Rational design, fabrication, characterization and in vitro testing of biodegradable microparticles that generate targeted and sustained transgene expression in HepG2 liver cells.
Poly(lactide-co-glycolide) (PLGA) microparticles have significant potential for sustained delivery of plasmid DNA (pDNA). However, unmodified PLGA microparticles have poor transfection efficiencies. In this study, we use several approaches to enhance the transfection efficiencies of PLGA microparticles in a HepG2 liver cell line. Polyethylenimine (PEI) is used to condense the pDNA prior to loading into the PLGA microparticles. This provides enhanced loading efficiencies and greater protection to the pDNA during the entrapment process. In addition, the pDNA used (ApoE) incorporates a hybrid liver-specific murine albumin enhancer/α1 antitrypsin promoter (AlbE/hAAT) to enhance transgene expression in human liver (HepG2) cells. The percentage of surfactant used in the preparation of the microparticles, the polymer composition of the PLGA, the ratio of the PEI to pDNA (N/P), the structure of the PEI and the potential utility of a galactose targeting ligand were then investigated to further optimize the efficacy of the cationic microparticle non-viral delivery system in transfecting HepG2 cells. For each PLGA PEI-pDNA microparticle formulation prepared, we evaluated particle size, ζ-potential, loading of pDNA, cytotoxicity, and transgene expression in HepG2 cells and control human embryonic kidney (HEK293) and monkey African green kidney fibroblast-like (COS7) cells. Loading PLGA particles with PEI-ApoE pDNA complexes resulted in a significant reduction in particle size when compared to PLGA microparticles loaded with ApoE pDNA alone. Scanning electron microscopy images showed that all the particle formulations were smooth and spherical in appearance. Incorporation of the cationic PEI in the PLGA particles changed the ζ-potential from negative to positive. Complexing PEI with ApoE pDNA increased the loading efficiency of the ApoE pDNA into the PLGA microparticles. The cytotoxicity of PLGA particles loaded with PEI-ApoE pDNA complexes was similar to PLGA particles loaded with ApoE pDNA alone. The transfection efficiency of all particle formulations prepared with ApoE pDNA was significantly higher in HepG2 cells when compared to HEK293 and COS7 cell lines. The release of PEI-pDNA complexes from particles prepared with different PLGA polymer compositions including PLGA 50-50, PLGA 75-25, and PLGA 85-15 was sustained in all cases but the release profile was dependent on the polymer composition. Transmission electron microscopy images showed that PEI-pDNA complexes remained structurally intact after release. The optimum formulation for PLGA particles loaded with PEI-ApoE pDNA complexes was prepared using 2% polyvinyl alcohol, 50-50 PLGA compositions and N/P ratios of 5-10. Strong sustained transgene expression in HepG2 cells was generated by PLGA PEI-ApoE pDNA particles up to the full 13 days tested.
Intra
Janjira
J
Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, USA.
Salem
Aliasger K
AK
D000818:Animals; D001057:Apolipoproteins E / Q000008:administration & dosage / Q000235:genetics; D001672:Biocompatible Materials / Q000737:chemistry*; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004337:Drug Carriers / Q000737:chemistry*; D004339:Drug Compounding; D015195:Drug Design; D005690:Galactose / Q000737:chemistry; D018014:Gene Transfer Techniques; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007097:Imines / Q000737:chemistry; D019344:Lactic Acid / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008863:Microspheres; D015394:Molecular Structure; D010316:Particle Size; D010957:Plasmids; D011095:Polyethylenes / Q000737:chemistry; D011100:Polyglycolic Acid / Q000737:chemistry; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D013499:Surface Properties; D014162:Transfection; D019076:Transgenes / Q000235:genetics*
D001057:Apolipoproteins E; D001672:Biocompatible Materials; D004337:Drug Carriers; D007097:Imines; D011095:Polyethylenes; C505405:poly(ethylene imine); D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D004247:DNA; D005690:Galactose