Peptide-mediated gene transfer. Effect of the size of complexes with DNA on the efficiency of transfection and receptor-specific binding with cellular target

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Item Type:	Article
Title:	Peptide-mediated gene transfer. Effect of the size of complexes with DNA on the efficiency of transfection and receptor-specific binding with cellular target
Creators Name:	Haberland, A., Zaitsev, S., Dalluege, R., Schneider, M., Zastrow, H., Sukhorukov, G.B., Vorob'ev, V.I. and Boettger, M.
Abstract:	The transfection activity of DNA complexes with cationic polypeptides including the RGD sequence capable of binding specifically to the integrin receptor has been studied. This approach allowed using receptor-mediated endocytosis for penetration of transfection complexes inside cells. An attempt was made to establish a correlation between the activity of these complexes and the size of their particles. The work used polylysine-based linear and cyclic amino acid sequences (16 consecutively arranged lysine residues, K 16), containing integrin-specific sites described earlier by other authors, as well as structurally similar, newly-synthesised sequences, whose interaction with integrin receptor has not been established. These sequences were used as controls. Complexes formed by these carriers with plasmid DNA were examined with respect to their solubility in physiological solutions, whereas sizes of the complexes were characterised by the method of dynamic light scattering and with the aid of atomic force microscopy. Experiments on transfection were performed on cell lines ECV304 and HeLa. Plasmid pCMV Luc, which carries the expressed liciferase gene, was used as vector DNA. The transfection complexes obtained were added to the cell cultures, and efficiency of penetration of the vector DNA into cells was studied using the intensity of expression of the luciferase gene as a criterion. Values of the transfection activity of DNA-peptide complexes were within the limits of 10 6-10 8 RLU (relative light units) per mg protein and followed the order: K 16-GGCKYPKYPCA > K 16-GGCRADMFGCA > K 16-GGARGDMFGAA > K 16-GGCRGDMFGCA > K 16-GGCRGEMFGCA. This sequence in the distribution of activities casts doubts on the fact of the integrin-dependent caption of transfection complexes in the pathway of receptor-mediated endocytosis. At the same time, the data obtained indicate a certain correlation between the transfection activity and size of transfection complexes. The particle sizes were within the limits of > 1500 - 500 nm for the above sequence of DNA-peptide complexes. A reduction of sizes of the complexes led to a decrease of the transfection efficiency. The maximal activity was revealed in the complexes formed by DNA with polypeptides, which had no affinity to the integrin receptor, but showed the highest level of aggregation. Thus, the crucial factor for the transfection activity turned out to be the final size of aggregates formed by the carrier with DNA, rather than the receptor-binding properties of the carrier. The data obtained do not rule out a possibility of receptor-mediated transfection. However, they show that for analysis of the transfection activity of complexes of DNA with a receptor-specific or unspecific carrier, it is complexes with identical or similar physico-chemical properties, which should be compared.
Source:	Biologicheskie Membrany
ISSN:	0233-4755
Publisher:	Mezhdunarodnaya Kniga
Volume:	20
Number:	4
Page Range:	278-287
Date:	2003

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