Polymeric Gene Carriers Research Articles

An Angiogenic, Endothelial-Cell-Targeted Polymeric Gene Carrier

An Angiogenic, Endothelial-Cell-Targeted Polymeric Gene Carrier

An Angiogenic, Endothelial-Cell-Targeted Polymeric Gene Carrier

Targeting is one of the primary considerations in designing a specific and efficient gene delivery system. Here, an angiogenic endothelial cell-targeted polymeric gene delivery carrier was developed by conjugating an alpha(v)beta3/alpha(v)beta5 integrin-binding RGD peptide, ACDCRGDCFC, into the cationic polymer polyethyleneimine (PEI) via a hydrophilic poly(ethylene glycol) (PEG) spacer. The incorporation of PEG into PEI improved the poor physicochemical properties of PEI-DNA complexes. At a neutral charge ratio, DNA complexes with PEI were polydisperse and substantially aggregated, whereas DNA complexes with PEI-g-1PEG-RGD were homogeneous with 100-200 nm effective diameter. Their surface charge was also significantly reduced due to the charge shielding effect of PEG. However, the extensive grafting of PEI with PEG was shown to inhibit the DNA condensation process, significantly decreasing transfection efficiency. In in vitro transfection experiments with angiogenic endothelial cells, PEI-g-1PEG-RGD showed an approximately fivefold increase in transfection efficiency over PEI, due to an integrin-mediated internalization pathway. PEI-g-1PEG-RGD also exhibited high specificity to angiogenic endothelial cells compared with normal endothelial cells, which was confirmed by in vitro transfection experiments with non-targeting PEI-g-1PEG-RAE in angiostatic endothelial cells.

A new synthesis of galactose-poly(ethylene glycol)-polyethylenimine for gene delivery to hepatocytes

A synthesis method of conjugating polyethylenimine (PEI) derivatives with terminally galactose-grafted poly(ethylene glycol) (PEG) was developed by using a bifunctional PEG derivative containing both an α-vinyl sulfone (VS) and an ω- N-hydroxysuccinimidyl (NHS) ester groups (VS-PEG-NHS). VS-PEG-NHS is commonly used as a crosslinker to modify proteins with ligands by first coupling amine groups to the NHS ester, followed by coupling sulfhydryl groups to the VS ester, because the reaction of VS groups with amine groups of proteins is suppressed below pH 8. However, the 1H-NMR determination of the conjugated products of branched PEI ( M w=25 kDa) with VS-PEG-NHS at pH 6.0–8.0 indicated that the VS groups were completely bound to the amine groups of PEI as well as the NHS groups. At pH 7.0, all VS groups reacted with the primary, secondary, or tertiary amine groups of PEI in 2 h. Such different reaction behaviors would be due to a higher density of amine groups of PEI as compared with those of proteins. In contrast, the reactions with a small molecular monoamine, such as p-aminophenyl β- d-galactopyranoside, showed that the NHS groups selectively coupled with the amine groups, and the VS groups remained completely intact. The NHS groups of VS-PEG-NHS were selectively conjugated to amine groups of p-aminophenyl β- d-galactopyranoside (VS-PEG-Gal). Then, the VS groups of Gal-PEG unit were completely conjugated with the primary, secondary, or tertiary amine groups of PEI. Thus, the use of only two reaction steps could conveniently carry out the conjugation of terminally galactose-grafted PEG to 1 and 5 mol.% of amine functions in PEI. The gel retardation assay of the complexes between Gal-PEG-PEI and plasmid DNA indicated that these polymeric gene carriers possess the potent ability to condense plasmid DNA electrostatically as well as PEI. The transfection efficiency with 1% Gal-PEG-PEI in human hepatocyte-derived cell lines (HepG2), a model of parenchymal cells in liver (hepatocytes), was superior to that of PEI at their corresponding optimal ratios of polymer to plasmid DNA. In HepG2 cells, luciferase activity with 1% Gal-PEG-PEI at an N/P ratio of 20 was 2.1-fold greater than that of PEI at an N/P ratio of 5. In mouse fibroblasts (NIH3T3) that have no ASGP receptors, the transfection efficiency with 1% Gal-PEG-PEI drastically decreased to 1/40 of that with PEI. These data indicate that a new synthesis method can produce polyethylenimine derivatives with terminally galactose-grafted poly(ethylene glycol) for specific gene targeting to the liver.

Influential factors on temperature-controlled gene expression using thermoresponsive polymeric gene carriers

Thermoresponsive random copolymers, poly[N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)], were synthesized in three compositions, and their in vitro gene transfection efficiency to cultured cells at different incubation conditions was measured using β-galactosidase as a marker gene. These three copolymers increased gene expression when the cell incubation temperature was lowered below 37°C. This increase in transfection efficiency when temperature was lowered is considered to be due to enhanced DNA release from the polymer-DNA complex in cells. The influence of complex preparation conditions (charge ratio and temperature) and cell incubation conditions (incubation period with the complex, cooling temperature, timing, and period) was analyzed to elucidate the mechanism of this gene expression control as well as to maximize the increase in gene expression on lowering of the cell incubation temperature. One copolymer showed a nine-fold increase in gene expression on lowering of the temperature to 20°C for 3 h. In analyses of temperature effects in the complex formation procedure on the gene expression, tight complex formation was considered to contribute to efficient cell uptake and/or evasion from enzymatic DNA degradation.

Water-soluble lipopolymer for gene delivery.

The use of biocompatible polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity, and mutagenesis. Nontoxic water-soluble lipopolymer (WSLP), poly(ethylenimine)-co-[N-(2-aminoethyl) ethyleneimin]-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine) was synthesized using branched poly(ethylenimine) (PEI, mw 1800) and cholesteryl chloroformate. Following synthesis and purification, the structure and molecular weight of WSLP were confirmed by (1)H NMR and MADI-TOF mass spectrometry, respectively. The percentage of cholesterol conjugated to PEI was about 47%, and the average molecular weight of WSLP was approximately 2000 Da. WSLP/pDNA complexes were prepared at different N/P (nitrogen atoms of WSLP/phosphate of plasmid DNA) ratios and characterized in terms of particle size, zeta potential, osmolarity, surface morphology, and cytotoxicity. WSLP condensed plasmid DNA when N/P ratio reached 2.5/1 and no free DNA was detected at N/P ratio of 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was in the range of 25.9 to 148.5 nm and was dependent on N/P ratios. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of approximately 50 nm in diameter. WSLP/pDNA complexes or WSLP itself were nontoxic to CT-26 colon adenocarcinoma and 293 T human embryonic kidney transformed cells when formulated at the N/P ratio of 10/1 and below as determined by MTT assay. In contrast, PEI25000/pDNA complexes were toxic to these cells. Erythrocytes aggregated when incubated with PEI25000/pCMV-Luc complexes at high DNA concentrations, but there was little aggregation with WSLP/pCMV-Luc complexes. WSLP/pCMV-Luc complexes demonstrated higher transfection efficiency in both CT-26 and 293 T cells compared to PEI25000- or PEI1800-based formulations. WSLP/pCMV-Luc complexes are nontoxic and showed enhanced in vitro transfection. Thus, WSLP will be a suitable carrier for in vivo gene delivery.

Development of Biomaterials for Gene Therapy

Novel biocompatible polymeric gene carriers have been examined for their potential in treating various genetic and acquired diseases. The use of polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity, and mutagenesis. However, effective polymer-based gene therapy requires the control of cellular access and uptake, intracellular trafficking, and nuclear retention of plasmid DNA. Inefficient endosomal release, cytoplasmic transport, and nuclear entry of plasmids are currently limiting factors in the use of polymers for effective plasmid-based gene therapy. Therefore, several different polymeric gene carriers have been designed recently in an attempt to overcome these problems. This review explores the conceptual and experimental aspects of polymer-based gene delivery and presents an overview on the recent use of polymers to enhance the effectiveness of plasmid-based systems. Despite their current limitations, polymeric carriers have significant potential as commercially viable gene medicines.

Gene expression control by temperature with thermo-responsive polymeric gene carriers

A thermo-responsive copolymer, poly( N-isopropylacrylamide (IPAAm)- co-2-(dimethylamino)ethyl methacrylate (DMAEMA)- co-butylmethacrylate (BMA)), was synthesized and its in vitro gene transfection efficiency at different incubation temperatures was evaluated. A copolymer containing 8 mol% DMAEMA and 11 mol% BMA (P(IP-8DA-11BM)) had a lower critical solution temperature (LCST) at 21°C, therefore the copolymer was insoluble above 21°C and soluble below 21°C. The LCST of P(IP-8DA-11BM) solution was not affected by the presence of salmon DNA. This copolymer was complexed with plasmid DNA, and the stability of the complex was analyzed by gel electrophoresis. DNA was completely retained in the complex, which was observed in the gel loading slot at 37°C. At 20°C, DNA was found to be partially dissociated from the complex by the appearance of the same band as DNA in the control experiment. These results clearly show that complex formation/dissociation was modulated by temperature alteration. The transfection efficiency of polymer–plasmid complexes was evaluated in COS-1 cells using pCMV-lacZ plasmid, encoding for β-galactosidase as a reporter gene. The transfection efficiency of PDMAEMA homopolymer incubated at 37°C for 48 h was greater than that incubated at 20°C for 3 h and 37°C for 45 h. In contrast, the transfection efficiency of P(IP-8DA-11BM) incubated at 20°C for 3 h and 37°C for 45 h was much higher than that incubated at 37°C for 48 h. Such an increased transfection efficiency on lowering the temperature is considered to be due to appropriate formation/dissociation control of P(IP-8DA-11BM)–DNA complexes.

Transfection efficiency increases by incorporating hydrophobic monomer units into polymeric gene carriers

The water soluble terpolymer, poly( N-isopropylacrylamide (IPAAm)- co-2-(dimethylamino)ethyl methacrylate (DMAEMA)- co-butylmethacrylate (BMA)) was synthesized, and its efficiency in in vitro gene transfection was evaluated. Copolymers with different compositions were synthesized by radical polymerization. For a series of copolymers containing 60 mol% of DMAEMA, the plasmid bands were retained within the gel loading slot, independent of polymer/plasmid weight ratios or BMA monomer content. In contrast, for a series of copolymers containing 20 mol% DMAEMA, plasmid bands of complexes were retarded with increasing weight ratios. For the copolymer with 10 mol% BMA content, the plasmid was completely retained within the gel loading slot. The transfection efficiency of polymer/plasmid complexes was evaluated in COS-1 cells using a pCMV-lacZ plasmid, encoding for β-galactosidase as a reporter gene. Transfection efficiency of a series of copolymers containing 20 mol% of DMAEMA varied with BMA content. The transfection efficiency of the copolymers with 0, 2, and 5 mol% of BMA was low. The transfection efficiency of the copolymers with 10 mol% of BMA was about 2-fold higher than that of the PDMAEMA control homopolymer. The transfected cells were observed at a very wide range of polymer/plasmid weight ratios. The transfection efficiency of all copolymers containing 60 mol% of DMAEMA was lower than that of the PDMAEMA homopolymer.

Biodegradable polyester, poly[alpha-(4-aminobutyl)-L-glycolic acid], as a non-toxic gene carrier.

The aim of this study was to develop a non-toxic polymeric gene carrier. For this purpose, biodegradable cationic polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) was synthesized. PAGA was designed to have ester linkage because polyesters usually show biodegradability. Degradation of PAGA in an aqueous solution was followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PAGA/DNA complexes were characterized by gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS). The transfection was measured by using the beta-galactosidase reporter gene. PAGA was degraded in aqueous solution very quickly and the final degradation product was a monomer (L-oxylysine). Formation of self-assembling biodegradable complexes between PAGA and DNA at a charge ratio 1:1 (+/-) was confirmed by gel band shift assay and AFM. In these studies, controlled release of DNA from the complexes could be seen. The complexes showed about 2-fold higher transfection efficiency than DNA complexes of poly-L-lysine (PLL), a structural analogue of PAGA, which is the most commonly used poly-cation for gene delivery. The polymer did not show cytotoxicity, possibly because of its degradability and the biocompatibility of the monomer. The use of the biodegradable poly-cation, PAGA, as a DNA condensing agent will be useful in safe gene delivery.

Lactose-poly(ethylene glycol)-grafted poly-L-lysine as hepatoma cell-tapgeted gene carrier.

To investigate the delivery of DNA into cells, lactose-poly(ethylene glycol)-grafted poly-L-lysine (Lac-PEG-PLL) polymers were synthesized as polymeric gene carriers. The new synthetic carriers, varying the substitution ratio of lactose-poly(ethylene glycol) (lactose-PEG), were characterized by NMR spectroscopy and size-exclusion chromatography. Electrophoretic mobility assay confirmed that the new gene carrier makes a complex with plasmid DNA. The attached poly(ethylene glycol) gives better solubility properties to gene/carrier complex. Transfection experiments showed that Lac-PEG-PLL efficiently delivers DNA to a hepatoma cell line in vitro; the best efficiency was achieved at a 1:3 weight ratio of DNA to carrier. As the lactose-PEG substitution content increased up to 30%, the transfection efficiency increased, which demonstrates that the lactose serves as a targeting moiety. No considerable cytotoxicity was observed due to Lac-PEG-PLL or its complex with DNA within the concentration range for this experiment. The use of chloroquine increased transfection efficiency that indicates the involvement of hydrolytic degradation of the system in lysosome. It is likely that plasmid DNA/Lac-PEG-PLL complexes enter the cells through a receptor-mediated endocytosis mechanism. These results show that Lac-PEG-PLL can form a complex with plasmid DNA and serve as an efficient gene delivery carrier with lower cytotoxicity compared to that of poly-L-lysine. Therefore, it is expected that our Lac-PEG-PLL carrier can be used as an in vivo gene delivery vector.

Polyethylene glycol-grafted poly- l-lysine as polymeric gene carrier

A new series of gene carriers, polyethylene glycol (PEG)-grafted poly- l-lysine (PLL, mol. wt.=25 000) with three different PEG-grafted ratios (5, 10 and 25 mole%, which means 5, 10 and 25% of ϵ-amino group of PLL was modified by PEG), was synthesized. These new gene carriers, named comb-shaped PEG- g-PLL copolymer, showed a 5- to 30-fold increase in transfection efficiency compared to PLL alone on a human carcinoma cell line. It is likely that Hep G2 cells were transfected by plasmid DNA/PEG- g-PLL complexes through an endocytosis mechanism due to the fact that chloroquine increased transfection efficiency. Although Lipofectin™, a cationic lipid formulation, showed slightly higher transfection efficiency than PEG- g-PLL in Hep G2 cells, our designed PEG- g-PLL demonstrated lower cytotoxicity, early gene expression and maintenance of gene expression for up to 96 h.