Synthesis and Characterization of Biodegradable Cholesteryl End

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Biomacromolecules 2005, 6, 524-529

Synthesis and Characterization of Biodegradable Cholesteryl End-Capped Polycarbonates

Scheme 1. Synthesis of Chol-(DTC)n

Tao Wan, Tao Zou, Si-Xue Cheng,* and Ren-Xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, Peoples Republic of China Received October 18, 2004

Introduction Biodegradable polymers form a class of the most attractive biomaterials because of their wide applications in biomedical fields. Among them, aliphatic polycarbonates are a series of biodegradable polymers attracting great interest due to the surface erosion degradation mechanism and the good biocompatibility because their degradation products are less acidic comparing with the conventional biodegradable polyesters such as poly(lactic acid) (PLA). Owing to their desirable properties, aliphatic polycarbonates have great potentials in tissue engineering and drug controlled release.1-6 Stimulated by the need for biomedical materials with improved properties, modification strategies have been used to optimize some specific properties such as bioactivity and biocompatibility. Incorporation of the bioactive or biocompatible compounds including lipids, vitamins, hormones, and peptides to the polymer chains is one of the most important modification strategies.7-10 Cholesterol is a fundamental composition of mammalian membranes. Stupp et al. reported that using cholesteryl oligo(L-lactic acid) to modify PLA can obviously promote cell adhesion.8 In their study, cholesteryl oligo(L-lactic acid) was synthesized by the ring-opening polymerization of L-lactide initiated by aluminum alkoxide, which was formed by the reaction between triethyl-aluminum with cholesterol.8,9 Although biodegradable polycarbonates are currently considered as the promising materials for biomedical applications such as nerve guides,2 studies on modification of aliphatic polycarbonates by incorporation of bioactive compounds are very limited. As far as we know, the modification of polycarbonates by the cholesteryl moiety has never been reported. In this study, we used cholesterol with a hydroxyl group as an initiator to initiate the bulk ring-opening polymerization of 2,2-dimethyltrimethylene carbonate (DTC) without adding any catalyst. A series of polymers/oligomers end-capped by the cholesteryl moiety were obtained by a one-step reaction conveniently. Cholesterol was selected in our molecular design due to its high thermodynamic affinity for the cell membrane, universally important function in all eukaryotic cells, and homeostasis for cell survival.11,12 Because these properties are undoubtedly attractive and of importance for cell attachment and proliferation, the cholesteryl end-capped polymers/oligomers have promising applications in tissue engineering. Another consideration to * To whom correspondence should be addressed. Fax: 86-27-68754509. E-mail: [email protected].

incorporate the cholesteryl moiety to our polymers/oligomers is laid upon the mesogenic nature induced by the cholesteryl moiety, which may have potentials in preparation of selfassembling drug delivery systems.13 Moreover, because the polymers/oligomers were obtained by the ring-opening polymerization without adding any catalyst, this synthesis route is very useful in biopolymer fields since it permits the synthesis of biodegradable polymers with good biocompatibility without toxic impurities. Experimental Section Materials. Cholesterol (Bio Life Science & Technology Co. Ltd. Shanghai China) was recrystallized from ethanol and dried in a vacuum oven. 2,2-Dimethyl-1,3-propanediol (Shanghai Chemical Co.) was dried in a vacuum oven at 60 °C for 2 h. Triethylamine (Shanghai Chemical Co.) was distilled over CaH2. Ethyl chloroformate (Shanghai Chemical Co.) was of analytical grade and used as supplied. All solvents (Shanghai Chemical Co.) were freshly distilled before use. Synthesis of Cholesteryl End-Capped Polymers/Oligomers. Monomer 5,5-dimethyl-1,3-dioxan-2-one (2,2-dimethyltrimethylene carbonate, DTC) was synthesized according to a literature procedure.6 Cholesteryl end-capped polymers/oligomers were synthesized by the ring-opening polymerization of DTC using cholesterol with a hydroxyl group as an initiator without adding any catalyst (Scheme 1). End-capped polymers/ oligomers with different feed ratios of the initiator cholesterol to the monomer DTC were prepared. The cholesterol/DTC (mol/mol) ratios for end-capped polymers/oligomers coded Chol-(DTC)n-1, Chol-(DTC)n-2, Chol-(DTC)n-3, Chol(DTC)n-4, and Chol-(DTC)n-5 were 1/4, 1/10, 1/20, 1/40, and 1/80, respectively. The details of the polymerization reactions are as follows. For each polymerization, the mixture of cholesterol and DTC with a certain molar ratio was well mixed and was placed in a dried silanized glass flask with a

10.1021/bm049340t CCC: $30.25 © 2005 American Chemical Society Published on Web 12/15/2004

Notes

magnetic stirring bar. The flask was evacuated, purged with nitrogen three times, sealed, and then immersed in an oil bath preheated to 150 °C to carry out the polymerization. The reaction was allowed to proceed for 8 h. After the polymerization, to obtain Chol-(DTC)n-1, the product was dissolved in THF and precipitated in water. To obtain Chol(DTC)n-2, Chol-(DTC)n-3, Chol-(DTC)n-4, and Chol(DTC)n-5, the products were dissolved in THF and precipitated in methanol. The precipitated polymers/oligomers were carefully washed by ether to remove unreacted cholesterol and then dried in a vacuum oven. For comparison, un-capped poly(2,2-dimethyltrimethylene carbonate) (PDTC) was synthesized by the ring-opening polymerization under the same conditions without adding cholesterol and other initiators. Characterizations. The Fourier transform infrared (FTIR) spectra of the polymers/oligomers (in KBr pellets) were recorded on a PerkinElmer-2 spectrometer. The 1H nuclear magnetic resonance (1H NMR) spectra were recorded on a Mercury VX-300 spectrometer at 300 MHz, using CDCl3 as a solvent and TMS as an internal standard. Gel permeation chromatography (GPC) was used to determine the molecular weights of polymers/oligomers. GPC analysis was performed on a Waters HPLC system equipped with a 2690D separation module and a 2410 refractive index detector. Polystyrene standards were used as calibration standards for GPC. Chloroform was used as an eluent and the flow rate was 1.0 mL/min. Differential scanning calorimetry (DSC) was carried out with a Perkin-Elmer DSC 7 thermal analyzer. Samples were heated from 40 to 130 °C at a heating rate of 5 °C/min and then cooled to 40 °C at a cooling rate of 5 °C/min. The morphologies of polymers/oligomers were observed by a polarizing light microscope (PLM) (Olympus BX51) with a heating stage (Linkam THMS-600). The samples were viewed using crossed polarizers between which a retardation plate of 530 nm was inserted. The optical images were recorded by software Linksys 2.43. The in vitro hydrolytic degradation for the cholesteryl end-capped polymers/oligomers was carried out in the phosphate buffer solution with pH ) 7.4 at 37 °C in a shaking water bath (Grant OLS 200). Oligomer/polymer pellet samples with a thickness of 0.5 mm and a diameter of 10 mm were prepared by compression molding at 130 °C, followed by rapid cooling at room temperature. Surface morphologies of samples before and after degradation for 3 months were observed using a Hitachi ×650 scanning electron microscope (SEM). Results and Discussion Ring-opening polymerization is an efficient method for producing aliphatic polycarbonates with high molecular weights. For aliphatic cyclic esters and carbonates, their ringopening polymerizations can be proceeded in the presence of hydroxyl compounds such as water, alcohol, and poly(ethylene glycol) (PEG).1,14-16 To achieve well-defined structures for the resulting polymers, the reactivity of the monomers should be high enough and/or the catalyst-initiator, which can afford the requisite control over molecular weight and polydispersity for the specific monomer, must be identified.

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Figure 1. FTIR spectra of cholesterol, Chol-(DTC)n-1 and PDTC.

From our experimental results, we found the initiation by cholesterol was effective and the reactivity of the monomer, 2,2-dimethyltrimethylene carbonate (DTC), was high enough for undergoing the ring-opening polymerization without additional catalysts. In our study, all polymers/oligomers were synthesized under rigorously anhydrous conditions. The monomer DTC and the initiator cholesterol were carefully dried to avoid the initiation by water, which would lead to a mixture of poly(2,2-dimethyltrimethylene carbonate) (PDTC) and Chol-(DTC)n. The optimum ring-opening polymerization conditions were identified to be 8 h at 150 °C. Under the optimized reaction conditions, the polymerizations of DTC could be efficiently initiated by the -OH group of cholesterol, forming cholesteryl end-capped polymers/oligomers, Chol-(DTC)n after the polymerizations. The chemical structure of the cholesteryl end-capped polymers/oligomers was verified by FTIR and 1H NMR. As shown in the FTIR spectra (Figure 1), for PDTC, the band at 1747 cm-1 is attributed to CdO in DTC repeating units. For Chol-(DTC)n-1, the presence of CdO of the carbonate leakage is supported by the strong absorption peak at 1743 cm-1. Figure 2 shows the 1H NMR spectra of cholesterol, PDTC, and Chol-(DTC)n-1 as a typical example. In the spectrum of Chol-(DTC)n-1, the typical signals from the cholesteryl moiety and the DTC repeating units can be observed at 0.68, 0.86, 0.88, 0.94 (cholesteryl moiety: -CH3), 5.34 (cholesteryl moiety: sCHdC), 4.40 (cholesteryl moiety: >CHO-CO), 1.00 (DTC repeating unit: -CH3), 3.95 (DTC repeating unit: -CH2-), and 3.35 ppm (DTC repeating unit: -CH2-OH). Because the cholesteryl moiety has been incorporated to the polymer chain after the polymerization, the signal at 3.51 ppm for cholesterol (>CH-OH) cannot be found in the spectrum of Chol-(DTC)n-1 anymore, and a new signal appears at 4.40 ppm (cholesteryl moiety connected to DTC repeating units: >CH-O-CO). These 1H NMR spectra imply that the initiation by the -OH group is successful. To further confirm cholesterol is an effective initiator, we calculated the degree of end-capping for all of the products of ring-opening polymerizations initiated by cholesterol. The degree of end-capping is defined as the end-capped oligomer/

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Figure 2.

1H

Notes

NMR spectra of cholesterol, Chol-(DTC)n-1 and PDTC. The solvent used was CDCl3.

Table 1. Synthesis and Molecular Weights of End-capped Polymers/Oligomers NMR

polymer/oligomer

cholesterol/DTC feed ratio (mol/mol)

n

degree of end-capping

yield (%)

Chol-(DTC)n-1 Chol-(DTC)n-2 Chol-(DTC)n-3 Chol-(DTC)n-4 Chol-(DTC)n-5

1/4 1/10 1/20 1/40 1/80

3.8 12 18 32 70

0.96 1.00 1.00 1.00 1.00

69 60 71 67 69

polymer as a percentage of all polymerization product, which could be estimated via the 1H NMR spectrum by comparing the integral of the signal at 4.40 ppm (>CH-O-CO in the cholesteryl moiety connected to DTC repeating units) and the integral of the signal at 3.35 ppm (-CH2-OH in both oligomers/polymers with and without the end-capped cholesteryl moiety). As shown in Table 1, the degrees of endcapping for all polymers are very closed to 1, indicating that the initiations by water and other impurities are very limited. Because the degrees of end-capping for all polymers are very close to 1, from the 1H NMR spectra, we can further

GPC Mn (g/mol) 1.67 × 103 3.12 × 103 4.87 × 103 7.33 × 103 1.08 × 104

Mw/Mn 1.25 1.35 1.45 1.56 1.58

calculate the degrees of polymerization by comparing the integration values of the peaks of DTC repeating units and the peaks of the cholesteryl moiety. As listed in Table 1, the values of the number of DTC repeating units, n, calculated from 1H NMR are consistent with the DTC/ cholesterol feed ratios. This further suggests that the -OH groups in cholesterol molecules are effective propagation centers during the ring-opening polymerizations. However, we have to notice that the n values are lower than the DTC/ cholesterol feed ratios for samples Chol-(DTC)n-3, Chol(DTC)n-4, and Chol-(DTC)n-5. This result maybe caused

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Notes

Figure 3. DSC characterization of end-capped polymers/oligomers and PDTC.

Figure 4. Morphologies of polymers/oligomers observed by polarizing light microscope. (1) Chol-(DTC)n-1, (2) Chol-(DTC)n-2, (3) Chol(DTC)n-3, (4) Chol-(DTC)n-4, (5) Chol-(DTC)n-5, and (6) PDTC.

by the following reason. Because the initiator content in these reaction systems is low, almost all cholesterol molecules become initiation centers, whereas a small portion of monomers may still remain in the reaction systems after the polymerizations, and the unreacted monomers are removed during the purification of the polymerization products later.

In an ideal case, the degree of polymerization is controlled by the monomer/cholesterol molar ratio. In our system, however, these two values are not exactly the same. The possible reasons are as follows. Although most cholesterol molecules react with DTC and form end-capped polycarbonates, not 100% of the cholesterol molecules react with

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Table 2. Properties of End-Capped Polymers/Oligomers and PDTC PLM polymer/oligomer Chol-(DTC)n-1 Chol-(DTC)n-2 Chol-(DTC)n-3 Chol-(DTC)n-4 Chol-(DTC)n-5 PDTC

DSC

temperature range in which liquid crystallinity exibits (°C)

Tc (°C)

∆Hc (J/g)

64-88 83-94 87-98 no obvious liquid crystallinity no obvious liquid crystallinity no liquid crystallinity

does not crystalize does not crystalize does not crystalize 72 86 111

-15.4 -17.2 -20.5

Figure 5. Surface morphologies of polymers/oligomers observed by SEM. (1a) Chol-(DTC)n-1, before degradation; (1b) Chol-(DTC)n-1, after degradation for 3 months; (2a) Chol-(DTC)n-2, before degradation; (2b) Chol-(DTC)n-2, after degradation for 3 months; (3a) Chol-(DTC)n-3, before degradation; (3b) Chol-(DTC)n-3, after degradation for 3 months; (4a) Chol-(DTC)n-4, before degradation; (4b) Chol-(DTC)n-4, after degradation for 3 months; (5a) Chol-(DTC)n-5, before degradation; (5b) Chol-(DTC)n-5, after degradation for 3 months.

DTC monomers. For Chol-(DTC)n-1 and 2, the loss of a very low molecular weight fraction by the purification process may also exist. The loss of products during the purification procedure may not be ignored. According to the pervious studies of other researchers as well as our group, the -OH group can effectively initiate cyclic ester monomers such as lactide14 and cyclic carbonate monomers such as DTC15,16 to produce the polymers with well-defined structures. It was also reported that adding alcohol in excess to DTC monomers allowed functionalized polycarbonates to be synthesized, and in most cases, the degree of polymerization was controlled by the monomer/ alcohol ratio.15 Our studies are in accordance with pervious literature. However, we would like to point out the possible existence of carbonate exchange, although the major reaction is propagation. If the polymerization involves carbonate exchange side reactions, because a portion of carbonate exchange reactions gives the polycarbonate with two -CH2OH ends and the polycarbonate with two cholesteryl ends which will not result in the changes in signals in 1H NMR, the side reactions cannot be detected from 1H NMR. GPC results (Table 1) show that all end-capped polymers/ oligomers have unimodal molecular weight distributions, with the molecular weight distributions ranging from 1.25 to 1.58. The molecular weight Mn increases with decreasing initiator content. In our polymerization systems, each cholesterol molecule has one -OH group to initiate the polymerization of DTC, and as a consequence, the chain length of the resultant polymer/oligomer increases with decreasing -OH content within the studied initiator concentration range. Because of the mesogenic nature of the cholesteryl moiety, theoretically the end-capped polymers/oligomers with certain molecular weights should exhibit liquid crystallinity in particular temperature ranges. To examine the liquid crystal-

linity of Chol-(DTC)n, we characterized our samples by DSC and PLM. The characterization results are shown in Figures 3 and 4 and summarized in Table 2. The samples Chol(DTC)n-1, Chol-(DTC)n-2, and Chol-(DTC)n-3 show liquid crystal textures at certain temperature ranges. For end-capped polymers Chol-(DTC)n-4 and Chol-(DTC)n-5 with the low cholesteryl content, no obvious liquid crystalline state can be observed during heating. From PLM characterization, we can find that Chol-(DTC)n-1 shows a transition of solid state to liquid crystalline state at 64 °C and a transition of liquid crystalline state to isotropic state at 88 °C. Figure 4(1) shows a typical liquid crystal texture observed in this temperature range. Similar phenomena can be observed for Chol(DTC)n-2 and Chol-(DTC)n-3. With an increase in molecular weight, the transition temperature increases and the temperature range in which liquid crystallinity exhibits becomes narrow. DSC results (Figure 3) are in accordance with PLM images. For Chol-(DTC)n-1, Chol-(DTC)n-2, and Chol(DTC)n-3, we can identify two transition peaks during the heating. However, the two peaks are not well separated. This is explainable in terms of the polydispersity of the oligomer/ polymer samples. The transition temperatures of the oligomers/polymers with different molecular weights are different. The transition peaks may overlap each other, and as a result, the overall DSC curve cannot show two narrow transition peaks. When cooling the polymer samples at a cooling rate of 5 °C/min, samples Chol-(DTC)n-1, Chol-(DTC)n-2, and Chol-(DTC)n-3 do not crystallize. As shown in Figure 3, two transition peaks are present during the first heating of PDTC without the end-capped cholesteryl moiety. According to pervious studies,5 the peak around 90 °C is caused by the crystalline modification and the peak around 115 °C is the crystal melting peak. For endcapped polymer Chol-(DTC)n-5, similarly, we can observe two peaks that appeared during the heating, which implies

Notes

that the crystalline modification and melting also occur for Chol-(DTC)n-5 because its cholesteryl content is low. For Chol-(DTC)n-4, only one melting peak can be detected during heating. For samples PDTC, Chol-(DTC)n-5 and Chol-(DTC)n-4, crystallization can be observed during cooling. DSC results (Table 2) show that the absolute value of crystallization enthalpy ∆Hc and the crystallization temperature Tc decrease with increasing cholesteryl content in the polymer. Consistently, from the PLM images in Figure 4(4-6), we can find the crystals formed in PDTC are large, the crystals of Chol-(DTC)n-5 are smaller, and the crystals of Chol-(DTC)n-4 are even smaller. This is mainly due to the existence of the cholesteryl moiety, which disturbs the regularity of the molecular structure, frustrates crystal perfection, and inhibits the crystallization. It is well-known that conventional aliphatic polycarbonates such as poly(1,3-trimethylene carbonate) (PTMC) and PDTC degrade slowly under hydrolytic conditions. In our study, we used SEM to visualize the surface morphologies of cholesteryl end-capped polymers/oligomers before and after hydrolytic degradation. Although the degradations for all samples are slow as expected and no obvious weight losses could be detected, after degradation for 3 months, SEM images in Figure 5 still clearly show that the surfaces of all samples become unsmooth and some holes appear on the film surfaces, indicating the occurrence of degradation. Conclusions Cholesteryl end-capped biodegradable polycarbonates were successfully synthesized by the ring-opening polymerization of 2,2-dimethyltrimethylene carbonate (DTC) initiated by cholesterol bearing a hydroxyl group without adding any catalyst. The polymers/oligomers with different molecular weights can be obtained by adjusting the initiator/monomer feed ratio. Because of the incorporation of the cholesteryl moiety, some of these polymers/oligomers exhibit liquid

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crystallinity in particular temperature ranges. Compared with the high crystallizibility of PDTC, the crystallization of endcapped polymers/oligomers is inhibited. Acknowledgment. This research was supported by Grant (20204010) from the National Natural Science Foundation of China. One of the authors, S.-X.C., is grateful to the Ministry of Education of China for the financial support of “Trans-Century Training Program Foundation for the Talents” and Wuhan University for the grant of “Innovation Project Foundation for Young Scientists”. Thanks are also due to Ms. Qing-Rong Wang for the GPC measurement. References and Notes (1) Rokicki, G. Prog. Polym. Sci. 2000, 25, 259. (2) Peˆgo, A. P.; Van Luyn, M. J. A.; Brouwer, L. A.; van Wachem P. B.; Poot, A. A.; Grijpma, D. W.; Feijen, J. J. Biomed. Mater. Res. 2003, 67A, 1044. (3) Zhu, K. J.; Hendren, R. W.; Jensen, K.; Pitt, C. G. Macromolecules 1991, 24, 1736. (4) Matsuda, T.; Kwon, K.; Kidoaki, S. Biomacromolecules 2004, 5, 295. (5) Yu, C.; Zhang, L.; Shen, Z. J. Mol. Catal. A Chem. 2004, 212, 365. (6) Ariga, T.; Takata, T.; Endo, T. Macromolecules 1997, 30, 737. (7) Kricheldorf, H. R.; Kreiser-Saunders I. Polymer 1994, 35, 4175. (8) Hwang, J. J.; Iyer, S. N.; Li, L. S.; Claussen, R.; Harrington, D. A.; Stupp, S. I. Proc. Natl. Acad. Sci. U.S.A. 2003, 99, 9662. (9) Klok, H. A.; Hwang, J. J.; Stupp, S. I. Macromolecules 2002, 35, 746. (10) Klok, H. A.; Hwang, J. J.; Hartgerink, J. D.; Stupp, S. I. Macromolecules 2002, 35, 6101. (11) Simons, K.; Ikonen, E. Science 2000, 290, 1721. (12) Sugiyama, K.; Hanamura, R.; Sugiyama, M. J. Polym. Sci. Polym. Chem. 2000, 38, 3369. (13) Liu, X.-M.; Pramoda, K. P.; Yang, Y.-Y.; Chow, S. Y.; He, C. Biomaterials 2004, 25, 2619. (14) Molina, I.; Li, S.; Martinez, M. B.; Vert M. Biomaterials 2001, 22, 363. (15) Colomb, E.; Novat, C.; Hamaide, T. Macromol. Chem. Phys. 1999, 200, 2525. (16) Peng, T.; Cheng, S. X.; Zhuo, R. X. J. Polym. Sci. Chem. 2004, 42, 1356.

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