(W1030-05-30) Drug Delivery Optimization and Nutraceutical as Adjuvant with Antiretroviral Therapy to Suppress HIV-1 in CNS Reservoirs

Purpose: The CNS serves as a sanctuary for ongoing HIV-1 replication, leading to chronic inflammation, cytokine release, and cellular damage, resulting in HAND. One major hurdle is the limited penetration of anti-retroviral therapy (ART) drugs, such as ART01, across the blood-brain barrier (BBB), which hinders their efficacy in CNS reservoirs. Moreover, viral replication-induced neuronal damage and potential neurotoxicity associated with ART further complicate the treatment. To overcome these obstacles, we have developed a poly(lactic-co-glycolic acid) (PLGA)-based formulation to improve the transmigration of ART01 into the CNS. Our objective is to enhance the antiviral efficacy of ART01 by incorporating NC01, a nutraceutical with proven anti-inflammatory and antioxidant activities, as an adjuvant. We aim to target CNS HIV reservoirs, particularly macrophages and microglia, to optimize therapeutic interventions for reducing HAND. We hypothesize that administering ART01 through the intranasal (IN) route will increase its brain concentration, minimizing off-target effects. Additionally, NC01 is expected to enhance ART01 brain concentration by suppressing efflux transporters. By combining an FDA-approved ART01 with NC01 in our PLGA nanoformulation, we envision a potential breakthrough in effectively treating HIV in the CNS while protecting against neuronal damage. This approach holds promise for improved therapeutic outcomes and better management of HAND-related complications.

Methods: In this study, we developed PLGA nanoformulations containing ART01 and NC01, and characterized them for particle size, zeta potential, and drug encapsulation. The particle size and zeta potential of the PLGA nanoformulations were determined using dynamic light scattering (DLS) analysis. Drug encapsulation efficiency was assessed using LC-MS/MS, quantifying the concentrations of ART01 and NC01 encapsulated within the PLGA nanoparticles to evaluate drug-loading capacity. Additionally, U1 cells were exposed to ART01 (1µM) with or without NC01 (5µM) for 24 hours to examine ART01 accumulation. Total antioxidant capacity and ROS levels were measured through the Total Antioxidant Capacity Assay Kit and flow cytometry, respectively, while western blot analysis was employed to determine antioxidant enzyme levels (catalase and SOD1) after drug exposure. Furthermore, inflammatory response in U1 cells was investigated by treating them with Control (DMSO), ART01 (1µM), and NC01 (5µM) for 24 hours, and cytokine and chemokine levels were measured in the culture media using Human Custom Procartaplex 9-plex. In vivo biodistribution studies were conducted in Balb/c mice, administering ART01 (25 mg/kg) with and without NC01 (20 mg/kg) via intraperitoneal (IP) and intranasal (IN) routes, and analyzing ART01 concentrations in the plasma, brain, lungs, and liver.

Results: Our findings demonstrated improved brain drug concentration for ART01, an FDA-approved drug, when used along with NC01, a nutraceutical with proven anti-inflammatory and antioxidant activities, using an intranasal (IN) delivery in wild-type mice. The results were comparable whether the ART01/NC01 were used alone or upon encapsulation in PLGA. When compared the biodistribution of ART01 with two different concentrations with two administration routes, IN administration, compared to intraperitoneal administration demonstrated a significantly improved ART01 penetration in the brain at both low (5mg/kg) and high (25mg/kg) concentrations. We also observed modulation of cytokine profile with significant decrease in proinflammatory cytokines (IL-1β, TNF-α, IL-18) and increase in anti-inflammatory cytokines (IL-10) after ART01 and NC01 treatment in U1 cells.

Conclusion: In this study, we demonstrated the promising potential of NC01 treatment, either alone or encapsulated with PLGA nanoparticles, in enhancing the therapeutic efficacy of ART01 in both in vitro and in vivo settings. In U1 cells, NC01 treatment showed significant improvements by enhancing the intracellular concentration of ART01, augmenting total antioxidant capacity, sustaining oxidative stress, and ameliorating inflammation. Moreover, in in vivo experiments with BALB/c mice, NC01 treatment, whether administered alone or encapsulated with PLGA nanoparticles, led to significant enhancements in biodistribution, particularly in the brain. These combined effects of NC01 in both settings effectively complemented the therapeutic outcomes achieved by ART01 alone. Thus, our findings highlight the potential of NC01 as an adjuvant to ART01, offering a promising strategy for improving HIV-1 treatment outcomes and reducing associated neurocognitive complications, such as HIV-associated neurocognitive disorder (HAND).

Acknowledgements: Funding Agency/Grant: NIH MH125670 and AG081140.

Figure 1. (A). Concentration-time profiles for the accumulation of ART01 in U1 cells with or without NC01. (B) Area under the curve of ART01 and ART01+NC01. Results are expressed as means ± SEM of the cellular concentrations measured (ng/mg of protein).
Figure 2: (A) Total antioxidant capacity using the Total Antioxidant Capacity Assay Kit and (B) ROS levels using flow cytometry (C,D) antioxidant enzymes (catalase and SOD1) levels measured by western blot after 24 h drug exposure (ART01-1µM; NC01-5µM).
Figure 3. Measurement of cytokines in U1-cell media treated with ART01 and NC01: The U1 cells were exposed to a single dose of Control (DMSO), ART01 (1µM), and NC01 (5µM) and observed for 24 hrs. The protein levels of various cytokines and chemokines were measured from the culture media using Human Custom Procartaplex 9-plex.
Figure 4. Biodistribution of ART01 (25 mg/kg) with and without NC01 (20 mg/kg) administered via intraperitoneal (IP) and intranasal (IN) in Balb/c mice. ART01 concentrations were analyzed in the plasma (A, B), brain (D), lungs (E), and liver(F). Concentrations of ART01 were measured as ng/g in tissues and ng/ml in plasma. Statistical analyses were carried out by using ANOVA (multiple comparisons) and t-test (two groups). Results are expressed as means ± S.E.M. . * represents p < 0.05 when compared with ART01. # represents p < 0.05 when compared with ART01+NC01 (IP).

Figure 5. (A). Concentration-time profiles for the accumulation of ART01 in U1 cells with or without NC01. (B) Area under the curve of ART01 and ART01+NC01. Results expressed as means ± SEM of the cellular concentrations measured (ng/mg of protein).
Figure 6: (A) Total antioxidant capacity using the Total Antioxidant Capacity Assay Kit and (B) ROS levels using flow cytometry (C,D) antioxidant enzymes (catalase and SOD1) levels measured by western blot after 24 hrs PLGA-NPs exposure (ART01-1µM; NC01-5µM).
Figure 7. Measurement of cytokines in U1-cell media treated with PLGA-ART01 and PLGA-NC01: The U1 cells were exposed to a single dose of Control (DMSO), ART01 (1µM), and NC01 (5µM) and observed for 24 hrs. The protein levels of various cytokines and chemokines were measured from the culture media using Human Custom Procartaplex 9-plex.
Figure 8. Biodistribution of PLGA-ART01 (25 mg/kg) with and without PLGA-NC01 (20 mg/kg) administered via intraperitoneal (IP) and intranasal (IN) in Balb/c mice. ART01 concentrations were analyzed in the plasma (A, B), brain (D), lungs (E), and liver(F). Concentrations of ART01 were measured as ng/g in tissues and ng/ml in plasma. Statistical analyses were carried out by using ANOVA (multiple comparisons) and t-test (two groups). Results are expressed as means ± S.E.M. . * represents p < 0.05 when compared with ART01. # represents p < 0.05 when compared with ART01+NC01 (IP).

Depiction of Neuropathogenic mechanisms that contribute to HAND along with
limitations of conventional ART drugs and proposed advantages of nanoformulation