Preparation of poly(ethylene glycol) grafted poly(3-hydroxyalkanoate) networks

Preparation of poly(ethylene glycol) grafted

poly(3-hydroxyalkanoate) networks

41470 Kocaeli, Turkey

3_{University of Massachusetts, Polymer Science and Engineering Department, Amherst, MA 01003, USA}

(Received: October 23, 1998; revised: December 23, 1998)

SUMMARY: Poly(3-hydroxyalkanoate)-g-poly(ethylene glycol) crosslinked graft copolymers are described. Poly(3-hydroxyalkanoate)s containing double bonds in the side chain (PHA-DB) were obtained by co-fee-ding Pseudomonas oleovorans with a mixture of nonanoic acid and anchovy (hamci) oily acid (in weight ratios of 50/50 and 70/30). PHA-DB was thermally grafted with a polyazoester synthesized by the reaction of poly(ethylene glycol) with MW of 400 (PEG-400) and 4,49-azobis(4-cyanopentanoyl chloride). Sol-gel analy-sis and spectrometric and thermal characterization of the networks are reported.

Introduction

Poly(3-hydroxyalkanoate)s, PHAs, are naturally biode-gradable polyesters produced as intracellular energy and carbon storage materials by a wide variety of microorgan-isms1–3)_{, and have the following general structure:}

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in which R =1(CH2)nCH3 for most naturally occurring PHAs. Among the bacteria capable of synthesizing PHAs with higher alkyl groups at the three position, Pseudomo-nas oleovorans has been investigated very extensively4)

. This bacterium produces medium chain length PHA ran-dom copolymers of R-3-hydroxy repeating units contain-ing 6 to 12 carbon atoms. These PHAs need chemical modification for some medical and industrial applica-tions. To improve the mechanical and viscoelastic proper-ties of biodegradable PHAs, several fine attempts have been reported5–10)

. Hazer5, 6)

has reported grafting reactions of poly(3-hydroxynonanoate) (PHN) with polystyrene and poly(methyl methacrylate) peroxidic initiators. c-Irra-diation of the mixture of PHN and methyl methacrylate (MMA) also yielded PHN-g-PMMA graft copolymers7)_. Lenz et al.8–10)

obtained PHAs with double bonds from P.oleovorans co-fed with 10-undecenoic acid and non-anoic or octnon-anoic acid. Irradiation8)_{and epoxidation}10)_of the PHAs containing double bonds have also been reported. Our recent work11) _{included PHA production} from P.oleovorans fed with edible oily acids to obtain PHAs containing double bonds.

The present work deals with the grafting reactions of PHAs containing double bonds with polyazoesters

obtained from PEG and 4,49-azobis(4-cyanopentanoyl chloride) (ACPCL) as shown in Scheme 1.

Experimental part

Bacterial poly(3-hydroxyalkenoate), PHA-DB

Anchovy(hamci) oil was obtained from hamci, which is a fish taken from the Black Sea. This oil was hydrolyzed in a 10% solution of KOH in ethanol, after which the solution was neutralized with a 10% solution of sulfuric acid in water to obtain the carboxylic acid substrates. The acid mixture contained 27% of unsaturated acid11)_.

Stock cultures of P.oleovorans (ATCC 29347) were used in all growth and polymer production experiments as reported before11)_{. The strains were maintained at 4}8C on

nutrient agar plates using the modified mineral E* medium described below, with 20 mM nonanoic acid as the carbon source. The culture was grown in a 1-L solution of mineral Macromol. Chem. Phys. 200, No. 8 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 1999 1022-1352/99/0808–1903$17.50+.50/0 Scheme 1: Reaction design of polyazoester synthesis from PEG and ACPCL

medium containing (NH4)2HPO4 (1.1 g), K2HPO4 (5.8 g),

KH2PO4 (3.7 g), 0.1 M MgSO4 and 1.0 mL of a

microele-ment solution. (This microelemicroele-ment solution contained FeSO4N 7H2O (2.78 g), MnCl2N 4H2O (1.98 g), CoSO4N

7 H2O (2.81 g), CaCl2N 2H2O (1.67 g), CuCl2N 2H2O

(0.17 g), and ZnSO4N 7H2O (0.29 g) in 1 L of 1 N HCl). The

mixture of anchovy oily acid and octanoic acid (or nonanoic acid) was used as a sole carbon source at a concentration of 20 mM, and the pH was adjusted to 7.00. The cells were grown under aerobic conditions in 3-L cultures, which were agitated at 250 rpm at 308C in a rotary shaker. Usually, these batch cultures were harvested after 24 h. The cells were har-vested by centrifugation (48C, 12000 rpm). The cells were washed with methanol to discard unreacted oily acids and lyophilized on a Freeze Dry System. The dry cell weights were determined gravimetrically. The polymer was extracted from lyophilized cells in a Soxhlet extractor with 200 mL of chloroform. After the solvent was evaporated to 4 – 5 mL, the solution was filtered through glass wool and the polymer was precipitated into 300 mL of vigorously stirred methanol. After two precipitation cycles, the polymer was dried under vacuum for two days. Total biomass and polymer yield are listed in Tab. 1.

Methanolysis and GC-MS analysis

The methanolysis reaction was carried out in chloroform/ methanol/sulfuric acid (1 mL/0.85 mL/0.15 mL) at 1008C for 140 min, following a procedure identical with that described previously11)_{. The methyl esters obtained were}

assayed by gas chromatography and mass spectroscopy (GC-MS analysis) using a Hewlett Packard HP 5890 gas chroma-tograph with He carrier gas. After injection, the column was maintained at 608C for 4 min and then heated at 108C/min to 2708C. A temperature program was used which efficiently separated the different methyl 3-hydroxyalka(e)noates. Each peak in the chromatogram was analyzed by mass spectro-scopy. The data were processed with a Hewlett-Packard laboratory data system.

Polyazoester synthesis

The poly(ethylene glycol) PEG-400 (the number 400 refers to the molecular weight of the PEG) was received from Fluka. Polyazoester, PAE-400, was prepared from PEG-400 and 4,49-azobis(4-cyanovaleryl chloride)12)_{or 2,2}

9-azobis(iso-butyronitrile) via Pinner synthesis13)_{. In the preparation of}

PAE-400, chain extension may occur even if an excess amount of one reagent is used. Thus a small amount of poly-azoester with higher molecular weight can also occur.

Grafting reactions

In a typical grafting procedure reported elsewhere14)_{, a}

solu-tion was prepared from a mixture of 0.5 g of PHA-DB, 0.5 g of PAE-400 and 30 ml of chloroform (as a solvent). The solution was stirred for 24 h, spread onto aluminium mould (radius: 10 cm) and air dried. Gel formation was carried out with this aluminium mould by introducing in an oven pre-heated to 1008C for 1 h. The grafted polymer mixture was

extracted with chloroform for 24 h for sol-gel analysis. The gels were removed, washed with methanol and dried under vacuum at 308C for two days.The results of the grafting pro-cedure are listed in Tab. 2.

Characterization of PHA-g-PEG graft copolymer networks Gel permeation chromatography (GPC) chromatograms of the biopolyesters were taken using a Waters-510 HPLC pump with Waters-410 differential refractometer and Styra-gel HR1 + HR2 column system, THF was used as the elution solvent at 408C.

IR and NMR spectra of the polymer samples were recorded on KBr pellets (150 mg + 2 mg sample) using a Perkin Elmer 177 IR spectrometer and a 200 MHz Bruker-AC 200L NMR spectrometer in CDCl3, respectively.

Ther-mal analysis was carried out on 6 – 8 mg samples on a DuPont 910 differential scanning colorimeter (DSC). The polymer samples were heated at a rate of 108C/min from –70 to 1308C, quickly cooled, and then scanned a second time using the same heating rate and temperature range.

Scanning electron micrographs (SEM) were taken on a JEOL JXA-840A electron microscope. The specimens were frozen under liquid nitrogen, and were then fractured, mounted and coated with gold (300 A˚ ) on an Edwards S 150B sputter coater. SEM measurements were operated at 15 kV, and the electron images were recorded directly from the cathode ray tube on a Polaroid film. The magnification range was varied up to 11 0006.

Swelling degrees of polymers at equilibrium were deter-mined by gravimetry at room temperature in CHCl3or in

H2O. Swelling ratios, qv, were calculated using the volume

ratio of swollen polymer (vswollen polymer) to dry polymer

(vdry polymer)14).

Results and discussion

Synthesis and characterization of PHA-DB

Pseudomonas oleovorans was grown on a mixture of anchovy oily acid and nonanoic acid in two different ratios, 30/70 and 50/50, to obtain poly(3-hydroxyalkanoic acid)s containing unsaturated groups, with M—n ranging from 66104_{to 9}6104_{(see Tab. 1). The structure analysis} of the PHAs was carried out using GC-MS after methano-lysis of the PHAs (see the Experimental section). The more anchovy oily acid in the initial feed, the higher the side-chain unsaturation in PHA-DB samples (see Tab. 1). The saturated units varying from C-12 to C-18 in the PHA samples increase with the increase of the initial feed ratio of anchovy oily acid (A) (30% of such units for the initial feed ratio of A/N = 30/70 and 47% for the ratio of 50/50), while smaller saturated units varying from C-7 to C-9 decrease (C-7: 13%, C-9: 54% for an initial feed ratio of 30/70 and C-7: 9%, C-9: 38% for an initial feed ratio of 50/50). Fig. 1 a and 1 b illustrate the GC-MS spectra of the two PHAs containing double bonds. The double

bonds of PHAs were also observed through the chemical shiftsd = 2.0 (for CH2protons next to the double bonds) and 5.4 (for protons of double bonds) in the 1_{H NMR} spectra shown in Fig. 2.

Characterization of graft copolymer networks

The amount of the unsaturated groups in PHA-DB ranged from 3.5 to 5.5% (Tab. 1 and 2). Free radical crosslinking occurred in graft copolymer samples of PHA-DB

contain-ing 5.5% of unsaturated units. However, PHA-DB con-taining 3.5% of unsaturated units did not give observable crosslinked polymer. This may be caused by the lower concentration of unsaturated groups in PHA-DB obtained from 70/30 feed ratio. In fact, in our recent work, we stu-died crosslinking in dependence of the amount of double bonds in macromonomeric initiators15)

. We observed that a minimum vinyl content is needed for crosslinking. (The minimum critical vinyl concentration for gelation is 5% for macromonomeric initiators, MIM-400.) The grafting reactions of PHA copolymer containing 5.5% of double bonds (run no. 101, 111 and 121) with PAE-400 gave crosslinked copolymers, as depicted in Scheme 2.

Swelling degrees of the polymers at equilibrium were determined by gravimetry at room temperature in CHCl3

Tab. 1. Biosynthesis of PHA-DB from P.oleovorans co-fed with anchovy oily acid (A) and nonanoic acid (N) Run. no. Fermentation

time in h Wt. ratio A/N Dry cell in gN L–1 PHA in dry cell in wt.-% Unsaturated groups in mol-% M—n6104 P.Da) 16 17 30/70 0.54 24 3.5 6 1.4 28 23 50/50 0.75 35 5.5 9 1.7 a) _{Polydispersity index.}

Fig. 1. GC-MS spectra of PHA-DBs. (a) run no. 16, (b) run no. 28 in Tab. 1

Fig. 2. 1_{H NMR spectrum of PHA-DB (run no. 28 in Tab. 1)}

Tab. 2. Results and conditions of grafting onto PHA-DB (no. 28 in Tab. 1)

Run no.

Feed composition Cross-linked Swelling ratio PHA-DB in g PAE in g polymer in g in CHCl3 in H2O 101 0.470 0.107 0.156 11.3 1.1 111 0.472 0.308 0.230 19.2 1.6 121 0.475 0.769 0.395 15.7 1.3 191 0.157 0.193 0.190 19.8 1.9 141a) _{0.489 0.247 No crosslinking – –}

or in H2O. There are high swelling ratios of the cross-linked polymers in CHCl3, which can be attributed to a very large network structure, i. e., a high molecular weight of crosslinking chains (Mc). The small swelling degrees of the copolymer networks in H2O indicate that the additional hydrophilicity coming from the grafted PEO is limited.

IR spectra of the graft copolymer samples obtained clearly indicated the 1OH band at 3420 cm–1

which belongs to the PEG segments. For comparison, the IR spectra of the unsaturated polyester PHA-DB and the graft copolymer PHA-g-PEG are displayed in Fig. 3.

As to the thermal characterization of the graft copoly-mers, small amounts of PEG did not affect considerably their thermal properties. The maximum weight loss rates of the PHA-g-PEG samples 101 and 121 in Tab. 2 were 275 and 2858C, respectively (pure PHA: 2858C). Melting (Tm) and glass transition (Tg) temperatures of the graft copolymer networks were most the same as those of PHA-DB (Tg: –508C, Tm: 508C). Because of 12 and C-18 saturated repeating units in the copolymer, its melting and glass transition temperatures were somewhat lower than those of PHA: Tg: –458C, Tm: 558C.

SEM analysis of the fractured surfaces of the copoly-mer networks was also performed. PEG globules in the PHA matrix were clearly observed in the SEM micro-graph (see Fig. 4 a). This is comparable with the SEM micrograph (Fig. 4 b) of polybutadiene-g-PEG (in ref.14)

, Fig. 6 a and 6 b) on which the drops of PEG segments covered the polybutadiene surface. In this case, because of the abundance of vinyl groups in polybutadiene, a huge amount of PEG drops on the surface was observed.

In conclusion, insertion of double bonds into PHAs is possible by co-feeding or feeding the bacterium P.oleo-vorance with substrates containing double bonds. Poly(3-hydroxyalkanoate)s containing double bonds can be crosslinked via free radical mechanism for the potential uses as drug delivery systems and biodegradable food Scheme 2: Crosslinking reactions of PHA containing double

bonds with polyazoester

Fig. 3. IR spectra of PHA-DB (run no. 28 in Tab. 1) (a) and PHA-g-PEG (run no. 121 in Tab. 2) (b)

Fig. 4. (a) SEM micrograph of PHA-g-PEG (run no. 121 in Tab. 2), (b) polybutadiene-g-PEG in ref.14) _{(Magnifications:}

packing. Because PEG units in graft copolymers of PHAs were expected to improve hydroswelling, polyazoesters based on PEG were used for this purpose.

1)_{Ch. Sasikala, Ch. Ramana, Adv. Appl. Microbiol. 42, 97}

(1996)

2)_{H. Brandl, R. A. Gross, R. W. Lenz, R. C. Fuller, Adv.} Bio-chem. Eng. Biotechnol. 41, 77 (1990)

3)_{R. W. Lenz, Adv. Polym. Sci. 107, 1 (1993)}

4)_{R. A. Gross, C. De Mello, R. W. Lenz, H. Brandl, R. C.}

Fuller, Macromolecules 22, 1106 (1989)

5)_{B. Hazer, Polym. Bull. 33, 431 (1994)}

6)_{B. Hazer, Macromol. Chem. Phys. 197, 431 (1996)}

7)_{M. S. Erogˇlu, T. C¸ aykara, B. Hazer, Polym. Bull. 41, 53}

(1998)

8)_{G. N. Babu, W. J. Hammar, D. R. Rutherford, R. W. Lenz, R.}

Richards, S. D. Goodwin, 1996 International Symposium on Bacterial Polyhydroxyalkanoates, pp. 48 – 55

9)_{K. D. Gagnon, R. W. Lenz, R. J. Farris, R. C. Fuller, Polymer} 35, 4358 (1995)

10)_{M. Bear, M. L. Durand, V. Langlois, R. W. Lenz, S.}

Good-win, P. Guerin, Reac. Funct. Polym. 34, 65 (1997)

11)_{B. Hazer, O. Torul, M. Borcakli, R. W. Lenz, R. C. Fuller, S.}

D. Goodwin, J. Environ. Polym. Degradation 6, 109 (1998)

12)_{L. J. Laverty, Z. G. Gardlund, J. Polym. Sci., Polym. Chem.} Ed. 15, 2001 (1977)

13)_{R. Walz, B. Bo¨mer, W. Heitz, Makromol. Chem. 178, 2527}

(1977)

14)_{B. Hazer, Macromol. Chem. Phys. 196, 1945 (1995)} 15)_{U. Yıldız, B. Hazer, Macromol. Chem. Phys. 199, 163 (1998)}