Mn ) 900 and 2000 Da; Aldrich) was used as received. The propylene glycol and ethylene glycol units were 3.5 and 15.5 for PLX with molecular weight of 900; and 3.5 and 40.5 for PLX with molecular weight of 2000, respectively. L-Alanine carboxy anhydrides (M & H Laboratory, Korea), Lphenylalanine carboxy anhydrides (M & H Laboratory, Korea), and 1,6-diphenyl-1,3,5-hexatriene (Aldrich) were used as received. Toluene was dried over sodium before use. Chloroform and N,N-dimethyl formamide (anhydrous) were treated with magnesium sulfate before use. Synthesis. The PAF-PLX-PAF was synthesized by ring-opening polymerization of the N-carboxy anhydrides of L-alanine and N-carboxy anhydrides of L-phenylalanine in the presence of PLX.20,21 To synthesize PIII in Table 1, PLX (3.00 g, 1.50 mmol; Mn ) 2000 Da; Aldrich) was dissolved in toluene (50 mL) and the residual water was removed by azeotropic distillation to a final volume of about 5 mL. Anhydrous chloroform/dimethyl formamide (15 mL; 2/1 v/v), Ncarboxy anhydrides of L-alanine (3.24 g, 28.42 mmol), and N-carboxy anhydrides of L-phenylalanine (1.34 g, 7.05 mmol) were added to the

10.1021/bm9004436 CCC: $40.75  2009 American Chemical Society Published on Web 07/28/2009

Gelation of PAF-PLX-PAF Block Copolymer Solution

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Table 1. List of Polymers Studied polymer

PAF-PLX-PAFa

Ala/Phea

Mnb

Mw/Mnb

PI PII PIII PIV PV

680-2000-680 680-2000-680 850-2000-850 290-900-290 490-900-490

83/17 76/24 82/18 80/20 80/20

2380 2550 2800 1340 1590

1.2 1.1 1.2 1.3 1.3

a Determined by 1H NMR in CF3COOD. b Determined by gel permeation chromatography using N,N-dimethyl formamide as an eluting solvent. Poly(ethylene glycol)s were used as the molecular weight standards.

reaction mixture. They were stirred at 40 °C for 24 h under a dry nitrogen atmosphere. The polymer was purified by precipitation into diethyl ether, followed by evaporation of the residual solvent under vacuum. The yield was 60%. Other polymers with different composition and block length were similarly prepared. Table 1 summarizes the list of polymers studied in this paper. 1 H and 13C NMR Spectroscopy. 1H NMR spectra of PAF-PLXPAF (PIII) in CF3COOD (500 MHz NMR spectrometer; Varian) was used to determine the composition of the polymer. 13C NMR spectral changes of the PAF-PLX-PAF (PIII; 8.0 wt. % in D2O) were investigated as a function of temperature. The solution temperature was equilibrated for 20 min at each temperature. Gel Permeation Chromatography. The gel permeation chromatography system (Waters 515) with a refractive index detector (Waters 410) was used to obtain the molecular weights and molecular weight distributions of the polymers. N,N-Dimethyl formamide was used as an eluting solvent. The poly(ethylene glycol)s (Polyscience, Inc.), with a molecular weight range of 200-20000 Da and polydispersity index (Mw/Mn) of 1.05-1.15, were used as the molecular weight standards. An OHpak SB-803QH column (Shodex) was used. Phase Diagram. The sol-gel transition of the polymer aqueous solution was investigated by the test tube inverting method.22 The aqueous polymer solution (1.0 mL) was put in the test tube with an inner diameter of 11 mm. The transition temperature was determined by flow (sol)-no flow (gel) criterion with a temperature increment of 1 °C per step. Each data point is an average of three measurements. Dynamic Mechanical Analysis. Changes in storage modulus of the polymer aqueous solutions were investigated by dynamic rheometry (Thermo Haake, Rheometer RS 1).23 The aqueous polymer solution was placed between parallel plates of 25 mm diameter and a gap of 0.5 mm. To minimize the water evaporation during the experiment, the plates were enclosed in a water saturated chamber. The data were collected under a controlled stress (4.0 dyn/cm2) and a frequency of 1.0 rad/s. The heating rate was 0.5 °C/min. Circular Dichroism Spectroscopy. Ellipticity of the PAF-PLXPAF (PIII) aqueous solution was obtained by the circular dichroism instrument (J-810, JASCO) as a function of concentration at a fixed temperature of 10 °C. The shift in the band position of a negative band was plotted as a function of polymer concentration. The crossing point of the two extrapolated lines was defined as the critical micelle concentration. In addition, ellipticity of the PAF-PLX-PAF (PIII) aqueous solution was obtained as a function of temperature at a fixed concentration of 0.05 wt % in a range of 10-50 °C by an increment of 10 °C per each step. The aqueous solution was equilibrated for 20 min at each temperature. Hydrophobic Dye Solubilization. Critical micelle concentration of PAF-PLX-PAF (PIII) was investigated by UV-vis spectroscopy (S3100, SCINCO) at 10 °C. 1,6-Diphenyl-1,3,5-hexatriene solution in methanol (10 µL at 0.4 mM) was injected into a polymer aqueous solution (1.0 mL) in a polymer concentration range of 1.0 × 10-3∼5.0 × 10-1 wt %. The UV-vis spectra of the solutions were recorded from 300 to 400 nm. The absorbance at 378 nm relative to 400 nm was plotted against the polymer concentration, and the crossing point of the two extrapolated lines was defined as the critical micelle concentration.24 Dynamic Light Scattering. The apparent size of PAF-PLX-PAF (PIII) aggregates in water (0.05 wt %) was studied by a dynamic light

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scattering instrument (ALV 5000-60x0) at 10 °C. A YAG DPSS-200 laser (Langen, Germany) operating at 532 nm was used as a light source. Measurements of scattered light were made at an angle of 90° to the incident beam. The results of dynamic light scattering were analyzed by the regularized CONTIN method. The decay rate distributions were transformed to an apparent diffusion coefficient. From the diffusion coefficient, the apparent hydrodynamic size of a polymer aggregate can be obtained by the Stokes-Einstein equation. FTIR Spectroscopy. IR spectra (FTIR spectrophotometer FTS-800; Varian) of the PAF-PLX-PAF (PIII) aqueous solution (8.0 wt % in D2O) were investigated as a function of temperature in a range of 10-50 °C by an increment of 10 °C per step. The polymer aqueous solution was injected between two ZnSe cells separated with a Teflon spacer in a temperature-controlled chamber and was equilibrated for 20 min at each temperature. The resolution of the FTIR spectra was 1 cm-1. Transmission Electron Microscopy (TEM). The PAF-PLX-PAF (PIII) aqueous solution (10 µL) was placed on the carbon grid and the excess solution was blotted with filter paper. The grids were dried at room temperature for 24 h. The microscopy image was obtained by JEM-2100F (JEOL) with an accelerating voltage of 200 kV. Phosphotungstanate was used as a staining agent. Scanning Electron Microscopy (SEM). The PAF-PLX-PAF aqueous solution (8.0 wt %) was dropped on a carbon grid. An excess amount of solution was removed by filter paper and the grid was airdried at 37 °C for 24 h. The SEM image was obtained from a field emission scanning electron microscope (JMS-6700F, JEOL) operated at 10.0 keV. In Vitro Gel Duration. The PAF-PLX-PAF (PIII) aqueous solution (0.5 mL; 8.0 wt %) and F127 aqueous solution (0.5 mL; 30 wt %) formed a gel by keeping the vials (diameter ) 11 mm) at 37 °C for 2 min. Phosphate buffered saline (3.0 mL) at 37 °C was added on top of the gel and the whole medium (3.0 mL) was replaced every day. The mass (by volume) of the remaining gel was measured every day.

Results and Discussion PAF-PLX-PAF block copolymers with different compositions and block length were prepared by varying a ratio of N-carboxy anhydrides of amino acids to PLX, a ratio of N-carboxy anhydrides of L-Ala to N-carboxy anhydrides of L-Phe, and PLX length. The composition of the polymer was determined by 1H NMR spectra in CF3COOD (Supporting Information, Figure S1a). The synthesis scheme and detailed structure of the polymer are shown in Scheme 1: l + n ) 3.5; m ) 15.5 and 40.5 for PLX 900 and 2000, respectively. The areas of the 1H NMR spectra at 1.0-2.0, 3.0-5.1, and 7.0-7.6 ppm were used to calculate the composition and the molecular weight of the PAFPLX-PAF. A1.0-2.0, A3.0-5.1, and A7.0-7.6 are the areas of the peaks at 1.0-2.0 ppm {Ala: -(CH(CH3)CONH)-; PLX: -NHCH(CH3)CH2-, -(OCH(CH3)CH2)-}, 3.0-5.1 ppm {Phe: -(CH(CH2C6H5)CONH)-; Ala: -(CH(CH3)CONH)-; PLX: -NH CH(CH3)CH2-, -(O CH(CH3)CH2)-, -(O CH2CH2)-}, and 7.0-7.6 ppm {-(CH(CH2C6H5)CONH)-}, respectively. Based on the above assignment, A1.0-2.0/A7.0-7.6 ) (13.5 + 6x)/10y, and A3.0-5.1/A7.0-7.6 ) (75.5 + 2x + 6y)/10y for PAF-PLX 900-PAF. A1.0-2.0/A7.0-7.6 ) (13.5 + 6x)/10y and A3.0-5.1/A7.0-7.6 ) (175.5 + 2x + 6y)/ 10y for PAF-PLX 2000-PAF. The molecular weight and molecular weight distribution determined by gel permeation chromatogram were in a range of 1340-2800 Da and 1.1-1.3, respectively. Unimodal distribution of the polymer molecular weight in gel permeation chromatograms of PAF-PLX-PAF and PLX (Mn ) 2000 Da) shows that the polymers are well prepared (Supporting Information, Figure S1b). Table 1 summarizes the list of polymers studied in this paper. Aqueous solutions of the PAF-PLX-PAF (PIII) undergo solto-gel transition as the temperature increases in a concentration

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Scheme 1. Synthesis of the PAF-PLX-PAF

range of 6.0-10.0 wt %. The phase diagram determined by the test tube inverting method is shown in Figure 1a. Photos of the PAF-PLX-PAF aqueous solutions (8.0 wt %) in a sol state at 20 °C and a gel state at 37 °C are also shown. When the concentration was lower than 6.0 wt %, the polymer aqueous solution showed an increase in viscosity as the temperature increased, however, they were not strong enough to resist the flow, and thus, they were defined as a sol state. At polymer concentrations higher than 10.0 wt %, the polymer aqueous system was a gel and did not undergo sol-gel transition over 0-100 °C. Therefore, sol-to-gel transition was observed in a

Figure 1. (a) Phase diagram of the PAF-PLX-PAF (PIII) aqueous solutions determined by the test tube inverting method. Photos are PAF-PLX-PAF (PIII) aqueous solutions (8.0 wt %) at 20 °C (sol state) and 37 °C (gel state). (b) Increases in storage modulus (G′) and loss modulus (G′′) of the PAF-PLX-PAF (PIII) aqueous solutions (8.0 wt %) during the sol-to-gel transition. The plot on a linear scale is inserted. A PLX aqueous solution (8.0 wt %) was compared as a control.

polymer concentration range of 6-10 wt %. As the temperature of the polymer solution in a concentration range of 6-10 wt % increased, the sol-to-gel transition was observed at a specified temperature, and the gel phase persisted until 100 °C and did not show gel-to-precipitation, which was observed for the thermogelling polyester aqueous solution.6,23,27,28 Similar trends were observed for the diblock copolymer of the mPEG-PAF aqueous solution.20 The increases in the storage modulus (G′) and loss modulus (G′′) of PAF-PLX-PAF aqueous solutions (8.0 wt %) were observed as the temperature increased. G′ is an elastic component of the complex modulus and is a measure of the gel-like behavior of a system, whereas G′′ is a viscous component of the complex modulus and is a measure of the sol-like behavior of the system. The crossover of G′ and G′′ at 30 °C is an indication of the sol-to-gel transition (Figure 1b).23,25 To prove that the increases in the moduli are caused by the sol-to-gel transition, the change in the storage modulus and loss modulus of the PLX aqueous solution (8.0 wt %) were compared. There was no noticeable change in the moduli of the PLX aqueous solution, and the fact that G′ was smaller than G′′ indicated a sol state of the PLX aqueous solution in this temperature range. The PAF-PLX-PAF consisting of hydrophilic PLX and hydrophobic PAF blocks tend to self-assemble in water. The assembly process was traced by circular dichroism spectra and hydrophobic dye (1,6-diphenyl-1,3,5-hexatriene) solubilization. In a low polymer concentration range of 0.001-0.01 wt %, CD spectra of PAF-PLX-PAF show a broad negative Cotton band at 210-225 nm. As the polymer concentration increased to 0.05-0.5 wt %, the negative Cotton band sharpened and increased its magnitude. At the same time, a red-shift of the negative Cotton band was observed from 210 to 235 nm (Figure 2a). Such a red-shift of the Cotton band is caused by the selfassembly of the polypeptides to a high order structure as reported for poly(ethylene glycol)-poly(L-alanine) and poly(ethylene glycol)-poly(L-benzyl glutamate) block copolymers.18,26 The CD spectra of PAF-PLX-PAF in relation to the secondary structure of polypeptide will be discussed in detail in the next section. To confirm the self-assembly of the PAF-PLX-PAF in water as the polymer concentration increases, 1,6-dipheny-1,3,5hexatriene was dissolved in the polymer aqueous solution. The dye concentration was fixed at 4.0 µM and the PAF-PLX-PAF concentration varied from 0.001 to 0.5 wt %. As the PAF-PLXPAF concentration increased, the absorbance of dye at 342, 356, and 380 nm increased, which is the typical pattern of hydrophobic domain (micelle) formation of the polymers in water (Figure 2b).24,27 Critical micelle concentration (CMC) was determined by extrapolation of the lines at low and high concentration regions (Figure 2c). CMC determined by CD spectra and the dye solubilization method coincided at 0.02-0.05 wt %. The apparent size and size distribution of the PAF-PLX-PAF (PIII) assemblies in an aqueous solution (0.05 wt %) are

Gelation of PAF-PLX-PAF Block Copolymer Solution

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Figure 4. TEM image of the PAF-PLX-PAF (PIII). The polymer aqueous solution (0.05 wt %) was air-dried at 10 °C. The scale bar is 100 nm.

Figure 2. (a) CD spectra of PAF-PLX-PAF (PIII) aqueous solution at 10 °C as a function of concentration (wt %) in water. (b) Absorbance of hydrophobic dye (1,6-diphenyl-1,3,5-hexatriene) at 10 °C as a function of PAF-PLX-PAF (PIII) concentration in water. The legends in a and b are the concentration (wt %) of PAF-PLX-PAF in water. (c) Determination of CMC of the PAF-PLX-PAF (PIII) in water at 10 °C by the CD and the dye solubilization method (DSM). A crossing point of the extrapolated lines defines the CMC.

Figure 3. Apparent size distribution of PAF-PLX-PAF (PIII) at 0.05 wt % in water at 10 °C.

measured by dynamic light scattering (Figure 3). The most probable size of the polymer assemblies is in a range of 25-50 nm. The assembly pattern of the PAF-PLX-PAF developed from the polymer aqueous solution (0.05 wt %) is shown by TEM images (Figure 4). Even though the micelle images can be partially distorted during the solvent evaporation, TEM images showed spherical micelles with an average size of 20-50 nm,

Figure 5. (a) CD spectra of PAF-PLX-PAF (PIII) aqueous solution (0.05 wt %) as a function of temperature by an increment of 10 °C from 10 to 50 °C. The subtracted spectra at a temperature from the spectra at 10 °C (∆Θ) show the increase in the magnitude of positive band at 200 nm and negative band at 218 nm. (b) FTIR spectra of PAF-PLX-PAF (PIII) aqueous solution (8.0 wt %) as a function of temperature by an increment of 10 °C from 10 to 50 °C. The subtracted spectra at a temperature from the spectra at 10 °C (∆Αbsorbance) show the decrease in absorbance at 1640 cm-1 and increase in absorbance at 1620 cm-1.

which can be correlated with the results observed by dynamic light scattering. To see the change in the secondary structure of polypeptides as a function of temperature, CD and FTIR spectra of the PAFPLX-PAF aqueous solution were investigated. As the temperature increased, the negative Cotton band at 210-230 nm and the positive Cotton band at 190-205 nm in CD spectra were partially increased in magnitude (Figure 5a). The direct interpretation of the CD spectra of PAF-PLX-PAF is interfered with the πfπ* absorption of Phe moiety in this spectral range (Supporting Information, Figure S2: CD spectra of (Phe)(Ala)4 pentapeptide (purchased from Peptron, Korea) aqueous solution).20,29 However, subtracted CD spectra (inset in Figure 5a) gave useful information on the secondary structural change as the temperature increased. The subtracted CD spectra clearly demonstrate the increase in the magnitude of the positive band at 200 nm and the negative band at 218 nm as the temperature increased. The two characteristic CD bands are an indication of a β-sheet structure of a polypeptide.30,31 Therefore, CD spectra suggest that the fraction of β-sheet structure of PAF increases as the temperature increases. As the temperature increased from 10 to 50 °C, the infrared absorption band at 1620 cm-1 (amide I band related to β-sheet

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Figure 6. 13C NMR spectra of the PAF-PLX-PAF (PIII) in D2O (8.0 wt %) as a function of temperature.

Kim et al.

Figure 8. Schematic presentation of the sol-to-gel transition of PAFPLX-PAF aqueous solutions. The thin blue lines and thick green lines indicate the fully hydrated PLX at low temperature and partially dehydrated PLX at high temperature, respectively. Brown lines indicate PAF.

Figure 7. SEM image of the PAF-PLX-PAF hydrogel (xerogel). The polymer aqueous solution (8.0 wt %) was air-dried at 37 °C. The scale bar is 2 µm.

structure) increased, whereas the broadband around 1640 cm-1 (amide I band related to random coil structure) decreased (Figure 5b).32,33 The subtracted FTIR spectra (inset in Figure 5b) clearly demonstrated that the band centered at 1640 cm-1 decreased and the band centered at 1620 cm-1 increased as the temperature increased. Both CD and FTIR spectra suggest that the fraction of β-sheet structure of PAF partially increases as the temperature increases. 13 C NMR spectra of PAF-PLX-PAF (PIII) aqueous solution (8.0 wt % in D2O) showed a significant broadening of a peak at 72.0-72.8 ppm as the temperature increased from 20 to 40 °C (Figure 6). The sol-to-gel transition of the PAF-PLX-PAF aqueous solution was observed in this temperature range. The broadening of the NMR peak is an indication of a decrease in the molecular motion of the of PLX that has been claimed for the dehydration of the poly(ethylene glycol) block.5,10,11 SEM image of the gel was developed by air-drying the 8.0 wt % aqueous PAF-PLX-PAF (PIII) solution on a carbon grid at 37 °C. The image shows that the polymer assemblies with spherical or short cylindrical structures form a percolating network with interaggregate connections (Figure 7). Based on the above observation, the sol-to-gel transition mechanism was suggested (Figure 8). PAF-PLX-PAFs are selfassembled to micelles when the polymer concentration is higher than the critical micelle concentration. The hydrophobic PAFs form a core and hydrophilic PLXs form a shell of the micelle, where the PAFs are self-assembled into a β-sheet structure in the micelle core. As the temperature increases, the PLX dehydrates and β-sheet structure of PAF is partially strengthened to form a percolating network and water is entrapped among the interaggregated connections, which is a gel.

Figure 9. (a) Structure-property relationship of phase diagram of the PAF-PLX-PAF aqueous solutions determined by the test tube inverting method (n ) 3). (b) FTIR spectra of PAF-PLX-PAF aqueous solution (8.0 wt %) in D2O.

The tuning of the sol-to-gel transition could be realized by varying the PAF block length, PLX length, and the Ala/Phe ratio of the PAF (Figure 9). As the ratio of Ala/Phe of the PAF decreases from 83/17 to 76/24 at a similar block length of PAF (680 Da) and PLX (2000 Da; PI and PII), FTIR spectra of the two polymers showed similar secondary structures in water, however, the sol-to-gel transition temperature decreases. This fact suggests that phenylalanine facilitates the sol-to-gel transition by making the polymer more hydrophobic. As the PAF block length increased from 680 to 850 Da at a fixed PLX length (2000 Da; PI and PIII), FTIR spectra showed that a sharp absorption band at 1623 cm-1 increased, whereas a broad absorption band centered at 1640 cm-1 decreased. This fact suggests that a β-sheet structure of PAF is strengthened as the PAF block length increases from 680 to 850 Da. In addition, the sol-to-gel transition temperature decreased as the PAF block length of the PAF-PLX-PAF increased from 680 to 850 Da (PI and PIII). The increase in β-sheet structure of PAF as well as the increase in hydrophobicity of the polymer contributes to the decrease in the sol-to-gel transition temperature.

Gelation of PAF-PLX-PAF Block Copolymer Solution

As the PLX molecular weight of PAF-PLX-PAF decreased from 2000 to 900 Da, the solubility of the PAF-PLX-PAF was significantly decreased. PV with block length of 490-900-490 (PAF-PLX-PAF) developed a dominantly β-sheet structure (FTIR in Figure 9b) and was partially soluble in water as a 6.0 wt % at 10 °C. PIV with block length of 290-900-290 (PAFPLX-PAF) showed sol-to-gel transition at a much lower temperature than the above polymers. Similar decreases in solto-gel transition temperatures with increasing the hydrophilic block length were reported for poly(ethylene glycol)/poly(lactic acid-co-glycolic acid), poly(ethylene glycol)/polycaprolactone, and poly(ethylene glycol)/poly(trimethylene carbonate).5,10,27,28 The in vitro gel stability of PAF-PLX-PAF (PIII) was compared with a typical reverse thermogelling poloxamer (F127). F127 is an (ethylene glycol)99-(propylene glycol)65(ethylene glycol)99 triblock copolymer of which aqueous solutions undergo sol-to-gel transition as the temperature increases. In vitro study showed that the PAF-PLX-PAF (PIII) gel prepared from an initial polymer concentration of 8.0 wt % in water are eliminated less than 30% over one month, whereas F127 prepared from an initial polymer concentration of 30.0 wt % completely disappeared within 5 days (Supporting Information, Figure S3).

Conclusions The reverse thermal gelation and aqueous solution behavior of the PAF-PLX-PAF were studied as a function of temperature. Various instrumental methods such as circular dichrosim spectroscopy, FTIR spectroscopy, hydrophobic dye solubilization, dynamic light scattering, TEM, dynamic mechanical analysis, 13C NMR spectroscopy, and SEM suggest that the polymers form micelles in water and the sol-to-gel transition accompanies a partial dehydration of PLX and secondary structural change of PAF from random coil to β-sheet. The solto-gel transition temperature decreases as the ratio of Ala to Phe decreases, PAF length increases, and the PLX length decreases. The polypeptide-based thermogelling aqueous solution is suggested to be a promising in situ depot forming system with durability as a gel in the excess amount of water. Acknowledgment. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST; Grant No. 2009-0080447), the World Class University (WCU) project of the Ministry of Education, Science and Technology (MEST), the National Research Foundation of Korea (NRF) through Ewha Womans University (Grant No. R31-2008-000-10010-0), and Korea Research Foundation Grant funded by the Korean government (MOEHRD, Basic Research Promotion Fund, Grant No. KRF2006-005-J04003). Supporting Information Available. 1H NMR of PLX and PAF-PLX-PAF (PIII) in CF3COOD, GPC chromatogram of PLX (Mn ) 2000 Da) and PAF-PLX-PAF (PIII), circular dichroism spectra of (Phe)(Ala)4 aqueous solutions (0.01 wt %), in comparison of the in vitro duration of the in situ formed gel

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PAF-PLX-PAF and F127. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Jeong, B.; Kim, S. W.; Bae, Y. H. AdV. Drug DeliVery ReV. 2002, 54, 37–51. (2) Loh, X. J.; Li, J. Expert Opin. Ther. Pat. 2007, 17, 965–977. (3) He, C.; Kim, S. W.; Lee, D. S. J. Controlled Release 2008, 127, 189– 207. (4) Yu, L.; Ding, J. Chem. Soc. ReV. 2008, 37, 1473–1481. (5) Hwang, M. J.; Suh, J. M.; Bae, Y. H.; Kim, S. W.; Jeong, B. Biomacromolecules 2005, 6, 885–890. (6) Yu, L.; Zhang, H.; Ding, J. Angew Chem., Int. Ed. 2006, 45, 2232– 2235. (7) Nagahama, K.; Ouchi, T.; Ohya, Y. AdV. Funct. Mater. 2008, 18, 1220–1231. (8) Chenite, A.; Chaput, C.; Wang, D.; Combes, C.; Buschmann, M. D.; Hoemann, C. D.; Leroux, J. C.; Atkinson, B. L.; Binette, F.; Selmani, A. Biomaterials 2000, 21, 2155–2161. (9) Lee, B. H.; Lee, Y. M.; Sohn, Y. S.; Song, S. C. Macromolecules 2002, 35, 3876–3879. (10) Kim, S. Y.; Kim, H. J.; Lee, K. E.; Han, S. S.; Sohn, Y. S.; Jeong, B. Macromolecules 2007, 40, 5519–5525. (11) Choi, B. G.; Sohn, Y. S.; Jeong, B. J. Phys. Chem. B 2007, 111, 7715– 7718. (12) Sosnik, A.; Cohn, D. Biomaterials 2004, 25, 2851–2858. (13) Loh, X. J.; Tan, Y. X.; Li, Z.; Teo, L. S.; Goh, S. H.; Li, J. Biomaterials 2008, 29, 2164–2172. (14) Gezimati, J.; Singh, H.; Creamer, L. K. J. Agric. Food Chem. 1996, 44, 804–810. (15) Lao, U. L.; Sun, M.; Matsumoto, M.; Mulchandani, A.; Chen, W. Biomacromolecules 2007, 8, 3736–3739. (16) Megeed, Z.; Cappello, J.; Ghandehari, H. AdV. Drug DeliVery ReV. 2002, 54, 1075–1091. (17) Pochan, D. J.; Schneider, J. P.; Kretsinger, J.; Ozbas, B.; Rajagopal, K.; Haines, L. J. Am. Chem. Soc. 2003, 125, 11802–11803. (18) Choi, Y. Y.; Joo, M. K.; Sohn, Y. S.; Jeong, B. Soft Matter 2008, 4, 2383–2387. (19) Oh, H. J.; Joo, M. K.; Sohn, Y. S.; Jeong, B. Macromolecules 2002, 41, 8204–8209. (20) Jeong, Y.; Joo, M. K.; Bahk, K. H.; Choi, Y. Y.; Kim, H. T.; Kim, W. K.; Sohn, Y. S.; Lee, H. J.; Jeong, B. J. Controlled Release 2009, 137, 25–30. (21) Lecommandoux, S.; Achard, M. F.; Langenwalter, J. F.; Klok, H. A. Macromolecules 2001, 34, 9100–9111. (22) Tanodekaew, S.; Godward, J.; Heatley, F.; Booth, C. Macromol. Chem. Phys. 1997, 198, 3385–3395. (23) Jeong, B.; Windisch, C. F.; Park, M. J.; Sohn, Y. S.; Gutowska, A.; Char, K. J. Phys. Chem. B 2003, 107, 10032–10039. (24) Booth, C.; Attwood, A. Macromol. Rapid Commun. 2000, 21, 501– 527. (25) Sarvestani, A. S.; He, X.; Jabbari, E. Biomacromolecules 2007, 8, 406– 415. (26) Ding, W.; Lin, S.; Lin, J.; Zhang, L. J. Phys. Chem. B 2008, 112, 776–783. (27) Jeong, B.; Bae, Y. H.; Kim, S. W. Macromolecules 1999, 32, 7064– 7069. (28) Yu, L.; Chang, G.; Zhang, H.; Ding, J. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 1122–1133. (29) Hamley, I. W.; Krysmann, M. J.; Castelletto, V.; Noirez, L. AdV. Mater. 2008, 20, 4394–4397. (30) Hwang, J.; Deming, T. J. Biomacromolecules 2001, 2, 17–21. (31) Hamley, I. W.; Ansari, I. A.; Castelletto, V.; Nuhn, H.; Rosler, A.; Klok, H. A. Biomacromolecules 2005, 6, 1310–1315. (32) Ozbas, B.; Kretsinger, J.; Rajagopal, K.; Schneider, J. P.; Pochan, D. J. Macromolecules 2004, 37, 7331–7337. (33) Yamada, N.; Ariga, K.; Natio, M.; Matsubara, K.; Koyama, E. J. Am. Chem. Soc. 1998, 120, 12192–12199.

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