Phosphoester-Containing Polymers For Drug And Gene Delivery

Development of drug and gene delivery carriers with fine properties is a hot topic of biomedicine research. In the thesis, two polymers with unique properties were designed and synthesized as the carriers of drug and gene delivery systems, respectively. The structures and properties of the two polymers were well characterized.A novel glutathione-responsive amphiphilic block copolymer consisting of polycaprolactone and polyphosphoester with intervening disulfide bond, termed as PCL-SS-PEEP, was synthesized. The block copolymer was well characterized by 1H NMR and gel permeation chromatograph. The amphiphilic copolymer formed micelles in aqueous solution and the critical micelle concentration was 3.1×10-3 mg mL-1. Transmition electron microscopy and dynamic light scattering (DLS) measurements demonstrated that the micelles were relatively uniform in size with the average diameter ca. 100 nm. The micelles displayed glutathione-dependent size changes according to DLS measurement. Doxorubicin [1] was encapsulated in the micelles and its in vitro release profile demonstrated that more DOX was released at a faster rate in the presence of glutathione (GSH). At cellular level, the cells were pretreated with glutathione monoester (GSH-OEt) at different concentrations to manipulate the intracellular GSH concentration. The confocal laser scanning microscopic and flow cytometric analyses indicated that more intensive fluorescence of DOX were detected in cells pretreated by GSH-OEt due to the GSH-triggered DOX release. The DOX-loaded micelles also exhibited higher cytotoxicity in GSH-OEt-pretreated A549 cells. This indicated that the GSH-responsive PCL-SS-PEEP micelles are promising for intracellular drug delivery and it also provides a new paradigm for designing drug delivery vehicles.Cyclic phosphate monomer ethyl ethylene phosphate (EEP) modified poly(ethylenimine) (PEI), denoted as PEI-EEP, was synthesized for gene delivery. Three PEI-EEP polymers were synthesized and their structures were characterized by 1H and 31P NMR methods. The buffering capabilities of PEI-EEPs were examined by acid–base titration and the titration profiles showed that all of the PEI-EEPs had the similar buffering capability as unmodified PEI. Physiochemical characteristics of PEI-EEP/DNA complexes were analyzed by agarose gel electrophoresis, and particle size and zeta potential measurements. All of the PEI-EEPs were able to condense DNA efficiently at N/P ratios higher than 0.5:1. The particle sizes of PEI-EEPs/DNA complexes were around 160–250 nm, while the surface charges were around 30-50 mV at the N/P ratios ranging from 10:1 to 50:1. In vitro cell viability and transfection ability were evaluated in HEK293 and HeLa cells using PEI as the control. The cytotoxicity of PEI-EEPs and PEI-EEPs/DNA complexes were lower than that of PEI and its complexes with DNA. The in vitro transfection experiments indicated that the transfection efficiency of PEI-EEP/DNA complexes were related to the modification degrees by the phosphate. When the phosphate units per PEI were 52 and 75, the PEI-EEP/DNA complexes exhibited comparable or even higher transfection ability than PEI/DNA complex did at its optimal N/P ratio of 10:1 in the absence of serum. However, when the phosphate units increased to 116 per PEI, the transfection efficiency dramatically reduced.