Studies On Functional Gene And Drug Delivery Systems Based On Biomacromolecule/Calcium Carbonate Hybrid Nanoparticles

In gene therapy for the treatments genetic diseases, gene delivery systems can introduce exogenous genetic materials into the target cells to achieve a therapeutic effect. The purpose of drug delivery systems is to maximize the efficacy of drugs minimize the side effects. In gene and drug delivery, efficient and safe gene and drug carriers have an important significance. This thesis focuses on the functional biomacromolecule/calcium carbonate gene or drug delivery systems with improved delivery efficiencies, which achieved by introducing protamine sulfate and functional peptides to the delivery systems.In Chapter1, the recent progress of non-viral vectors for gene delivery is reviewed, with the emphasis on targeting delivery. In addition, the strategies to reverse multi-drug resistance in cancer treatments are introduced.In Chapter2, by introducing a biocompatible cationic and naturally occurring polypeptide, protamine sulfate (PS), to the delivery system, PS/CaCO3/DNA ternary nanoparticles were prepared for efficient gene delivery. By adding the cationic polypeptide PS in the co-precipitation system of calcium carbonate and DNA, PS/CaCO3/DNA nanoparticles could be formed by self-assembly facilely. The effect of PS on the properties of the ternary nanoparticles was studied by varying the PS amount in the nanoparticles. The size and zeta potential measurements indicated the PS/CaCO3/DNA nanoparticles with a proper PS amount exhibited a decreased size and an increased zeta potential. The in vitro gene transfections mediated by different nanoparticles in293T cells and HeLa cells were carried out in the presence of10%fetal bovine serum, using pGL3-Luc and pEGFP-C1as reporter plasmids. As compared with both PS/DNA nanoparticles and CaCO3/DNA nanoparticles, PS/CaCO3/DNA nanoparticles exhibited significantly enhanced gene delivery efficiency, which was higher than that of Lipofectamine2000/DNA. Confocal microscopy observation showed PS/CaCO3/DNA nanoparticles could efficiently deliver DNA to cell nuclei. These results indicated the PS/CaCO3/DNA nanoparticles have promising applications in gene delivery.In Chapter3, to enhance the gene delivery efficiency, protamine sulfate (PS) and a cell penetrating peptide (KALA) were introduced to the calcium carbonate based gene delivery system. The dual-functionalized gene delivery system, KALA/PS/CaC03/DNA nanoparticles were prepared by a facile co-precipitation method in an aqueous medium. For comparison, mono-functionalized gene delivery systems were also prepared to study the individual effect of PS and KALA. The size and ζ-potential measurements indicated KALA/PS/CaCO3/DNA nanoparticles exhibited a decreased size and an increased ζ-potential as compared with CaCO3/DNA nanoparticles. The in vitro gene transfections mediated by different nanoparticles in293T cells and HeLa cells were carried out in the presence of10%fetal bovine serum, using pGL3-Luc as a reporter plasmid. As compared with un-modified CaCO3/DNA nanoparticles and mono-functionalized nanoparticles, dual-functionalized nanoparticles exhibited significantly enhanced gene delivery efficiency. Confocal microscopy observation showed dual-functionalized nanoparticles could deliver DNA more efficiently through enhanced cellular uptake and nuclear localization. These results indicated the KALA/PS/CaCO3/DNA nanoparticles prepared in this study have promising applications in gene delivery.In Chapter4, to enhance the gene delivery efficiency, a functional peptide, GS-14, with a RGD sequence for tumor targeting and a R8sequence for enhanced cell uptake was introduced to the calcium carbonate based gene delivery system containing heparin-biotin (HPB) through biotin-avidin interaction. GS-14/Avidin/HPB/PS/CaCO3/DNA nanoparticles were prepared by self-assembly in an aqueous medium. The size and ζ-potential of the nanoparticles were measured, and the in vitro gene transfections mediated by different nanoparticles in COS-7cells and HeLa cells were carried out in the presence of10%fetal bovine serum, using pGL3-Luc as a reporter plasmid. As compared with CaCO3/DNA nanoparticles, GS-14/Avidin/HPB/PS/CaCO3/DNA nanoparticles exhibited significantly enhanced gene delivery efficiency and tumor cell targeting properties. These results indicated the GS-14/Avidin/HPB/PS/CaCO3/DNA nanoparticles have promising applications in targeted gene delivery.Chapter5focuses on the drug delivery systems which can overcome multidrug resistance (MDR). PS/CaCO3/DOX nanoparticles loaded with an anticancer drug, doxorubicin hydrochloride (DOX), and PS/CaCO3/DOX/TQR nanoparticles co-loaded with DOX and a MDR inhibitor, tariquidar (TQR), were prepared. The size and ζ-potential of nanoparticles was measured, and the in vitro tumor cell inhibition was evaluated in MCF-7/Adr cells. The results showed that PS/CaCO3/DOX nanoparticles exhibited stronger cell inhibition as compared with both free DOX and CaCO3/DOX nanoparticles. With the presence of MDR inhibitor, the dual drug loaded CaCO3/DOX/TQR nanoparticles exhibited enhanced cell inhibition as compared with the mixture of free DOX and free TQR. In addition, among different nanoparticles, the dual drug loaded PS/CaCO3/DOX/TQR nanoparticles exhibited the strongest cell inhibitory effect and could effectively inhibit drug resistance in MCF-7/Adr cells. These results indicated the PS/CaCO3/DOX/TQR nanoparticles could be a promising drug delivery platform for the co-delivery of different drugs to overcome multidrug resistance.