Synthesis And Properties Of Novel Biodegradable And Electroactive Polyphosphazenes

As a new kind of functional material, conducting polymers not only possess good biocompatiblity but also can modulate cellular activities, including cell adhesion, migration, DNA synthesis, and protein secretion, implying a promising application in biomedical field. In this dissertation, a novel electroactive, biocompatible and biodegradable polyphosphazenes were designed and synthesized for peripheral nerve regeneration. The structure and properties of the synthesized polymer were characterized by different methods. Biological evaluation and electrical activation of materials were carried out, via electrical stimulation, by cultivating RSC96 Schwann cells on the surface of the materials. The idea of tethering conducting polymer and biodegradable materials together to create a new kind of polymer explores a new way for regenerating nerve tissue engineering. The major work is summarized as the following:Oligoaniline of aniline tetramer and pentamer were synthesized successfully in this part. The influences of reaction temperature, dosage of oxidant and concentration of different proton acid on yield of aniline tetramer were investigated. Two synthetic routes for the preparation of aniline pentamer had been studied. The results showed that the high yield and purity of pentamer should be synthesized through step-by-step method. Chemical structure of aniline tetramer and pentamer was characterized by FT-IR,¹H-NMR, ¹³C-NMR and UV-Vis. The electrochemical properties of the as-synthesized oligoaniline also were investigated by CV and Four-Point probe method. Reactive poly(dichlorophosphazene) was prepared from hexachlorocyclotriphosphazene by thermal initiated ring-open polymerization. Polymerization conditions were investigated, including polymerization temperature, polymerization time and water concentration in respect to the yield of poly(dichlorophosphazene).Monosubstituted polyphosphazene of Poly[(glycine ethyl ester)phosphazene] (PGEE) and poly[(methylphenoxy) phosphazene] (PMPP) were prepared by grafting glycine ethyl ester and methoxyphenol on polyphosphazene respectively. Poly[(methylphenoxy)(aniline tetramer) phosphazene] (PMAP) and poly[(glycine ethyl ester)(aniline pentamer) phosphazene] (PGAP) were further synthesized by grafting oligoaniline and biodegradable groups on polyphosphazene as side groups. Some factors affecting yields of two mixed-substituent poly (organophosphazenes), such as reaction temperature and order of substitutional group added, were studied and discussed. FT-IR,¹H-NMR,UV-Vis> ³¹P-NMR were used to characterize the chemical structure of as-synthesized polymers. The electrochemical properties of PGAP and PMAP were characterized by CV and Four-point probe method. The conductivity of PGAP and PMAP is 2x10^-5S/cm and 1.45x10(-6)S/cm respectively, which confirmed that the conductivity of PGAP is better than PMAP. In addition, the molecular weight of PGAP determinated by GPC is Mw= 2.1 x10⁵, Mn=6.2x104, and the heterogeneity is d=3.42.The pH value of degradation medium, and the variation of molecular weight were determinated and the variation of the morphology and microstructure of PGEE,PMPP,PGAP and PMAP was observed during the degradation in vitro. The results showed that the biodegradability of poly (organophosphazenes) is relevant to the properties of substituent groups. The pH value of degradation medium tended to rise but still in the range of neutral during the whole degradation time of PGAP and PGEE. The results also illustrated that PGAP and PGEE is biodegradable.After cultivating RSC96 Schwann cells on the surface of PGAP and PMAP materials, Biological evaluation was carried out through cell proliferation, cytotoxicity, hemolysis test and SEM observation. The viability using MTT assay of RSC96 cells seeded on PGAP films showed that PGAP could promote cells adhesion and proliferation. Fluorescent probe technology indicated that cytotoxicity of PGAP is similar to PDLLA, which suggested PGAP is non-toxic. The results of hemolysis test showed PGAP meets the requirements of medical materials. The SEM results showed that PGAP has better cell affinity than PDLLA. All of the above results suggested that the PGAP copolymer was non-toxic and could support the cell attachment and proliferation.A novel adjustable direct current regulated power supply was designed and prepared for stimulating cell growth. In order to elucidate the effect of electrical stimulation through PGAP on RSC96 cells proliferation, experiments were performed in which RSC96 cells grown on PGAP were subjected to a potential through the film, through the solution and not subjected to any stimulation, respectively. The effects of non-conducting material, such as PDLLA and PGEE, with or without electrical stimulation on RSC96 cell proliferation were also evaluated. Different constant potential and stimulation time on proliferation of RSC96 cells were also studied. The results showed that electrical stimulation through the PGAP films could accelerate cells proliferation. Cells grown on PGAP materials in which charge was passed through the solution possessed medican pseudopodium length similar to cells without an electrical stimulus. As for non-conducting materials, medican pseudopodium length is no significantly different between with or without electrical stimulation. In addition, the cell proliferation results using MTT assay showed that there is no significant difference when stimulation potential and time exceed to 100 mV and 72 h, respectively.