Article pubs.acs.org/Macromolecules
PEG-L-PAF and PEG-D-PAF: Comparative Study on Thermogellation and Biodegradation Eun Young Kang, Bora Yeon, Hyo Jung Moon, and Byeongmoon Jeong* Department of Bioinspired Science (WCU), Department of Chemistry and Nano Science, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-750, Korea S Supporting Information *
ABSTRACT: L-Polypeptides and D-polypeptides can be prepared from natural L-amino acids and non-natural Damino acids, respectively. In this study, poly(ethylene glycol)− poly(L-alanine-co-L-phenyl alanine) (PEG-L-PAF) and poly(ethylene glycol)−poly(D-alanine-co-D-phenyl alanine) (PEGD-PAF) with similar molecular weight and composition, but different stereochemistry were investigated, focusing on thermogelling behavior and biodegradation. The sol-to-gel transition temperature of both PEG-L-PAF and PEG-D-PAF aqueous solutions decreased from 26 to 7 °C as the concentration increased from 4.0 wt % to 9.0 wt %. Dynamic light scattering, transmission electron microscopy, circular dichroism spectra, and 13C NMR spectra suggested that the sol-to-gel transition involved changes in molecular assemblies resulting from dehydration of PEG for both PEG-L-PAF and PEG-D-PAF. In particular, the significant differences between PEGL-PAF and PEG-D-PAF were observed for histocompatibility as well as in vitro/in vivo degradation. Only PEG-L-PAF was significantly degraded by cathepsin B and elastase, as well as under in vivo conditions. The histocompatibility assayed by the H&E staining method showed that formation of the collagen capsule around the PEG-D-PAF gel was thicker than the PEG-LPAF gel, indicating that acute inflammation was milder with PEG-L-PAF gel than with PEG-D-PAF gel. Current study emphasizes the significance of stereochemistry in biomaterial development.
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INTRODUCTION Polypeptides have recently been drawing attention for uses as specific building blocks, drug delivery carriers, and tissue engineering scaffolds, due to the fact that they can be used to produce specific secondary structures or nanostructures by modulating their amino acid composition and sequences.1−5 Amino acids, except for glycine, have a chiral center, which can be a natural L-form or a non-natural D-form. The application of polypeptides as biopharmaceutical intermediates or vaccines is based on the chemical or enzymatic conversions among this stereospecific species.6−8 From a biomaterial point of view, a polypeptide can be degraded by proteolytic enzymes and the degradation of the polypeptide can be controlled by varying the stereospecificity of the constituent amino acids.9−11 Aqueous solutions of thermogelling polymer undergo sol-togel transition as the temperature increases. They are lowviscous aqueous solutions at low temperatures and become a semisolid gel at high temperatures, typically at the body temperature of warm-blooded mammal (37 °C). The phase transition polymeric systems have been extensively investigated as minimally invasive drug delivery depots, injectable tissue engineering scaffolds, and biomaterials that prevent postsurgical adhesion, due to the simple procedures required for encapsulating pharmaceutical agents or cells, followed by facile implantation of the depot at a target site.12−17 In this case, the duration of the in situ formed gel is a very important parameter © 2012 American Chemical Society
for practical applications. Ideally, the duration should be matched to the biomedical function of the system. Therefore, a gel should be eliminated after releasing all the encapsulated drug from the drug delivery system, and the tissue growth rate should be matched to the gel elimination rate for a tissue engineering scaffold.18 In this study, we synthesized thermogelling poly(ethylene glycol)−poly(alanine-co-phenyl alanine) (PEG−PAF) block copolymers of PEG-L-PAF and PEG-D-PAF with a similar molecular weight, but different chiralities. Alanine−phenyl alanine and alanine−alanine sequences are degraded by proteolytic enzymes in the subcutaneous layer of mammals such as cathepsin B, cathepsin C, elastase, and metalloproteinase-2 (MMP-2).10,19,20 Therefore, the PAF was chosen as a degradable polypeptide block. As the sequence and other chemical factors affect thermogelling behavior, biodegradability, and biocompatibility,21−23 we herein examine the effect of chirality of a thermogel consisting of PEG and PAF. The PEG-L-PAF and PEG-D-PAF consist of naturally occurring L-amino acids and non-natural D-amino acids, respectively. The physicochemical properties, in vitro/in vivo Received: December 29, 2011 Revised: February 10, 2012 Published: February 16, 2012 2007
dx.doi.org/10.1021/ma202809c | Macromolecules 2012, 45, 2007−2013
Macromolecules
Article
elastase; in phosphate buffered saline (150 mM) with 0.02 wt % Triton X-100, 0.1 mM ethylene diamine tetraacetic acid, and 5 mM glutathione at pH = 5.5 for cathepsin B; and in Tris buffered solution (0.05 M) with 10 mM CaCl2 at pH = 7.4 for MMP-2. The polymer concentration was fixed at 1.0 wt %. Five units of each enzyme were used for the in vitro study except for MMP-2, where a concentration of 2.5 μg/mL was used because information concerning the number of enzyme (MMP-2) units was not available. The recovered samples were extracted with N,N-dimethylformamide for molecular weight analysis by gel permeation chromatography. In Vivo Degradation. Aqueous polymer solutions (0.5 mL; 5.0 wt % of PEG−PAF) contained in prefilled syringes were injected into the subcutaneous layer of rats using a 25 gauge syringe needle. Rats were sacrificed 1 day, 5 days, and 15 days after injection for the gel duration study. The remaining gel was removed from the rats and stored at −20 °C. Histology. The histology around the implant site was investigated for 5 days after implantation. Neutral buffered formalin (NBF) solution was prepared by mixing formaldehyde solution (37−40%; 100 mL), sodium phosphate monobasic (4.0 g), sodium phosphate dibasic (6.5 g), and deionized water (900 mL). The tissue surrounding the gel was stored in NBF solution at −20 °C. After 24 h, the tissue was embedded in a frozen section compound (Leica, USA) and stored at −80 °C. Microcryotomed sections of tissue with a thickness of ∼8 μm were stained with hematoxylin and eosin (H&E), and examined by microscopy (Olympus lX71-F22PM, Japan). Animal Procedures. All experimental procedures using animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Committee of Ewha Womans University.
degradation profiles, and tissue compatibility of the PEG-L-PAF and PEG-D-PAF were compared.
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EXPERIMENTAL SECTION
Materials. α-Amino-ω-methoxy-poly(ethylene glycol) (PEG) (M.W ∼ 2000 Da) (ID Bio, Korea), N-carboxy anhydrides of Lalanine, N-carboxy anhydrides of D-alanine, N-carboxy anhydrides of Lphenyl alanine, and N-carboxy anhydrides of D-phenyl alanine (KPX Life Science, Korea) were stored at 4 °C in a desiccator filled with drying agents under high vacuum and used as needed. Toluene (Aldrich, USA) was dried over sodium before use. Chloroform (Aldrich, USA) and N,N-dimethylformamide (anhydrous) (Aldrich, USA) were treated with anhydrous magnesium sulfate before use. Synthesis of PEG−PAF. PEG-L-PAF was prepared by ringopening polymerization of the N-carboxy anhydrides of L-alanine and N-carboxy anhydrides of L-phenyl alanine in the presence of α-aminoω-methoxy-poly(ethylene glycol).10,24,25 PEG-D-PAF was similarly prepared by using N-carboxy anhydrides of D-alanine and N-carboxy anhydrides of D-phenyl alanine in the presence of α-amino-ω-methoxypoly(ethylene glycol). 1 H and 13C NMR Spectroscopy. 1H NMR spectra of the PEG− PAF (in CF3COOD) (500 MHz NMR spectrometer; Varian, USA) were used to determine the composition of the polymer. 13C NMR spectral changes of the PEG−PAF (5.0 wt % in D2O) were investigated as a function of temperature. The solution temperature was equilibrated for 20 min at each temperature. Phase Diagram. The sol−gel transition of the polymer aqueous solution was investigated by the test tube inverting method. The aqueous polymer solution (1.0 mL) was added into a vial with an inner diameter of 11 mm. The transition temperature was determined by the flow (sol)−nonflow (gel) criterion with a temperature increment of 1 °C per step. Each data point is an average of 3 measurements. Dynamic Mechanical Analysis. Changes in modulus of the polymer aqueous solutions were investigated by dynamic rheometry (Thermo Haake, Rheometer RS 1). The aqueous polymer solution was placed between parallel plates of 25 mm diameter with a gap of 0.5 mm. To minimize water evaporation during the experiment, the plates were enclosed in a water saturated chamber. The data were collected under conditions of controlled stress (4.0 dyn/cm2) and a frequency of 1.0 rad/s. The heating rate was 0.5 °C/min. Transmission Electron Microscopy (TEM). The PEG−PAF aqueous solution (10 μL; 0.01 wt %) at 10 °C 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. Dynamic Light Scattering. The apparent size of PEG−PAF aggregates in water (1.0 wt %) was studied by a dynamic light scattering instrument (ALV 5000−60 × 0) as a function of temperature. The aqueous solution was equilibrated for 20 min at each temperature. A YAG DPSS-200 laser (Langen, Germany) operating at 532 nm was used as a light source. 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 could be obtained by the Stokes−Einstein equation. Circular Dichroism (CD) Spectroscopy. A CD spectrophotometer (J-810, JASCO) was used to study the ellipticity of the PEG−PAF aqueous solution as a function of polymer concentration at a fixed temperature of 20 °C in a polymer concentration range of 0.01−1.0 wt %. In addition, ellipticity of the PEG−PAF aqueous solution was obtained as a function of temperature at a fixed concentration of 0.01 wt % in a range of 10−50 °C at increments of 10 °C per step. The aqueous solution was equilibrated for 20 min at each temperature. In Vitro Degradation. The in vitro degradation of PEG−PAF was studied over a 3 day period in phosphate buffered saline (150 mM) at pH = 7.4 for the control and chymotrypsin; in phosphate buffered saline (150 mM) with 1.0 mM CaCl2 at pH = 7.4 for collagenase and
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RESULTS AND DISCUSSION The N-carboxy anhydrides of alanine and the N-carboxy anhydrides of phenyl alanine were copolymerized on the amine-terminated PEG to prepare the PEG−PAF.10,19,20 The molecular weight of PEG−PAF was calculated by 1H NMR spectra (CF3COOD) (Figure 1). The end group of alanine
Figure 1. 1H NMR spectra (CF3COOD) of PEG-L-PAF and PEG-DPAF.
(methyl) (1.7−1.9 ppm) was distinguished from internal alanine (methyl) (1.2−1.7 ppm) of the PEG−PAF in the 1H NMR spectra. The integration of the peaks in 1H NMR spectra at 1.2−1.9 ppm (−CH 3 of alanine), 3.8−4.1 ppm (−CH2CH2O− of PEG), and 7.0−7.5 ppm (phenyl group of phenylalanine) was used to calculate the composition and the molecular weight of the PEG−PAF by the following equations. CH3O(CH2CH2O)n CH2CH2{[NHCOCH(CH3)]x [NHCOCH(CH2C5H5)]y } − NH2D+ 2008
dx.doi.org/10.1021/ma202809c | Macromolecules 2012, 45, 2007−2013
Macromolecules
Article
sol-to-gel transition (Figure 2b).26,27 G′ and G″ are the measures of an elastic component and a viscous component of a complex modulus (G*). Therefore, the elastic component dominates the viscous component above the sol-to-gel transition temperature. The aqueous solution (5.0 wt %) of PEG with a molecular weight of 2000 Da that did not show a significant change in G′ or G″ were also compared. The G′ was smaller than G″ over the temperature range studied, suggesting that the PEG aqueous solution maintained its sol state (Figure 2b). PEG−PAF consists of the hydrophilic PEG block and the hydrophobic PAF block, therefore, the polymers self-assembled into micelles in water. The apparent sizes of the self-assemblies of PEG−PAF were measured by dynamic light scattering at a 1.0 wt % concentration. Particles with a size of 10−60 nm were observed at 10 °C, and random molecular aggregation led to formation of large particles (100−800 nm) with a broad size distribution as the temperature increased to 30 °C (Figure 3a
n = 44 for PEG with a molecular weight of 2000 Da. A1.2 − 1.9 /A3.8 − 4.1 = 3x /180 A7.0 − 7.5/A3.8 − 4.1 = 5y/180
A1.2−1.9, A3.8−4.1, and A7.0−7.5 are the areas of the peaks at 1.2−1.9 ppm, 3.8−4.1 ppm, and 7.0−7.5 ppm in the 1H NMR of PEG− PAF. The number 180 is the number of protons related to PEG appearing at A3.8−4.1. On the basis of the assignments, the structures of PEG-L-PAF and PEG-D-PAF were calculated to be EG44-L-A12F4 and EG44-D-A13F4, respectively. The molecular weight distribution determined by GPC was 1.1−1.2. The unimodal distribution of the polymer molecular weight in GPC shows that the PEG−PAF was well prepared (Supporting Information, Figure S1). PEG−PAF aqueous solutions underwent sol-to-gel transition as the temperature increased. The phase diagram of the PEG− PAF aqueous solution was determined by the test tube inverting method (Figure 2a). A sol-to-gel transition of the
Figure 2. (a) Phase diagram of PEG−PAF aqueous solutions determined by the test tube inverting method (n = 3). (b) Modulus of the PEG−PAF aqueous solutions (5.0 wt %) as a function of temperature. G″ of α-amino-ω-methoxy−poly(ethylene glycol) (PEG) aqueous solution (5.0 wt %) is compared as a control. G′ of the PEG aqueous solution was
