The role of dextran conjugation in transfection mediated by dextran-grafted polyethylenimine.
BACKGROUND
Conjugation through primary amines is one of the most commonly used methods to modify polycationic vectors for gene delivery. A better understanding of the effect of the conjugation on the mechanisms of transgene expression can help design efficient polycationic vectors.


METHODS
Dextran with a molecular weight of 1500 was grafted onto polyethylenimine (PEI) to produce various degrees of grafting in an effort to investigate how the conjugation affected the mechanisms of transgene expression. Flow cytometry was employed to quantitate the cellular entry of plasmid and the level of transgene expression, which were measured using ethidium monoazide labeled plasmid and green fluorescent protein (GFP), respectively. The buffering capacity of the grafted PEI was determined by titration, and the integrity of the DNA-polymer complexes were examined by exposure to heparin.


RESULTS
Grafting of dextran onto PEI was found to significantly diminish the cytotoxicity, buffering capacity, cellular entry, and the integrity of the DNA-polymer complexes. The reductions enlarged as the degree of grafting increased from 0 to 1.84%; however, at an optimal degree of grafting, the dextran-grafted PEI enhanced the percentages of GFP-positive cells to a level 3 times and 1.3 times of those mediated by unmodified PEI for CHO and MDA-MB-231 cells, respectively.


CONCLUSIONS
These results demonstrated that the conjugation of dextran onto the primary amines of PEI inhibited the entry of plasmid across the cell membrane, but the change in the structures of the DNA-polymer complexes was able to promote transgene expression when the degrees of conjugation fell below 0.64%.
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan. tsengwc@ch.ntust.edu.tw
Wen-Chi
WC
Tseng
Chien-Hsiang
CH
Tang
Tsuei-Yun
TY
Fang
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry; D003911:Dextrans / Q000737:chemistry*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D008970:Molecular Weight; D010957:Plasmids; D011094:Polyethyleneimine / Q000737:chemistry*; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D014407:Tumor Cells, Cultured
D003911:Dextrans; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA