Development of Multifunctional Polymeric Micelle-like Nanoparticles Based on Polyethyleneimine as Drug and Gene Delivery Carriers
| Autor: | Wei-Ti Kuo, 郭維珶 |
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| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 The absence or overproduction of a specific protein in the body can lead to a variety of clinical manifestations depending on its structural or functional role. Gene therapy is a method for treatment or prevention of disease by transferring genetic information to the patient’s somatic cells. Non-viral gene carriers composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation, and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer such that high molecular weight (>25kDa) of PEI can elevate the transfection efficiency. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, a multi-functional nano-micelle was developed for both drug and gene delivery application. PEI was modified by grafting stearic acid (SA) and formulated to polymeric micelles with positive surface charge to evaluate for gene delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil-in-water (o/w) solvent evaporation method. The success of PEI –SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential of 10k PEI-SA determined by photon correlation spectroscopy was 149.6 ± 1.2 nm and 64.1±1.5 mV in water, respectively. Gel retardation assay indicated that these self-assemble positive charge micelles can effectively bind to pDNA and siRNA for gene delivery. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed 10k PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results proved that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Furthermore, PEI-SA micelles not only can serve as gene carriers, but also offered physical barriers to protect DNA and siRNA against nuclease digestion. The siRNA delivered by PEI-SA carriers also demonstrated significantly higher cellular uptake efficiency and stability even in the presence of serum proteins than free siRNA. The polyplex formulated by 10k PEI-SA/siRNA also provided excellent post transcriptional gene silencing efficiency, in particular when co-delivered with lysosomotropic agent chloroquine. In the animal model study, the combination effect of co-delivering doxorubicin and VEGF siRNA by PEI-SA nanoparticles showed a promising synergistic effect towards anti-tumor growth. In summary, the amphiphilic structure of PEI-SA micelles can provide advantages for multifunctional tasks; where the hydrophilic shell modified with cationic charges can electrostatically interact with DNA or siRNA, and the hydrophobic core can serve as payloads for hydrophobic drugs, making it truly a promising multifunctional vehicle for both genetic and chemotherapy application. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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