February 5, 2008

Chemical Modifications of Bacterial Polyesters: From Stability to Controlled Degradation of Resulting Polymers

By Renard, E Langlois, V; Guerin, P

Polyhydroxyalkanoates (PHAs) form an enlarged family of biopolyesters, which are biocompatible, biodegradable and non- toxic. Polyhydroxyalkanoates biodegradation corresponds to a hydrolysis involving endo- or exo-enzymatic systems in the breaking cleavage of esters bonds. This type of degradation is needed for environmental applications. In the case of therapeutic and biomedical uses, a simple hydrolysis is required. Hydrolytic degradation of PHAs is not evident as shown on poly(3- hydroxyoctanoate) (PHO) and is depending on the structure of the polyester and more particularly on the nature of the side chains. In some cases, blending with others polymers has decreased PHAs crystallinity and has made easier hydrolysis. Another route consists in the preparation of unsaturated PHAs which can be chemically modified. Pendant double bonds have been turned into carboxylic, hydroxyl or epoxy groups. Moreover these reactive functions were used for grafting oligomers of hydrolysable polylactic acid (PLA) or hydrophilic polyethylene glycol (PEG). Otherwise block copolymers with polycaprolactone (PCL) have been prepared, aiming at nanoparticles formation in the view of drug release. Therefore, the hydrophilic/hydrophobic balance of these materials was controlled by chemical modification and their stability/hydrolysis has been studied. Results have shown that the most suitable products in regard to hydrolysis concern PHAs containing carboxylic groups in side chains, noted poly(3-hydroxyoctanoate-co-9-carboxy-3- hydroxydecanoate) and its derivatives. Carboxylic groups promote water penetration into the polymer and participate to ester groups hydrolysis through better water penetration and catalysis. Keywords: PHAs, Functionalised poly(3-hydroxyalkanoates), Poly(3- hydroxyoctanoate-co-9-carboxy-3-hydroxydecanoate), Graft copolymers, Diblock copolymers, Nanoparticles, Water stability, Hydrolysis


Polyhydroxyalkanoates (PHAs) are natural polyesters, that many organisms in the environment accumulate in the form of intracellular granules to store carbon and energy.1-3 They have a high degree of polymerisation, are highly crystalline, isotactic (only the enantiomer of absolute configuration R is present in these polymers) and insoluble in water. Although the most well studied PHA is poly(3- hydroxybutyrate) (PHB), over 140 constitutive monomer units4 have been investigated. Polyhydroxyalkanoates have been traditionally classified according the side chain length R (Fig. 1), i.e. as short chain length (SCL) (C4 and C5 carbon atoms in the monomer unit), medium chain length (MCL) (C6 to C14) and long chain length (LCL) (C>14).

This wide variety of monomers leads to PHAs with versatile properties. The accumulated polymers types are determined by the substrate specificities of the PHAs synthases and are depending on the carbon source. Among the great number of PHAs described in the literature only few of these polymers are produced in large quantities. Polyhydroxyalkanoates have received increasing interest due to their high potential for commodity and speciality plastic production and for medical or pharmaceutical applications such as drug delivery systems and tissue engineering.5-7

One of special interests of PHAs is their environmental biodegradation property compared with the petroleum based synthetic polymers. Enzymatic degradations of the common PHB and PHB copolymers have been widely studied as environment friendly polymeric materials.8-10 Intracellular depolymerase systems lead to CO2 and H2O when bacteria need energy or carbon sources. Aaerobic and anaerobic PHB degrading bacteria and fungi have been isolated from various environments such as soil, sludge, fresh water and sea water. The microorganisms excreted extracellular PHB depolymerases to degrade PHB and used the resulting compounds as nutrients. Several PHB depolymerases have been purified from some microorganisms and their properties have been characterised.11-13 Enzymatic degradation depends on (cristallinity) spherulite size and chemical composition of PHAs. The enzymatic degradation of PHB films was investigated using an extracellular PHB depolymerase from Alcaligenes faecalis T1. In a first step PHB depolymerase degraded the PHB chains in the amorphous state on the surface of the films and subsequently eroded the PHB chains in the crystalline state.14 Furthermore, the rate of enzymatic erosion of PHB samples (obtained by chemical polymerisation) containing both (R) and (S) monomer units is strongly dependent on the stereocomposition of the polymers. Seebach15 had demonstrated that the enzyme recognised only the ester bond between sequential (R)-HB units. The rate of PHAs biodegradation varied with the chemical structure of the polymer as illustrating by the stability towards enzymatic degradation of poly(3-hydroxyvalerate), poly(3-hydroxyoctanoate) (PHO), poly(3- hydroxyhexanoate) in regard to A. Faecalis PHB depolymerase, whereas (in compare with) poly(3-hydroxypropionate), poly(4- hydroxybutyrate) and poly(ethylene succinate)16 were completely degraded by this depolymerase.

1 Chemical structure of PHAs

All polyesters are susceptible to degrade by hydrolysis to some extent. Under normal conditions bacterial polyesters are water stable. The hydrolytic degradation rate of PHAs is very dependant on the chemical structure and materials crystallinity. Poly(3- hydroxybutyrate) has been hydrolysed in acidic and basic conditions, as a standard ester. Poly(3-hydroxybutyrate) and PHBHV copolymers degraded relatively slowly at physiological pH values. A random scission of the polymer chains occured first followed by weight loss of the samples caused by further decomposition.17 Poly(3- hydroxybutyrate) degradation was increased by the addition of polymers or plasticisers which disrupted the polymer cristallinity and consequently accelerated hydrolysis. Poly(3-hydroxybutyrate)/HV has been blended with a hydrophilic polymer, poly(ethyleneglycol) (PEG) or a hydrolysable polyester, racemic poly(-lactic acid) PLA50. The presence of PEG or PLA increased the hydrolytic rate.18

The study has been extended to a mcl PHA, the PHO. This polymer is synthetised by different bacteria, but the most common is Pseudomonas sp Gpol.19,20 This bacterium has been extensively studied because of its ability to produce PHAs from a large spectrum of carbon substrates. After several months in water at pH 7.4 no change has been observed. The degradations of PHO blending with PLA50 and PEG have been monitored by weight loss and by size exclusion chromatography.18 In the case of blending with oligomers (PLA50 and PEG respectively 750 and 2100 g mol^sup -1^), weight loss occured rapidly upon incubation in the buffer. The weight losses were assigned to the release of water soluble PEG or hydrolysis of short PLA chains. The weight losses strictly corresponded to the oligomer contents in the blends. After removing the oligomers from the polymeric matrices, no significant weight loss was noted after 100 days. In the case of high molecular weight PLA50 (42 500 g mol^sup -1^), the blend was resistant to hydrolytic degradation. This polymer remained trapped in the immiscible blend. Water absorption in polymer material was the most important factor influencing the hydrolytic process, and in the case of PHO the matrix was very hydrophobic and water penetration was limited.

2 Chemical structure of PHOU

One way to obtain more hydrophilic poly(3-hydroxyalkanoate)s consisted in the introduction of specific groups in the macromolecular side chains. Functional PHAs with hydrophilic groups such as carboxylic, amine or hydroxyl on the side chains were prepared by biotechnological syntheses but the fermentation conditions are difficult and generally polymers are obtained with very low yields.21 Some bacteria are able to synthesise polyesters with reactive pendant double bonds, which can be turned into hydrophilic functions. Polymers with enhanced hydrophilicity are of great interest because they are usually more biocompatible. These hydrophilic moieties can be also used for further modifications, as the preparation of graft copolymers. The combination of bioconversion and organic chemistry allowed modulating more precisely the physical properties of these bacterial polymers as solubility, hydrophilic/hydrophobic balance and bioavailability.

In this paper, the authors want to present these polymers, starting from bacterial polyesters containing unsaturated monomer units and their chemical modifications. The properties of water stability or hydrolysis are presented in the perspective of biomedical applications.

Results and discussion

Polymers and copolymers with unsaturated pendant groups

Only a few bacteria are able to produce unsaturated PHAs with a good yield. For example, Rhodospirillum Rubrum has been grown on 4- pentenoic acid.22 Three types of short monomer units were present, among which 3-hydroxy-4-pentenoic acid (10%), but the yield was

Polyhydroxyalkanoates containing epoxy groups22

To achieve specific physical properties, the unsaturated units were converted to other functional groups, such as epoxy, which could be further cross-linked to improve mechanical properties. Moreover, epoxidation of PHOUs is of great interest because the epoxide function is highly reactive under mild conditions with a variety of reagents to prepare derivatives with polar or ionic groups. The epoxidation reaction22 was carried out in the presence of metachloroperbenzoic acid (MCPBA) (two equivalents) on a series of PHOUs (Fig. 3). All different samples of the PHO^sub 100-x^U^sub x^ reacted totally at 20[degrees]C in 12 h, as determined by ^sup 1^H and ^sup 13^C NMR. Disappearance of signals corresponded to double bounds (140 and 115 ppm for ^sup 13^C NMR spectra) and presence of two peaks assigned to the oxirane carbon atoms (47 and 52 ppm).

Moreover, molecular weights of epoxidised PHOUs (EpPHOUs) have displayed, the macromolecular chains were not affected by this chemical reaction (Table 1). Results are identical with PHU^sub 100^.

All epoxidised polymers were completely soluble in organic solvents, including chloroform, tetrahydrofuran, methylene chloride and acetone, demonstrating that cross-linking reactions did not occur. All samples presented a melting point, but lower than PHOUs.

Because of the versatility of Pseudomonas sp. Gpol to produce particular PHAs, especially those containing functional groups on the side chains, it was interesting to investigate the growth capability of this bacterium and to synthesise PHAs with an epoxy substrate used alone or in mixture with a co substrate. A new carbon source 10-epoxyundecanoic acid was prepared by chemical modification of 10-undecenoic acid and was used either alone or in mixture with sodium octanoate in different proportions. The fermentation conditions have been changed; the oxygen flow was more important than in the case of PHO^sub 100^ and PHO^sub 100-x^U^sub x^. The objective was to oblige the bacteria to use oxygen gas for the metabolism instead of the oxygen atom included in the oxirane group and to increase the bacterial polyesters production to the detriment of the bacterial development. Surprisingly, experimental results showed that this bacterium is able to use 10-epoxyundecanoic as nutrient and to incorporate it in the polyester. Cells and polymer yields depended on the percentage of the epoxidised compound, which is a non-natural carbon source in the feed mixture. PHO^sub 100- x^E^sub x^ (Table 2) presented the same characteristics that EpPHOUs, concerning solubility in organic solvents and similar ^sup 1^H and ^sup 13^C NMR spectra (the presence of peaks corresponding to epoxy groups).

The fermentation conditions have been changed for using this new carbon source. The oxygen flow was more important to oblige the bacteria to use oxygen gas for their metabolism instead of the oxygen atom included in the oxirane group. The experimental results showed that the cells and polymer yields depended on the percentage of the epoxidised compound in the feed mixture, which is a non- natural carbon source. These polymers have a very low glass transition and high molecular weight (Table 3).

Table 1 PHO^sub 100-x^ U^sub x^ and Ep. PHO^sub 100-x^U^sub x^ molecular weights

4 Synthetic route to hydroxylation PHOU (only three double bonds were indicated on PHOU to simplify the scheme)

These results opened the route to further chemical modifications and to prepare new functionalised PHAs. An easy transformation consisted in the cross-linking of EpPHOUs by using ultraviolet (UV).25 Resulting compounds could be completely biodegradable because the cross-linkings are succinate esters, which are known to be susceptible to enzymatic attack. But for hydrolysis improvement, others types of chemical reactions have to be considered.

Polyhydroxyalkanoates modified with pendant hydroxylic groups26

Two methods have been investigated to increase hydrophilicity of PHAs by hydroxylation of unsaturated pendant groups. Lee et al.27 have carried out the conversion of double bonds to diol function by oxidation with basic KMnO^sub 4^.The degree of hydroxylation was about 55-60% for PHO^sub 100-x^U^sub x^ with x varying from 45 to 93. The conversion rate was about 60% and no significant molecular weight decrease of modified PHOUs was observed after hydroxylation. An alternative route leading to the introduction of hydroxyl groups in PHOUs was based on the hydroboration-oxydation of alkenes.26 Unsaturated PHO^sub 75^U^sub 25^ was chemically modified with Borane- Tetrahydrofuran (BH^sub 3^-THF) complex. Reaction led to respective PHOU with hydroxyl groups (PHOU-ol) in the side chains as shown in Fig. 4. Borane is well known to readily add to alkenes to yield trialkylboranes. The reaction was carried out in dried THF at 0[degrees]C with BH^sub 3^-THF complex. The reaction yielded to observable cross-linked polymer indicating the formation of trialkylboranes. The cross-linking reaction was caused by the high concentration of unsaturated groups in PHOU (25 mol.-%). Then, the alkylboranes were in situ converted into alcohols by oxidation with alkaline peroxide involving the destruction of the cross-linked product. Polyhydroxyalkanoates containing hydroxyl groups were very soft and sticky materials so it was more difficult to handle them compared with the corresponding PHOU.

Table 2 Preparation and characteristics of PHAs with P. sp. Gpo1 grown on sodium octanoate (Oct) and 10epoxyundecanoic acid (Epox.)

Table 3 PHO^sub 100-x^U^sub x^ and PHE molecular weight and thermal characteristics

5 ^sup 1^H NMR spectrum of PHOU-ol in CDCl^sub 3^ (spectrum up 13 mol.-% of OH groups, spectrum below 25 mol.-% of OH groups)

The conversion of unsaturated groups in PHOU is dependant of BH^sub 3^ amounts. Reaction conversion was monitored by ^sup 1^H NMR. PHOU-ol obtained with 0.5 and four equivalents of BH^sub 3^ per unsaturated group respectively were studied by ^sup 1^H NMR (Fig. 5). In the first case, the product contained unreacted olefin groups at delta=4.95 ppm and delta=5.75 ppm suggesting incomplete conversion of the alkene functions. In addition, a new peak appeared at delta=3.6 ppm corresponding to methylene protons neighboring hydroxyl group. In contrast in the ^sup 1^H NMR spectrum of a PHOU- ol sample obtained with an excess of BH^sub 3^ (4 eq.), the signals centred at delta=4.95 ppm and delta=5.75 ppm for the unsaturated groups disappeared attesting complete hydroxylation. For this work, BH^sub 3^/unsaturated units molar ratio ranging from 0.5 to 4 were used. Results were reported in Table 4. As expected, OH content was found to increase with increasing BH^sub 3^ amounts. Results showed that OH contents in the range from 10 to 25% were adjusted by changing BH^sub 3^/unsaturated units molar ratio.

A comparison of the data reported in Table 5 showed that the molecular weights of hydroxylated PHOU decreased with the hydroxyl groups content in the polymer.

The differences may be interpreted in terms of differences in the hydrodynamic volumes of the products in THF due to the presence of polar groups. But, this change is more reasonably explained as being due to a reduction in the molecular weight of the polymer during the hydroboration-oxydation process. Furthermore, as previously mentioned, hydrogen bonding occured to form interchain bonds that make difficult the determination of molecular weight distribution.

The thermal properties of PHOU-ol samples were investigated by differential thermal analysis. As shown in Table 5, the transformation of the olefin groups in PHOU into hydroxyl groups caused a slightly increase in the glass transition temperatures (T^sub g^) and the loss of the crystallinity. Since the T^sub g^ did not vary drastically with the extent of OH content, T^sub g^ can be considered as a suitable parameter to reveal the effect of PHOU hydroxylation. The polar hydroxyl groups formed intramolecular interactions that affected backbone mobility. All PHOU-ol samples were amorphous, which agreed with the sticky feature of these polymers.

Table 4 Effect of BH^sub 3^ molar ratio on hydroxylation of PHOU* Table 5 Characterisation of PHOU-ol samples

Table 6 Solubility of PHOU-ol in different solvents

Possible changes of PHOU-ol solubilities after hydroxylation were investigated and listed in Table 6.

PHOU-ol, with 10% of hydroxyl groups had the same feature as the precursor PHOU. However, the presence of 25% polar groups modified the hydrophobicity/ hydrophilicity balance. The solubility was considerably different from that of the initial polymer, since polymer became soluble in polar solvent such as ethanol. The introduction of hydroxyl groups in the macromolecular chains seemed to not modify the stability of polymers. The interest of such polymers, besides the hydrophilic character, concerned the preparation of graft copolymers. Timbard28 has used hydroxyl pendant groups to initiate a ring opening polymerisation of epsilon- caprolactone with Sn(Oct)^sub 2^ (Fig. 6).

The analysis of graft sequences was made by ^sup 13^C NMR. P(HOU- ol-g-CL) displayed caprolactone graft sequences with M^sub n^ between 1400 and 750, and they had a powder aspect in contrast to PHOU-ol precursor which was an oil (Table 7).

Poly(3-hydroxyoctanoate) containing carboxylic groups: P(HOxDy- COOH)s

Carboxylic groups are of greatest importance to bind bioactive molecules, hydrolysable, hydrophilic oligomers or targeting proteins. Moreover, the presence of this functional group favoured the hydrolysis of corresponding polymers which could be used in drug delivery system. The chemical modification was first carried on PHO^sub 90^U^sub 10^ with KMnO^sub 4^ as oxidation reagent, in the presence of KHCO^sub 3^(27). But 50% of unsaturated groups were not transformed and molecular weights were significantly reduced. The oxidation reaction conditions were modified using KMnO^sub 4^ and 18- crown-6-ether as phase transfer and the dissociating agent of KMnO^sub 4^ (Fig. 7) PHO^sub 75^U^sub 25^ was totally oxidised in 16 h.29,30

6 Synthesis of PHOU-ol-g-PCL

7 Synthetic route to oxidation of PHOU

The complete oxidation into carboxylic groups was displayed by ^sup 1^H NMR. No signals due to diols or unsaturated groups were present. Molecular weights after oxidation were investigated by comparison between PHOUs and PHOxDy-COOHs (Table 8). Molecular weights of PHOxDy-COOHs were investigated by the comparison of COOH different contents (Table 8). Concerning PHO^sub 90^D^sub 10^-COOH, no significant modification of molecular weight was observed whereas an apparent reduction in molecular weight for PHO^sub 75^D^sub 25^- COOH was observed.

Table 7 Characteristics of PHOU-ol-g-PCL

The presence of 25% polar groups in PHO^sub 75^D^sub 25^-COOH modified the hydrophobicity/hydrophilicity balance. This amorphous sticky material (T^sub g^=-19[degrees]C) polymer is now soluble in methanol, acetone and in some acetone/water mixtures (Table 9).

Table 8 Molecular weights of PHOD

8 M^sub n^(t)/M^sub n^(t=0) variations of PHOCOOH at various incubation times

Furthermore, the PHOD-COOH solubility in polar mixtures acetone/ water depended on pH. At low pH, polymers are insoluble. Only in strong basic conditions (pH>/=11) the product is soluble, but the polymer was rapidly degraded in this condition (Table 10).

Hydrolytic degradation

This fast hydrolytic degradation under strong basic conditions was associated with the presence of carboxylic groups because the great stability of PHO and PHOU has been tested at basic pH.

For this reason, the weight loss of PHO^sub 75^D^sub 25^-COOH has been studied in a buffer solution (pH=10) and at room temperature. Weight loss decreased continuously, reaching 100% after ~2 h 30 min (Fig. 8).

These modified bacterial polyesters have been studied, essentially for drug delivery systems as nanoparticles. Nanoparticles were prepared by a nanoprecipitation-solvent evaporation method.31

Table 9 Solubility of PHOD-COOH

Table 10 Hydrolytic degradation

9 Photographs (TEM) of nanoparticles prepared by nanoprecipitation method of corresponding polyester

At first, nanoparticles were obtained from PHO,31 but they collapsed dramatically contrary to nanoparticles prepared with PHO^sub 75^D^sub 25^-COOH as observed by TEM (Fig. 9). The difference was due to the nanoparticles hydrophobicity/ hydrophilicity balance of the polyesters. Nevertheless, PHO^sub 100- x^D^sub x^-COOH has very poor mechanical properties and is very sticky.

10 Nanoparticles prepared with a 100%PHO, b P(HO-b-CL) (4%PHO) and c 100%PCL. (MET)

11 Hydrolytic degradation of PHO and block PHO polymers at pH 7.3 and 37[degrees]C

Block copolymers

In order to improve the stability of particles aimed at drug delivery, novel diblock copolymers have been synthesised on the basis of elastomeric PHO or PHO^sub 75^D^sub 25^-COOH as soft segment and a more crystalline polycaprolactone (PCL) segment.28,32 Copolymers were prepared through combining in a first step the preparation of poly(3-hydroxyalkanoate)s oligomers, having a hydroxyl end group and in a second step, the controlled coordination- insertion ring opening polymerisation of epsilon-caprolactone initiated by the OH-oligomer end group using a trialkyl metal as catalyst. Oligomers were obtained by methanolysis with different molecular weights according to the time reaction. A series of block copolymers were prepared by varying the oligomer molecular weights keeping the PCL block length constant. All the PHA-b-PCL copolymers were crystalline (Table 11) and corresponding nanoparticles were globally spherical without coalescence (Fig. 10)

Table 11 Characteristics of diblock copolymers P(HO-b-CL)

P(HO^sub 75^D^sub 25^-COOH-b-CL), PHO^sub 75^D^sub 25^COOH (48 300 g mol^sup -1^) and P(HO^sub 75^D^sub 25^-COOH-b-CL) with M^sub n,P(HO-COOH)^=4800 g mol^sup -1^ and M^sub n,PCL^=26 800 g mol^sup - 1^ films have been immersed in a buffer solution at pH=7.3 (37[degrees]C). The degradation is observed for both polymers, but with a different rate due to the presence of hydrophobic PCL sequences (Fig. 11).

Graft copolymers

It was interesting to use reactive carboxylic side groups of P(HOD-COOH) to prepare new grafted polymers with controlled compositions. The synthesis of grafted PHAs by direct esterification with poly(ethylene glycol) (PEG) or polyflactic acid) (PLA) oligomers were carried out.30 The hydrophilic PEG was selected because it could increase the particles hydrolysis. In contrast, the hydrophobic PLA could increase their stability by internalisation inside the core of the particles. Our aim was to use modified PHAs as a matrix to develop particles for drug delivery or drug targeting. The synthesis of grafted PHAs is outlined in Fig. 12.

The grafting reaction proceeded by direct condensation between carboxylic groups of P(HOD-COOH), noted PHOD and hydroxyl terminal groups of PEG or PLA oligomers. The most common activation for carboxylic functions was applied using dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridin (DMAP) as catalyst (Table 12). Monofunctional oligomers of PEG and PLA were selected to avoid cross- linking side reactions. Commercial monofunctional PEG was used. For PLA, the terminal carboxylic group was esterified with trimethylsilyldiazomethane.

Graft copolymers were characterised by SEC (Table 13). SEC analysis allowed us to confirm that unreacted oligomers were eliminated by purification. The molecular weight data for graft PHOD showed that obtained molecular weights of graft PHOD were very close to the molecular weights of the precursor PHOD. It can be explained by short chain lengths of both oligomers. In order to study the behaviour of PHOD-g-PEG in aqueous media, the grafting of PHOU was carried out with three monomethylated PEG with respectively M^sub n^= 350, 750, 2000 gmol^sup -1^. The maximum grafting percentage is about 50% for PEG350 and only 20% for PEG2000. This result can be explained by the steric hindrance with increasing chain length, and by a decrease of the hydroxyl terminal group.

12 Synthetic route to grafting oligomers of PEG and PLA

The hydrolytic degradation of PHOD-g-PEG films at 37[degrees]C and pH 7.3 was followed during 10 months. The weight loss was recorded and compared to the weight loss of the non graft polymer PHOD under similar conditions.33 The PHOD sample was degraded into soluble fragments after three months whereas the grafted films remained stable during the same period of time. The results are shown in Fig. 13.

A weight loss was observed during the initial two weeks and this reduction increased with PEG molecular weight. After this first period, the film weight remained approximately constant during 10 months. The sec analysis of the samples only showed a small shift (Fig. 14). The graft polymer is much more resistant than PHOD to hydrolysis at physiological pH. It is supposed that the hydrophobic polymer backbone is sheltered by the PEG grafts against the hydrolysis. The graft groups of PEG, although hydrophilic, do not favour the penetration of water towards the main chain and thus are capable to stabilise the functionalised polymer. PHOD-g-PEG could thus form particles with a hydrophobic core shell, which is functionalised with carboxyl groups, and has an outer layer constituted of PEG arms, which could make inaccessible the remaining carboxylic groups. This layer is able to prevent the aggregation of the particles and stabilise therefore their colloidal state.

Table 12 Results of PEG or PLA grafting reactions on PHOD

Table 13 Physical characteristics of graft PHOD determined by SEC


Bacterial polyesters are of great interest for biomedical permanent or non permanent applications, due to their excellent biocompatibility and non toxicity. Over the past years, PHAs have been used to develop devices for vascular system, orthopaedy, drug delivery, urology, fabrication of a trileaflet heart valve scaffold in tissue engineering.34 All polyesters are susceptible to hydrolysis to some extent, but as shown above, controlled simple hydrolysis is not evident and in many cases, it is necessary to use functionalised PHAs, which have to be chemically modified. It is evident that carboxylic pendant groups constitute, at present time, the most promising functions, necessary for polymer hydrolysis degradation. But to strictly control the material degradation, the situation is very complex. According to the required system, the percentage of carboxyl groups has to be adjusted, by varying the percentage of double bonds in the polymer precursor. Moreover well defined mechanical properties have been obtained by use of copolymers with a hydrophobic sequence. At last, it is important to note that the master of the hydrophobic/ hydrophilic balance is not enough to control all hydrolysis factors. Phenomena are more complex, and degradation has to be still studied, with other combinations of functionalised and not functionalised polyesters. 13 Weight loss of different PHOD-g-PEG films in phosphate buffer (pH 7.24, 37[degrees]C) during time: [black circle] - PHOD-g-PEG 350 (grafting 52%); [black square] - PHOD-g-PEG 350 (grafting 28%); [diamonds] - PHOD-g-PEG 750 (grafting 29%); [black triangle up] - PHOD-g-PEG 2000 (grafting 19%)

14 Change in molecular size of PHOD-g-PEG during hydrolytic degradation in phosphate buffer (pH 7.24, 37[degrees]C): [white circle] - PHOD-g-PEG 350 (grafting 52%); [white square] - PHOD-g- PEG 350 (grafting 28%); * - PHOD-g-PEG 750 (grafting 29%); [white triangle up] - PHOD-g-PEG 2000 (grafting 19%)

(c) 2007 Institute of Materials, Minerals and Mining

Published by Maney on behalf of the Institute

Received 12 September 2006; accepted 23 August 2007


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E. Renard, V. Langlois* and P. Guerin

LRP, UMR 7581, Universite Paris XII- CNRS , 2 a 8, rue Henri DUNANT - 94 320 Thiais, France

* Corresponding author, email [email protected]

Copyright Institute of Materials Dec 2007

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