Lipid-Polymer Hybrid Nanoparticles Incorporating Diverse HIV-1 Cure Agents for Latency Reversal

A proposed strategy to cure HIV, known as “shock and kill”, uses latency-reversing agents (LRAs) to reactivate latent proviruses for the purge of HIV reservoirs. A variety of small molecule LRAs have been identified, but none has yet been proven effective in actually reducing the reservoir size in vivo partially due to their low potency, poor solubility, and toxicity issues. Additionally, the diverse physicochemical properties of LRAs also limit their combination to achieve maximal latency reversal. Nanocarriers (NC) address these challenges by improving drug solubility, safety, and providing sustained drug release and simultaneous delivery of multiple drugs to target tissues and cells.

Here, we formulated lipid-coated poly lactic-co-glycolic acid (PLGA) nanoparticles (LCNPs) that incorporate physicochemically diverse LRAs. We prioritized LRA combinations that have been shown to achieve almost complete activation of resting CD4 T cells from HIV-1-infected individuals on suppressive combinational antiretroviral therapy (cART). These include binary combinations of ingenol-3-angelate or prostratin (PKC agonists), panobinostat (HDAC inhibitor), JQ1 (BET inhibitor), disulfiram (NF-κb inhibitor), and butyric acid. To incorporate LRAs into LCNPs, ingenol-3-angelate, JQ1 and disulfiram were physically encapsulated into LCNPs (LRA/LCNPs). We also chemically conjugated ingenol-3-angelate, prostratin and panobinostat to carboxyl-terminated PLGA, which was verified by H-NMR and HPLC following LCNP synthesis (LRA-LCNPs). Cholesteryl butyrate was used as the prodrug of butyric acid and was inserted into the lipid bilayer of LCNPs. LCNP size, polydispersity index (PDI), ζ-potential, and long-term colloidal stability of all formulations were measured by dynamic light scattering (DLS). Drug loading and sustained release were quantified by RP-HPLC. All LRA formulations showed monodisperse size with diameters of 160-220 nm and achieved drug loading up to 3wt%. Conjugation of LRAs compared to their physical encapsulation led to slower release from LCNPs with only 15% of the drug released after 1 week, while encapsulated LRAs were released completely within 24 hours in human serum. Initial high-throughput screening of activity and toxicity of our LCNP drug formulations was conducted in a latently infected J-Lat A1 cell line. All LRAs formulated in LCNPs retained their activity. Free drugs such as panobinostat and disulfiram both induced high levels of cell apoptosis at effective concentrations, however, their toxicity was significantly reduced once formulated in LCNPs. Using models to quantitate the combined drug effect of free versus LCNP formulations, we demonstrated that PKC agonists with JQ1 showed synergistic enhancement of latency reversal while leading to less cytotoxicity in LCNPs. Combination LRA formulations in LCNPs verified to induce latency reversal in resting CD4 T cells from HIV-infected patients on suppressive cART will be functionalized to target CD4 T cells and evaluated in non-human primates.