Water-soluble lipopolymer for gene delivery.
The use of biocompatible polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity, and mutagenesis. Nontoxic water-soluble lipopolymer (WSLP), poly(ethylenimine)-co-[N-(2-aminoethyl) ethyleneimin]-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine) was synthesized using branched poly(ethylenimine) (PEI, mw 1800) and cholesteryl chloroformate. Following synthesis and purification, the structure and molecular weight of WSLP were confirmed by (1)H NMR and MADI-TOF mass spectrometry, respectively. The percentage of cholesterol conjugated to PEI was about 47%, and the average molecular weight of WSLP was approximately 2000 Da. WSLP/pDNA complexes were prepared at different N/P (nitrogen atoms of WSLP/phosphate of plasmid DNA) ratios and characterized in terms of particle size, zeta potential, osmolarity, surface morphology, and cytotoxicity. WSLP condensed plasmid DNA when N/P ratio reached 2.5/1 and no free DNA was detected at N/P ratio of 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was in the range of 25.9 to 148.5 nm and was dependent on N/P ratios. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of approximately 50 nm in diameter. WSLP/pDNA complexes or WSLP itself were nontoxic to CT-26 colon adenocarcinoma and 293 T human embryonic kidney transformed cells when formulated at the N/P ratio of 10/1 and below as determined by MTT assay. In contrast, PEI25000/pDNA complexes were toxic to these cells. Erythrocytes aggregated when incubated with PEI25000/pCMV-Luc complexes at high DNA concentrations, but there was little aggregation with WSLP/pCMV-Luc complexes. WSLP/pCMV-Luc complexes demonstrated higher transfection efficiency in both CT-26 and 293 T cells compared to PEI25000- or PEI1800-based formulations. WSLP/pCMV-Luc complexes are nontoxic and showed enhanced in vitro transfection. Thus, WSLP will be a suitable carrier for in vivo gene delivery.
Han So
Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Mahato
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RI
Kim
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D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000737:chemistry; D004903:Erythrocyte Aggregation / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects; D018014:Gene Transfer Techniques / Q000592:standards*; D006801:Humans; D008055:Lipids / Q000138:chemical synthesis / Q000494:pharmacology* / Q000627:therapeutic use; D008081:Liposomes / Q000138:chemical synthesis / Q000494:pharmacology / Q000627:therapeutic use; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology* / Q000627:therapeutic use; D012995:Solubility; D014162:Transfection / Q000379:methods / Q000592:standards; D014407:Tumor Cells, Cultured; D014867:Water
D008055:Lipids; D008081:Liposomes; C438935:poly(ethylenimine)-co-(N-(2-aminoethyl) ethyleneimin)-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine); D014867:Water; D011094:Polyethyleneimine; D002784:Cholesterol