title: Water-soluble lipopolymer for gene delivery.
abstract: 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.
authors.affiliation: Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
authors.lastname: Han So
authors.forename: R I
authors.initials: RI
authors.lastname: Mahato
authors.forename: S W
authors.initials: SW
authors.lastname: Kim
mesh_terms: 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
chemical_list: 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