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Synthesis of Polylactide-Grafted Dextrans and Their Application as Biodegradable Biomedical Materials

Yuichi Ohya, Tomohiro Kontani, Toshifumi Saito, Rie Aoki, and Tatsuro Ouchi. Department of Applied Chemistry, Faculty of Engineering & High Technology Research Center, Kansai University, Yamate 3-3-35, Suita, Osaka 564-8680, Japan

Biodegradable, biocompatible polymers are of interest for use in several biomedical and pharmaceutical applications. Based on its biodegradability, biocompatibility, high mechanical strength, and good shaping and molding properties, polylactide (PLA) is frequently utilized as an implantable carrier for drug delivery systems, as well as a surgical repair material. However, its high crystallinity interferes with predictable degradation, reducing its compatibility with soft tissues and presenting an obstacle to its application as a biodegradable soft plastic. One promising approach to overcoming these problems in PLA is the introduction of hydrophilic segments and branched structures. Polysaccharides are biodegradable hydrophilic polymers that degrade enzymatically and possess relatively good biocompatibility properties, but are insoluble in common organic solvents. PLA is commonly synthesized by a ring-opening polymerization of lactic acid dimer (lactide, LA). The anionic ring-opening polymerization reaction of LA is performed in the presence of an alkali metal alkoxide to give PLA. Our group has succeeded in obtaining graft copolymers consisting of PLA and polysaccharides using the hydroxyl group alkoxides of the trimethylsilyl (TMS)-protected polysaccharides as initiators and subsequent removal of TMS groups. Using dextran as a polysaccharide, the obtained PLA-grafted dextran (Dex-g-PLA) (Fig) films were found to have lower values of Tg, Tm, crystallinity, and a higher viscosity compared with poly-L-lactide (PLLA) films due to the introduction of polysaccharide segments and branched structures into PLLA. The biodegradability and compatibility of Dex-g-PLA were depend on the molecular structure, length and number of graft chains, and proportion of hydrophilic to hydrophobic segments in the graft copolymers. In this presentation, the possibility of Dex-g-PLA as a new biodegradable soft-biomaterial was investigated through a preliminary investigation of the effect of molecular structure of Dex-g-PLA on cell attachment on the films prepared from the copolymer. Moreover, Dex-g-PLA has amphiphilic structure and should be useful drug release depot for hydrophilic macromolecules (proteins, nucleic acids). So we also report on the preparation of protein (BSA, bovine serum albumin)-loaded microspheres (MSs) from Dex-g-PLA. Dex-g-PLA was synthesized through the graft-polymerization of L-LA on TMS-protected dextran in THF using t-BuOK as an initiator and the subsequent deprotection of the TMS groups according to the method reported previously. The majority of the hydroxyl groups of dextrans were protected by TMS groups in order to achieve solubility in organic solvents and to control the number of reaction sites with the alkali metal initiator. All of the graft copolymers obtained were soluble in THF, chloroform, dimethylformamide, dimethylsulfoxide, and other organic solvents, but not soluble in water. We could obtain Dex-g-PLA with various length and numbers of graft chains and differential content of sugar unit. The water contact angles of the graft copolymer films and PLLA, PDLLA films were investigated. The annealing of films was performed in water at 37C over Tg of Dex-g-PLA (1619C) for certain period. Generally, the surface of amphiphilic polymer film is covered with hydrophobic domains in air, while it tends to change to the hydrophilic surface by annealing in water. The dynamic contact angle decreased with increasing the content of sugar units. Namely, the wettability of surface of Dex-g-PLA films increased with sugar content. This result suggested that the hydrophilic dextran segment were moved onto the film surface through micro-Brownian motion by annealing in water. Attachment of L929 cells to the film surfaces of graft copolymer and PLLA was measured in a medium containing serum for 112 h of incubation. All of the Dex-g-PLA films prepared exhibited lower cell attachment ability than PLLA film. Moreover, number of attached cells decreased with increase of the contents of sugar unit. Because Tg of used Dex-g-PLA is lower than the culture temperature (37C), the hydrophilic dextran segments were moved onto the film surface through micro-Brownian motion to afford hydrophilicity of Dex-g-PLA films surfaces. Namely, the cell attachments ability was suppressed by the introduction of sugar units. The morphology of the cells attached to the polymer films was observed by SEM. PLLA film and the graft copolymer film having lowest content of sugar unit exhibited a well-spread morphology, while the cells on graft copolymer having highest content of sugar unit exhibited almost round shapes. The cells on the other films exhibited between them. These results from SEM observation are in harmony with the results of the cell attachment test. Dex-g-PLA films annealed in water gain high hydrophilicity surface by the hydrophilic dextran segments were moved onto the film surface through micro-Brown motion. Consequently, serum protein and cells do not adhere to the Dex-g-PLA films. BSA-loaded MSs were produced according to a W/O/W emulsion solvent evaporation/extraction method. BSA was dissolved in ultra pure water. This internal aqueous phase was added into an organic solution (mixture of methylene chloride and DMSO) containing PLLA or Dex-g-PLA and sonicated at 0 C with stirring. This primary emulsion was poured into a 0.1% (w/v) external aqueous solution of polyvinyl alcohol (PVA) and stirred with a magnetic stirrer for 30 min at room temperature. Then, organic solution was completely evaporated under reduced pressure. The suspension was centrifuged to obtain the precipitated MSs. The obtained MSs were washed with ultra pure water and freeze-dried. To investigate the release behavior of BSA from MS and localization of BSA in MS, the preparation of FITC-BSA-loaded MSs was carried out according to the same method. The entrapped efficiency of BSA as water-soluble model protein drug with in MS(Dex-g-PLA) showed higher value than that within MS(PLLA). The release rate of BSA could be controlled by the use of Dex-g-PLA introduced hydrophilic dextran segment for PLLA. The stable entrapment of BSA within MS(Dex-g-PLA) was proved by CD measurement.