Novel Shielded Transferrin−Polyethylene Glycol−Polyethylenimine/DNA Complexes for Systemic Tumor-Targeted Gene Transfer

Abstract

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.

PEGylated quaternized copolymer/DNA complexes for gene delivery

“…The higher transfection efficacy exhibited by the large aggregated particles could result from the conjunction of several features: (i) they sediment onto the cell surface more rapidly than the small complexes thereby intensifying the contact with the cells which stimulates cellular internalization, (ii) since they contain a large proportion of free cationic polymers in addition to those complexed with DNA, they destabilize the membrane favoring their entry into cells, and (iii) their endosomolytic activity is far higher than that of the small particles (Zaric et al, 2004). The reduced transfection activity and lower cytotoxicity of PEGylated polyplexes relative to unshielded polyplexes could be due to the formation of complexes with improved colloidal stability and reduced size (as demonstrated in Table 2) and to a reduced cellular interaction and internalization (Kursa et al, 2003;Merdan et al, 2003;Deshpande et al, 2004). Hence, the cellular uptake of unshielded and PEGylated polyplexes was evaluated at different time intervals using flow cytometry.…”

Epidermal Growth Factor–PEG Functionalized PAMAM-Pentaethylenehexamine Dendron for Targeted Gene Delivery Produced by Click Chemistry

“…22 PEG modification minimizes these disadvantages and elongates the circulation retention time of polyplexes. 23,24 On the other hand, PEG shielding suppresses transfection efficiency by reducing intracellular uptake and endosome escape of polyplexes. 25,26 Targeting ligands such as serum protein transferrin, antibodies, or growth factors have been incorporated into polyplexes to obtain higher targeted cell specificity and recovery the depressed transfection efficacy by PEG shielding.…”

Delivery of CD44 shRNA/Nanoparticles within Cancer Cells

“…The Tf-PEG-PEI was prepared as described previously (41). Briefly, transferrin was linked with N-hydroxysuccinimide/ PEG/maleimide and then allowed to react with a mercaptopropionate-modified branched PEI to form Tf-PEG-PEI (molecular mass of ϳ400 kDa).…”

“…These unfavorable effects can be minimized by "shielding" of the positive surface charge of the vectors with polyethylene glycol (PEG). PEGylation of PEI polyplexes can prevent the systemic degradation of the plasmid DNA and reduce the toxicity of polyplexes (41). To increase the transfection efficiency of the shielded particles (plasmid DNA/PEG-PEI), different targeting ligands, such as peptide, growth factors and proteins, or antibodies, have been incorporated into the vectors (42).…”