Biodegradable Polyester For Anti-cancer Drug And Gene Delivery

Current chemotherapy is far from satisfactory:drug treatments often have limited effectiveness and patients suffer from serious side effects. Gene therapy represents a novel form of medical treatment that is expected to have major impact on human health in 21st century since a large number of human diseases are caused by genetic disorders. The success of chemotherapy or gene therapy is largely dependent on the development of a vehicle or vector that can selectively and efficiently deliver drug and gene to target cells with minimal toxicity. The use of nano-sized particles in cancer therapy and gene delivery is particularly exciting, as these materials can increase drug solubility and stability, as well as improve pharmacological effect and gene transfection efficiency. Herein, we reported that a series of biodegradable aliphatic polyester with diverse structures were designed and synthesized via enzymatic copolymerization using Candida antarctica lipase B (CALB) as the catalyst. Combing nano-fabrication technology, we developed several biodegradable polymeric drug and gene delivery systems, which would significantly improve the cancer treatment and gene transfection. There are three parts in this dissertation, including:PartⅠ, we show that degradable particles of a hydrophobic polymer can effectively deliver camptochecin to tumors after i.v. administration. Free-standing nanoparticles with diameters of 100-300 nm were successfully fabricated from highly hydrophobic, biodegradable poly(ω-pentadecalactone-co-butylene-co-succinate) (PPBS) copolyesters. PPBS copolymers with various compositions were synthesized via copolymerization of o-pentadecalactone (PDL), diethyl succinate (DES), and 1,4-butanediol (BD) using CALB as the catalyst. Camptothecin (CPT,12-22%) was loaded into PPBS nanoparticles with high encapsulation efficiency (up to 96%) using a modified oil-in-water single emulsion technique. The CPT-loaded nanoparticles had a zeta potential of about-10 mV. PPBS particles were non toxic in cell culture. Upon encapsulation, the active lactone form of CPT was remarkably stabilized and no lactone-to-carboxylate structural conversion was observed for CPT-loaded PPBS nanoparticles incubated in both phosphate-buffered saline (PBS, pH=7.4) and DMEM media for at least 24 hr. In PBS at 37℃, CPT-loaded PPBS nanoparticles showed a low burst CPT release (20-30%) within the first 24 hrs followed by a sustained, essentially complete, release of the remaining drug over the subsequent 40 days. Compared to free CPT, CPT-loaded PPBS nanoparticles showed a significant enhancement of cellular uptake, higher cytotoxicity against Lewis lung carcinoma and 9L cell lines in vitro, a longer circulation time, and substantially better antitumor efficacy in vivo. These results demonstrate the potential of PPBS nanoparticles as long-term stable and effective drug delivery systems in cancer therapy.PartⅡ, we have developed a polymeric nanoparticles system for both Daunorubicin and siRNA delivery by using poly(PDL-co-DO). First, mice test experiments showed that random poly(PDL-co-DO) copolymers had good biocompatibility. No significant abnormal tissue growth was observed polymer films were implanted in mice for up to four weeks. Second, free-standing nanoparticles with 200 nm size in average were successfully prepared from poly(PDL-co-DO). The degradation rate of the nanoparticles was studied over a 70-day period in phosphate buffered saline (PBS) solution at 37℃. On average, the molecular weight (Mn) of the particles decreased by approximately 0.84% per day. Furthermore, daunorubicin was encapsulated into poly(PDL-co-DO) nanoparticles with high loading. DNR released from the loaded nanoparticles exhibited a biphasic trend consisting of an initial burst followed by a gradual sustained release. Finally, high encapsulation efficiency siRNA/poly(PDL-co-42%DO) nanoparticles were successfully prepared at optimize condition (spermindine, as a counterion, was added to condense siRNA). In vitro gene knockdown experiments indicated that siRNA/poly(PDL-co-42%DO) nanoparticles gave a significant enhanced luciferase gene silence. With the further development, poly(PDL-co-DO) nanoparticles could be potentially used as hydrophilic drug and gene delivery vehicles.PartⅢ, a series of biodegradable poly(amine-co-esters) were synthesized in one step via enzymatic copolymerization of lactone, diesters and amino-substituted diols. More specifically, C4-C12 diesters (i.e., from succinate to dodecanedioate) and diethanolamine comonomers with either an alkyl (methyl, ethyl, n-butyl,t-butyl) or an aryl (phenyl) substituent on nitrogen were successfully incorporated into the poly(amine-co-ester) chains. Upon protonation at slightly acidic conditions, these poly(amine-co-esters) readily turned to cationic polyelectrolytes, which are capable of condensing with polyanionic DNA to form polyplex nanoparticles. In vitro cell transfection screening revealed that three of the copolymers, poly (ω-pentadecalactone-N-methyldiethyleneamine sebacate) (PDL-PMSC), poly(N-methyldiethyleneamine sebacate) (PMSC) and poly(N-ethyldi-ethyleneamine sebacate) (PESC), possessed comparable or even higher transfection efficiency in delivering pLucDNA compared to that of Lipofectamine 2000. Studies on the physical properties and morphology of PESC/pLucDNA, PMSC/pLucDNA and PDL-PMSC/pLucDNA nanoparticles showed that both polyplexes had desirable particle sizes (< 100 nm) for cellular uptake and are capable of functioning as proton sponges to facilitate endosomal escape after cellular uptake. These polyplex nanoparticles exhibited extremely low cytotoxicity. Furthermore, gene transfection experiments performed using mouse tumor models showed that PDL-PMSC and PMSC are substantially more effective gene carrier than PEI in delivering pLucDNA to the tumor cells in vivo. All these properties make the poly(amine-co-esters) to be promising non-viral vectors for safe and efficient DNA delivery in gene therapy.