*3.6. Ethanol Injection*

Unilamellar liposomes can be prepared by the Ethanol Injection (EI) method which can be used for NABDs encapsulation. This method provides the rapid injection of an ethanolic solution, in which lipids are dissolved, into an aqueous medium containing nucleic acids to be encapsulated, through the use of a needle. Vesicles are spontaneously formed when the phospholipids are dispersed throughout the medium. By utilizing this method Stabilized Antisense-Lipid Particles (SALPs) can be obtained [87], i.e., Saffari and collaborators showed that by the Ethanol injection method DOTAP-liposomes loaded with an antisense oligonuclotide (AsODN) against protein kinase C alpha, with a size of 115 nm, not requiring downsizing with extrusion or other methods, can be achieved. They obtained an encapsulation efficiency of around 90% that was about seven times more than that of the TFH method, also tried out in their work [88]. In a work of Saad and collaborators cationic liposomes were produced by the EI method for the co-delivery of doxorubicin and siRNA targeted to MRP1 and BCL2 mRNA (suppressors of pump and nonpump cellular resistance) to multi-drug resistance (MDR) lung cancer cells, enhancing the chemotherapy efficiency [89].
