Enhancement of gene delivery by an analogue of α-MSH in a receptor-independent fashion

Abstract

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.

A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene

“…Although the addition of the TfRsvFv targeting moiety to the PEG terminus increased the transfection efficiency, this increase still did not bring the transfection level up to that observed with the complex lacking PEG (TsLP), but only made it comparable to the level of unmodified lipoplex (LP). This finding is similar to other reports 5,7 of decreased transfection efficiency after Tumor targeting by TfRscFv-PEG-lipoplex W Yu et al PEGylation, and increased efficiency after ligand conjugation to the PEG terminus using standard precoating methods. These results suggest that at least in vitro, the inclusion of PEG in the complex decreases gene delivery.…”

“…12,26 It has also been proposed that PEG may hinder the electrostatic interaction between negatively charged DNA and positively charged lipids during the forming of the lipoplex, whose structure is critical for efficient gene delivery. 27 Thus, to avoid interference of PEG during the lipoplex formation, a postcoating strategy was utilized instead of a widely used precoating preparation strategy 5,7 ( Figure 1). Using a standard precoating scheme the PEGylated lipoplex (PLP) has been reported to have a gene delivery efficiency which is 100-times 5 to 10 000-times 7 lower than that of the lipoplex without PEGylation (LP).…”

“…27 However, to date, most targeting sterically stabilized gene delivery vehicles have been studied in vitro. [3][4][5][6][7][8][9] As the ultimate goal of any potential gene therapy is delivery in vivo, a comparison study of TsPLP both in vitro and in vivo was undertaken to elucidate the influence of the PEGylation on gene delivery efficiency. PLP showed the lowest in vitro transfection activity (Figure 2), and this is likely due to the PEG molecule preventing association of the lipoplex with the target cell 2,12 and/or due to PEG inhibition of fusion between lipid and endosomal membrane, which is necessary for the intracellular release of exogenously delivered DNA.…”

“…2 The application of this modification (PEGylation) to nonviral gene delivery systems has drawn considerable interest. [3][4][5][6][7][8][9] Benefits of PEGylation have been described and include improvement of systemic circulation of lipoplexes or polyplexes; 1,10 improvement of the solubility of polyplexes thereby allowing the preparation of higher therapeutic concentrations without subsequent aggregation; 10,11 and reduction of in vivo toxicity of the polyplex. 10 However, disadvantages of PEGylation have also been observed.…”

Relevance of bi-functionalized polyelectrolyte multilayers for cell transfection

“…The binding of the hormone or its analogue to specific cell receptors activates the production of melanin as the final product of the respective signal transduction. Due to its ability to bind to and internalize with specific cell surface receptors, this peptide was previously considered as a candidate for melanoma cells targeting and melanoma transfection by receptor-mediated endocytosis [16,17]. It was also shown to enhance gene delivery in solution in a receptor-independent fashion due to its membrane-destabilizing properties [16].…”