Endosomal Trafficking of Nanoformulated Antiretroviral Therapy Facilitates Drug Particle Carriage and HIV Clearance

Abstract

Limitations of antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance, and failure to penetrate viral reservoirs. Recent developments in nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at or adjacent to the sites of the viral replication cycle. Poloxamer 407-coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were ϳ1,000 times higher in cells than those that could be achieved by the native drug. ATV particles in late and recycling endosome compartments were seen following pulldown by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of the compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1 p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000-fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The colocalization of nano-ATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long-acting nanoART can serve as a drug carrier in both cells and subcellular compartments and, as such, can facilitate viral clearance. IMPORTANCEThe need for long-acting ART is significant and highlighted by limitations in drug access, toxicity, adherence, and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells, and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly, and increasing ART concentrations in such sites enhances viral clearance. The current work uncovers a new mechanism by which nanoART can enhance viral clearance over native drug formulations.

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

“…In URMC-099-treated humanized mice, LC3B puncta in CD68 + macrophages were also readily apparent ( Figure 7C). These results suggest that URMC-099 modulates HIV-1-induced cytokine responses in vivo through autophagy (6,47). …”

“…Subsequently, ATV concentrations in total cells or LC3B-positive autophagosomal compartments were determined. LC3B-positive compartments were immunoisolated using LC3B antibody as described elsewhere (6,56). See the Supplemental Experimental Procedures for additional details.…”

“…We previously developed, characterized, and tested therapeutic responses of long-acting nanoformulated ART (nanoART) (4)(5)(6)(7)(8)(9)(10)(11). Recent successes have come from encasing hydrophobic crystalline antiretroviral drugs (ARVs) into excipients, decorating the formed particles with ligands that target cells and tissue reservoirs or by pharmacologically boosting intracellular depot formations (4,(11)(12)(13).…”

“…However, the production of more hydrophobic and protein-bound ARVs provides only a partial solution. Controlling the dynamics of particle trafficking inside the cell and within early, recycling, and late endosomes is necessary to sustain intracellular drug particle depots and limit hepatic and renal metabolism and drug excretion (4,6,11,14,15).…”

Why Novel Nanoparticle-based Delivery Platforms Hold Key for HIV/AIDS Treatment and Prevention?

“…The concentrations of nanoATV were found to be approximately higher in cells compared to those that could be achieved by the native drug. 11 From this experiment, it can be deduced that the nanocarrier enhances the retention of drugs in subcellular compartments as compared to the native drug.…”