Document Detail

Kinetic analysis of the initial steps involved in lipoplex--cell interactions: effect of various factors that influence transfection activity.
MedLine Citation:
PMID:  11342154     Owner:  NLM     Status:  MEDLINE    
We investigated the mode of interaction of lipoplexes (DOTAP:DOPE/DNA) with HeLa cells, focusing on the analysis of the initial steps involved in the process of gene delivery. We evaluated the effect of different factors, namely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin into the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were performed upon cell treatment with inhibitors of endocytosis. Our results indicate that a decrease of the net charge of the complexes (upon addition of DNA) generally leads to a decrease in the extent of binding, cell association and fusion, except for the neutral complexes. Association of transferrin to the lipoplexes resulted in a significant enhancement of the interaction processes referred to above, which correlates well with the promotion of transfection observed under the same conditions. Besides triggering internalization of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was reduced upon their incubation with cells in the presence of serum, suggesting that serum components limit the transferrin fusogenic properties. Results were analyzed by using a theoretical model which allowed to estimate the kinetic parameters involved in lipoplex--cell interactions. The deduced fusion and endocytosis rate constants are discussed and compared with those obtained for other biological systems. From the kinetic studies we found a twofold enhancement of the fusion rate constant (f) for the ternary lipoplexes. We also concluded that HeLa cells yield a relatively low rate of endocytosis. Overall, our results estimate the relative contribution of fusion of lipoplexes with the plasma membrane, endocytosis and fusion with the endosomal membrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies.
M T da Cruz; S Simões; P P Pires; S Nir; M C de Lima
Related Documents :
16973744 - The herpesvirus glycoproteins b and h.l are sequentially recruited to the receptor-boun...
18979254 - Quantitative assays for cell fusion.
2999444 - Proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent diffe...
7662964 - Classification of fanconi anemia patients by complementation analysis: evidence for a f...
23878824 - Saliva and wound healing.
22840454 - Inhibition of pim-1 attenuates the proliferation and migration in nasopharyngeal carcin...
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  1510     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  2001 Feb 
Date Detail:
Created Date:  2001-05-08     Completed Date:  2001-05-24     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  136-51     Citation Subset:  IM    
Department of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, Portugal.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Cell Membrane / chemistry
DNA / chemistry*
Fatty Acids, Monounsaturated / chemistry
Gene Therapy
Gene Transfer Techniques*
Glycerophospholipids / chemistry
Hela Cells / chemistry*
Intracellular Membranes / chemistry
Liposomes / chemistry*
Membrane Fusion
Quaternary Ammonium Compounds / chemistry
Transferrin / chemistry
Reg. No./Substance:
0/1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; 0/Fatty Acids, Monounsaturated; 0/Glycerophospholipids; 0/Liposomes; 0/Phosphatidylethanolamines; 0/Quaternary Ammonium Compounds; 11096-37-0/Transferrin; 113669-21-9/1,2-dioleoyloxy-3-(trimethylammonium)propane; 9007-49-2/DNA

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Previous Document:  A photophysical study of the polyene antibiotic filipin. Self-aggregation and filipin--ergosterol in...
Next Document:  Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids:...