(M1530-12-71) Nanoparticle-Based Drug Delivery Approach Increased the Bioavailability and Efficacy of the ART Therapy in Vitro

Purpose: The life expectancy of people with HIV increased after the introduction of antiretroviral therapy (ART) in the 1990s. However, the adherence rate of HIV treatment in the US is generally low, and those HIV patients will risk drug resistance and treatment failure. Pill burden, dosing frequency, and adverse events are the three main factors affecting HIV patients’ compliance. To improve the adherence rate of HIV patients, a formulation with an extended-release profile, higher bioavailability, and efficacy is needed. Our group showed that Poly (D, L-lactide-co-glycolide) (PLGA) can endow the extended release of ART drugs. Also, it has been proved that the widely used nutraceutical-NC01 can inhibit the P-glycoprotein efflux transporter, which is located on the membrane of most HIV-infected cells. NC01 can inhibit inflammation conditions, which further enhances the bioavailability of ART02 and reduces the production of inflammation factors. Here we studied whether loading an FDA-approved ART drug-ART02 and NC01 in PLGA to form the nanoparticles will increase the bioavailability and efficacy of ART drugs in HIV-infected macrophages.

Methods: We loaded an ART drug-ART02 in PLGA using nanoprecipitation and nutraceutical NC01 by nanoprecipitation and emulsion techniques. The physicochemical properties of nanoparticles were determined by transmission electron microscopy and dynamic light scattering. Human monocyte cells (U937) and HIV chronically infected promonocytic cells (U1) were differentiated with 100 nM phorbol-12-myristate-13 acetate (PMA) for 72 h before the treatment with 5.5µM ART02 and 5µM NC01 in the form of either the free drug or PLGA formulations. The cellular concentrations of the drugs were measured by a liquid chromatography system coupled to a tandem mass spectrometer.

Results: We successfully loaded ART02 in PLGA with an encapsulation efficiency %(EE%) of 81.25 ± 5.194% and NC01 with 54.83 ± 1.101% (Mean ± SEM). The size of PLGA-Ctrl, PLGA-ART02, and PLGA- NC01 is 201.6 ± 0.6119nm, 175.1 ± 1.906 nm, and 351.6 ± 4.768 nm. The zeta potential of PLGA-Ctrl, PLGA-ART02, and PLGA-NC01 is 0.32 ± 0.5215 mV, 0.28 ± 0.0265 mV, and -1.943 ± 0.1964 mV. U937 cells were differentiated using PMA and treated with ART02 for 1h, 3h, 9h, and 24h. After normalized by protein amount, cellular concentrations of ART02 in U937 were higher in the cells treated with the PLGA formulation without significant cytotoxicity. The area under the curve (AUC) of U937 cellular concentration as depicted in Figure 2A indicated that U937 cells in the PLGA-ART02 group experienced more exposure to ART02. The efficacy of PLGA-ART02 and its combination with NC01 were determined in HIV-infected cells (U1). HIV replication in U1 cells treated with ART02 was attenuated as evidenced by the reduction of viral p24 protein expression. In the presence of PLGA-NC01 or PLGA-ART02 HIV replication in U1 cells was further decreased.

Conclusion: This is the first study to determine the effect of the combination of PLGA nano-formulation of the ART therapy and a nutraceutical compound on HIV replication. We found that the PLGA formulation could improve drug absorbance and enhance HIV inhibition in vitro, suggesting that PLGA nano-formulation can increase the bioavailability and efficacy of ART drugs in HIV-infected macrophages. Future investigation is necessary to determine whether the combination of PLGA formulations of the ART drug and the nutraceutical can be used as a strategy to improve HIV patients’ compliance and lower the risk of drug resistance in HIV patients.

References: 1. Y. Gong, K. Zhi, P. K. B. Nagesh, N. Sinha, P. Chowdhury, H. Chen, S. Gorantla, M. M. Yallapu and S. Kumar, "An Elvitegravir Nanoformulation Crosses the Blood–Brain Barrier and Suppresses HIV-1 Replication in Microglia," Viruses, vol. 12, no. 5, pp. 564, 2020.
2. K. L. Schaecher, "The Importance of Treatment Adherence in HIV," Supplements and Featured Publications, vol. 19, no. 12, 2013.

Acknowledgements: This study was funded by NIH grant (MH125670) to Dr. Santosh Kumar.
Disclosure: Due to the potential intellectual property right, codes for the drug and the nutraceutical were used in this abstract.


Figure 1 A schematic coverage of this study.


Figure 2. A&B. Transmission electron microscopy (TEM) images of PLGA-ART02 and PLGA-NC01 NPs of which sizes are ~100nm. C. Size of nanoparticles. The size of PLGA-Ctrl is 201.6 ± 0.6119nm. PLGA-ART02 is 175.1 ± 1.91nm and NC01 is 351.6 ± 4.77 nm. D. The zeta potential of PLGA-Ctrl. is 0.32 ± 0.5215 mV PLGA-ART02 is -0.28 ± 0.03 mV and PLGA-NC01 is -1.94 ± 0.20 mV. TEM, DLS, and zeta potential measurements were conducted at room temperature.


Figure 3. A. Time-dependent cellular concentration of ART02 in U937 cells. U937 cells were incubated with ART02 and PLGA-ART02 for 1h, 3h, 9h, and 24h. Mean ± SEM were graphed from three independent experiments. B. Free drug form of ART02 reduced the p24 expression in U1 after treatment of ART02 for 9h. C. p24 expression was significantly reduced in U1 treated with PLGA-ART02+PLGA-NC01. The mean ± SEM were graphed from three independent experiments. (*P < 0.05, **P < 0.01)