Mansoor M. Amiji
Samuel Gatley, Rebecca L. Carrier
Date of Award
Master of Science
Department or Academic Unit
Bouve College of Health Sciences. Department of Pharmaceutical Sciences
Immunology, Genetics, Emulsions (Pharmacy), Gene therapy, Macrophages, Nanoparticles, Transfection
Pharmacy and Pharmaceutical Sciences
Purpose:Multi-compartmental delivery systems can effectively deliver therapeutic payloads by overcoming different extra- and intra-cellular barriers. The main objective of this study was to develop DNA-encapsulated solid nanoparticles-in-emulsion formulation to enhance gene delivery and transfection in macrophages.
Methods:Type B gelatin based nanoparticles were prepared by ethanol water solvent displacement method. Safflower oil-containing water in oil in water multiple emulsion was prepared by a two step emulsification technique with the help of Silverson homogenizer L4RT®. EGFP-N1 plasmid DNA that expresses enhanced green fluorescent protein (GFP) was encapsulated in solid type B gelatin nanoparticles (GNP) which were further encapsulated in the innermost aqueous phase of safflower oil containing water in oil in water (W/O/W) multiple emulsion (ME). Fluorescence microscopy and GFP-specific ELISA were performed for qualitative and quantitative determination of plasmid EGFP-N1 gene transfection in murine adherent alveolar macrophages J774A.1.
Results:Particle size of the NiE formulations was found to be less than or equal to 5 um in diameter. Results obtained from gene transfection evaluations showed that plasmid EGFP-N1 or mIL-10 loaded NiE formulations offered higher and sustained GFP expression compared GFP expression observed with plasmid EGFP-N1 or mLI-10 loaded ME and GNP formulations or plasmid DNA complexed with Lipofectin® , a cationic lipid based standard gene transfection reagent in J774A.1 murine adherent alveolar macrophage cell lines.
Conclusion:The results of this study show that DNA-containing solid gelatin nanoparticles can be encapsulated in the innermost aqueous phase of the W/O/W multiple emulsions to form NiE formulations. EGFP-N1 or mIL-10 plasmid-loaded NiE formulations were capable producing sustained gene transfection in J774A.1 murine adherent alveolar macrophage cell lines. In addition mIL10 transfection using NiE formulation showed highest amount of TNFα and IL-1β suppression upon LPS stimulation.
Attarwala, Husain, "In vitro evaluations of macrophage-targeted anti-inflammatory gene delivery and transfection using nanoparticles-in-emulsion formulations" (2010). Pharmaceutical Science Master's Theses. Paper 17. http://hdl.handle.net/2047/d20000800
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