Study On Natural Polymer And Peptide Modified CaCO₃Based Co-precipitates For Gene And Drug Delivery

Efficient and safe carriers are of imporatance in gene delivery and drug controlled release. For gene delivery purpose, compared with viral vectors which suffer from the safety concern, the non-viral vectors exhibit advantages including high safety, and low immunity. Nevertheless, among different non-viral vectors, the biocompatibility of the widely investigated cationic polymers and cationic liposomes is still not satisfactory. Compared with them, the delivery systems based on the co-precipitates of inorganic compounds (such as CaCO3and CaP) and DNA have better biocompatibility. In addition, for drug delivery purpose, the nano-and microparticles based on inorganic compounds (such as CaCO3and CaP) are a class of important drug carriers.The focus of this thesis research is natural polymer and peptide modified inorganic compound based co-precipitate delivery systems. Their performances in gene and drug delivery were evaluated.In Chapter1, the recent research progress of gene and drug delivery systems is reviewed, with the emphasis on non-viral gene vectors and the co-delivery of gene and drug.Chapter2reports natural polymer modified CaCO3based gene and drug delivery systems. To improve the performance of nanostructured calcium carbonate in gene and drug delivery, a hydrophilic polysaccharide, alginate, was added to the co-precipitation systems to form alginate/CaCO3/DNA nanoparticles. The size and zeta potential of the nanoparticles were measured by a Zetasizer. Due to the existence of alginate chains which retarded the crystallization of calcium carbonate and form the hydrophilic outer shells, the alginate/CaCO3/DNA nanoparticles exhibited a decreased size and enhanced stability in the aqueous solution. To evaluate the of gene and drug co-delivery ability, doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was loaded in the nanoparticles to form alginate/CaCO3/DNA/DOX nanoparticles. The in vitro gene transfections mediated by different nanoparticles in293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. With an appropriate amount of alginate, the gene transfection efficiency of alginate modified nanoparticles could be significantly enhanced as compared with the nanoparticles without alginate modification for the gene delivery systems, as well as the gene and drug co-delivery systems. The study on in vitro cell inhibition effects showed the cell viability decreased with increasing DOX amount loaded in alginate/CaCO3/DNA/DOX nanoparticles. The alginate modification is a useful strategy to improve the calcium carbonate co-precipitation technique for the preparation of gene and drug delivery systems, and the nanoparticles prepared in this study have promising applications in gene and drug delivery.Chapter3focuses on the alginate modified CaCO3based delivery systems for antitumor gene and drug co-delivery. A facile strategy for efficient co-delivery of gene and drug was developed. Using a co-precipitation method, doxorubicin hydrochloride (DOX), an anti-tumor drug, and p53expression plasmid were encapsulated in alginate/CaCO3/DNA/DOX nanoparticles with high encapsulation efficiency. The in vitro cell inhibition effect of the alginate/CaCO3/DNA/DOX nanoparticles was evaluated by MTT assay in HeLa cells. The alginate/CaCO3/DNA/DOX nanoparticles exhibited a high cell inhibition rate about80%, indicating that the alginate/CaCO3/DNA/DOX nanoparticles could effectively mediate gene transfection and deliver the drug to the cells. Compared with the co-delivery of gene and drug, the treatments by alginate/CaCO3/DOX nanoparticles and alginate/CaCO3/DNA nanoparticles separately led to much lower cell inhibition rates. Compared with the CaCO3/DNA/DOX nanoparticles without alginate modification, the alginate/CaCO3/DNA/DOX nanoparticles with a decreased particle size exhibited enhanced delivery efficiency. The alginate/CaCO3/DNA/DOX nanoparticles have promising applications in cancer treatments.Chapter4reports peptide modified CaCO3based gene and drug delivery systems. To improve the gene transfection efficiency, a cationic cell-penetrating peptide, KALA, was introduced to the co-precipitation systems to form CaCO3/KALA/DNA nanoparticles. The size and zeta potential of the nanoparticles were measured by a Zetasizer, the size of CaCO3/KALA/DNA nanoparticles increased with increasing KALA amount, and the zeta potential of CaCO3/KALA/DNA nanoparticles increased with increasing KALA amount because of the addition of positively charged KALA. Due to the existence of KALA, the gene transfection efficiencies of CaCO3/KALA/DNA nanoparticles could be significantly enhanced both in HeLa and293T cells. The cellular uptake study showed that the cellular uptake of CaCO3/KALA/DNA nanoparticles was obviously enhanced compared to the CaCO3/DNA nanoparticles without peptide modified. To evaluate the of gene and drug co-delivery ability, doxorubicin hydrochloride (DOX), an anti-tumor drug and p53expression plasmid were loaded in the nanoparticles to form CaCO3/KALA/DNA/DOX nanoparticles. The in vitro cell inhibition effect of the CaCO3/KALA/DNA/DOX nanoparticles was evaluated by MTT assay. The CaCO3/KALA/DNA/DOX nanoparticles exhibited a high cell inhibition rate higher than80%, indicating that the CaCO3/KALA/DNA/DOX nanoparticles could effectively mediate gene transfection and deliver the drug to the cells. Compared with the CaCO3/DNA/DOX nanoparticles without KALA modification, the CaCO3/KALA/DNA/DOX nanoparticles with enhanced cellular uptake exhibited enhanced delivery efficiency. The CaCO3/KALA/DNA/DOX nanoparticles have promising applications in cancer treatments.Chapter5is concentrated on composite CaCO3/CaP gene delivery systems. To control the particle size and improve the stability of the nanoparticles, carbonate and phosphate ions were simultaneously introduced to the co-precipitation systems to form CaCO3/CaP/DNA nanoparticles. The size of nanoparticles was measured by a Zetasizer. With an appropriate molar ratio of carbonate ion/phosphate ion, the CaCO3/CaP/DNA nanoparticle with a decreased size and improved stability could be obtained. The in vitro gene transfections mediated by different nanoparticles in293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. The gene transfection efficiency of CaCO3/CaP/DNA nanoparticles could be significantly enhanced as compared with CaCO3/DNA and CaP/DNA co-precipitates. The cellular uptake study indicated that with an appropriate molar ratio of carbonate ion/phosphate ion, the cellular uptake efficiency of CaCO3/CaP/DNA nanoparticles could be significantly enhanced as compared with CaCO3/DNA and CaP/DNA co-precipitates.