With the aim to improve the specificity and to reduce the cytotoxicity of polyethylenimine (PEI), we have synthesized the conjugates of the branched PEI (25 kDa) with transferrin. The transferrin-PEI (TP) conjugates with five compositions were synthesized using periodate oxidation method and confirmed by FT-IR spectroscopy and gel permeation chromatography. The free amine contents of TP conjugates, which were able to condense and deliver DNA, increased as the amount of PEI increased. TP/DNA polyplexes were characterized by measuring gel electrophoresis, ethidium bromide fluorescence quenching, particle size and zeta potential of complexes. Complete complexation of the polyplexes was observed above the N/P ratio of 5 in TP/ DNA, and above 3 in PEI/DNA, respectively. The zeta potential of the complexes decreased as the amount of transferrin in TP conjugates increased. Transfection efficiency of TP conjugates was evaluated in HeLa cell and Jurkat cell systems. Among the five compositions of TP conjugates, TP-2 system mediated a higher beta-galactosidase gene expression than PEI system in Jurkat cell which was known to express elevated numbers of transferrin receptors. From the results of the cell viability based on MTT assay, TP conjugates showed lower cytotoxicity compared with the PEI system. We expect that the TP conjugate can be used efficiently as a nonviral gene delivery vector.
[Show abstract][Hide abstract] ABSTRACT: Novel cationic pentablock copolymers based on poly(2-diethylaminoethylmethacrylate) (PDEAEM) and Pluronic F127 were evaluated as non-viral gene delivery vectors from a physiochemical point of view for stability and transfection efficiency in complete growth media. A novel strategy was introduced to sterically stabilize the polyplexes of such Pluronic-based cationic polymers against aggregation with serum proteins. As cationic pentablock copolymers condense plasmid DNA into nanoplexes of 100-150 nm diameter, unmodified Pluronic added to the formulation self-assemble with the pentablock copolymers on the surface of polyplexes and shield the cationic PDEAEM chains of pentablock copolymers sterically with its long poly(ethyleneoxide) chains. These coated polyplexes formed colloidally stable dispersions of 150-250 nm diameter in serum-supplemented buffers. Cryo-TEM micrographs also showed that coating polyplexes with unmodified Pluronic reduced aggregation in serum proteins. Pentablock copolymers preserved the integrity of plasmid DNA condensed inside the polyplexes and provided efficient resistance to its degradation by nucleases. Though the total amount of DNA retained by ExGen 500 polyplexes after nuclease digestion was more than that retained by pentablock copolymers, the amount of plasmid retained in supercoiled form was not significantly different. Polyplexes coated with unmodified Pluronic provided efficient transfection in SKOV3 cells in complete growth media, comparable to that provided by ExGen 500 in terms of number of cells transfected, and one order less in terms of total transgene protein expressed. These sterically shielded polyplexes also exhibited much lower cytotoxicities than uncoated polyplexes of pentablock copolymers, and significantly lower than the cytotoxicity of ExGen 500 at relevant concentrations. This colloidally stable, versatile, multi-component gene delivery system also forms thermo-reversible injectable hydrogels like Pluronics at physiological temperatures that can be used for sustained delivery of polyplexes, and is promising for systemic applications.
[Show abstract][Hide abstract] ABSTRACT: In this study lactoferrin (Lf) was investigated as a targeting ligand for receptor-mediated gene delivery to human bronchial epithelial cells. A high number of lactoferrin receptors (LfRs) were detected on bronchial epithelial (BEAS-2B), but not on alveolar epithelial (A549) cells by fluorescence microscopy and FACS measurements, suggesting potential targeting selectivity for bronchial epithelial cells. Molecular conjugates with ratios of Lf to branched polyethylenimine 25 kDa (PEI) ranging from 4:1 to 1:40 (mol/mol) were synthesized and analyzed for complexation of plasmid DNA (pDNA), transfection efficiency, and cytotoxicity. Whereas particle size increased with the degree of Lf coupling from 45 to 225 nm, surface charge was not significantly influenced. Transfection studies on BEAS-2B cells revealed that Lf-PEI 1:20 exhibited the highest luciferase gene expression which was 5-fold higher at an N/P ratio (molar ratio of PEI nitrogen to pDNA phosphate) of 4 than PEI and could be inhibited by an excess of free Lf. With A549 cells, no significant enhancement in transfection efficiency between Lf-PEI/pDNA and PEI/pDNA complexes could be observed. Increasing the degree of Lf coupling to PEI resulted in reduced transfection efficiency in both alveolar and bronchial epithelial cells. Cell viability assays resulted in significantly lower cellular toxicity of Lf-PEI/pDNA compared with PEI/pDNA complexes. We suggest that Lf represents a potent targeting ligand for receptor-mediated gene delivery to bronchial epithelial cells and might be a promising candidate for lung gene transfer in vivo.
[Show abstract][Hide abstract] ABSTRACT: Exon-skipping oligonucleotides (ESOs) with 2'-O-methyl modifications are promising compounds for the treatment of Duchenne muscular dystrophy (DMD). However, the usefulness of these compounds is limited by their poor delivery profile to muscle tissue in vivo. We previously established that copolymers made of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) enhanced ESO transfection in skeletal muscle of mdx mice, resulting in widespread distribution of dystrophin-positive fibers, but limited dystrophin expression by Western blot. In an attempt to improve ESO delivery and dystrophin expression, a new formulation of PEG-PEI copolymer was used, along with functionalized derivatives containing either the cell-penetrating peptide TAT (trans-activator of transcription), adsorbed colloidal gold (CG), or both TAT and CG. Tibialis anterior muscles were given three intramuscular injections of various PEG-PEI-ESO polyplexes (3 days apart; 5 microg of ESO per injection) and muscles were harvested 3 weeks after the first injection. Surface modifications of PEG-PEI copolymers with TAT showed the highest level of dystrophin recovery, with a 6-fold increase in dystrophin-positive fibers compared with ESO alone and up to 30% of normal dystrophin expression by Western blot. The adsorption of CG to either PEG-PEI or TAT-PEG-PEI copolymers showed no further improvement in dystrophin expression. Our data indicate that TAT-modified PEG-PEI copolymers are effective carriers for delivery of ESOs to skeletal muscle and are promising compounds for the therapeutic treatment of DMD.
Human gene therapy 08/2008; 19(8):795-806. DOI:10.1089/hum.2007.129 · 3.76 Impact Factor
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