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 ∼1,000 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 nanoATV 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.

Importance: The 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.

FIG 1

Comparison of native and nanoATV cellular drug uptake and antiretroviral activities. (A) Time course for MDM uptake of native or nanoATV. MDM were treated with native or nanoATV for 24 h. (B) Computational simulation of the time course of MDM drug uptake and retention. After 24 h treatment with native or nanoATV, cells were washed with PBS and treated with fresh medium for 24 h. (C and D) HIV-1 DNA (C) and RNA (D) levels were quantitated 14 days after viral infection (MOI = 0.1) of MDM loaded with various concentrations of native or nanoATV. The units for viral DNA and RNA are copies/10³ cells and copies/cell, respectively. (E and F) HIV-1 RT activity was determined 14 days after HIV-1 infection in MDM loaded with various concentrations of native ATV (E) or nanoATV (F). The data are expressed as averages ± SEM for 3 replicate wells. The data presented are from one of three representative experiments. *, significantly different from non-drug-treated cells (P < 0.05); †, significantly different from the respective native-ATV treatment (P < 0.05).

FIG 2

HIV-1 p24 staining of virus-infected MDM pretreated with native or nanoATV. MDM were treated with native or nanoATV for 16 h and then challenged with HIV-1 (MOI = 0.1). Fourteen days later, the cells were fixed and stained for HIV-1 p24 antigen. The treatment groups were HIV-1-infected controls (A); 1, 10, and 100 μM native ATV (B to D, respectively); 1, 10, and 100 μM nanoATV (E to G, respectively); and uninfected MDM (H). Red, HIV-1 p24 staining; blue, cell nuclei stained with DAPI. (I) Quantitation of immunostaining by densitometry using ImagePro Plus v. 4.0. The data are expressed as averages and SEM. *, significant differences from HIV-1-infected controls (P < 0.05); †, significant differences from native-ATV treatment (P < 0.05).

FIG 3

NanoATV subcellular distribution. (A and B) Uninfected MDM (A) and HIV-1-infected MDM (B) were treated with 100 μM CF568-labeled nanoATV (red) (28) for 16 h and then immunostained with Rab5, Rab7, Rab11, or Rab14 antibodies and Alexa Fluor 488-labeled secondary antibody (green) to visualize nanoparticle and organelle coregistration. The arrowheads indicate overlap (yellow) of nanoATV and Rab compartments. DAPI (blue) stain indicates cell nuclei. (C and D) Quantitation of the percent overlap of nanoATV and Rab compartments in uninfected (C) and HIV-1-infected (D) MDM. The data are expressed as averages and SEM of 10 replicates.

FIG 4

Kinetics of nanoparticle trafficking in subcellular compartments. (A) The purity of Rab5, Rab7, Rab11, and Rab14 isolated compartments was confirmed by Western blotting using antibodies to Rab7 and Rab14 proteins. (B) ATV levels in different endosomal compartments in MDM exposed to 100 μM nanoATV for 48 h. (C) Simulated subcellular nanoATV uptake in different endosomal compartments over the same time frame. (D) Simulated subcellular nanoATV uptake pathways. The data are expressed as averages ± SEM of 3 replicates.

FIG 5

Intracellular pathways for HIV-1 progeny virion production and nanoATV trafficking. (A) Schematic diagram of HIV-1 and nanoATV trafficking. (B) HIV RT activity in subcellular endosomal compartments. MDM were infected with HIV-1 for 4 h and then treated with 100 μM native or nanoATV for 16 h. Endosomal compartments were isolated using specific Rab antibody-coated magnetic beads on day 7 and day 14 after infection, and RT activity was measured in the endosomal compartments. R5, R7, R11, and R14 represent Rab5, Rab7, Rab11, and Rab14, respectively. The data are expressed as the averages of 5 experimental replicates.