Development and Characterization of siRNA Lipoplexes: Effect of Different Lipids, In Vitro Evaluation in Cancerous Cell Lines and In Vivo Toxicity Study

Abstract

Cationic liposomes have long been used as non-viral vectors for small interfering RNA (siRNA) delivery but are associated with high toxicity, less transfection efficiency, and in vivo instability. In this investigation, we have developed siRNA targeted to RRM1 that is responsible for development of resistance to gemcitabine in cancer cells. Effect of different lipid compositions has been evaluated on formation of stable and less toxic lipoplexes. Optimized cationic lipoplex (D2CH) system was comprised of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000-DSPE). D2CH lipoplexes have shown particle size (147.5 ± 2.89 nm) and zeta potential (12.26 ± 0.54 mV) characteristics essential for their in vivo use. In vitro cytotoxicity study has shown low toxicity of developed lipoplexes as compared with lipofectamine-2000 up to N/P ratio as high as 7.5. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. In addition, developed lipoplexes also showed ~3 times less hemolytic potential as compared with DOTAP/DOPE lipoplexes at lipid concentration of 5 mg/mL. Lipoplexes also maintained particle size less than 200 nm on exposure to high electrolyte concentration and showed >70% siRNA retention in presence of serum showing siRNA protection conferred by lipoplexes. Furthermore, in vivo acute toxicity studies in mice showed that formulation was non-toxic up to a dosage of 0.75 mg of siRNA/kg as lipoplexes and 300 mg lipid/kg as blank liposomes indicating tolerability of lipoplexes at a dose much higher than required for therapeutic use. Promising results of this study warrant further investigation of developed siRNA lipoplexes for cancer treatment.

Luminescent/magnetic PLGA-based hybrid nanocomposites: a smart nanocarrier system for targeted codelivery and dual-modality imaging in cancer theranostics

“…Although commercial transfection reagents Lipofectamine 2000 with VEGF shRNA exhibited a higher VEGF downregulation effect, apparent cytotoxicity could be caused by Lipofectamine 2000 at relatively high dosages, consistent with previous reports. 66 The expression of VEGF at the protein level was further measured by ELISA as shown in Figure 11B and was similar to the results obtained by qPCR. PLGA/PPF/VEGF shRNA showed a knockdown of VEGF expression (3,765 pg/mL) in comparison with the control (4,236 pg/mL) and PLGA/PPF/SC shRNA (4,411 pg/mL) groups.…”

siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery

“…D2CH lipoplexes showed high cellular uptake of 83.83 ± 1.01% in A549 cells and 67.07 ± 0.86% in HT1299 cells which was five to six fold higher than that obtained with naked siRNA. As well, at 5 nM concentration D2CH lipoplexes showed 27.15 ± 1.85% gene expression while naked siRNA has 83.50 ± 2.5% gene expression in A549 cells (similar values were obtained in H1299 cells) -results that emphasize siRNA carriers importance (Khatri et al, 2014).…”

Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization

“…14,26,36 Data have suggested that the disaccharides interact directly with the polar groups of the lipids to prevent particle fusion during freezing and can also prevent ice formation by increasing the glass transition temperature of the solution. 34,37 Overall, freezing the LNPs at -80°C could maintain their stability with the help of trehalose or sucrose; however, deep freezers are not always available, particularly during transport.…”