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Abstract
The ATP binding-cassette (ABC) transporters are a protein superfamily that efflux endogenous molecules and drugs through ATP hydrolysis. This superfamily has five subfamilies, but the most studied and ubiquitous ABC transporter isoform is ABCB1 (P-glycoprotein or P-gp). This isoform plays a critical role in drug disposition, serves as a gatekeeper across many critical biological barriers, and plays a major role in human disease. The protein expression within the superfamily is highly regulated on genetic, translational, and post-transcriptional levels. In particular, short oligonucleotides called microRNAs are known to have significant effects on ABC transporter gene expression. A lot of interest exists in therapeutic applications for microRNAs since they can potentially reduce disease-causing effects from these transporters. Specific inhibitors have been developed for P-gp and other ABC transporters to combat disease, but they have been plagued by unacceptable toxicity in the clinic. To overcome this therapeutic roadblock, we have developed a novel method to bioengineer miRNA delivering vesicles to specific tissues called the “Functionalized Lipid Insertion Method.” The method produces miRNA delivering vehicles from extracellular vesicles (mEVs) and liposomal nanoparticles (mLNPs), which can be loaded with microRNA by electroporation. In vitro experiments with hepatoma HepG2 cells show that microRNA uptake and functional effects are significantly enhanced with these modified vesicles over other approaches. Functionalized vesicles were designed for in vivo experiments with different antibodies to determine the organ miRNA delivery specificity in mice. Functionalized vesicles made with non-targeting antibodies delivered microRNA to the spleen due to the intraperitoneal route of administration. Functionalized vesicles targeting the endocytotic liver receptor delivered microRNA specifically to the mouse liver, while functionalized vesicles targeting a non-endocytotic kidney receptor were less specific. To determine the immunogenic effects of these vesicles, functionalized vesicles were designed with antibodies that are known to target multiple organs. Mice treated with these functionalized vesicles exhibited no physical manifestations of an immune response or showed significant changes in their cytokine levels. Ultimately, our technology has a lot of potential to treat devastating diseases such as cancer as well as the ability to scale up production for the pharmaceutical industry.