(M1030-12-83) Application of Hybrid Extracellular Vesicle-Liposome (EV-Lip) to Improve the Encapsulation Efficiency of Antiretroviral Drugs

Purpose: EVs are lipid bilayer-enclosed particles released from various cell types, which endows its target delivery to its parental cells¹. In our previous study, we found that antiretroviral drugs can be loaded in the EVs derived from U937 cells. However, the low encapsulation efficiency (EE%) of the EV formulation poses a challenge for in vitro and in vivo studies. Liposomes are a small synthesis nanoparticle, composed of at least one phospholipid bilayer. It has been approved by the FDA as a safe carrier for active molecules with excellent EE%. The lipid components of EVs are similar to liposomes. In this study, we would like to integrate both EVs and liposomes, which can leverage the benefits of both components and serve as a potential clinical treatment to inhibit HIV in the brain.

Methods: Monocytic cells (CL01) were differentiated by 100 nM phorbol-12-myristate-13 acetate (PMA) for 72 h followed by exchanging the media with EV- free media for 3-4 days. EVs were collected from 150-200ml of CL01 media. The amount of EVs used for loading was measured by protein level. The media went through series of centrifugation, 2000g for 30 minutes, 10,000g for 1h, and 100,000g for 4h at 4°C. 2 mg of a mixture of two lipids (L1 and L2) and ART02 were dissolved in chloroform and a thin film was formed using rotary evaporation. 10ml of EV resuspension in 1XPBS was added into the flask to rehydrate the thin film. After sonication, the suspension was extruded in a jacket extruder (Genizer). The encapsulation efficiency was measured by LC-MS/MS. The morphology of the EV-Liposome hybrid nanoparticle has been observed under transmission electron microscopy (TEM). The size and zetapotential of the EV-Lip nanoparticles were measured by dynamic light scattering (DLS). TEM, and DLS measurements were conducted at room temperature.

Results: We isolated approximately 8mg of EVs from 180ml of media. The average size of EV was 181.6nm (Figure 1) and the zetapotential of EV was -15.5mV before extrusion. ART02 was successfully incorporated into the hybrid EV-Liposome structure with an EE% of 48%, a size of 151.8 nm (Figure 2), and a zeta potential of -7.76mV. The TEM as showed in Figure 3 revealed the unified structure of the EV-Liposome complex.

Conclusion: The size of EV-Lip falls within the range of size of exosomes with slightly negative charge. The integrity of membrane was maintained after extrusion. FRET-based lipid mixing assay will be performed to determine the fusion between EVs and liposomes. The EE% of EV-Lip was significantly improved compared to that of direct loading in EVs. Nanoparticles with a diameter below 200 nm and a neutral or slightly negative surface charge (close to zero zeta potential) are more likely to avoid rapid clearance by the mononuclear phagocytic system (MPS) or reticuloendothelial system (RES)². With appropriate EE%, we can apply the novel drug delivery system to the in-vitro and in-vivo study to investigate its safety and efficacy. These studies can support our primary objective of development of Lip-EV is to increase the permeability of antiretroviral drug into brain to inhibit the HIV replication in CNS reservoir.

References: 1. de Jong OG, Kooijmans SAA, Murphy DE, Jiang L, Evers MJW, Sluijter JPG, Vader P, Schiffelers RM. Drug Delivery with Extracellular Vesicles: From Imagination to Innovation. Accounts of Chemical Research. 2019;52(7):1761-70. doi: 10.1021/acs.accounts.9b00109.
2. Mazumdar S, Chitkara D, Mittal A. Exploration and insights into the cellular internalization and intracellular fate of amphiphilic polymeric nanocarriers. Acta Pharmaceutica Sinica B. 2021;11(4):903-24. doi: https://doi.org/10.1016/j.apsb.2021.02.019.

Acknowledgements: This study was funded by NIH grants AG081140, and MH125670 to Dr. Santosh Kumar. Disclosure: Due to the potential intellectual property right, codes were used to indicate the EV source, ART drug, and lipids in this abstract.

Figure 1. Size distribution profile of EV from DLS.

Figure 2. Size distribution profile of Hybrid EV-Lip from DLS.

Figure 3. Transmission electron microscopy (TEM) of Hybrid EV-Lip. Size: ~100nm Magnification: 150,000X.