A reducible polycationic gene vector derived from thiolated low molecular weight branched polyethyleneimine linked by 2-iminothiolane.
To improve transfection efficiency and reduce the cytotoxicity of polymeric gene vectors, reducible polycations (RPC) were synthesized from low molecular weight (MW) branched polyethyleneimine (bPEI) via thiolation and oxidation. RPC (RPC-bPEI(0.8 kDa)) possessed MW of 5 kDa-80 kDa, and 50%-70% of the original proton buffering capacity of bPEI(0.8 kDa) was preserved in the final product. The cytotoxicity of RPC-bPEI(0.8 kDa) was 8-19 times less than that of the gold standard of polymeric transfection reagents, bPEI(25 kDa). Although bPEI(0.8 kDa) exhibited poor gene condensing capacities (∼2 μm at a weight ratio (WR) of 40), RPC-bPEI(0.8 kDa) effectively condensed plasmid DNA (pDNA) at a WR of 2. Moreover, RPC-bPEI(0.8 kDa)/pDNA (WR ≥2) formed 100-200 nm-sized particles with positively charged surfaces (20-35 mV). In addition, the results of the present study indicated that thiol/polyanions triggered the release of pDNA from RPC-bPEI(0.8 kDa)/pDNA via the fragmentation of RPC-bPEI(0.8 kDa) and ion-exchange. With negligible polyplex-mediated cytotoxicity, the transfection efficiencies of RPC-bPEI(0.8 kDa)/pDNA were approximately 1200-1500-fold greater than that of bPEI(0.8 kDa)/pDNA and were equivalent or superior (∼7-fold) to that of bPEI(25 kDa)/pDNA. Interestingly, the distribution of high MW RPC-bPEI(0.8 kDa)/pDNA in the nucleus of the cell was higher than that of low MW RPC-bPEI(0.8 kDa)/pDNA. Thus, the results of the present study suggest that RPC-bPEI(0.8 kDa) has the potential to effectively deliver genetic materials with lower levels of toxicity.
Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, 421 Wakara way, Suite 318, Salt Lake City, UT 84108, USA.
Han Chang
HC
Kang
Ho-Jung
HJ
Kang
You Han
YH
Bae
D003432:Cross-Linking Reagents / Q000737:chemistry*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007096:Imidoesters / Q000737:chemistry*; D008422:Materials Testing; D015394:Molecular Structure; D008970:Molecular Weight; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011094:Polyethyleneimine / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014162:Transfection
D003432:Cross-Linking Reagents; D007096:Imidoesters; D011073:Polyamines; D000071228:Polyelectrolytes; D011094:Polyethyleneimine; D013438:Sulfhydryl Compounds; C005905:methyl 4-mercaptobutyrimidate