lipofectamine and Inflammation

Neuroinflammation is a hallmark of acute and chronic neurodegenerative disorders. The main aim of this study was to evaluate the therapeutic efficacy of intranasal cationic nanoemulsion encapsulating an anti-TNFα siRNA, for potential anti-inflammatory therapy. TNFα siRNA nanoemulsions were prepared and characterized for particle size, surface charge, morphology, and stability and encapsulation efficiency. Qualitative and quantitative intracellular uptake studies by confocal imaging and flow cytometry, respectively, showed higher uptake compared to Lipofectamine® transfected siRNA. Nanoemulsion significantly lowered TNFα levels in LPS-stimulated cells. Upon intranasal delivery of cationic nanoemulsions almost 5 fold higher uptake was observed in the rat brain compared to non-encapsulated siRNA. More importantly, intranasal delivery of TNFα siRNA nanoemulsions in vivo markedly reduced the unregulated levels of TNFα in an LPS-induced model of neuroinflammation. These results indicate that intranasal delivery of cationic nanoemulsions encapsulating TNFα siRNA offered an efficient means of gene knockdown and this approach has significant potential in prevention of neuroinflammation.. Neuroinflammation is often seen in patients with neurodegenerative disorders and tumor necrosis factor-alpha (TNFα) plays a significant role in contributing to neuronal dysfunction. As a result, inhibition of TNFα may alleviate disease severity. In this article, the authors investigated using a cationic nanoemulsion system carrying TNFα siRNA intra-nasally to protect against neuroinflammation. This new method may provide a future approach in this clinical setting.

Topics: Administration, Intranasal; Animals; Anti-Inflammatory Agents; Brain; Emulsions; Flow Cytometry; Gene Silencing; Humans; Inflammation; Lipids; Macrophages; Nanoparticles; Neurons; Particle Size; Rats; RNA, Small Interfering; Tumor Necrosis Factor-alpha

The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous cells. In conclusion, non-specific effects should not be overlooked in the application of RNAi for mesenchymal cell transfection and may need to be overcome for its effective therapeutic application.

Topics: Aggrecans; Cartilage, Articular; Cell Cycle; Cell Survival; Cells, Cultured; Chitosan; Chondrocytes; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type II; Core Binding Factor Alpha 1 Subunit; Cyclin-Dependent Kinase Inhibitor p21; Cyclooxygenase 2; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Humans; Hyaluronic Acid; Inflammation; Intervertebral Disc; Knee Joint; Lipids; Lumbar Vertebrae; Mesenchymal Stem Cells; Osteopontin; Polyethyleneimine; Proliferating Cell Nuclear Antigen; RNA Interference; RNA, Small Interfering; Transfection

Interleukin (IL)-10 is an anti-inflammatory cytokine that has great potential for use in the treatment of inflammatory and immune illnesses. In this study, gene transfer was used to induce IL-10 transgene expression in murine lungs for treatment of endotoxin-induced lung inflammation. Gene transfer was performed with a cytomegalovirus (CMV)-IL-10 plasmid with the aid of the liposomal agents LipofectAMINE and N-[1-(2,3-dioleoyl)propyl]-N,N, N-trimethylammonium methylsulfate (DOTAP). Administration of the endotoxin caused a marked increase in lung inflammation as indicated by increased tumor necrosis factor (TNF)-alpha release and neutrophil count. Pretreatment of the mice with IL-10 plasmid with and without LipofectAMINE had no inhibitory effect on lung inflammation and IL-10 transgene expression. LipofectAMINE by itself induced lung inflammation, an effect that was not observed with DOTAP. IL-10 plasmid when codelivered with DOTAP expressed biologically active IL-10 protein and caused a reduction in endotoxin-induced inflammation. Transgene expression was observed as early as 3 h after administration, peaked at 12 h, and declined thereafter. We conclude that IL-10 gene transfer is a feasible approach for the treatment of lung inflammation.

Topics: Animals; Cation Exchange Resins; Cytomegalovirus; Endotoxins; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Inflammation; Interleukin-10; Lipids; Lipopolysaccharides; Liposomes; Lung; Male; Mice; Mice, Inbred BALB C; Plasmids; Quaternary Ammonium Compounds