Toward Synthetic Viruses: Endosomal pH-Triggered Deshielding of Targeted Polyplexes Greatly Enhances Gene Transfer in vitro and in vivo

Abstract

Nonviral vectors should undergo "virus-like" changes compatible with the steps of gene delivery. Poly(ethylene) glycol (PEG) shielding of DNA/polycation polyplexes protects from nonspecific interactions with the extracellular environment. pH-triggered removal of the shield within the endosome may be advantageous. Polycation and PEG were linked via acylhydrazides or pyridylhydrazines. The pyridylhydrazone prepared from polylysine and propionaldehyde-PEG showed the greatest acid-dependent hydrolysis; at pH 5, 37 degrees C for 10 min, 90% hydrolyzed, while at pH 7.4 the half-life was 1.5 h. Particle size and zeta potential measurements of the polyplexes showed complete deshielding within 1 h at pH 5, while at pH 7.4 the shield remained at 4 h, 37 degrees C. For gene transfection a targeting conjugate was also included in the polyplex, transferrin as ligand for K562 and Neuro2A cells and epidermal growth factor for HUH-7 and Renca-EGFR cells. Marker gene expression showed that the reversibly shielded polyplexes exhibited up to 2 log orders of magnitude higher gene expression in vitro and 1 log magnitude higher gene expression in an in vivo mouse model, compared to the stably shielded control polyplexes. Engineering of polyplexes with more dynamic domains is an encouraging new direction in nonviral vector design.

Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid

“…To improve the stability in the systemic circulation and the tumor accumulation, various groups have devised non-viral gene carriers with PEG. [13][14][15][16][17][18][19][30][31][32] Recently, cleavable PEG systems were developed, which are cleaved in response to an intracellular environment (e.g. low pH in endosome/ lysosomes, thiolytically small molecules, etc.…”

“…Most of the current cleavable PEG devices have been designed to be cleaved in response to the intracellular microenvironment, such as low pH or a specific enzyme in the endosome/lysosome and the reductive conditions in the cytoplasm. [14][15][16][17][18][19] Here, we report the development of a novel gene delivery system for cancer gene therapy, using a MEND modified with an enzymatically cleavable PEG-lipid. The cleavable PEG-lipid is composed of a PEG/matrix metalloproteinase (MMP)-substrate peptide 20 /1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ternary conjugate (PPD), which is specifically cleaved by MMP in the extracellular space in tumor tissues.…”

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

“…HuH-7 hepatocellular carcinoma cells (JCRB 0403, Tokyo, Japan) were cultured in DMEM/HAM F-12 (1:1) medium containing 10% FCS, 100 U 3 mL À1 penicillin and 100 μg 3 mL À1 streptomycin as described in our previous study. 25 All the cells were cultured in 37°C incubator with 5% CO 2 supply and humidified atmosphere. at a density of 5000 cells per well in 100 μL of cell culture medium.…”

“…Bioreversible PEGylation might be one possible solution. 25,53 PAMAM dendrimer in higher generations usually generate improved transfection yield via enhanced endosome escape capacity. 20 Alternatively, the transfection efficiency of our developed EGF-PEG-PAMAM-PEHA polyplexes could possibly be improved by selecting PAMAM dendrons with higher generations than that applied in current study, with the PEG shielding preventing enhanced cytotoxicity.…”

“…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. 27À29 The epidermal growth factor receptor (EGFR) is highly expressed in a wide variety of human tumors, such as lung, breast and hepatocellular cancers.…”