Biomacromolecules 2005, 6, 885-890
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Caprolactonic Poloxamer Analog: PEG-PCL-PEG Min Ji Hwang,† Ju Myung Suh,† You Han Bae,‡ Sung Wan Kim,‡ and Byeongmoon Jeong*,† Department of Chemistry, Division of Nano Science, Ewha Womans University, Daehyun-Dong, Seodaemun-Ku, Seoul, 120-750, Korea, and Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah Received October 16, 2004; Revised Manuscript Received December 11, 2004
The aqueous solution of poly(ethylene glycol)-poly(caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG) triblock copolymers (>15. wt. %) undergoing “clear sol-gel-turbid sol” transition as the temperature increases from 20 to 60 °C has been developed. Light scattering and 13C NMR study suggested that the transition mechanisms are the micellar aggregation for the clear sol to gel transition (lower transition), whereas the increase in PCL molecular motion for gel to turbid sol transition (upper transition). In contrast to the previous thermogelling biodegradable polymers with a sticky paste morphology, the powder form of the PEG-PCL-PEG triblock copolymers makes it easy to handle and allows fast dissolution in water. Therefore, the lyophilization into a powder form followed by facile reconstitution was possible. This system is believed to be promising for drug delivery, cell therapy, and tissue engineering. Introduction Due to the advances in biotechnology during the previous decades, the formulation of biopharmaceuticals and cellbased therapy have been hot issues.1 However, most biopharmaceuticals such as erythropoietin, human growth hormone, insulin, interferon, etc. are still administered through the frequent subcutaneous or intramuscular injections.2 There is an urgent need for a delivery system which can deliver the drug in a patient-friendly manner. As for cell therapy, the cells taken out of a patient are cultured in a medium to produce a tissue. The tissue is transplanted in the patient by a surgical procedure. To meet the above needs for protein drug delivery and cell therapy, an in situ gel-forming polymer has been suggested as a promising material for a minimally invasive therapy.3 Such a system enables pharmaceutical agents or cells to be easily entrapped and form a depot by a simple syringe injection at a target site, where the depot acts as a sustained drug delivery system or cell-growing matrix to produce the tissue in situ. In particular, the thermogelling polymer which is an aqueous solution at room temperature or lower, and forms a gel at body temperature (37 °C) has been suggested for the delivery of the biopharmaceuticals or cells which are susceptible to the heat or organic solvent.4,5 The currently reported thermogelling systems are poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock copolymer (PEG-PPG-PEG; Poloxamers),6 poly(ethylene glycol)/poly(lactic acid-co-glycolic acid) (PEG/ PLGA) triblock and graft copolymers,7-9 poly(ethylene glycol)/poly(propylene fumarate) (PEG/PPF),10 chitosan/ glycerol phosphate,11 and polyphosphazene/PEG/oligopep* Corresponding author. E-mail:
[email protected]. Fax: 82-2-32773411. † Ewha Womans University. ‡ University of Utah.
tides.12 However, the poloxamer forms a fast-eroding gel and can be used for 1∼2 days delivery systems at best.7,13 The other polymers are in a sticky paste form and cannot be lyophilized into a powder. Therefore, they are difficult to weigh and to transfer. In particular, it takes several hours to dissolve them to make an injectable aqueous solution. Most pharmaceutical formulations are needed to be lyophilized to a powder form to increase the stability of the drug. Therefore, the lyophilization followed by reconstitution or redissolution of a thermogelling system has been a concern for the practical application. As a poloxamer (PEG-PPG-PEG) analogue, we introduced a polycaprolactone instead of PPG to solve the problems of the above thermogelling polymers. The crystalline nature of PCL14 is supposed to give the PEG-PCL-PEG triblock copolymer a brittle powder morphology in addition to the thermogelling property. In addition, PCL is already approved by the U. S. Food and Drug Administration (FDA) as a contraceptive implant (Capronor).15 The phase transition behavior, mechanism of transition, and structure-property relationship on the phase transition of the PEG-PCL-PEG triblock copolymer aqueous solution were investigated. Experimental Section Materials. -Caprolactone (Aldrich), stannous octoate (Aldrich), and monomethoxy poly(ethylene glycol) (MPEG) (MW ) 550 and 750; Aldrich), 1,6-diphenyl-1,3,5-hexatriene (DPH; Aldrich), hexamethylene diisocyanate (HMDI) (Aldrich), and anhydrous toluene (Aldrich) were used as received. Synthesis. The PEG-PCL-PEG triblock copolymers were similarly prepared with PEG-PLGA-PEG triblock copolymers.7 The synthetic scheme is shown in Figure 1. To synthesize the PEG-PCL-PEG (550-2100-550), the monomethoxy poly(ethylene glycol) (10.0 g, 18.0 mmol, Mn
10.1021/bm049347a CCC: $30.25 © 2005 American Chemical Society Published on Web 01/13/2005
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Figure 1. Synthetic scheme showing the PEG-PCL-PEG triblock copolymer preparation.
) 550) was dissolved in anhydrous toluene (80 mL), and the solvent was distilled off to a final volume of 30 mL to remove the residual water adsorbed to the polymer. -Caprolactone (19.1 g, 160 mmol) and stannous octoate (52 µL, 0.16 mmol) were added to the reaction mixtures, and stirred at 120 °C for 24 h. The progress of the reaction could be traced by the GPC chromatogram for this polymer. HMDI (1.47 mL, 9.0 mmol) was added to the reaction mixture, and it was stirred at 60 °C for 7 h. The product was isolated by precipitation into diethyl ether. The polymer was dissolved in 30 mL of methylene chloride and fractionally precipitated by slowly adding diethyl ether. Two times of the fractional precipitation separated the triblock copolymer with a final yield of 60%. The residual solvent was removed under vacuum. 1 H NMR (CDCl3) of PEG-PCL-PEG: δ 1.35 (-OCH2CH2CH2CH2CH2CO- and -OOCNHCH2CH2CH2CH2CH2 CH2NHCOO-), δ 1.62 (-OCH2CH2CH2CH2CH2CO and -OOCNHCH2CH2CH2CH2CH2CH2NHCOO-), δ 2.30 (OCH2CH2CH2CH2CH2CO), δ 3.13 (-OOCNHCH2CH2CH2CH2CH2CH2NHCOO-), δ 3.38 (CH3O end group), δ 3.60 (-OCH2CH2-), δ 4.06 (-OCH2CH2CH2CH2CH2CH2CO-), δ 4.20 (-CH2CH2O-COCH2CH2CH2CH2CH2CO-). Gel Permeation Chromatography. The gel permeation chromatography (GPC) system (Waters 515) with a refractive index detector (Waters 410) was used to obtain molecular weight and molecular weight distributions. Tetrahydrofuran (THF) was used as an eluting solvent. The PEGs in a molecular weight range of 400∼10 000 Da were used as the molecular weight standards because THF is a good solvent not only for the PEG in this molecular weight range but also for the PEG-PCL-PEG. Styragel HMW 6E and HR 4E columns (Waters) were used in series. NMR Study. A 500 MHz NMR spectrometer (Varian) was used for 1H NMR (in CDCl3) to study composition of the polymer and 13C NMR (in D2O) to see the spectral change of the PEG-PCL-PEG triblock copolymer as a function of temperature. The solution temperature was equilibrated for 20 min before the measurement. Sol-Gel Transition. The sol-gel transition was determined by the test tube inverting method and dynamic mechanical analysis. For the test -tube inverting method, the 4 mL vials (diameter 1.1 cm) containing 0.5 mL of PEG-
PCL-PEG triblock copolymer solutions were immersed in a water bath at a designated temperature for 20 min. The transition temperatures were determined by flow (sol)-no flow (gel) criterion when the vial was inverted using a temperature increment of 1 °C per step.16 The accuracy of the sol-gel transition temperature was (1 °C. The sol to gel transition of the polymer aqueous solution was also investigated by dynamic rheometry (Thermo Haake, Rheometer RS 1).9,17 The aqueous polymer solution was placed between parallel plates of 25 mm diameter and a gap of 0.5 mm. The data were collected under a controlled stress (4.0 dyn/cm2) and a frequency of 1.0 rad./s. The heating rate was 0.2 °C/min. Critical Micelle Concentration. Critical micelle concentration (CMC) was determined by the dye solubilization method at room temperature (20 °C).19 DPH solution in methanol (10 µL at 0.4 mM) was injected into an aqueous polymer solution (1.0 mL) in a concentration range of 0.0005∼0.5 wt. %. The absorption spectra of these solutions were recorded from 320 to 400 nm. The absorbance at 378 nm relative to that at 400 nm was plotted against polymer concentration and the crossing point of the two extrapolated straight lines was defined as the CMC of the polymer. Dynamic Light Scattering. The size of a micelle was studied by a dynamic light scattering (DLS) instrument (ALV 5000-60 × 0) as a function of concentration at 30 °C and as a function of temperature at 0.1 wt. %. A YAG DPSS200 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 DLS were analyzed by the regularized CONTIN method. The decay rate distributions were transformed to an apparent diffusion coefficient (D). From the apparent diffusion coefficient, the hydrodynamic radius (r) of a micelle can be obtained by the Stokes-Einstein equation. Differential Scanning Calorimetry. A differential scanning calorimeter (DSC; Perkin-Elmer) was used to study the melting and recrystallization temperatures of the polymers in a temperature range of 10∼60 °C with a heating and cooling rate of 5 °C/min. A polymer (about 5.0 mg) was loaded in a cell and heat exchange was recorded during the heating and cooling cycle.
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PEG-PCL-PEG Table 1. List of PEG-PCL-PEG Triblock Copolymers Studied
PI PII PIII
PEG-PCL-PEGa
Mna
CL/EGa
Mnb
Mw/Mnb
(EG)12.5-(CL)17.5-(EG)12.5 (EG)12.5-(CL)20.1-(EG)12.5 (EG)17.0-(CL)24.6 -(EG)17.0
550-2190-550 550-2370-550 750-2800-750
0.70 0.81 0.72
3390 3650 4690
1.1 1.2 1.1
a Determined by 1H NMR in CDCl based on ethylene glycol (EG) unit (4H, 3.6 ppm) and caprolactone (CL) unit (2H, 2.2 ppm) of the polymers. 3 Determined by GPC. In the GPC, tetrahydrofuran was used as an eluting solvent and PEGs in a molecular weight range of 400∼10 000 were used as the molecular weight standards. b
Figure 2. GPC chromatogram to showing the triblock copolymer (PI) preparation. The reaction proceeds from PEGs to PEG-PCL diblock copolymers and finally to PEG-PCL-PEG triblock copolymers.
Results and Discussion The PEG-PCL diblock copolymer was synthesized by ringopening polymerization of -caprolactone onto the MPEG in the presence of stannous octoate as a catalyst. The PEGPCL diblock copolymer with a terminal hydroxyl group was coupled by HMDI to the PEG-PCL-PEG triblock copolymer. The progress of the reaction, that is, PEG, PEG-PCL diblock copolymer, and PEG-PCL-PEG triblock copolymer, could be traced by gel permeation chromatography (Figure 2). The ethylene peak of the ethylene glycol (CH2CH2O) unit at 3.6 ppm and the methylene peak of the caprolactone (COCH2CH2CH2CH2CH2O) unit at 2.2 ppm in the 1H NMR spectra were used for the determination of number average molecular weight (Mn) of the PEG-PCL-PEG triblock copolymer. The molecular weight and molecular weight distribution determined by GPC were in a range of 3300∼4700 and 1.1∼1.2, respectively. Table 1 summarizes the polymers investigated in this study. The phase diagram of the PEG-PCL-PEG triblock copolymer (PI) aqueous solutions determined by a test tube inverting method is shown in Figure 3. The lower sol is a transparent solution or liquid phase, whereas the upper sol is a turbid suspension of the polymer. In this paper, the sol (flow) and gel (nonflow) are defined by the flow characteristics when a solution containing the vial is inverted at a given temperature. This definition is also well correlated with the falling ball method or dynamic mechanical analysis.17 The sol-to-gel transition was accompanied by a sharp increase in viscosity. The critical gel concentration (CGC) above which the gel phase appears was about 15 wt %. The phase transition of the PEG-PCL-PEG triblock copolymer aqueous solution was similar to that of the PEG-PLGA-PEG triblock copolymer aqueous solution except for the presence of a transparent sol phase over 52.5∼53 °C when the concentration is larger than 25 wt % for the PEG-PCL-PEG system. When the concentration of the polymer is less than 25 wt %, the upper sol was turbid similar to that of the PEG-
Figure 3. Phase diagram of the PEG-PCL-PEG triblock copolymer (PI) in deionized water (top). The transition temperature was measured by a test tube inverting method. The absorbance at 630 nm of the PEG-PCL-PEG triblock copolymers (30 wt %) as a function of temperature is shown (bottom).
PLGA-PEG triblock copolymer system.7 Typically, as the temperature increases, the transparent solution (lower sol) undergoes transparent gel formation f turbid gel formation f turbid sol (upper sol) formation. Micellar aggregate formation was suggested for the sol to gel transition (lower transition), whereas increase in the PLGA molecular motion and disruption of micellar structure was proposed for the gel-to-sol transition (upper transition) for the PEG-PLGAPEG triblock copolymer aqueous solution.7 The transparent sol phase at 52.5∼53 °C that is just above the gel-to-sol transition temperature seems to be related to the dehydration of PEG. The PEG melting point of the PCL/ PEG block copolymer, in a neat state without water, decreases as the PCL molecular weight increases, suggesting the phase mixing of PEG and PCL in a neat state.20 The Raman shift of carbonyl stretching abruptly changed from 1723 to 1729 cm-1 for current PEG-PCL-PEG aqueous solution (25 wt %) at the transparent sol phase. The same change was observed in a neat state at the melting point of current PEG-PCL-PEG where the phase mixing between PEG and PCL is possible as a liquid. This finding suggests that the phase mixing between PEG and PCL is involved in the transparent sol phase at 52.5∼53 °C. Therefore, as a ternary system of PEG/PCL/water, it becomes a transparent sol over some composition range at 52.5∼53 °C. With a further increase in temperature, the PEG is dehydrated more, and the triblock copolymer becomes insoluble in water as a whole; that is, syneresis occurs. The detailed structural change of the polymer is to be reported using various
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Figure 4. Dynamic mechanical analysis of the PEG-PCL-PEG triblock copolymer (PI) aqueous solutions as a function of temperature and concentration.
Figure 5. Ratio of PCL (34 ppm) peak height to PEG (70 ppm) in the 13C NMR spectra of PEG-PCL-PEG triblock copolymer (PI) aqueous solutions (25 wt %) in D2O as a function of temperature.
instrumental methods including X-ray scattering, neutron scattering, and Raman spectra. The modulus in sol state was less than 0.1 Pa. The modulus increased in a very abrupt manner by the sol-to-gel transition (lower transition) as the temperature increased (Figure 4).9,17 The sol-to-gel transition temperature varied depending on the concentration. The decrease in the G′ at 43 C (20%) or at about 50 °C (30 or 35 wt. %) indicates the gel-to-sol transition (upper transition) of the polymer solution. To understand the molecular mechanism of the phase transition, 13C NMR of the PEG-PCL-PEG triblock copolymer (PI) in D2O (25 wt %) was studied. The sharp peak of PEG (CH2CH2O) at 70 ppm and the rather collapsed peak of PCL (COCH2CH2CH2CH2CH2O) at 34 ppm indicates the core-shell structure of the triblock copolymer in the lower sol state as well as in the gel state.7-9,18 The peak height ratio of PCL to PEG can give a rough estimate of the relative molecular motion of each block. The ratio is small for lower sol and gel states, indicating the core-shell structure in the lower sol and gel states, whereas the ratio increases at higher temperature or at the upper sol state (Figure 5). This fact indicates that the increase in the molecular motion of PCL drives the upper gel-to-sol transition as in the case of the PEG-PLGA-PEG triblock copolymer.7 The core-shell structure of the PEG-PCL-PEG triblock copolymer in water was confirmed by the hydrophobic dye solubilization method. The hydrophobic dye partitioned into a hydrophobic domain such as the core of a micelle shows higher absorptivity at 340∼378 nm as a characteristic triplet band compared with the dye in water. The relative absorbance at 378-400 nm was used to determine the CMC at 20 °C.9 The CMC of PEG-PCL-PEG triblock copolymer (PI) was 0.005 wt % (Figure 6). To study the micellar aggregation, the micellar size of the PEG-PCL-PEG triblock copolymer (PI) was studied as a function of temperature at 0.1 wt % (Figure 7). The radius
Figure 6. Critical micelle concentration of PEG-PCL-PEG triblock copolymer (PI) aqueous solutions at 20 °C. The absorbance at 378 nm increases by increasing polymer concentration at a fixed dye (1,6diphenyl-1,3,5-hexatriene) concentration (top). CMC was determined by the two extrapolated lines of the absorbance at 378 nm (below).
Figure 7. Micellar size of PEG-PCL-PEG triblock copolymer (PI) aqueous solutions (0.1 wt %) as a function of temperature.
of 10 and 23 nm of micelles or micellar aggregates coexist at 20 °C at 0.5 wt % in water. However, the population of 23 nm micelles are dominant in a temperature range of 25∼45 °C. At above 47 °C, significant aggregates with larger size were noted and the solution became apparently turbid at this concentration (0.1 wt %). The change in the micellar size was also studied as a function of concentration at 30 °C (Figure 8). When the concentration is lower than 1.0 wt %, the micelles with a radius of 23 nm were dominant. When the concentration increased to more than 2.0%, the micellar aggregate with a radius of about 500 nm was dominant and there was a small population of micelles with 23 nm. When the concentration is 5.0%, much larger aggregates of 1500 nm radius species were dominated. From these results, the micellar aggregation mechanism might be responsible for the sol-to-gel transition at 30 °C. The presence of the gel phase at around body temperature (37 °C) indicates that the PEG-PCL-PEG triblock copolymer
PEG-PCL-PEG
Figure 8. Micellar size of PEG-PCL-PEG triblock copolymer (PI) aqueous solutions as a function of concentration at 30 °C.
Figure 9. Control of phase transition of PEG-PCL-PEG triblock copolymers. The transition temperature was measured by the test tube inverting method. The molecular weight of each block was given for easy comparison instead of PI, PII, and PIII.
is a promising candidate for an injectable depot system that can be formulated at room temperature or lower, and forms a gel depot in situ upon subcutaneous or intramuscular injection. However, the control of phase behavior of the thermogelling polymer is critical when we design a specific application such as drug delivery or cell therapy. This is because the transition temperature determines the formulation temperature and injectability at that temperature. The gel window, the temperature range at which a gel phase exists, determines the temperature range of a potential application. The critical gel concentration (CGC) determines the minimal concentration that can be used for a depot system. Figure 9 shows that the phase transition temperature, critical gel concentration, and the gel window could be controlled by varying the molecular weight of each block. As the PCL length increased, the CGC lowered and the sol-to-gel transition temperature decreased, indicating that the hydrophobic interactions drive the sol-to-gel transition as in the case of the PEG-PLGA-PEG triblock copolymer system. As the PEG length increases from 550 to 750, the sol-to-gel transition temperature significantly increased even though the mole ratio of CL to EG is similar (Table 1). When the PEG molecular weight was larger than 1 000, the sol-to-gel transition was not observed in a biomedically significant temperature range of 0∼50 °C. With larger PEG length, the PCL length should increase at the same time to give the polymer with appropriate thermogelling properties. However, the polymer could not be dissolved in water in this case. Interestingly, the PEG-PLGA-PEG (550-2320-550) triblock copolymer can be compared with the PEG-PCL-PEG (550-2370-550) triblock copolymer.7 The numbers are the molecular weights of each block. Aqueous solutions of both polymers showed “clear sol-gel-turbid sol” transition as the temperature increased. However, the CGC of PEG-PLGAPEG was 25 wt % whereas that of PEG-PCL-PEG was 10
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Figure 10. DSC thermogram of PEG-PCL-PEG triblock copolymers. HP1, HP2, and HP3 are heating curves for the PEG-PCL-PEG triblock copolymer of PI, PII, and PIII, respectively. CP1, CP2, and CP3 are cooling curves for the PEG-PCL-PEG triblock copolymer of PI, PII, and PIII, respectively. Heating and cooling rate were 5 °C/min.
wt %. The gel window of the 25 wt % of the former was 38∼48 C whereas that of the latter was 18∼50 C. This fact indicates the hydrophobicity of the PCL compared with PLGA, resulting in the difference in the sol-gel transition behavior. Based on the critical micelle concentration, glycolide/lactide (2/8 by mole ratio), lactide, and caprolactone units were calculated to be 3, 4, and 10 times more hydrophobic than the propylene oxide unit, respectively.6 To conclude, the phase transition could be controlled by controlling the block length as well as the nature of each block. To investigate the thermal transition of the PEG-PCL-PEG triblock copolymers, a differential scanning calorimeter was used. The two melting transitions at 34 and 40 °C were observed during the heating cycle. The cooling curve shows one exothermic peak at about 20 °C. The single exothermic peak at 20 °C on the cooling curve and two endothermic peaks at 34 and 40 °C on the heating curve indicate the melting of PCL at 34 °C followed by melting of recrystallized PCL domain during the heating cycle (Figure 10).18 In the case of the multiblock PEG-PCL copolymer consisting of low molecular weight PEGs (MW) 150, 400, 600, and 1000) and PCL (MW)1250) showed two similar endothermic peaks between 30 and 45 °C which were assigned for PCL melting transitions related to melting and recrystallization of PCL during the heating cycle.18 The presence of melting transition at above 34 °C indicates the lyophilizability of the PEG-PCL-PEG triblock copolymer aqueous solution to a powder form. The most important characteristic of the PEG-PCL-PEG triblock copolymer compared with a previous thermogelling biodegradable system including PEG/PLGA, polyphosphazene, and PEG/PPF is the powder morphology. This means not only convenience in handling, weighing, and transferring but also lyophilizability to the powder form which is a critical issue in pharmaceutical formulation of a drug. In addition, the lyophilized powder was dissolved in water within 1 min by heating at 50 °C for 10 s followed by quenching at 0 °C for 30 s. Conclusions The aqueous solution of the PEG-PCL-PEG triblock copolymers showed a clear sol-to-gel transition as the temperature increased. With further increase in temperature,
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the gel underwent a gel-to-turbid sol transition, forming a closed loop “clear sol-gel-turbid sol” transition. The clear sol-to-gel transition (lower transition) seems to be driven by micellar aggregation through hydrophobic interactions, whereas the gel-to-turbid sol transition (upper transition) is driven by increased molecular motion of PCL. Compared with the thick paste form of the thermogelling PEG-PLGA-PEG triblock copolymer, the thermogelling PEG-PCL-PEG triblock copolymer is a powder at room temperature or below and convenient to handle and lyophilized to powder form. In particular, the PEG-PCL-PEG can be quickly dissolved in water, indicating that it is very promising for practical applications. Acknowledgment. This work was supported by the Ministry of Science and Technology (MOST) of Korea and Korea Science and Engineering Foundation (KOSEF; Grant Number R01-2002-000-00274-0). References and Notes (1) Daga, A.; Muraglia, A.; Quarto, R.; Cancedda, R.; Corte, G. Gene Ther. 2002, 9, 915-921. (2) Packhaeuser, C. B.; Schnieders, J.; Oster, C. G.; Kissel, T. Eur. J. Pharm. Biopharm. 2004, 58, 445-455. (3) Heller, J.; Barr, J.; Ng, S. Y.; Shen, H. R.; Abdellaoui, S.; Gurny, R.; Castioni, N. V.; Loup, P. J.; Baehni, P.; Mombelli, A. Biomaterials 1999, 23, 4397-4404. (4) Jeong, B.; Lee, K. M.; Gutowska, A.; An, Y. H. Biomacromolecules 2002, 3, 865-868.
Hwang et al. (5) Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules 1999, 32, 7370-7379. (6) Booth, C.; Attwood, D. Macromol. Rapid Comm. 2000, 21, 501527. (7) Jeong, B.; Bae, Y. H.; Kim, S. W. Macromolecules 1999, 32, 70647069. (8) Jeong, B.; Wang, L. Q.; Gutowska, A. Chem. Commun. 2001, 16, 1516-1517. (9) Jeong, B.; Kibbey, M. R.; Birnbaum, J. C.; Won, Y. Y.; Gutowska, A. Macromolecules 2000, 33, 8317-8322. (10) Behravesh, E.; Shung, A. K.; Jo, S.; Mikos, G. Biomacromolecules 2002, 3, 153-158. (11) 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. (12) Lee, B. H.; Lee, Y. M.; Sohn, Y. S.; Song, S. C. Macromolecules 2002, 35, 3876-3879. (13) Jeong, B.; Bae, Y. H.; Kim, S. W. J. Controlled Release 2000, 63, 155-163. (14) Pitt, C. G.; Chasalow, F. I.; Hibionada, Y. M.; Klimas, D. M.; Schindler, A. J. Appl. Polym. Sci. 1996, 26, 3779-3785. (15) Armani, D.; Liu, C. S. J. Micromech. Microeng. 2000, 10, 80-84. (16) Tanodekaew, S.; Godward, J.; Heatley, F.; Booth, C. Macromol. Chem. Phys. 1997, 198, 3385-3395. (17) Chung, Y. M.; Simmons, K.; Gutowska, A.; Jeong, B. Biomacromolecules 2002, 3, 511-516. (18) Ferruti, P.; Mancin, I.; Ranucc, E.; Felice, C. D.; Latin, G.; Laus, M. Biomacromolecules 2003, 4, 181-188. (19) Alexandrisdis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414-2425. (20) Cerrai, P.; Tricoli, M.; Andruzzi, F.; Paci, M. Polymer 1989, 30, 338-345.
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