544. PLGA Nanoparticles for DNA Vaccination–Waiving Complexity and Increasing Efficiency (2006)

Immunization with naked plasmid DNA is emerging as a novel vaccination strategy. This approach involves the introduction of pDNA encoding antigenic proteins into host cells. DNA vaccination advantage over antigen vaccination is mainly due to stimulation of CD8+ cytotoxic T cells. Several non-viral methods have been developed to deliver DNA. However, these methods also suffer from drawbacks including: lower efficiency, short duration of gene expression and lack of targeting. PLGA nanoparticles (NPs) containing pDNA could serve as an efficient gene delivery system for DNA vaccination. PLGA-NPs were formulated using a modified double-emulsion technique. Size distribution was measured using Cryo-TEM and Coulter Counter and was found to be 300 nm in average. PLGA protected pcDNA from shear stresses during sonication as was seen by comparing gel electrophoresis images. We have optimized time and amplitude of sonication to minimize the destructive effect on pcDNA integrity. We achieved 85% entrapment efficiency of pcDNA. Plasmid was released from NPs in a sustained manner for over a month.Uptake and distribution of the NPs in various cell lines, including macrophages, were studied by labeling particles and DNA with fluorescent dyes: 6-coumarin, Lysotracker Red and DRAQ5. Confocal images taken at the nucleus z-section proved that NPs are inside cells and not attached to their outer membrane. Uptake of NPs was found to be dependant on particles diameter. Minimal uptake was visualized in cells when experiments were performed at 4°C. Localization of NPs was mainly inside the endo-lysosomes as was evident in the overlay images of fluorescent NPs (green) and Lysotracker (red) staining (Fig.1). The localization of NPs post uptake and high uptake dependency on temperature may indicate an active uptake process and not a passive one. Cells were incubated with increasing concentrations of PLGA-NPs for 48 hrs and their viability was evaluated using AlamarBlue assay. The effect of NPs on cells viability was negligible. We transfected several cell lines with pGL3-Luciferase-NPs (Fig.2). Expression levels were found to be up to 400-fold higher compared to experiments with naked plasmid. Protein expression was maintained for at least 3 weeks. Uptake was found to be dependant on cytochalasin B concentration indicating uptkae via phagocytosis. Our results demonstrate that PLGA-NPs can serve as an efficient gene delivery system. PLGA-NPs do not affect cells viability and phagocytosed by cells of the immune system. Therefore, they may serve for various gene therapy applications including DNA vaccinationFig. 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunization with naked plasmid DNA is emerging as a novel vaccination strategy. This approach involves the introduction of pDNA encoding antigenic proteins into host cells. DNA vaccination advantage over antigen vaccination is mainly due to stimulation of CD8+ cytotoxic T cells. Several non-viral methods have been developed to deliver DNA. However, these methods also suffer from drawbacks including: lower efficiency, short duration of gene expression and lack of targeting. PLGA nanoparticles (NPs) containing pDNA could serve as an efficient gene delivery system for DNA vaccination. PLGA-NPs were formulated using a modified double-emulsion technique. Size distribution was measured using Cryo-TEM and Coulter Counter and was found to be 300 nm in average. PLGA protected pcDNA from shear stresses during sonication as was seen by comparing gel electrophoresis images. We have optimized time and amplitude of sonication to minimize the destructive effect on pcDNA integrity. We achieved 85% entrapment efficiency of pcDNA. Plasmid was released from NPs in a sustained manner for over a month.Uptake and distribution of the NPs in various cell lines, including macrophages, were studied by labeling particles and DNA with fluorescent dyes: 6-coumarin, Lysotracker Red and DRAQ5. Confocal images taken at the nucleus z-section proved that NPs are inside cells and not attached to their outer membrane. Uptake of NPs was found to be dependant on particles diameter. Minimal uptake was visualized in cells when experiments were performed at 4°C. Localization of NPs was mainly inside the endo-lysosomes as was evident in the overlay images of fluorescent NPs (green) and Lysotracker (red) staining (Fig.1). The localization of NPs post uptake and high uptake dependency on temperature may indicate an active uptake process and not a passive one. Cells were incubated with increasing concentrations of PLGA-NPs for 48 hrs and their viability was evaluated using AlamarBlue assay. The effect of NPs on cells viability was negligible. We transfected several cell lines with pGL3-Luciferase-NPs (Fig.2). Expression levels were found to be up to 400-fold higher compared to experiments with naked plasmid. Protein expression was maintained for at least 3 weeks. Uptake was found to be dependant on cytochalasin B concentration indicating uptkae via phagocytosis. Our results demonstrate that PLGA-NPs can serve as an efficient gene delivery system. PLGA-NPs do not affect cells viability and phagocytosed by cells of the immune system. Therefore, they may serve for various gene therapy applications including DNA vaccination