Liposomes with asymmetric bilayers produced from inverse emulsions for nucleic acid delivery

Abstract

Asymmetrical lipid nanoparticles are interesting nanocarriers for charged molecules, like nucleic acids. They promise control over inner and outer charge. High charge density on the inside is favourable for efficient condensation and charge neutralisation of highly charged biopharmaceuticals, while a neutral or slightly negative outer layer promotes biocompatibility. The main goal of this work was the development and characterisation of asymmetric liposomes, prepared using water-in-oil (w/o) nanoemulsions of phospholipids (PLs) and squalene in a centrifugal field. This method enables the control over the lipid composition of each monolayer.Liposomes were prepared by passing PL w/o nanoemulsions through an oil-water interface previously saturated with PLs. We used N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) or N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3phosphocholine (NBD-PC) as a fluorescent marker for either the inner or outer lipid layer and plasmid DNA (pDNA) as nucleic acid payload. The final liposomes had sizes below 200 nm and polydispersity indexes of 0.3 and had a bilayer asymmetry of 70%, thus shielding the charge of positive PLs in the inner bilayer leaflet. Final formulations were examined using negative staining transmission electron microscopy (TEM). Plasmid encapsulation efficiency of the method was 10-15%. Our results indicate that the w/o nanoemulsion-centrifugation method allows the successful production of liposomes with tailored features for encapsulation of nucleic acid therapeutics. ARTICLE HISTORY

The Phospholipid Research Center: Current Research in Phospholipids and Their Use in Drug Delivery

“…Here, the group of Hermann Nirschl (Karlsruhe Institute for Technology, Karlsruhe, Germany) developed a novel process using centrifugation of water-in-oil (w/o) emulsions, based on the use of phospholipids as emulsifiers, avoiding the use of aggressive treatments and allowing asymmetric membrane functionalities. While the previous project addressed the characterization of the applied substances to produce liposomes [55,[117][118][119][120][121], the follow-up study focuses on the optimization of liposome production process [122,123]. The proof-of-concept of the flotation of aqueous droplets from a w/o nano-emulsion to an aqueous phase by centrifugation shows advantages when compared to known liposome production methods; the use of solvents is redundant, and the encapsulation efficiency of hydrophilic model substances is higher than anything described in the literature so far.…”

How to Achieve High Encapsulation Efficiencies for Macromolecular and Sensitive APIs in Liposomes

“…While this method works for large uni-lamellar vesicles of between 1 and 10 µm, several limitations exist such as an appropriate size for pharmaceutical applications, the stability of nano-emulsions, and oranogel formation between the three phases (water, oil and phospholipids). A smaller size of liposome was achieved by de Matos et al [17]. However, the phase transfer and encapsulation efficiencies were still insufficient.…”

“…The question remains as to whether a high encapsulation efficiency is possible by applying the centrifugation process. While De Matos et al used the centrifugation method to produce asymmetric liposomes and encapsulated nucleic acids, they used squalene for the preparation of nano-emulsions leading to a plasmid encapsulation of 10-15% [17]. Hence, an evaluation of the encapsulation efficiency of the formed liposomes from the water-in-fluorocarbon (w/fc) nano-emulsion remains to be carried out.…”

Eudragit S100 Entrapped Liposome for Curcumin Delivery: Anti-Oxidative Effect in Caco-2 Cells

“…They can be easily marked both with organic fluorescent molecules and quantum dots with a high quantum yield, emitting in a wide range of wavelengths, ideal for theranostic applications [17,18]. In addition, liposomes can carry fully active proteins (including integral membrane proteins), genetic material, and other biomacromolecules [19][20][21]. Finally, liposomes can be covered with both natural and synthetic polymers that increase their steric and biological stability, adapting them to specific experimental needs [22,23].…”