(M1430-03-17) Targeted Antiretroviral Therapy Using Controlled Release Polymeric Nanoparticles for Brain Viral Load Reduction and Management of HIV Associated Neurocognitive Disorder (HAND)

Purpose: Human immunodeficiency virus (HIV) is neurotropic, enabling it to breach the blood-brain barrier, leading to a spectrum of neurocognitive dysfunctions collectively termed HIV-associated neurocognitive disorder (HAND). These impairments span from mild to severe cognitive deficits and afflict 39-70% of HIV-positive individuals. The blood-brain barrier presents a formidable obstacle to the delivery of antiretroviral drugs to the brain, hindering the maintenance of therapeutic concentrations over time. To mitigate this challenge, there is a pressing need for a brain-targeted controlled-release delivery system capable of efficiently transporting antiretroviral drugs to the brain parenchyma. In response, this study introduces functionalized poly(lactic-co-glycolic acid) nanoparticles loaded with the antiretroviral drugs Emtricitabine and Bictegravir, engineered to facilitate controlled drug release within the brain and potentially eradicate viral reservoirs. These nanoparticles are surface-modified with mannose to target GLUT-1 transporters on the blood-brain barrier and incorporate cell-penetrating peptides such as Penetratin to enhance cellular uptake. In vitro assessments encompass cytocompatibility, cellular uptake, transport across a blood-brain barrier model, and efficacy against Eco HIV-infected microglia cells. Additionally, in vivo investigations encompass biodistribution, pharmacokinetics, biocompatibility, and the ability to reduce brain viral load in infected mice.

Methods: To accommodate high payloads of both hydrophilic and hydrophobic drugs, we optimized the double emulsion technique for nanoparticle preparation. The resultant formulation underwent characterization for size, zeta potential, and polydispersity index (PDI) using dynamic light scattering (DLS). In vitro release and entrapment efficiency of the nanoparticles were assessed via RP/HPLC-UV. Additionally, cytocompatibility and cellular uptake of the functionalized nanoparticles were evaluated in bEnd.3 cells, astrocytes, and microglia cells. Hemolytic activity of both functionalized and plain nanoparticles was examined against red blood cells (RBCs). The formulation's transport capabilities were evaluated using an in vitro blood-brain barrier model. To assess its potential to reduce viral reservoirs in vitro, the formulation was tested on Eco-HIV-infected microglia cells, with viral load quantified using a p24 viral protein ELISA kit. C57BL/6 mice received intravenous injections of 40 mg/kg of Emtricitabine-equivalent PLGA nanoparticles or a free drug mixture. Animals were sacrificed at various time points (1h, 8h, 16h, 24h, 72h, 168h, and 240h) for organ collection. Biocompatibility of the nanoparticles was evaluated through histological examination of tissue sections collected after 24 hours and 10 days of nanoparticle, free drug, and PBS treatment. In the in vivo infection study, different treatment groups were intravenously administered to infected mice, with plasma and major organs collected after 7 days for further analysis. Viral burden was determined by quantifying HIV gag RNA using real-time PCR.

Results: The optimized dual-functionalized, dual drug-loaded nanoparticles exhibited a uniform size distribution and spherical morphology, as evidenced by transmission electron microscopy (TEM) images. These nanoparticles possessed a particle size of 235.14 ± 14.6 nm and a zeta potential of -11.29 ± 1.84 mV, with a polydispersity index (PDI) of 0.086 ± 0.019. Encouragingly, controlled release profiles were observed for both drugs, with 90.3 ± 10.7% of Emtricitabine and 85 ± 4.7% of Bictegravir released over a 10-day period. Assessment of cell viability confirmed the non-toxic nature of the formulations to bEnd.3 and glial cells. Importantly, the percent hemolysis associated with these nanoparticles remained below 10%, affirming their safety for intravenous administration. Furthermore, the modified nanoparticles exhibited significantly (p < 0.05) higher cellular uptake and transport across an in vitro blood-brain barrier (BBB) model compared to plain nanoparticles and free drugs. In vitro efficacy studies underscored the superiority of the dual-functionalized (PenMan) treatment group, demonstrating significantly lower p24 levels compared to the free drug-treated group (p < 0.01). In vivo investigations in C57BL/6 mice indicated that drug concentrations delivered via nanoparticles peaked at 16 hours post-treatment and remained elevated for up to 10 days. Remarkably, PenMan nanoparticles exhibited significantly higher brain to plasma ratios at 16-hour time point (p < 0.01). Moreover, analysis revealed that PenMan nanoparticles significantly reduced gag RNA expression in both the brain and spleen of infected mice compared to control groups (p < 0.01). The comparative CT method was employed to quantify the data, ensuring robustness and accuracy in the assessment of treatment efficacy.

Conclusion: The double emulsion PLGA nanoparticles had uniform size distribution. The modification of the nanoparticles with mannose and Penetratin (PenManNP) displayed superior cellular uptake, transport across in-vitro BBB barrier model, and efficacy against Eco-HIV infection, while having good biocompatibility with RBCs, bEND.3 cells, astrocytes and primary glial cells. The nanoparticles successfully targeted the brain tissue and maintained treatment concentrations in the brain for a lengthy duration of 10 days. This sustained release profile offers the advantage of continuous therapeutic efficacy, potentially minimizing the need for frequent dosing. These results indicate that the dual functionalized polymeric nanoparticles can effectively deliver both drugs across the blood-brain barrier to the brain in a controlled release fashion to thwart the HIV reservoir and potentially manage HIV-associated neurocognitive disorder (HAND).

Acknowledgements: This research was supported by the National Institutes of Health (NIH) Grants R01 AG051574, RF1 AG068034 and 3RF1AG068034-01A1S1.


Figure 1: In-vitro cytocompatibility of different PLGA nanoparticles formulations and free drugs in (A) brain endothelial cells, (B) primary astrocytes and (C) microglia cells. Cellular viability of free drug and PLGA at different concentration was evaluated upon exposure for 24h Data represented as mean ± S.D. (n = 6). (D) Fluorescence microscopic images (20X magnification) showing cellular uptake of rhodamine labeled PenMan NP in bEnd.3 cells after 2 h, 4 h and 6 h of incubation with treatment. The nuclei of the cells were stained with Hoechst 33342. The images show the overlap of rhodamine (red) and nuclei of the cells (blue).


Figure 2: A) Transport through BBB model over a period of 24 h. (B) Endothelial cell permeability coefficient (Pe, expressed in 1x10-6 cm/s) of different nanoparticles after 24h treatment. (C) In-vitro viral suppression after treatment with different PLGA nanoparticles and free drug. Data represented as mean ± S.D. (n = 4). Statistically significant differences (p < 0.05) are shown as (*) with Free drug and (#) Plain nanoparticles


Figure 3: (A) Biodistribution of different nanoparticle and free drugs after 1 h, 8h, 16h, 24 h, 72 h, 168 h, and 240 h of intravenous injection in C57BL/6 mice in Brain, (B) Biodistribution of different nanoparticle and free drugs in brain after 16h of intravenous injection in C57BL/6 mice in Brain. (C) Comparing the brain to plasma ratio of PenMan NP and free drugs at 16 h after injection. (n=6) (D) Expression of gag RNA levels in EcoHIV infected mice brain after treatment with different PLGA nanoparticles and free drugs. Fold change in gag RNA expression was quantified using RT-qPCR and comparative CT method. Data are expressed as mean ± SD of injected dose percentage (%ID) per gram of tissue. Statistically significant (p < 0.01) differences are shown as (*) with the free drug treatment group and (#) with Plain nanoparticles.