A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro and in vivo in mice

Abstract

Nano-based delivery systems have greatly enhanced our ability to administer and target drugs and macromolecules to their therapeutic targets. Oligonucleotide drugs have great therapeutic potential but have off-target effects and stability issues, so they are often encapsulated in functionalized vesicles with targeting ligands such as antibodies (Ab). Herein, we describe a novel, scalable and straightforward approach to produce functionalized vesicles called the “Functionalized Lipid Insertion Method.” This method differs significantly from an older approach referred to as the “Detergent-Dialysis Method.” The older method requires excess detergent and extensive dialysis over many hours to produce the functionalized vesicles. With the “Functionalized Lipid Insertion Method,” only the functionalized lipid is detergent-solubilized during the formation of the functionalized vesicle. The approach reduces the dialysis time, keeps the vesicle intact, and orients the functionalized lipid to improve targeting compared to the older method. The dynamic light scattering (DLS) technique demonstrated that vesicle size is sensitive to the initial detergent-solubilized component mixture by the older method. In contrast, functionalized vesicle size increases are consistent with functionalized lipid insertion into the vesicle. In vitro, functionalized vesicles using our approach are able to deliver oligonucleotides selectively and can functionally affect liver cancer HepG2 cells. Functionalized vesicles produced by this method can also achieve targeted delivery of oligonucleotides in mice without inducing a significant immune response through cytokine production or showing physical signs of an immune response. The industrial and therapeutic significance and implications of functionalized vesicles produced by our method are also discussed.