Synthesis and Characterization of Polymeric Soybean Oil-g-Methyl

Biomacromolecules 2005, 6, 1750-1758

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Synthesis and Characterization of Polymeric Soybean Oil-g-Methyl Methacrylate (and n-Butyl Methacrylate) Graft Copolymers: Biocompatibility and Bacterial Adhesion Birten Ç akmaklı,† Baki Hazer,*,† I˙ shak O ¨ zel Tekin,‡ and Fu¨sun Begˇ endik Co¨mert§ Department of Chemistry, Faculty of Arts and Sciences, and Departments of Immunology and Medical Microbiology, Faculty of Medicine, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey Received January 27, 2005

Peroxidation, epoxidation, and/or perepoxidation reactions of soybean oil under air at room temperature resulted in cross-linked polymeric soybean oil peroxides on the surface along with the waxy soluble part, sPSB, with a molecular weight of 4690, containing up to 2.3 wt % peroxide. This soluble polymeric oil peroxide, sPSB, initiated the free radical polymerization of either methyl methacrylate (MMA) or n-butyl methacrylate (nBMA) to give PSB-g-PMMA and PSB-g-PnBMA graft copolymers. The polymers obtained were characterized by 1H NMR, thermogravimetric analysis, differential scanning calorimetry, and gel permeation chromatography techniques. Polymeric oil as a plasticizer lowered the glass transition of the PSB-g-PMMA graft copolymers. PSB-g-PMMA and PSB-g-PnBMA graft copolymer film samples were also used in cell culture studies. Fibroblast and macrophage cells were strongly adhered and spread on the copolymer film surfaces, which is important in tissue engineering. Bacterial adhesion on PSB-g-PMMA graft copolymer was also studied. Both Staphylococcus epidermidis and Escherichia coli adhered on the graft copolymer better than on homo-PMMA. Furthermore, the latter adhered much better than the former. Introduction Much attention has been paid to studying and developing environmentally biodegradable plastics to retard or eradicate plastic pollution.1,2 Current interest in cheap biodegradable polymeric materials has encouraged the development of such materials from readily available, renewable, inexpensive natural sources such as starch, polysaccharides, and edible oils.3 Today natural oils and fats are considered to be the most important class of renewable resources for the production of biodegradable polymers in two ways. The first is the production of poly(3-hydroxyalkanoate)s (PHAs) as an energy reserve material by some microorganisms by using plant oils and fish oils.4 The second is direct polymerization of the oils, for example, a copolymerization with divinylbenzene and styrene, leading to thermoset copolymers,5 or polymerization of vinyl,6 maleic anhydride,7 glycidyl ether,8 and norbornyl9 derivatives of the oil. Our efforts have recently focused on grafting reactions of monomers on naturally occurring peroxidized polymeric drying oils such as linseed oil.10 Oxidation of linseed oil in the air involves hydrogen abstraction from a methylene group between two double bonds in a polyunsaturated fatty acid chain.11-13 This leads to peroxidation, perepoxidation, hydroperoxidation, epoxidation, and then cross-linking via radical recombination. * To whom correspondence should be addressed. Phone: 011 90 372 322 17 03. Fax: 011 90 372 323 86 93. E-mail: [email protected], [email protected]. † Department of Chemistry, Faculty of Arts and Sciences. ‡ Department of Immunology, Faculty of Medicine. § Department of Medical Microbiology, Faculty of Medicine.

Soybean oil is a triglyceride with two dominant fatty acid residues, linoleic acid and oleic acid, and an average number of double bonds per molecule of 4.6. The average MW of soybean oil is around 874, and the oil contains linoleic acid (51%), oleic acid (25%), palmitic acid (11%), linolenic acid (9%), and stearic acid (4%) residues.14,15 Scheme 1 indicates the structure of soybean oil. Poly(methyl methacrylate) (PMMA) is used widely in medical practices, especially in intraocular lenses and bone cement. Biodegradable plastics are also of interest for medical applications because of their biocompatibility. The cell adhesion and spreading on a surface are the most indicative processes to assess the biocompatibility of a synthetic polymer.16 Because of their strong ability to adhere on different polymeric surfaces, L-929 fibroblast cells are the main cell type widely used in biocompatibility studies. Macrophages are important components of the mammalian immune system as professional antigen-presenting cells and nonspecific killers of a wide variety of pathogens. In vivo use of synthetic and biologically derived polymers in biomedical applications such as tissue engineering and drug delivery introduces an interaction with the host immune system that can determine the efficacy of the particular application.17 In many biomedical applications the adhesion of bacteria to biomaterials causes undesirable inflammation or infection. Bacterial adherence to polymer surfaces varied significantly depending on the polymer type as well as the strain of the bacteria. In recent years various groups have therefore focused on the development of bioinert, biocompatible coatings which can be used to minimize protein adsorption

10.1021/bm050063f CCC: $30.25 © 2005 American Chemical Society Published on Web 03/25/2005

Synthesis of PSB-g-PMMA and PSB-g-PnBMA Copolymers

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Scheme 1. Chemical Structure of Soybean Oil

Table 1. Results and Conditions of the Polymerization and the Peroxidization of Soybean Oil

run

soybean oil mass, g

polym time, days

total yield, g

soluble part concn, wt %

60-1 60-2 60-3 60-4 60-5 60-6 60-7

5.01 5.00 5.25 5.21 5.14 5.05 5.10

30 40 50 60 70 80 150

5.01 5.05 5.25 5.21 5.07 5.05 5.10

96 90 93 86 72 69 62

and bacterial adhesion while maintaining the mechanical and physical properties of the underlying substrate.18 In this work, as a cheap and abundant renewable source, soybean oil was converted to polymeric peroxide under atmospheric conditions at room temperature. Then it was used to initiate the graft copolymerization of MMA or n-butyl methacrylate (nBMA) to obtain a new biodegradable material. These newly synthesized graft copolymers are tested for their biocompatibilities and effects on bacterial adhesion.19 Experimental Section Materials. Soybean oil was locally supplied and used as received. MMA and nBMA were supplied from Aldrich and

molecular weight (GPC)

Mw

MWD

1887

1.26

4690

1.52

9428

2.38

peroxygen concn, wt %, in soluble polymer 2.0 2.1 2.3 2.3 2.3 1.5 1.3

freed from inhibitor by vacuum distillation over CaH2. All other chemicals were reagent grade and used as received. Formation of Polymeric Soybean Oil under Laboratory Conditions. For the formation of polymeric soybean oil, 5.0 g of soybean oil spread out in a Petri dish (L ) 16 cm) was exposed to sunlight in the air at room temperature. After a given time, a gel polymer film associated with a waxy and viscous liquid was formed. Chloroform extraction of the crude polymeric oil for 24 h at room temperature allowed separation of the soluble part of the polymeric soybean oil (sPSB) from the gel (gPSB). The results and conditions of polymer formation from soybean oil are listed in Table 1. Peroxygen contents of the sPSB samples varied from 1.3 to

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Table 2. Results and Conditions for the Polymerization of MMA and nBMA Initiated by PSB (Sample 60-4 in Table 1) at 80 °C

run

PSB mass, g

MMA mass, g

56-1 56-3 56-4 56-2 56-5 56-6 61-1 62-1 62-2 62-3 62-4

1.015 1.000 1.000 1.031 1.501 3.003 0.926 0.51 1.03 2.03 3.01

0.562 1.030 2.012 3.005 3.005 3.005

a

nBMA mass, g

polym time, h

2.42 2.42 2.42 2.42 2.42

4.5 5 5 4.5 5 5 6.5 8 8 8 8

polymer yield total, soluble, g wt % 0.80 0.84 1.47 2.93 3.37 4.72 2.57 1.15 1.54 2.52 3.31

67 76 53 80 79 43 20 48 78 36

molecular weight

Mw × 104

MWD

31 35 30

1.6 1.6 1.8

40 29 52 44 41

1.2 1.7 1.6 1.5 1.7

[PSB]a in copolymer, mol %

4 6 8 12 4

4

Calculated from their 1H NMR spectra (by comparison with peaks at 3.7 ppm (PMMA) and 4.2 ppm (PSB)).

Figure 1. 1H NMR spectrum of the PSB sample (no. 60-4 in Table 1). (The characteristic peaks of the precurser soybean oil have been marked on the spectrum.)

2.3 wt %. The Mw of sPSB sample 60-4 was 4690 with MWD ) 1.52. Peroxygen Analysis. Peroxide analysis of soluble PSB fractions was carried out by refluxing a mixture of 2-propanol (50 mL)/acetic acid (10 mL)/saturated aqueous solution of KI (1 mL) and 0.1 g of the polymeric sample for 10 min and titrating the released iodine against thiosulfate solution according to the literature.20 Peroxide contents of the soluble polymeric soybean oil fractions are listed in Table 1. Graft Copolymerization. For graft copolymerization of PSB peroxides with a vinyl monomer, a given amount of PSB and methyl methacrylate or n-butyl methacrylate were charged separately into a Pyrex tube. Argon was introduced through a needle into the tube for about 3 min to expel the air. The tightly capped tube was then put into a water bath at 80 °C. After the required time, the contents of the tube were coagulated in methanol. The graft copolymer samples were dried overnight under vacuum at 30 °C. The characteristic data for MMA and nBMA copolymerization initiated by the PSB peroxides are listed in Table 2. Fractional Precipitation of the Graft Copolymers. In a typical fractional precipitation procedure,21a-c 0.5 g of

Figure 2. 1H NMR spectrum of the PSB-g-PMMA block copolymer sample (56-6).

polymer sample was dissolved in 10 mL of CHCl3. Methanol was used as a nonsolvent and kept in a 50 mL buret. Afterward methanol was added to the polymer solution with continuous stirring, until the polymer began to precipitate. At this point, the γ value was calculated by taking the volume ratio of the nonsolvent (methanol) consumed to the solvent (chloroform, 10 mL). Polymer Characterization. 1H NMR spectra were recorded in CDCl3 at 17 °C with a tetramethylsilane internal standard using a 400 MHz NMR AC 400 L. The molecular weight of the polymeric samples was determined by gel permeation chromatography (GPC) with a Waters model 6000A solvent delivery system with a model 401 refractive index detector and a mode 730 data module and with two Ultrastyragel linear columns in series. Chloroform was used in the elution at a flow rate of 1.0 mL min-1. A calibration curve was generated with polystyrene standards. Differential scanning calorimetry (DSC) thermograms were obtained on a Netzsch DSC 204 with a CC 200 liquid nitrogen cooling system to determine the glass transition temperatures (Tg), and thermogravimetric analysis (TGA) of



Figure 3. 1H NMR spectrum of the PSB-g-PnBMA block copolymer sample (62-3). Table 3. Thermal Analysis Results of the Graft Copolymers and the Related Homopolymers DSC polymer PSB-g-PMMA 56-2 56-5 56-6 PSB-g-PnBMA 62-2 62-3 PSB (60-4) PnBMA PMMA

TGA

Tg1, °C

Tg2, °C

Tg3, °C

Tm, °C

-37 -35 -32

-17 -13 -11

94 83 88

1 3

25 24 -44

-18

Td2, °C

142

370

136 2

19 110

Td1, °C

375 365 420 320 370

the polymers obtained was performed on a PL TGA 1500 instrument to determine thermal degradation. For DSC analysis, samples were heated from 20 to 200 °C at a rate of 10 °C/min (first heating) and held at the final temperature for 1 min to eliminate the thermal history applied to the samples. After being cooled to -100 °C, they were then reheated to 200 °C at a rate of 10 °C/min (second heating). Cell Culture and Cell Adhesion Studies. The murine fibroblast cell line (L-929) and macrophage cell line (RAW 264.7) were purchased from the American Type Culture Collection (ATCC; Rockville, MD). The cell culture stock solution (RPMI-1640), which contains 10% (v/v) heatinactivated fetal bovine serum with 100 units/mL penicillin and 100 µg/mL streptomycin, was supplied from Gibco. In a typical cell culture study, a Petri dish (L ) 60 mm) was coated with the graft copolymer film (thickness ∼1 mm) by means of chloroform solution casting. The polymer-filmcoated Petri dish was sterilized by using ethylene oxide. A 6 mL sample of cell culture containing 0.8 × 105/mL L-929 (or 0.5 × 105/mL RAW 264.7) was poured into the polymercoated Petri dish and incubated at 37 °C in humidified air containing 5% (v/v) CO2. For a given time, the cells on the polymer surface were observed with an inverted microscope (Nikon Eclipse TE 300, Tokyo, Japan) and photographed with a Minolta Dimage 7i camera (magnification 200×). The cell photographs taken with this camera associated with the above microscope are given in Figures 7 and 8.

Figure 4. GPC chromatograms of the fractionally precipitated PSBg-PMMA and PSB-g-PBMA graft copolymers.

Bacterial Adherence. One Staphylococcus epidermidis strain and one Escherichia coli strain obtained from two different patients who had infections related to intravascular catheters were used for the adherence tests. The bacteria were kept at -80 °C in skim milk. A 10 µL sample of the bacterium culture was inoculated onto a blood agar plate (Oxoid, U.K.; tryptone (14.0 g/L), peptone (4.5 g/L), yeast extract (4.5 g/L), sodium chloride (5.0 g/L), agar (12.5 g/L), and sheep blood (7 wt %)) and kept overnight at 37 °C. Bacterial suspensions of 108 colony-forming units (CFUs)/ mL were prepared for each bacterium for the adherence tests according to the method cited in ref 22. Method: A polymer disk (thickness ∼1 mm, L ) 6 mm) was placed under sterile conditions in 1 mL of bacterial suspension and incubated at 37 °C for 30 min. The polymer disk was removed and rinsed with 2 mL of sterile phosphatebuffered solution (PBS) three times for 60 s to eliminate nonadhering bacteria. The polymer disk was transferred into 1 mL of PBS in a glass tube and agitated for 3 min via vortex at 2400 rpm/min. A 10 µL sample of PBS containing dislodged bacteria was seeded onto blood agar plates and

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spread to facilitate subsequent colony counting. Ten-fold dilutions were made to calculate an accurate count of bacteria adhered to the polymer disk surfaces. Ten-fold-diluted colonies were counted by the naked eye after 24 h of incubation at 37 °C. The bacterial density per polymer type ((CFUs/mL)/mm2) was calculated by dividing the colony number mean by the total surface area (mm2) of the polymer disk. Results and Discussion Polymeric Soybean Oil Containing Peroxide Groups. Polymeric soybean oil containing peroxide/hydroperoxide groups was obtained in the air at room temperature for eight weeks. PSB samples contained cross-linked films associated with the waxy soluble part (sPSB), which was isolated with the chloroform extraction of the cross-linked film. The viscous soluble part formed under the cross-linked film on the surface can also be separated easily. As expected, the cross-linked soybean oil amount increases to 38 wt % at 150 days of polymerization time. The results and conditions of the peroxidized polymeric soybean oil are listed in Table 1. The molecular weight of sample 60-4 was 4690 with MWD ) 1.52, and peroxygen contents were found to be between 1.3 and 2.3 wt %. Figure 1 shows the 1H NMR spectrum of the PSB sample (no. 64 in Table 1). The characteristic peaks of the precurser soybean oil have been marked on the spectrum which confirms the PSB segments in the copolymer structure. Graft Copolymerization. Because of their peroxide groups, sPSB samples initiated the copolymerization of MMA or nBMA at 80 °C to obtain PMMA-g-PSB and PnBMA-g-PSB in high yield. Copolymerization conditions and copolymer analysis results are listed in Table 2. The higher concentration of PSB in monomer solution yields a higher amount of cross-linked graft copolymer except for runs 56-6 and 62-4. Interestingly, the highest concentration of PSB feeding (ca. 3.00 g of PSB) yields a lower amount of cross-linked copolymer. Cross-linked and soluble graft copolymer fractions were isolated by means of chloroform extraction. Soluble fractions of the graft copolymers were fractionally precipitated to determine the γ values of the graft copolymers and their related homopolymers. Homo-PMMA and homoPnBMA were precipitated in the γ ranges 3.0-3.8 and 2.84.1, respectively, while PSB-g-PMMA and PSB-g-PnBMA copolymer fractions were precipitated in the γ ranges 2.53.8 and 2.5-3.9, respectively. Because γ values of the graft copolymers and related homopolymers were almost superimposed, fractional precipitation was useful only to determine the γ values of oil-grafted copolymers instead of to isolate pure graft copolymers from the related homopolymers. As we discuss below, unimodal GPC curves can be attributed to the pure graft copolymers freed from the related homopolymers. Homo-sPSB, a pale yellow viscous liquid, was already eliminated by staying in the solution during the precipitation procedure. 1H NMR spectra of the soluble copolymer samples of PSBg-PMMA (run 56-6, γ ) 2.0-4.0) contained characteristic

Figure 5. DSC traces of PSB-g-PMMA (runs 56-2, 56-6, and 56-5) and PSB-g-PnBMA (runs 62-2 and 62-3).

Figure 6. Thermogravimetric traces of PSB, PnBMA, and PSB-gPMMA (run 56-2) and PSB-g-PnBMA (run 62-3) graft copolymers.

peaks as indicated in Figure 2 (δ, ppm): -COOCH3 of MMA at 3.7 and -CH2- of SB at 2.8, 2.4, 1.9, 1.4, and 0.9; the peaks at 4.1-4.4 ppm originate from the protons in the methylene groups of the triglyceride. The vinylic protons are detected at 5.3 ppm. For this sample, PSB inclusion was found to be 12 mol % by taking the ratio of the signals at 3.7 and 4.2 ppm. PSB inclusion of the graft copolymers is proportionally increased with the PSB macroinitiator in the feed (compare runs 56-2, -5, and -6 in Table 2). 1H NMR spectra of the soluble copolymer samples of PSBg-PnBMA (run 62-3, γ ) 2.0-4.0) contained characteristic peaks as indicated in Figure 3 (δ, ppm): -COOCH3 of nBMA at 4.0 (shifted to higher field than that of PMMA) and -CH2- of SB at 2.8, 2.4, 1.9, 1.4, and 0.9; the peaks at



Figure 7. L-929 cell growth and adhesion test on the polymer samples: (a) 1 h, (b) 16 h.

4.1-4.4 ppm originate from the protons in the methylene groups of the triglyceride (which partially overlapped with the peak of PnBMA at 4.0 ppm). The vinylic protons are detected at 5.3 ppm. GPC was used to determine the molecular weights and polydispersity of the copolymers. Fractionated samples of PSB-g-PMMA and PSB-g-PnBMA gave unimodal traces which can be attributed to the graft copolymer structure without homopolymer impurities (see Figure 4). Thermal analysis of graft copolymers was performed by DSC and TGA. Table 3 lists the glass transition (Tg), melting transition (Tm), and decomposition (Td) temperatures. Figure 5 indicates the thermogravimetric traces of sPSB and soluble PMMA-g-PSB graft copolymers. A considerable plastization effect of polymeric oil in the PMMA graft copolymers has been observed by lowering the glass transition to 83 °C compared with 110 °C for homoPMMA. There is no big difference in the case of PnBMA

graft copolymers. Tg values of the graft copolymers were around of that of homo-PnBMA. Figure 5 also indicates DSC traces of the polymers. Tg values of PSB were shifted to higher values in PMMA graft copolymers, while they were not observed in PnBMA copolymers because the aliphatic side chain of PnBMA may help the compatibilization of polymeric oil. Figure 6 indicates the thermogravimetric traces of sPSB, PnBMA, and soluble PnBMA-g-PSB graft copolymers. The first temperature region at around 130-280 °C (stage I) is related to evaporation and decomposition of the unreacted free oil in the bulk polymer. It is interesting that the TGA traces of the graft copolymers were in the middle of the related homopolymers. When we compare the Td values of homo-PMMA and homo-PnBMA with the Td values of the graft copolymer of soybean oil, the latter are shifted to higher values (see Table 3). Cell Culture and Adhesion. We have chosen L-929 cells as the fibroblast and RAW 264.7 cells as the macrophage

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Figure 8. RAW 264.7 growth and adhesion test on the polymer samples: (a) 1 h, (b) 16 h.

cell line. Figures 7 and 8 show L-929 fibroblast and RAW 264.7 macrophage cell adhesion and proliferation on homocopolymers and graft copolymers, respectively. PSB-g-

PMMA and PSB-g-PnBMA film samples, PMMA- and PnBMA-coated Petri dishes, and a standard PS Petri dish set for the fibroblast cell culture and 62-2- and 62-3-coded

Synthesis of PSB-g-PMMA and PSB-g-PnBMA Copolymers Table 4. Adherence of Bacteria to PMMA and PSB-g-PMMA Determined by Direct Counting of Viable Adherent Bacteria Released by Vortex Agitation ((CFUs/mL)/mm2)a polymer

S. epidermidis

E. coli

PMMA PSB-g-PMMA 56-2 56-5 56-6

46428

42857

39285 28214 31321

1250 132 892

a The bacterial density ((CFUs/mL)/mm2) (CFUs ) colony-forming units) was calculated by dividing the colony number mean by the total surface area of the polymer disk.

copolymers, PMMA- and PnBMA-coated Petri dishes, and a standard PS Petri dish set for the macrophage cell culture were used. There were abundant spherical cells at the beginning in all dishes (Figures 7a and 8a). In the first hour, L-929 cells in the copolymer 56-5 dish were adhered significantly better than the other cells, and they turned into their authentic shapes, except RAW cells on PnBMA. The cell adhesion ability of RAW macrophages in PnBMA graft copolymer (62-3) was found to be greater than that of the others (Figure 8). These results were similar to those of the standard PS dish. After 16 h of incubation, the amounts of adhered L-929 (Figure 7b) and RAW (Figure 8b) cells were significantly increased for all polymeric surfaces. At this time, the amounts of adhered L-929 cells in 56-5, 62-2, and 62-3 were found to be greater than those of the others. The RAW cells did not have their own authentic shape on the PnBMA surface for 16 h. It is important that PnBMA-g-PSB is suitable for macrophage growing and adhering while homo-PnBMA is not suitable. However, the macrophage cell adhesion ability in 62-3 was found to be greater than that of the others. It was also observed that the macrophage cell adhesion ability in 62-2 was found to be higher than that of PnBMA. These findings were similar to those of standard PS Petri dish usage and supported the biocompatibility of the graft copolymers obtained. Bacterial Adhesion. Bacterial adhesion on any surface is necessary for development of infection. Despite significant advances, bacterial adhesion to polymer surfaces is still a significant problem. There are many studies involved in bacterial adhesion on different polymer surfaces.23-25 In this study, the adherence of bacteria to copolymer PMMAs was compared with that of PMMA. While the adherences of S. epidermidis and E. coli to PMMA disks were similar, a significant decrease in the adherence of E. coli to the PSB-PMMA graft was observed. The greatest decrease (1/324) in the adherence of E. coli was observed with 56-5 (Table 4). The adherence was 34 and 48 times lower in 56-2 and 56-6, respectively. Although the decrease in the adherence of S. epidermidis was far less than that of E. coli, there was a 1.1-, 1.6-, and 1.4-fold reduction in adherence to PSB-g-PMMA graft copolymers 56-2, 56-5, and 56-6, respectively. More significant reduction of adhesion was observed with E. coli, especially to polymer 56-5. Similary, Tunney et al. reported adhesion of the hydrophilic E. coli isolates to the copolymers increased with decreasing copolymer hydrophobicity. A relationship was not apparent between copolymer hydrophobicity and adherence of the


hydrophobic Enterococcus faecalis isolate in their study.26 The development of surfaces that reduce adherence of bacteria may have several applications, for instance on medical devices used in the urogenital tract, where catheterassociated infections are rampant and E. coli constitutes the most important causative organism for infection. According to our results PSB-g-PMMA is a promising novel copolymer of PMMA for use in medical devices, with the advantage of decreasing bacterial adherence. Conclusion Naturally peroxidized polymeric soybean oil as a macroinitiator initiates the free radical polymerization of MMA and nBMA, without any additional catalyst, which may be important for medical applications of the graft copolymers. Soybean oil inclusion acts as a plasticizer and can make PMMA and PnBMA partially biodegradable and biocompatible. Fibroblast and macrophage cells strongly adhered on the graft copolymers, especially PSB-g-PnBMA. Bacterial adherence to PSB-g-PMMA was found to be lower than that to PMMA. Acknowledgment. This work was financially supported by the Zonguldak Karaelmas University Research Fund. References and Notes (1) Uyama, H.; Kuwabara, T.; Tsujimoto, T.; Kobayashi, S. Biomacromolecules 2003, 4, 211. (2) Hazer, B. Chemical Modification of Synthetic and Biosynthetic Polyesters. In Biopolymers; Steinbuchel, A., Ed.; Wiley-VCH: Weinheim, Germany, 2003; Vol. 10, Chapter 6, pp 181-208. (3) (a) Lenz, R. W. AdV. Polym. Sci. 1993, 1, 107. (b) Doi, Y. Microbial Polyesters; VCH: New York, 1993. (c) Li, F.; Marks, D. W.; Larock, R. C.; Otaigbe, J. U. Polymer 2000, 41, 7925. (d) Yu, J.; Gao, J.; Lin, T. J. Appl. Polym. Sci. 1996, 62, 1491. (4) (a) Ashby, R. D.; Foglia, T. A. Appl. Microbiol. Biotechnol. 1998, 49, 431. (b) Hazer, B.; Torul, O.; Borcakli, M.; Lenz, R. W.; Fuller, R. C.; Goodwin, S. D. J. EnViron. Polym. Degrad. 1998, 6, 109. (c) Eggink, G.; van der Wal, H. M.; Huijberts, G. N.; de Waard, P. P. Ind. Crops and Prod. 1993, 1, 157. (d) van der Walle, G. A. M.; Huisman, G. J. H.; Weusthuis, R. A.; Eggink, G. Int. J. Biol. Macromol. 1999, 25, 123. (e) Majid, M. I. A.; Hori, K.; Akiyama, M.; Doi, Y. In Biodegradable Plastics and Polymers; Doi, Y., Fukuda, K., Eds.; Elsevier B.V.: Amsterdam, 1994; pp 417-424. (5) (a) Li, F.; Hanson, M. V.; Larock, R. C. Polymer 2001, 42, 1567. (b) Li, F.; Parreroud, A.; Larock, R. C. Polymer 2001, 42, 10133. (c) Li, F.; Larock, R. C. J. Appl. Polym. Sci. 2000, 78, 1044. (d) Li, F.; Larock, R. C. J. Polym. Sci., B: Polym. Phys. 2001, 39, 60. (e) Li, F.; Hanson, M. V.; Larock, R. C. Biomacromolecules 2003, 4, 1018. (6) (a) Guner, F. S.; Usta, S.; Erciyes, A. T.; Yagci, Y. J. Coat. Technol. 2000, 72, 107. (b) Bunker, S. P.; Wool, R. P. J. Polym. Sci., A: Polym. Chem. 2002, 40, 451. (7) Domb, A. J.; Nudelman, R. J. Polym. Sci., A: Polym. Chem. 1995, 33, 717. (8) Thames, S. H.; Yu, H.; Subramanian, R. J. Appl. Polym. Sci. 2000, 77, 8. (9) Chen, J.; Soucek, M. D.; Simonsick, W. J.; Celikay, R. W. Polymer 2002, 43, 5379. (10) Cakmakli, B.; Hazer, B.;, Tekin, I. O ¨ .; Kizgut, S.; Koksal, M.; Menceloglu, Y. Macromol. Biosci. 2004, 4, 649-655. (11) Tallman, K. A.; Roschek, B., Jr.; Porter, N. A. J. Am. Chem. Soc. 2004, 26, 9240. (12) (a) Wold, C. R.; Soucek, M. D. Chem. Mater. 2001, 13, 3032. (b) Wold, C. R.; Soucek, M. D. J. Coat. Technol. 1998, 70, 43. (13) Singleton, D. A.; Hang, C.; Szymanski, M. J.; Meyer, M. P.; Leach, A. G.; Kuwata, K. T.; Chen, J. S.; Greer, A.; Foote, C. S.; Houk, K. N. J. Am. Chem. Soc. 2003, 125, 1319. (14) Ilter, S.; Hazer, B.; Borcakli, M.; Atici, O. Macromol. Chem. Phys. 2001, 202, 2281.

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(15) Li, F.; Larock, R. C. J. Polym. Sci., B: Polym. Phys. 2000, 38, 2721. (16) Satriano, C.; Carnazza, S.; Guglielmino, S.; Marletta, G. Nucl. Instrum. Methods Phys. Res., B 2003, 208, 287. (17) Panilaitis, A. B.; Altman, G. H.; Chen, J.; Jin, H.-J.; Karageorgiou, V.; Kaplan, D. L. Biomaterials 2003, 24, 3079. (18) Ishihara, K. Novel polymeric materials for obtaining bloodcompatible surfaces. Trends Polym. Sci. 1997, 5, 401-7. (19) Langer, R.; Vacanti, J. P. Science 1993, 260, 920. (20) Swern, D., Ed. Organic Peroxides; Wiley: New York, 1970; Vol. II. (21) (a) Hazer, B.; Erdem, B.; Lenz, R. W. J. Polym. Sci., A: Polym. Chem. 1994, 32, 1739. (b) Hazer, B.; Baysal, B. M. Polymer 1986, 27, 961. (c) Wu, B.; Lenz, R. W.; Hazer, B. Macromolecules 1999, 32, 6856.

(22) Isenberg, H. D. Use of MacFarland Standards. Clinical Microbiology Procedures Handbook; ASM: Washington, DC, 1992; Vol. 1. (23) Speranza, G.; Gottardi, G.; Pederzolli, C.; Lunelli, L.; Canteri, R.; Pasquardini, L.; Carli, E.; Lui, A.; Maniglio, D.; Brugnara, M.; Anderle, M. Biomaterials 2004, 25, 2029. (24) Kodjikian, L.; Burillon, C.; Chanloy, C.; Bostvironnois, V.; Pellon, G.; Mari, E.; Freney, J. Jean; Roger, T. InVest. Ophthalmol. Visual Sci. 2002, 43 (12), 3717. (25) Pinna, A.; Zanetti, S.; Sechi, L. A.; Usai, D.; Falchi, M. P.; Carta, F. Ophthalmology 2000, 107, 1042. (26) Tunney, M. M.; Jones, D. S.; Gorman, S. P. Int. J. Pharm. 1997, 151, 121.

BM050063F

Synthesis and Characterization of Polymeric Soybean Oil-g-Methyl

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