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. 2011 Feb;32(4):1193-203.
doi: 10.1016/j.biomaterials.2010.08.079. Epub 2010 Nov 10.

A reducible polycationic gene vector derived from thiolated low molecular weight branched polyethyleneimine linked by 2-iminothiolane

Han Chang Kang  1 , Ho-Jung Kang, You Han Bae
Affiliations

A reducible polycationic gene vector derived from thiolated low molecular weight branched polyethyleneimine linked by 2-iminothiolane

Han Chang Kang et al. Biomaterials. 2011 Feb.

Abstract

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.

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Figures

Fig. 1
Fig. 1
The synthesis of bPEI-based reducible polycations (RPC-bPEI).
Fig. 2
Fig. 2
Cytotoxicity of RPC-bPEI0.8kDa and bPEI (bPEI0.8kDa and bPEI25kDa) in (a) HEK293 cells and (b) MCF7 cells (n=6, Mean±SEM).
Fig. 3
Fig. 3
Acid-base titration curves of RPC-bPEI0.8kDa and bPEI0.8kDa (n=3, Mean±SD).
Fig. 4
Fig. 4
Condensation of pDNA with RPC-bPEI0.8kDa in agarose gel.
Fig. 5
Fig. 5
(a) Particle size and (b) surface charge of RPC-bPEI0.8kDa/pDNA complexes in HBG.
Fig. 6
Fig. 6
DTT-triggering pDNA release from RPC-bPEI0.8kDa/pDNA complexes (WR 2) in the presence of a solution of heparin in 150 mM NaCl.
Fig. 7
Fig. 7
Normalized transfection efficiencies of RPC-bPEI0.8kDa/pDNA-, bPEI0.8kDa/pDNA- and bPEI25kDa/pDNA in (a) HEK293 cells and (b) MCF7 cells (the transfection efficiency of bPEI25kDa/pDNA (N/P=5)-transfected cells was set to 100.) (n≥4, Mean±SEM).
Fig. 8
Fig. 8
RPC-bPEI0.8kDa/pDNA- and bPEI25kDa/pDNA-uptake of (a) HEK293 cells and (b) MCF7 cells.
Fig. 9
Fig. 9
Intracellular localization of pDNA delivered by RPC-bPEI0.8kDa, bPEI25kDa, and bPEI0.8kDa in polyplex-transfected HEK293 cells 4 hr after transfection: (a) accumulated images and (b) center-sectional images. Nuclei, pDNA, and acidic vesicles were distinguished using Hoechst 33342 (blue), YOYO-1 (green), and LysoTracker® dye (red).
Fig. 9
Fig. 9
Intracellular localization of pDNA delivered by RPC-bPEI0.8kDa, bPEI25kDa, and bPEI0.8kDa in polyplex-transfected HEK293 cells 4 hr after transfection: (a) accumulated images and (b) center-sectional images. Nuclei, pDNA, and acidic vesicles were distinguished using Hoechst 33342 (blue), YOYO-1 (green), and LysoTracker® dye (red).
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References

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