Promote Gene Transfection Research Articles

β-Cyclodextrin-Based Polycationic Amphiphilic “Click” Clusters: Effect of Structural Modifications in Their DNA Complexing and Delivery Properties

Monodisperse facial amphiphiles consisting of a β-cyclodextrin (βCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hydrophobic chains at the secondary oxygens have been prepared by implementing two very robust "click" methodologies, namely cuprous cation-catalyzed azide-alkyne cycloaddition (CuAAC) and thiourea-forming reaction. Most interestingly, the use of solid-supported Cu(I) catalysts was found to be very well suited for multiple CuAAC while facilitating purification of the C(7)-symmetric macromolecular triazole adducts. The strategy is compatible with molecular diversity-oriented approaches, which has been exploited to generate a small library of click polycationic amphiphilic CDs (paCDs) for assessing the influence of structural modifications in the ability to complex, compact, and protect pDNA and the efficiency of the resulting paCD:pDNA nanocomplexes (CDplexes) to deliver DNA into cells and promote transfection. The results indicate that fine-tuning the hydrophilic/hydrophobic balance is critical to achieve optimal self-assembling properties and stability of the resulting CDplexes in saline- and serum-containing media. Triazole-type paCDs were, in general, less efficient in promoting gene transfection than thiourea-type derivatives. Nevertheless, the current body of results support that the "dual click" approach implying sequential CuAAC and thiourea-forming reactions represents a versatile strategy to optimize the gene delivery capabilities of cyclodextrin-based facial amphiphiles.

Nanoparticles for Gene Delivery: A Brief Patent Review

Gene therapy, as therapeutic treatment to genetic or acquired diseases, is attracting much interest in the research community, leading to noteworthy developments over the past two decades. Nonviral vectors have recently received an increasing attention in order to overcome the safety problems of viral counterpart. Nanoparticles with their special characteristics such as small particle size, large surface area and the capability of changing their surface properties have numerous advantages compared with other gene delivery systems. This article reviews the advances of nanoparticles in patents for gene delivery and emphasizes methods to promote gene transfection efficiency.

Nonviral Vectors with a Biosurfactant MEL-A Promote Gene Transfection into Solid Tumors in the Mouse Abdominal Cavity

Recently, we showed that a biosurfactatnt 4-O-[(4',6'-di-O-acethyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl] meso-erythritol A (MEL-A) greatly increased the efficiency of gene transfection mediated by cationic liposomes in vitro. We then studied whether the high transfection efficiency of these liposomes is maintained in vivo for tumor cells in the mouse abdominal cavity. When a complex of the liposomes and plasmid DNA was injected intraperitoneally into C57BL/6J mice bearing B16/BL6 tumors, we found that the biosurfactant significantly increased liposome-mediated gene transfection to the mouse tumor cells. The transfection efficiency of the plasmids into the solid tumors by the cationic liposomes of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol) with MEL-A increased by about 100-fold compared with that by the cationic liposomes of DC-Chol (commercially available) without MEL-A. The results suggest that nonviral vectors with MEL-A are very useful for gene transfection in vivo.

Preparation of Cationic Gold Nanoparticles for Gene Delivery

Cationic gold nanoparticles modified with 2-aminoethanethiol, 8-amino-1-octanethiol, and 11-amino-1- undecanethiol were prepared by NaBH4 reduction of HAuCl4 in the presence of thiols in water or a water/ethanol mixture solvent. The formation of gold nanoparticles depended on the kind of solvent, and the concentration of NaBH4, HAuCl4, and thiols. A low concentration of HAuCl4 and a high concentration of NaBH4 facilitated the formation of gold nanoparticles. A high concentration of HAuCl4 solution resulted in aggregation and a low concentration of NaBH4 did not induce the formation of gold nanoparticles. The size of the gold nanoparticles increased with an increase of the concentration of the HAuCl4 solution, while it decreased with an increase of the concentration of the NaBH4 solution. When thiols were present, the HAuCl4/thiol ratio must be in the range of 1:1 to 1:2 for the formation of gold nanoparticles in both the water and water/ethanol mixture solvents. Modification with thiol molecules changed surface charge of the gold nanoparticles from negative to positive, and stabilized the gold nanoparticles. The cationic gold nanoparticles promoted gene transfection. A high ratio of gold nanoparticles to DNA increased transfection efficiency. However, the length of the alkyl groups had no evident effect.

The self-assembly of biodegradable cationic polymer micelles as vectors for gene transfection

Cationic micelles self-assembled from a biodegradable amphiphilic copolymer, poly{( N-methyldietheneamine sebacate)- co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate} (P(MDS- co-CES)) have recently been reported for efficient gene delivery and co-delivery of drug and nucleic acid. In this study, poly(ethylene glycol) (PEG) of various molecular weights ( M n=550, 1100 and 2000) was conjugated to P(MDS- co-CES) having different cholesterol grafting degrees to improve the stability of micelle/DNA complexes in the blood for systemic in vivo gene delivery. DNA binding ability, gene transfection efficiency and cytotoxicity of P(MDS- co-CES), PMDS, PEGylated PMDS and PEGylated P(MDS- co-CES) micelles were studied and compared. As with P(MDS- co-CES), PEG–P(MDS- co-CES) polymers could also self-assemble into stable micelles of small size. However, PMDS and PEG–PMDS without cholesterol could not form stable micelles but formed large particles. PEGylation of polymers significantly decreased their gene transfection efficiency in HEK293, HepG2, HeLa, MDA-MB-231 and 4T1 cells. However, increasing N/P ratio promoted gene transfection. An increased cholesterol grafting degree led to greater gene expression level possibly because of the more stable core–shell structure of the micelles. PEG550–P(MDS- co-CES) micelles induced high gene transfection level, comparable to that provided by P(MDS- co-CES) micelles. PEGylated polymers were much less cytotoxic than P(MDS- co-CES). PEGylated P(MDS- co-CES) micelles may provide a promising non-viral vector for systemic in vivo gene delivery.

Efficient gene transfection using chitosan ? alginate core-shell nanoparticles

Reverse microemulsion was used as a template to fabricate chitosan-alginate core-shell nanoparticles encapsulated with enhanced green fluorescent protein (EGFP)-encoded plasmids. The average size of DNA-entrapped nanoparticles measured by dynamic light scattering was increased proportionally, with the N/P ratios ranging from 5 to 20. These alginate-coated chitosan nanoparticles endocytosed by NIH 3T3 cells trigged swelling of transport vesicles which render gene escape before entering digestive endolysosomal compartment and concomitantly promote gene transfection rate. Results showed that DNA-encapsulated chitosan-alginate nanoparticles with average size of 64nm (N/P ratio of 5) could achieve the level of gene expression comparable with the one obtained by using polyethyleneimine-DNA complexes.

High-efficiency gene transfection of macrophages by lipoplexes

Macrophage transfection studies are crucial for understanding gene regulation and expression. However, gene transfection in macrophages is difficult. We have shown here that macrophages are more resistant to gene transfection compared with other cell types. To further develop an efficient gene delivery system for macrophages, we evaluated various liposomal and non-liposomal agents including LipofectAMINE®, Lipofectin®, DOTAP, DEAE-dextran, and the DNA condensing agent protamine sulfate for their ability to promote gene transfection. CMV-luciferase was used as a reporter plasmid. Macrophage transfection was maximal at the DNA:LipofectAMINE:protamine ratio of 1:12:1 μg/ml. The LipofectAMINE formulation showed a 10–12-fold increase in transfection efficiency over DOTAP and a 4–5-fold increase over Lipofectin. This transfection method showed minimal toxicity at the concentrations tested and was at least 20–25-fold superior to the most frequently used DEAE-dextran method for macrophage transfection.