Water-Soluble Lipopolymer for Gene Delivery

• Published in: Bioconjugate chemistry 2001-05, Vol.12 (3), p.337-345
• Main Authors: Han, Sang-oh, Mahato, Ram I, Kim, Sung Wan
• Format: Article
• Language: English
• Subjects: Cell Survival - drug effects; Cholesterol - chemistry; Erythrocyte Aggregation - drug effects; Erythrocytes - drug effects; Gene Transfer Techniques - standards; Humans; Lipids - chemical synthesis; Lipids - pharmacology; Lipids - therapeutic use; Liposomes - chemical synthesis; Liposomes - pharmacology; Liposomes - therapeutic use; Plasmids - metabolism; Polyethyleneimine - analogs & derivatives; Polyethyleneimine - chemical synthesis; Polyethyleneimine - pharmacology; Polyethyleneimine - therapeutic use; Solubility; Transfection - methods; Transfection - standards; Tumor Cells, Cultured; Water
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc000120w

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 1H 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, ζ 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 ∼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.