Study On Functional Polymeric And Inorganic Drug And Gene Delivery Systems

The purposes of drug and gene delivery are to deliver the therapeutic agents including drugs and other bioactive agents to target organs, tissues, cells and organelles, by using carriers with specific structures and properties. Since the drugs and genes are delivered to target sites, the side effects of the therapeutic agents on the normal tissues and cells could be minimized with the enhanced bioavailability of the therapeutic agents. In the thesis research, different types of functional polymeric and inorganic drug and gene delivery systems with a good biocompatibility were prepared and their performances in gene and drug delivery were evaluated.In Chapter 1, the recent progress of drug and gene delivery systems is reviewed, with the emphasis on targeting delivery, non-viral gene vectors, and the substrate-mediated delivery systems loaded with vector/DNA complexes.Folic acid (FA) is a targeting group which is extensively used to deliver therapeutic and imaging agents to folate receptor (FR) overexpressed cancer cells. Chapter 2 reports the amphiphilic hyperbranched core-shell polymers with folate moieties as the targeting groups for targeted drug delivery. The core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic poly(s-caprolactone) (PCL) segments and hydrophilic poly(ethylene glycol) (PEG) segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The polymers were characterized by 1H NMR,13C NMR and combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis. The nanoparticles of the amphiphilic polymers prepared by dialysis method were characterized by transmission electron microscopy (TEM) and particle size analysis. Two antineoplastic drugs,5-fluorouracil (5-Fu) and paclitaxel, were encapsulated into the nanoparticles. The drug release property and the targeting of the drug loaded nanoparticles to different cells were evaluated in vitro. The results showed the drug loaded nanoparticles exhibited enhanced cell inhibition because folate targeting increased the cytotoxicity of drug loaded nanoparticles against folate receptor (FR) expressing tumor cells.KALA, a cationic endosomolytic and fusogenic peptide, has been extensively investigated as a gene vector to condense pDNA and siRNA, and an additive to be incorporated into vector/DNA complexes to achieve enhanced transfection efficiency. In Chapter 3, the effect of KALA on the substrate-mediated gene delivery was studied. A fast degrading cholic acid functionalized star poly(DL-lactide) was used to fabricate Ca-P/DNA/KALA co-precipitates deposited films and Ca-P/DNA/KALA co-precipitates encapsulated films for mediating substrate-mediated transfection. The in vitro gene transfections in HEK293T and HepG2 cells mediated by the different films were investigated. The effect of KALA content on the gene transfection was studied. The gene expression of film-mediated transfections could be significantly enhanced by the addition of KALA at a quite low content with DNA/KALA ratio of 10/1. Due to the surface erosion mechanism of the functionalized star poly(DL-lactide), the substrate-mediated transfection system could rapidly release Ca-P/DNA/KALA to mediate efficient gene expression. During the transfection, the degradation of the polymer films could be observed and the degradation did not show any unfavorable effects on the gene expression. The presence of KALA could significantly enhance the gene expression of substrate-mediated transfections.Among different non-viral gene delivery methods, the technique of co-precipitation of Ca2+ with DNA in the presence of inorganic anions is an attractive option because of the biocompatibility and biodegradability. In Chapter 4, nano-sized CaCO3/DNA co-precipitates for gene delivery were investigated. The effect of Ca2+/CO32- ratio on the gene delivery was investigated. The mechanism of the transfection mediated by CaCO3/DNA co-precipitates was studied by pretreatments of the cells with chloroquine, wortmannin and cytochalasin D, respectively. The in vitro gene transfections in 293T and HeLa cells were carried out for both solution-based transfection and solid-phase transfection. The gene expression of calcium carbonate based approach is strongly affected by the Ca2+/CO32- ratio because the size of CaCO3/DNA co-precipitates is mainly determined by the Ca2+/CO32- ratio. In addition, the encapsulation efficiency of DNA increases with decreasing Ca2+/CO32- ratio. With a proper Ca2+/CO32- ratio, CaCO3/DNA co-precipitates could effectively mediate gene transfection with the expression levels higher than that of Lipofectamine 2000 in the presence of serum. The mechanism study shows that CaCO3/DNA co-precipitates are internalized via endocytosis of the cells and macropinocytosis is the main route of internalization. Compared with the solution-based transfection, CaCO3/DNA co-precipitates in the solid-phase transfection exhibits a lower gene expression level. The calcium carbonate based approach has great potential in gene delivery.To achieve maximum therapeutic efficiency, the strategy of combined drug and gene therapy for tumor treatments has attracted more and more research interests. Chapter 5 focuses on the studies on the co-delivery of p53 gene and doxorubicin. With the presence of doxorubicin hydrochloride (DOX·HC1), co-precipitation of Ca2+ and pDsRed2-Nl-p53 resulted in formation of CaCO3/DNA/DOX nanoparticles. The tumor cell inhibition effect of the CaCO3/DNA/DOX nanoparticles was evaluated on HeLa cells. As compared with the treatments by p53 gene and the drug separately, the co-delivery of p53 gene and the drug could induce a higher cell inhibition and promote tumor cell apoptosis more effectively.