Optimal combination of cationic lipid and phospholipid in cationic liposomes for gene knockdown in breast cancer cells and mouse lung using siRNA lipoplexes

Abstract

Formulation of cationic liposomes is a key factor that determine the gene knockdown efficiency by cationic liposomes/siRNA complexes (siRNA lipoplexes). Here, to determine the optimal combination of cationic lipid and phospholipid in cationic liposomes for in vitro and in vivo gene knockdown using siRNA lipoplexes, three types of cationic lipid were used, namely 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB) and 11-[(1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino]- N , N , N -trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12). Thereafter, 30 types of cationic liposome composed of each cationic lipid with phosphatidylcholine or phosphatidylethanolamine containing saturated or unsaturated dialkyl chains (C14, C16, or C18) were prepared. The inclusion of phosphatidylethanolamine containing unsaturated and long dialkyl chains with DOTAP- or DDAB-based cationic liposomes induced strong luciferase gene knockdown in human breast cancer MCF-7-Luc cells stably expressing luciferase gene. Furthermore, the inclusion of phosphatidylcholine or phosphatidylethanolamine containing saturated and short dialkyl chains or unsaturated and long dialkyl chains into TC-1-12-based cationic liposomes resulted in high gene knockdown efficacy. When cationic liposomes composed of DDAB/1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE), TC-1-12/DOPE and TC-1-12/1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine were used, significant gene knockdown occurred in the lungs of mice following systemic injection of siRNA lipoplexes. Overall, the present findings indicated that optimal phospholipids in cationic liposome for in vitro and in vivo siRNA transfection were affected by the types of cationic lipid used.

DNA-AgNC Loaded Liposomes for Measuring Cerebral Blood Flow Using Two-Photon Fluorescence Correlation Spectroscopy

“…Cationic liposomes, similar to those used for this study, have been widely used for in vivo studies in mouse models and therapeutic delivery in humans . These liposomes have been found to accumulate at low levels in a variety of organs including the lungs, kidneys, and liver and have shown no indication of toxicity . Previous studies have also shown that DNA-AgNCs are nontoxic to mammalian cells. − To further investigate the biosafety of DNA-Ag 16 NC-loaded liposomes, the behavior and body weights of mice were monitored after the delivery of a mixture of DNA-Ag 16 NC-loaded liposomes and FITC-Dextran.…”

Efficient mRNA Delivery with mRNA Lipoplexes Prepared Using a Modified Ethanol Injection Method

“…A lipid-ethanol solution was prepared by dissolving cationic lipid (DOTAP, DDAB, DC-1-16, DC-1-14, DC-6-14, or TC-1-12), neutral helper lipid (DOPE, DOPC, or Chol), and PEG-Chol at a molar ratio of 49.5:49.5:1 in ethanol (Table 1) (2 mg/mL for cationic lipids; for example, 2 mg TC-1-12, 1.53 mg DOPE, and 0.08 mg PEG-Chol were dissolved in 1 mL ethanol) as previously reported [26]. For in vitro transfection, 0.5 µL of 1 mg/mL mRNA solution was transferred into a 1.5 mL tube containing 100 µL of PBS (pH 7.4), and the obtained solution was rapidly added to the lipid-ethanol solution (2.4 µL, 1.6 µL, 1.8 µL, 2.0 µL, 2.1 µL, and 3 µL for DOTAP, DC-1-14, DC-1-16, DDAB, DC-6-14, and TC-1-12 formulation) in another 1.5 mL tube at a charge ratio (+:-) of 4:1, based on previous reports [27,28]; subsequently, the mixture was vortex-mixed for 10 s.…”

“…mRNA lipoplexes with 0.05 µg of Fluc mRNA in 100 µL of culture medium (0.5 µg/mL mRNA) were added to HeLa, A549, or PC-3 cells at 50% confluency in 96-well plates. After 24 h of incubation, the number of viable cells was determined using the Cell Counting Kit-8 (Dojindo Laboratories, Inc., Kumamoto, Japan), as previously reported [28].…”

“…mRNA lipoplexes, such as 5 µg of CleanCap ® Cy5-mRNA and 10 µg of EZCap TM Cy5-mRNA, were administered by intramuscular (IM) and intravenous (IV) injections, respectively, into these mice. After 1 or 24 h of IM or IV injection, respectively, the mice were humanely sacrificed, and Cy5 fluorescence images of the lungs, heart, liver, spleen, and kidneys were acquired using a NightOWL LB981 NC100 system (Berthold Technologies, Bad Wildbad, Germany) as previously reported [28].…”

“…Three microliters of ice-cold luciferase cell culture lysis reagent (Promega Co., Madison, WI, USA) per 1 mg of tissue was added and homogenized immediately. The homogenized samples were centrifuged at 15,000 rpm for 3 min at 4 • C. Aliquots of 10 µL of the supernatant were mixed with 50 µL of PicaGene MelioraStar-LT Luminescence Reagent (Toyo Ink Co., Ltd., Tokyo, Japan), and counts per second (cps) were measured using a chemoluminometer (ARVO X2; Perkin Elmer Co., Ltd., Waltham, MA, USA) as previously reported [28]. The protein concentration of each supernatant was determined using a bicinchoninic acid (BCA) reagent (Pierce ™ BCA Protein Assay kit; Thermo Fisher Scientific, Inc.), and luciferase activity was calculated as cps/mg protein.…”