Spontaneous Entrapment of Polynucleotides upon Electrostatic Interaction with Ethanol-Destabilized Cationic Liposomes

Abstract

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.

Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation

“…The resulting cationic liposomes were 60 nm in diameter and exhibited a zeta potential of ~40 mV. As previously reported, 15,25 size was found to increase slightly on complexation with siRNA molecules and zeta potentials also decreased accordingly. Particle size showed a maximum at N/P ratio of 5.…”

“…15,25 Cationic liposomes were composed of the following lipids: DODMA, PEG-DSPE, DSPC and cholesterol in a molar ratio of 50:1.5:10:38.5. Lipids were dissolved in 3 mL ethanol and then 70.5 µL 1N HCl and 6.93 mL distilled water were added to this mixture (total lipids: 7.8 mg/mL).…”

Hypoxia-responsive ionizable liposome delivery siRNA for glioma therapy

“…MLP/siRNA complexes were made as previously reported. 10,35,37 As shown in Figure 2A, we found that MLP had strong ability to bind siRNA, which completely complexed siRNA at an N/P ratio of 5:1 (the molar ratio of nitrogen in MLP to phosphate in siRNA) through electrostatic interaction, leading to no free siRNA in the gel. The control liposome (DLP) was prepared by PD, DSPE-PEG2000, and cholesterol, and was mixed with siRNA in pH=3 citrate buffer at the optimal N/P ratio of 5:1 ( Figure S2).…”

Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis

“…Construction of siRNA-encapsulated Liposomes-siRNAencapsulated liposomes, called stable nucleic acid lipid particles (SNALPs), were prepared according to a method reported previously (25). 1,2-dioleyloxy-3-(dimethylamino)propane, 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine, cholesterol (2,15-dimethyl-14-(1,5-dimethylhexyl)tetracyclo-heptadec-7-en-5-ol), and PEG2000PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL) and dissolved in ethanol at a molar ratio of 40:10:47:3.…”