Nanoformulated antiretroviral drug combinations extend drug release and antiretroviral responses in HIV-1-infected macrophages: implications for neuroAIDS therapeutics

Abstract

We posit that improvements in pharmacokinetics and biodistributions of antiretroviral therapies (ART) for human immunodeficiency virus type one-infected people can be achieved through nanoformulationed drug delivery systems. To this end, we manufactured nanoparticles of atazanavir, efavirenz, and ritonavir (termed nanoART) and treated human monocyte-derived macrophages (MDM) in combination therapies to assess antiretroviral responses. This resulted in improved drug uptake, release, and antiretroviral efficacy over monotherapy. MDM rapidly, within minutes, ingested nanoART combinations, at equal or similar rates, as individual formulations. Combination nanoART ingested by MDM facilitated individual drug release from 15 to >20 days. These findings are noteworthy as a nanoART cell-mediated drug delivery provides a means to deliver therapeutics to viral sanctuaries, such as the central nervous system during progressive human immunodeficiency virus type one infection. The work brings us yet another step closer to realizing the utility of nanoART for virus-infected people.

Figure 1. NanoART morphology

Scanning electron microscopy analyses (magnification 30,000x) of nanoformulations shown include ATV in blue (A), EFV in green (B), and RTV in red (C) on top of a 0.2 μm polycarbonate filtration membrane. Measure bar equals 1μm (A,B,C) and 3μm (D,E). ATV(A) showed polygonal structures with sizes of approximately 1 μm; EFV (B) showed spherical structures of approximately 300 nm; RTV (C) showed rod structures with sizes of approximately 1 μm. Transmission electron microscope (magnification, 15,000x) demonstrated uptake of nanoART into MDMs exposed to a combination of ATV, EFV and RTV (D) compared to untreated cells (E). Within the cell, each type of nanoART is readily identifiable by its unique shape and an example has been outlined in blue (ATV), green (EFV), and red (RTV).

Figure 2. Simultaneous MDM uptake of nanoformulated ATV, RTV, and EFV

Fluorescence microscopy of MDM incubated with 100 μM each of ATV-H1045 (purple) labeled with Vybrant DiO cell-labeling solution, RTV-H1025 (green) labeled with Vybrant DiD cell-labeling solution, and EFV-P1044 (red) with rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine, triethylammonium salt. Overlay (white) represents colocalization of all three nanoART to cytoplasmic vesicles. Images were acquired after 2 h of incubation with nanoART combination.

Figure 3. MDM uptake and release of nanoformulated RTV, ATV, and EFV

Uptake: Levels of RTV-H1025, ATV-H1045, and EFV-P1044 in cell lysates of cultured MDM treated with 100 μM of each nanoART alone or in combination (nanoART*) and collected at specified times were determined by HPLC. Arrows indicate initiation time for release studies. Release: Levels of RTV, ATV, and EFV were determined over 20 days by HPLC in both cell lysates (cells) and extracellular media (media) at specified times. Data represent mean ± SD for n = 3 determinations/time point. Asterisks indicate significant differences (p< 0.05) between levels of drug when cells were treated with one nanoART compared to a combination of all three.

Figure 4. Antiretroviral activities for nanoART

Comparison of antiretroviral effects of ATV-H1045, RTV-H1025, and EFV-P1044 alone or in combination (nanoART*) 20 days after pretreatment with nanoART as measured by RT activity. RT activities were measured by ³H-TTP incorporation. Data represent mean ± SD for n = 6 determinations/treatment. “a” indicates significant difference (p< 0.001) from no nanoART treatment (Infected). “b” indicates significant difference (p< 0.001) from nanoART*.

Figure 5. NanoART effects HIV-1 p24 antigen expression in virus-infected MDM

Comparison of antiretroviral effects of ATV-H1045, RTV-H1025, and EFV-P1044 alone or combination (nanoART*) over 20 days in MDM pretreated with nanoART. Ten days after each viral challenge cells were immunostained for HIV-1 p24 antigen (brown). Images (40x) are representative of n = 6 determinations/time point. Cells treated with ATV-H1045, RTV-H1025, or EFV-P1044 alone showed decrease of antiretroviral protection and increased HIV p24 expression at 20 days; while cells treated with a combination of all three nanoART maintained complete suppression of viral p24 production.