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Fabrication of Supramolecular Hydrogels for Drug Delivery and Stem Cell Encapsulation De-Qun Wu,† Tao Wang,‡ Bo Lu,† Xiao-Ding Xu,† Si-Xue Cheng,† Xue-Jun Jiang,*,‡ Xian-Zheng Zhang,*,† and Ren-Xi Zhuo† Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan UniVersity, Wuhan 430072, China, and Department of Cardiology, Renmin Hospital of Wuhan UniVersity, Wuhan 430060, China ReceiVed March 4, 2008. ReVised Manuscript ReceiVed June 13, 2008 Supramolecular hydrogels self-assembled by R-cyclodextrin and methoxypolyethylene glycol-poly(caprolactone)(dodecanedioic acid)-poly(caprolactone)-methoxypolyethylene glycol (MPEG-PCL-MPEG) triblock polymers were prepared and characterized in Vitro and in ViVo. The sustained release of dextran-fluorescein isothiocyanate (FITC) from the hydrogels lasted for more than 1 month, which indicated that the hydrogels were promising for controlled drug delivery. ECV304 cells and marrow mesenchymal stem cells (MSC) were encapsulated and cultured in the hydrogels, during which the morphologies of the cells could be kept. The in Vitro cell viability studies and the in ViVo histological studies demonstrated that the hydrogels were non-cytotoxic and biocompatible, which indicated that the hydrogels prepared were promising candidates as injectable scaffolds for tissue engineering applications.
Introduction Polymeric hydrogels are three-dimensional networks with great potential for biomedical applications owing to their good biocompatibility.1-4 Hydrogels for cell encapsulation and proliferation are of importance in tissue engineering5-8 because hydrogels own the excellent property of permeability, which allows diffusion and transport of essential materials, such as oxygen and nutrients for cells. Chemical crosslinked poly(ethylene glycol) (PEG)-based hydrogels have been exploited extensively for biomaterials.9-13 However, because of covalent crosslinkages, the hydrogels are limited in implantables; furthermore, the time consumed in the incorporation of drugs also prohibits their applications. On the other hand, during the crosslinking reaction of the hydrogels, the properties of the drug could be destroyed. Thus, it is attractive to develop hydrogels where gelation and drug loading or cell encapsulation can be obtained simultaneously in aqueous solution without chemical crosslinking reaction. * Corresponding authors. Tel.: + 86-27-6875 4061. Fax: + 86-27-6875 4509. E-mail addresses:
[email protected] (X.-J.J.), or
[email protected] (X.-Z.Z.). † Wuhan University. ‡ Renmin Hospital of Wuhan University. (1) Shim, W. S.; Kim, J. H.; Park, H.; Kim, K.; Kwon, I. C.; Lee, D. S. Biomaterials 2006, 27, 5178–5185. (2) Gonen-Wadmany, M.; Oss-Ronen, L.; Seliktar, D. Biomaterials 2007, 28, 3876–3886. (3) Zhang, Y. J.; Wang, S. P.; Eghtedari, M.; Motamedi, M.; Kotov, N. A. AdV. Funct. Mater. 2005, 15, 725–731. (4) Liu, Y. C.; Shu, X. Z.; Prestwich, G. D. Biomaterials 2005, 26, 4737–4746. (5) Rowley, J. A.; Madlambayan, G.; Mooney, D. J. Biomaterials 1999, 20, 45–53. (6) Nguyen, K. T.; West, J. L. Biomaterials 2002, 23, 4307–4314. (7) Elisseeff, J.; Ferran, A.; Hwang, S.; Varghese, S.; Zhang, Z. Stem Cells DeV. 2006, 15, 295–303. (8) Iemma, F.; Spizzirri, U. G.; Puoci, F. Int. J. Pharm. 2006, 312, 151–157. (9) Zhu, A. P.; Chan-Park, M. B.; Gao, J. X. J. Biomed. Mater. Res., Part B: Appl. Biomater. 2006, 76B, 76–84. (10) Cho, C. S.; Han, S. Y.; Ha, J. H.; Kim, S. H.; Lim, D. Y. Int. J. Pharm. 1999, 181, 235–242. (11) Du, J. Z.; Sun, T. M.; Weng, S. Q.; Chen, X. S.; Wang, J. Biomacromolecules 2007, 8, 3375–3381. (12) Zhang, X. Z.; Chu, C. C. Polymer 2005, 46, 9664–9673. (13) Moffat, K. L.; Marra, K. G. J. Biomed. Mater. Res., Part B: Appl. Biomater. 2004, 71B, 181–187.
Although some physical hydrogels such as pluronic triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)poly(ethylene oxide)14,15 that undergo gel-sol transition within the temperature range were reported, they are not biodegradable. Triblock copolymers consisting of PEG and biodegradable blocks, such as poly(L-lactic acid) or poly(glycolic acid), which exhibit reversible sol-gel transitions in aqueous solutions, could be used as injectable systems.16-19 However, most physical hydrogels are not stable enough to sustain drug release for a long time. To date, supramolecular self-assembled hydrogels have attracted much interest in biological applications. Hydrogels formed by linear polymers threading the cavities of the cyclodextrins (CDs) are the typical examples. CDs are cyclic oligosaccharides, which mainly consist of six, seven, or eight R-D-glucopyranose units (R-CD, β-CD, and γ-CD, respectively). They are water-soluble but have hydrophobic internal cavities, with diameters through which some polymers can penetrate and form inclusion complexes.20-22 Here, we prepared a sort of hydrogel formed between R-CD and triblock polymers consisting of methoxypolyethylene glycol-poly(caprolactone)-(dodecanedioic acid)-poly(caprolactone)-methoxypolyethylene glycol (MPEG-PCL-MPEG) with different molecular weights. PEG is a water-soluble polymer that exhibits properties such as low toxicity, protein resistance, and immunogenicity. Furthermore, PEG can eliminate the immunogenicity and keep the biological properties of proteins. PEG has been frequently used for tissue engineering and drug delivery systems to improve the biocom(14) Alexandridis, P.; Hatton, T. A. Colloid Surf. 1995, 96, 1–46. (15) Bromberg, L. E.; Ron, E. S. AdV. Drug DeliVery ReV. 1998, 31, 197–221. (16) Yu, L.; Chang, G. T.; Zhang, H.; Ding, J. D. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 1122–1133. (17) Shim, W. S.; Kim, S. W.; Lee, D. S. Biomacromolecules 2006, 7, 1935– 1941. (18) Shim, W. S.; Yoo, J. S.; Bae, Y. H.; Lee, D. S. Biomacromolecules 2005, 6, 2930–2934. (19) Yu, L.; Zhang, H.; Ding, J. D. Angew. Chem., Int. Ed. 2006, 45, 2232– 2235. (20) Szejtli, J. Chem. ReV. 1998, 98, 1743–1754. (21) Harada, A.; Li, J.; Kamachi, M. Nature 1994, 370, 126–128. (22) Wenz, G. Angew. Chem., Int. Ed. 1994, 33, 803–822.
10.1021/la8006876 CCC: $40.75 2008 American Chemical Society Published on Web 08/05/2008
R-CD/MPEG-PCL-MPEG Supramolecular Hydrogels
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Scheme 1. Synthesis of MPEG-PCL-MPEG Triblock Polymer and Preparation of the r-CD/MPEG-PCL-MPEG Supramolecular Hydrogel
patibility of resultant polymers.23-25 Dodecanedioic acid (DDDA) is a carbon chain acid that is soluble in water with lower acidity, and used as a biocompatible material.26 Poly(ε-caprolactone) (PCL), a well known biodegradable aliphatic polyester with good biocompatibility, has also been widely explored for scaffolds in tissue engineering27,28 and matrices in drug controlled release.29 The hydrolysis degradation products of PCL are the same as the intermediates of cell metabolism.30 It is known that water-soluble polymers with molecular weight (Mn) above 10 000 are not suitable for filtration through the kidney membrane of a human owing to the large hydrodynamic radius.31 In this study, the average molecular weight of the moiety in the block polymers is below 10 000. In Vitro cell viability studies and in ViVo histological studies demonstrate that the hydrogel is non-cytotoxic and biocompatible, which indicates the hydrogels are promising injectable scaffolds for tissue engineering applications. The study of hydrogel scaffolds for MSC encapsulation for myocardial infarction therapy is currently in progress in our lab.
Materials and Methods Alpha-cyclodextrin (R-CD), methoxypolyethylene glycol 5000 (MPEG5000) and fluorescein isothiocyanate labeled dextran (dextranFITC 20 000) were obtained from Sigma Chemical Company (St. Louis, MO) and used without further purification. ε-Caprolactone (CL) was purchased from Sigma Chemical Company and dried over CaH2 for 48 h and then distilled under reduced pressure. Stannous 2-ethyl hexanoate (SnOct2) was obtained from Shanghai Chemical Reagent Co., China and used after distillation under reduced pressure. Toluene and tetrahydrofuran (THF) were dried by refluxing over CaH2 and Na complex and distilled just before use. Dichloromethane (DCM) was distilled before use. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GIBCO invitrogen corporation. (23) Won, C. Y.; Chu, C. C.; Lee, J. D. J. Polym. Sci, Part A: Polym. Chem. 1998, 36, 2949–2959. (24) Won, C. Y.; Chu, C. C.; Lee, J. D. Polymer 1998, 25, 6677–6681. (25) Chandy, T.; Mooradian, D. L.; Rao, G. H. J. Appl. Polym. Sci. 1998, 70, 2143–2153. (26) Guo, W. X.; Huang, K. X. Polym. Degrad. Stab. 2004, 84, 375–381. (27) Rhee, S. H.; Choi, J. Y.; Kim, H. M. Biomaterials 2002, 23, 4915–4921. (28) Kweon, H. Y.; Yoo, M. K.; Park, I. K.; Kim, T. H.; Lee, H. C.; Lee, H. S.; Oh, J. S.; Akaike, T.; Cho, C. S. Biomaterials 2003, 24, 801–808. (29) Prabu, P.; Dharmarj, N.; Aryal, S.; Lee, B. M.; Ramesh, V.; Kim, H. Y. J. Biomed. Mater. Res. Part A: 2006, 79A, 153–158. (30) Gilbert, R. D.; Stannett, V.; Pitt, C. G.; Schindler, A. In The Design of Biodegradable Polymers: Two Approaches in DeVelopment in Polymer Degradation; Grassie, N., Ed.; Elsevier: Amsterdam, 1982; pp 259–293. (31) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860–862.
Sodium azide, N,N′-dicyclohexylcarbodiimide (DCC), 4-(N,Ndimethylamino)-pyridine (DMAP) were purchased from Shanghai Chemical Reagent Co., China, and used without further purification. DDDA was purchased from Shanghai Chemical Reagent Co., China, and was recrystallized from ethanol at least twice. A total of 29 male adult Wistar rats with weights of 220-260 g were bought from the laboratory animal center, Tongji Medical Institute, Huazhong University of Science and Technology, China. Synthesis of MPEG-PCL-MPEG Triblock Copolymers. The synthesis of MPEG-PCL-MPEG triblock copolymers is schematically illustrated in Scheme 1. Carboxyl-terminated PCL was initially synthesized as follows. CL (9.12 g, 0.08 mol) and DDDA (0.0232 g, 0.01 mol) were reacted in the presence of catalyst SnOct2 (0.0972 g, 0.00024 mol, 3.0 mol % with respect to CL) under vigorous stirring in a glass ampoule under vacuum at 120 °C for 10 h. PCLDDDA-PCL was collected by dissolving the cooled reaction mixture in THF, precipitating at least twice in 10-fold ice-cold methanol, and filtrating under reduced pressure. 200 mL of ethanol was added to the resulting product. After vigorous stirring for 5 h, the product was obtained by filtering to remove the unreacted DDDA. The yield of the product was 78%. Then, PCL-DDDA-PCL (2.38 g, 0.005 mol, Mn ) 4760), DMAP (0.0915 g, 0.0075 mol), and MPEG5000 (7.5 g, 0.0015 mol) were dried in a 100-mL two-neck flask under vacuum at 60 °C over night. Then DCC (0.309 g, 0.0015 mol) was dissolved in 20 mL of anhydrous DCM and added to the flask. The mixture was stirred at room temperature under nitrogen for a day. Precipitated dicyclohexylurea (DCU) was removed by filtration, and the product was precipitated from diethyl ether two times. The product was further purified by redissolving in DCM, and dialysis against DCM (cut-off Mn 8000-12000) for 3 days to remove the unreacted PCL-DDDA-PCL and MPEG. The resulting copolymer was obtained by rotary evaporation, and the obtained solid was redissolved in THF, dialyzed against distilled water for 3 days, and freeze-dried in a Virtis Freeze-Drier under vacuum at -54 °C for 3 days. 1H NMR. The 1HNMR spectra of PCL-DDDA-PCL, MPEGPCL-MPEG, R-CD, and the inclusion complex formed between R-CD and the triblock polymers were recorded on a Mercury VX300 spectrometer at 300 MHz (Varian, U.S.A.) by using CDCl3 or deuterated dimethyl sulfoxide (DMSO-d6) as a solvent and tetramethylsilane (TMS) as an internal standard. Gel Permeation Chromatography. The molecular weight of the polymers was obtained by a gel permeation chromatographic (GPC) system equipped with a Waters 2690D separations module, and a Waters 2410 refractive index detector. THF was used as the eluent at a flow rate of 0.3 mL/min. Waters millennium module software was used to calculate the molecular weight on the basis of
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Table 1. Feed Compositions of the Supramolecular Hydrogels
DP0 DP1 DP2 DP3
polymer (wt %)
gel composition R-CD (wt %)
FITC-dextran (wt %)
H2O (wt %)
10.0 (MPEG5000) 10.0 (MPEG-PCL-MPEG, 5000-4760-5000) 10.0 (MPEG-PCL-MPEG, 5000-6310-5000) 10.0 (MPEG-PCL-MPEG, 5000-8170-5000)
10.0 10.0 10.0 10.0
0.50 0.50 0.50 0.50
79.5 79.5 79.5 79.5
a universal calibration curve generated by polystyrene standards of narrow molecular weight distribution. Fluorescence Spectroscopy. Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin-Elmer). Pyrene was used as a hydrophobic fluorescent probe. Aliquots of pyrene solutions (6 × 10-6 M in acetone, 1 mL) were added to containers, and the acetone was allowed to evaporate. Three-milliliter aqueous solutions of polymer (MPEG-PCL-MPEG (5000-4760-5000), MPEG-PCLMPEG (5000-6310-5000), MPEG-PCL-MPEG (5000-8170-5000), MPEG-PCL-MPEG (5000-9050-5000)) at different concentrations were added to the containers, which contained the pyrene residue. The aqueous sample solutions contained pyrene residue at the same concentration of 6 × 10-7 M. The solutions were kept at room temperature for 24 h to reach the solubilization equilibrium of pyrene in the aqueous phase. Emission was carried at 390 nm, and excitation spectra were recorded ranging from 240 to 360 nm. Both excitation and emission slit widths were 10 nm. From the pyrene excitation spectra, the intensities (peak height) of 337 nm (I337) were analyzed as a function of the polymer concentrations. A critical micellar concentration (CMC) value was determined from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentration. Preparation of Hydrogels. The aqueous solution of R-CD with a certain concentration was added to an aqueous solution of MPEGPCL-MPEG triblock copolymer with a certain concentration. The mixture was vigorously stirred followed by sonication for 2 min, then incubated in a 37 °C water bath for a few minutes. Through the self-assembly of the R-CD/MPEG-PCL-MPEG inclusion complexes, the hydrogel was formed. The feed compositions of the R-CD and MPEG-PCL-MPEG and other reactants were listed in Table 1, and resulting R-CD/MPEG-PCL-MPEG hydrogels were designated as DP0, DP1, DP2, and DP3, respectively, with different compositions. Wide-Angle X-ray Diffraction. The X-ray diffraction (XRD) patterns were obtained by a Shimadzu XRD-6000 diffractometer with a Ni filter and Cu KR1 radiation (λ: 1.54056 Å; voltage: 40 kV; current: 40 mA). Powder samples were mounted on a sample holder and scanned with a speed of 0.6° per minute. Rheological Studies. The dynamic rheology was measured on an ARES-RFS III rheometer (TA Instruments, U.S.A.). Double concentric cylinder geometry with a gap of 2 mm was used to measure steady viscoelastic parameters as functions of shear rate. Temperature control was established by connection with a julabo FS18 cooling/ heating bath kept within 0.2 °C. Morphology of the Hydrogels. The R-CD/MPEG-PCL-MPEG hydrogels were kept for 12 h after formation, then frozen quickly in liquid nitrogen and freeze-dried in a Virtis Freeze-Drier under vacuum at -54 °C for 2 days. The interior morphology of the hydrogels was studied by a scanning electron microscope (SEM). In Vitro Release of Dextran-FITC from the Hydrogels. DextranFITC was used as a model drug for the in Vitro drug release study. 2.0 mg of dextran-FITC (Mn 20 000) was dissolved in 200 mg of phosphate-buffered saline (PBS) solution (pH ) 7.4) containing of 20.0 wt % R-CD. The solution was added into a 1.0 mL cuvette with 40 mg of MPEG-PCL-MPEG triblock copolymer and 180 mg of PBS solution. The solution was mixed thoroughly and incubated in a 37 °C water bath overnight to form a viscous hydrogel with 10.0% of R-CD and 10.0% of MPEG-PCL-MPEG triblock copolymer. The cuvette was placed in a test tube with 50 mL of PBS solution. The test tube was shaken in a shaking water bath at 37 °C. At predetermined times, 4 mL of buffer medium was removed from the cuvette, and the volume of the PBS solution in the test tube was remained by adding the same volume of fresh PBS solution. The
concentrations of the dextran-FITC released from the hydrogel were analyzed using a LS55 luminescence spectrometer (Perkin-Elmer). Cytotoxicity Study. 1000 µL of ECV304 in DMEM with a concentration of 6.0 × 104 cells/mL was added to each well in a 24-well plate. After incubation for 24 h in an incubator (37 °C, 5% CO2), the culture medium was replaced by 1000 µL of DMEM containing the R-CD/MPEG-PCL-MPEG hydrogel (DP2) copolymer with particular concentrations, and the mixture was further incubated for 48 h. Then, DMEM with polymer was replaced by fresh DMEM, and 100 µL of MTT solution (5 mg/mL) was added. After incubation for 4 h, 800 µL of DMSO was added to each well and shaken at room temperature. Then the DMSO solution (150 µL) was transferred to a 96-well plate, and the optical density (OD) was measured at 570 nm with a Microplate Reader model 550 (BIO-RAD, U.S.A.). The viable rate was calculated by the following equation: viable rate ) ODtreated/ODcontrol, where ODcontrol was obtained in the absence of polymer, and ODtreated was obtained in the presence of polymer. Sterilization of the Hydrogel. Before in ViVo study, the MPEGPCL-MPEG (5000-6110-5000) triblock copolymer and R-CD were autoclaved at 120 °C for 20 min, respectively. Cell Encapsulation of the Hydrogel. ECV 304 cells and bone marrow MSCs in rats were centrifuged and mixed with the sterilized R-CD solution, respectively, then the solution with cells was transferred to the six-well plates. The sterilized MPEG-PCL-MPEG (5000-6110-5000) triblock polymer solution was dropped above the cell-contained R-CD solution in the well and agitated to form the hydrogel (DP2). Fresh medium was added to the wells, and the cells in hydrogels were incubated in an incubator (37 °C, 5% CO2). After incubation, the medium in the well was pipetted out, and the hydrogels with cells were washed twice in PBS (pH ) 7.4) solution, fixed in 2.5% glutaraldehyde at room temperature for 40 min, and washed again. Successively, the samples were dehydrated with increasing ethanol gradients, namely, 50%, 70%, 80%, and 95% for 5 minutes, and twice with 100% ethanol for 15 min. Finally, the hydrogels were frozen under liquid nitrogen and freeze-dried under vacuum at -54 °C for 2 days. The morphology of the cells in the hydrogels was observed by SEM. Intramuscular Injection of Hydrogel into Rats. The rats were acclimated for one week before being used. Rats were permitted to access standard food freely (laboratory animal center, Renmin Hospital of Wuhan University, China) and tap water. All care and handling of animals were performed with the approval of the Institutional Authority for Laboratory Animal Care. A total of 20 rats were randomly divided into two groups: group 1 (10 rats) for injection of 0.5 mL of 0.9 wt % sodium chloride physiological saline intramuscularly in the hind leg; group 2 (10 rats) for injection of 0.25 mL of MPEG-PCL-MPEG triblock polymer solution (10 wt %) and 0.25 mL of R-CD solution (10 wt %) intramuscularly in the hind leg through the supplied Duploject applicator, which holds the two components in separate syringes and provides simultaneous mixing and delivery. Rats were anesthetized by pentobarbital before injection. Blood (1.2 mL) was obtained from each rat one week after the injection for measuring the concentration of following biochemical indicator: aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), and blood urea nitrogen (BUN). All the indicators were measured by a Beckman automatic biochemistry analyzer in the department of clinical laboratory, Renmin Hospital of Wuhan University, China. Implants with surrounding tissues were obtained, then frozen and dissected by a SHANDON 20 °C freezing microtome immediately. Serial sections of 10 µm were stained with haematoxylin, erythrosin, and safran (HES). Photomicrographs of internal sections of each
R-CD/MPEG-PCL-MPEG Supramolecular Hydrogels
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Figure 2. The intensity of I337 in the excitation spectra as a function of the logarithm of MPEG-PCL-MPEG (5000-9050-5000) concentration. The CMC in aqueous medium is 17 mg/L.
Figure 1. 1H NMR spectra of (A) PCL-DDDA-PCL, (B) MPEG-PCLDDDA-PCL-MPEG, (C) R-CD, and (D) R-CD/MPEG-PCL-DDDAPCL-MPEG hydrogel. Table 2. The Molecular Weights and CMC Values for MPEG-PCL-MPEG Triblock Polymers block length (Mn) triblock polymer
Mn/PDI
PEG
PCL
CMC (g/L)
MPEG-PCL-MPEG (5000-4760-5000) MPEG-PCL-MPEG (5000-6310-5000) MPEG-PCL-MPEG (5000-8170-5000) MPEG-PCL-MPEG (5000-9050-5000)
14660/1.45
5000
4760
40
16770/1.36
5000
6310
27
17960/1.52
5000
8170
24
18220/1.39
5000
9050
17
sample were taken with a Zeiss Axioskop light microscope and a color video camera. Statistical Analysis. SPSS11.5 software was used for statistical analysis. Data was performed by one-way analysis of variance (ANOVA) and the Tukey-Kramer procedure for post hoc comparison. P < 0.05 was considered statistically significant.
Results and Discussion Synthesis and Characterization of MPEG-PCL-MPEG. The structure of PCL-DDDA-PCL and MPEG-PCL-MPEG was confirmed by 1H NMR as shown in Figure 1. As demonstrated in Figure 1A, the characteristic peaks of PCL chain appear at 2.21 ppm (f), 4.07 ppm (b), 1.41 ppm (d), 1.52 ppm (e), and 1.63 ppm (c). The methylene protons in DDDA are at 1.29 ppm (a). Figure 1B also demonstrates the characteristic peaks of the PCL chain and the methylene protons in MPEG at 3.61 ppm. In this work, before the ring-opening reaction, DDDA was dried under vacuum at 60 °C for 24 h, and CL was dried over CaH2 for 48 h and then distilled under reduced pressure. SnOct2 was used after distillation under reduced pressure. In order to exclude the water in the air, degassing through vacuum/nitrogen cycle was carried out three times, and each vacuum/nitrogen cycle lasted for half an hour. The pressure in the sealed glass ampoule was less than 10 Pa after degassing. Then, the reaction initiated by carboxylic acid occurred, and the resulted product, PCL-DDDA-PCL was obtained. With respect to the integrity ratio of PCL-DDDAPCL, as shown in Figure 1A, the integrity ratio of the peaks was A(f):A(b):A(c,e):A(d):A(a) ) 1:1.04:2.07:1.03:0.16. According to the GPC data as shown in Table 2, the molecular weight of
PCL-DDDA-PCL was 4760, and the integrity ratio of the peaks of PCL-DDDA-PCL should be A(f):A(g,h):A(b):A(c,e):A(d): A(a) ) 1:1:2:1:0.15, which is in agreement with the NMR data. In addition, with respect to the integrity ratio of MPEG-PCLDDDA-PCL-MPEG (shown in Figure 1B), the integrity ratio of the peaks was A(f):A(g,h):A(b):A(c,e):A(d):A(a) ) 1:10.05: 1.08:1.81:1.03:0.18. According to the GPC data in Table 2, the molecular weights of PCL-DDDA-PCL and MPEG was 4760 and 5000, respectively, and the integrity ratio of the peaks of MPEG-PCL-DDDA-PCL-MPEG should be A(f):A(g,h):A(b): A(c,e):A(d):A(a) ) 1:9.1:1:2:1:0.15, which is also in agreement with the NMR data. Thus, MPEG-PCL-DDDA-PCL-MPEG was prepared. Figure 1C and 1D demonstrated the 1H NMR spectra of R-CD and the R-CD/MPEG-PCL-DDDA-PCL-MPEG hydrogel, respectively. As shown in Figure 1D, all the signals of R-CD, PCL, and PEG of the resulting hydrogel appeared, and relative signals were also labeled. Four triblock polymers, MPEG-PCL-MPEG (5000-47605000), MPEG-PCL-MPEG (5000-6310-5000), MPEG-PCLMPEG (5000-8170-5000), and MPEG-PCL-MPEG (5000-90505000), were synthesized as listed in Table 2. These amphiphilic triblock polymers are able to form micelles in aqueous solution. Dye absorption technology was used to detect the formation of the micelles. The CMC values of the MPEG-PCL-MPEG triblock polymers in aqueous solution were confirmed by fluorescence excitation spectra of pyrene as a probe.32,33 As showed in Figure 2, the intensity of pyrene excitation spectra of I337 was plotted as a function of the logarithm of the triblock polymer concentrations to determine the CMC. As shown in Table 2, the CMC values of the four triblock polymers are 40, 27, 24, and 17 g/L, respectively. In generally, the CMC would decline with the increasing length of the hydrophobic part because longer hydrophobic blocks are easily removed from the aqueous environment in order to achieve a state of minimum free energy.34 In this study, as shown in Table 2, with the length of hydrophobic PCL increasing, the CMC of the MEPG-PCL-MPEG triblock copolymers decreases, which is consistent with the mechanism mentioned above. Hydrogel Formation. Neither MEPG-PCL-MPEG triblock copolymers nor R-CD can form hydrogels in aqueous solutions at room temperature separately. However, after mixing the aqueous solution of triblock copolymer and aqueous solution of R-CD at room temperature, hydrogels can be formed within a few minutes, as exhibited in Figure 3. It was reported that in aqueous solutions, PEG could form inclusion complexes with (32) Wilhelm, M.; Zhao, C.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J. L. Macromolecules 1991, 24, 1033–1040. (33) Li, J.; Ni, X.; Li, X.; Tan, N. K.; Lim, C. T.; Ramakrishna, S. Langmuir 2005, 21, 8681–8685. (34) Letchford, K.; Burt, H. Eur. J. Pharm. Biopharm. 2007, 65, 259–269.
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Figure 3. Optical photographs of MPEG-PCL-MPEG (5000-6310-5000) triblock polymer in aqueous medium (A) and R-CD/MPEG-PCL-MPEG (5000-6310-5000) supramolecular hydrogel (B).
Wu et al.
Figure 5. Viscosity of R-CD/MPEG-PCL-MPEG hydrogel (DP2) as a function of agitation time at a shear rate of 28.0 s-1.
Figure 4. X-ray diffractograms for (A) R-CD, (B) MPEG-PCL-MPEG (5000-4760-5000), (C) R-CD/MPEG-PCL-MPEG (5000-4760-5000) hydrogel after freeze-dried treatment, and (D) R-CD/MPEG-PCL-MPEG (5000-6310-5000) hydrogel after freeze-dried treatment.
R-CD by a stoichiometry of 2:1 (EG unit/R-CD).35 Here, the stoichiometry of EG unit/R-CD, calculated from the 1H NMR, is 1.8, which agrees well with the reported value. The inclusion complexes formed by R-CD and PEG blocks of MPEG-PCLMPEG triblock copolymers, which are thought to aggregate into microcrystals36 and act as physical crosslinkers, lead to the formation of a supramolecular polymer network. On the other hand, the micellization of the PCL block is another significant factor in the gelation process. The hydrophobic interactions between the PCL blocks promote the formation of the network of the polymer. Thus, the cooperation of the inclusion complexation between R-CD and PEG blocks and the micellization of the triblock copolymers induce the gelation in aqueous solutions. Wide-angle XRD was used to confirm the formation of the inclusion complex between the PEG-PCL-PEG triblock polymers and R-CD. Figure 4 shows the XRD patterns of R-CD, the triblock polymer, and the hydrogels after freeze-dried treatment. The characteristic peaks at ca. 19° and 23.5° are from PEG crystalline phase, and the two sharp reflections at 21.5° and 23.5° are from the PCL crystalline phase. There is no obvious peak from R-CD crystals in the pattern of the R-CD/MPEG-PCL-PEG complex (C,D), which indicates that a large number of R-CD molecules are threaded onto the MPEG-PCL-PEG triblock polymer. Rheological Studies. As shown in Figure 5, the R-CD/MPEGPCL-PEG supramolecular hydrogels are thixotropic, i.e., the viscosity of the hydrogels diminishes as they are sheared, which makes the hydrogels suitable for injection through needles. Before injection, drugs and bioactive agents, such as plasmid DNAs, proteins, and vaccines, can be encapsulated into the hydrogel at room temperature. Besides, stem cells can also be encapsulated in the hydrogel for myocardial infarction therapy. Once the hydrogel formed, the drug-loaded or cell-loaded formulation can (35) Harada, A.; Li, J. Macromolecules 1993, 26, 5698–5703. (36) Li, J.; Li, X.; Zhou, Z. H.; Xiping, Ni.; Leong, K. W. Macromolecules 2001, 34, 7236–7237.
Figure 6. SEM images of R-CD/MPEG hydrogel (DP0) and R-CD/ MPEG-PCL-MPEG hydrogels with different compositions (DP1, DP2, and DP3).
be injected into the specific tissues under pressure owing to the thixotropic property. Marcolongo et al.37 reported potential soft tissue repair by the use of thermosensitive hydrogels composed ofpoly-(N-isopropylacrylamide)-co-poly(ethyleneglycol)dimethacrylate, with the addition of trimethacryloxypropyltrimethoxysilane. Rochet et al.38 also exploited the hydrogel constituted of hydroxyapatite/tricalcium phosphate (HA/TCP) particles in suspension in a self-hardening Si-hydroxypropylmethylcellulose (HPMC) for ectopic bone formation. Compared with conventional implantable hydrogels, the injectable hydrogels for drug delivery or cell encapsulation would be much more attractive. Morphology of Hydrogels. The morphological structure of the freeze-dried hydrogels was investigated by SEM. The morphologies of the hydrogels formed by PEG and R-CD or triblock polymer MPEG-PCL-MPEG are shown in Figure 6. Owing to the amphiphilic property of the triblock polymer as well as the micellization of the triblock polymer in aqueous solution, phase-separated process occurs during the complexation of R-CDs and MPEG-PCL-MPEG. The hydrogels consisting of R-CD/MPEG or R-CD/MPEG-PCL-MPEG have porous structures. The pore size of the hydrogels varies with the content of hydrophilic and hydrophobic segments. As for the R-CD/MPEG hydrogel, the pore size is larger, while the one of R-CD/MPEGPCL-MPEG hydrogels is less. Among the DP1, DP2, and DP3 hydrogels, the pore size decreases with the increasing amount of hydrophobic moiety in the triblock polymer. The reason is that the micellization would aid to the formation of hydrogels, (37) Ho, E.; Lowman, A.; Marcolongo, M. Biomacromolecules 2006, 7, 3223– 3228. (38) Trojani, C.; Boukhechba, F.; Scimeca, J. C.; Vandenbos, F.; Michiels, J. F.; Daculsi, G.; Boileau, P.; Weiss, P.; Carle, G. F.; Rochet, N. Biomaterials 2006, 27, 3256–3264.
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Figure 7. In Vitro release profiles of dextran-FITC from R-CD/MPEG hydrogel (DP0) and R-CD/MPEG-PCL-MPEG hydrogels with different compositions (DP1, DP2, and DP3).
Figure 9. SEM micrographs of cells encapsulated in R-CD/MPEGPCL-MPEG hydrogel (DP2). (A) ECV304 cells (2 days); (B) ECV304 cells (2 days) with a higher magnification; (C) MSC cells (2 days), and (D) MSC cells (5 days).
Figure 8. Cytotoxicity of the R-CD/MPEG-PCL-MPEG (5000-63105000) hydrogel at different concentrations.
and the hydrophobic interactions are stronger for the triblock polymers with the longer hydrophobic segments. In Vitro Release Studies. The in Vitro controlled release of dextran-FITC from the supramolecular hydrogels is shown in Figure 7. Dextran-FITC has been widely used as a model drug for macromolecular drug release, such as proteins and peptides.39-43 As shown in Figure 7, R-CD/MPEG5000 hydrogels and R-CD/MPEG-PCL-MPEG hydrogels can sustain the release of dextran-FITC. However, R-CD/MPEG-PCL-MPEG hydrogels exhibit outstanding controlled release property with sustaining release for about one month. With the increasing hydrophobic moieties of the triblock polymers, the cumulative release of dextran-FITC from the hydrogels decreases, suggesting that the hydrophobic PCL moieties play a critical role in the stability of the supramolecular hydrogels formulation. The results further imply that the complexation of R-CD with PEG segments and the hydrophobic interactions between PCL blocks result in longer sustained drug release. After being immerged in the PBS solution for a long time, the supramolecular hydrogels dissociate, indicating that the release is mainly controlled by dissolution and dissociation, rather than by diffusion. Cytotoxicity of Hydrogel. MTT assay was performed to investigate the cytotoxicity of the hydrogels. The effect of the hydrogel concentrations on the proliferation of ECV304 was studied (Figure 8). The results demonstrate there is a nonsignificant decrease in cell viability when the concentration of the hydrogel is between 2 g/L and 12 g/L. Obviously, the supramolecular hydrogel has no apparent cytotoxicity. In brief, few of ECV 304 cells died with the concentration of the hydrogel increasing. The possible reason might be that with more hydrogel covering on the plates, the cells could not get enough air and (39) Metzmacher, I.; Radu, F.; Bause, M.; Knabner, P.; Friess, W. Eur. J. Pharm. Biopharm. 2007, 67, 349–360. (40) Li, J.; Li, X.; Ni, X. P.; Wang, X.; Li, H. Z.; Leong, K. W. Biomaterials 2006, 27, 4132–4140. (41) Roos, A.; Klee, D.; Schuermann, K.; Hocker, H. Biomaterials 2003, 24, 4417–4423. (42) Burke, M. D.; Park, J. O.; Srinivasarao, M.; Khan, S. A. J. Controlled Release 2005, 104, 141–153. (43) Koennings, S.; Sapin, A.; Blunk, T.; Menei, P.; Goepferich, A. J. Controlled Release 2007, 119, 163–172.
Figure 10. The sections of tissues after subcutaneous injection of 0.9 wt % sodium chloride physiological saline and intramuscular injection of R-CD/MPEG-PCL-MPEG hydrogel (DP2) (arrows indicate the injected hydrogel) for a week. (A) intramuscular injection of 0.9 wt % sodium chloride physiological saline (100×); (B) intramuscular injection of sodium chloride physiological saline (400×); (C) intramuscular injection of DP2 hydrogel (100×); (D) intramuscular injection of DP2 hydrogel (200×). All the sections were stained with HES.
nutrients from the medium, resulting in the decreased viability of ECV304 cells. Cells Cultured in the Supramolecular Hydrogel. In order to evaluate whether the ECV 304 and MSC were encapsulated in the supramolecular hydrogel, SEM was used to observe the inner cross-section of the hydrogel after freeze-drying. As demonstrated in Figure 9A,B, ECV 304 cells were observed in the hydrogel network and compared with the normal elliptic shape of ECV 304 cells, and, after culturing for 2 days, the morphologies of the ECV 304 cells did not change. Figure 9C,D shows the MSC cells encapsulated in the hydrogel. It can be seen that MSC cells kept the spindly morphologies in the hydrogel although the hydrogel network degraded at some degree. The property of rapid gelation kinetics is attractive because of its entrapment of biologically active additives such as drug and cells in situ injection. It’s inferred that the hydrogel is a potential biomaterial for the scaffold in tissue engineering. In ViWo Biocompatibility of the Supramolecular Hydrogel. During the in ViVo experiment period, neither reduction of locomotor activity of hind limbs, nor change of food intake was found for all rats. No regional cutaneous necrosis around the injection site was found, and no red swelling of the skin or necrosis was detected in both groups. As shown in Figure 10, compared to subcutaneous injection of 0.9 wt % sodium chloride physiological saline, the injection of R-CD/MPEG-PCL-MPEG
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compatibility in our investigation, and the blood indices between the control and intramuscular injection have no significance. This is due to the degradable PCL segments, which could be degraded by enzymes in ViVo, and the PCL chain could be licked up by macrophage. At the same time, because of the good biocompatibility and suitable aqueous radius of PEG, DDDA, and R-CD, the degradable products of the supramolecular hydrogel can penetrate the kidney of the rats. Figure 11. The comparison of biochemical indicators of the two groups of rats after intramuscular injection of 0.9 wt % sodium chloride physiological saline (control) and intramuscular injection of R-CD/ MPEG-PCL-MPEG hydrogel (DP2) one week later. (A) BUN (P ) 0.149), Cr (P ) 0.736), (B) AST (P ) 0.937), ALT (P ) 0.370). P > 0.05 indicates there is no statistical significance between the two groups.
hydrogel (DP2) shows slight inflammatory response. Liver and kidney function as well as blood indicators after injection of the biodegradable DP2 hydrogel in the tissues were investigated. Figure 11 demonstrates the concentrations of AST, ALT, BUN, and Cr a week after injection. The concentrations of control and intramuscular groups are listed as follows: (A) BUN: 7.89757.675 U/L (P ) 0.149), Cr: 44.45/50.35 U/L (P ) 0.736); (B) AST: 211.25/233.425 U/L (P ) 0.937), ALT: 64.65/57.925 U/L (P ) 0.370). The comparison between the control group and the intramuscular group shows all the P values listed above are higher than 0.05, which indicates there is no statistical significance between the groups. It is well-known that PCL is biodegradable in Vitro and in ViVo.44,45 The hydrogels show excellent bio(44) Fukuzaki, H.; Yoshida, M.; Asano, M.; Kumakura, M. Polymer 1990, 31, 2006–2014. (45) Ponsart, S.; Coudane, J.; Saulnier, B.; Morgat, J. L.; Vert, M. Biomacromolecules 2001, 2, 373–377.
Conclusions Supramolecular hydrogels consisting of R-CD and MPEGPCL-MPEG triblock polymers were prepared and characterized in Vitro and in ViVo. The hydrogels were suitable for injection through a small-diameter aperture. The sustained release of the dextran-FITC from the hydrogels, which lasted for more than one month, indicated the hydrogels are promising for drug delivery. Because of the rapid gelation property, the hydrogels could effectively entrap biologically active additives such as drugs and cells for in situ injection. ECV 304 and MSC cells were encapsulated in hydrogels, and the cell morphologies could be kept the during the cell culture. The in Vitro cytotoxicity and the in ViVo histological studies demonstrated that the hydrogels were promising as injectable scaffolds for tissue engineering applications because of their good biocompatibility. Acknowledgment. The Financial support from National Natural Science Foundation of China (50633020 and 20504024) and Ministry of Science and Technology of China (2005CB623903) is gratefully acknowledged. LA8006876
