Macrophage endocytic trafficking of antiretroviral nanoparticles

Abstract

Aim: Nanoformulated antiretroviral therapy can improve drug compliance for people infected with HIV. Additional benefits would include specific drug deliveries to viral reservoirs and reduction in systemic toxicities.

Methods: In this article, we describe mechanisms of crystalline antiretroviral nanoparticle (NP) uptake, intracellular trafficking and release in human monocyte-derived macrophages.

Results: Following clathrin-dependent endocytosis NPs bypassed lysosomal degradation by sorting from early endosomes to recycling endosome pathways. Disruption of this pathway by siRNAs or brefeldin-A impaired particle release. Proteomic and biological analysis demonstrated that particle recycling was primarily Rab11 regulated. Particles were released intact and retained complete antiretroviral efficacy.

Conclusion: These results suggest possible pathways of subcellular transport of antiretroviral nanoformulations that preserve both particle integrity and antiretroviral activities demonstrating the potential utility of this approach for targeted drug delivery.

Figure 1. Characterization of the ritonavir nanoparticle and its cellular interactions

(A) RTV-NP with measurements of physical properties and depicting coating of an inner layer of mPEG₂₀₀₀-DSPE/188 and an outer layer of DOTAP. Size and charge were determined by dynamic light scattering. At least four iterations for each reading were taken with <2% variance. (B) Scanning electron microscopy (magnification, 15,000×) of RTV-NP on top of a 0.2-μm polycarbonate membrane shows typical morphology resembling short rods with smooth edges. (C) Uptake of RTV-NP in monocyte-derived macrophages (MDMs) over 12 h and (D) retention of RTV-NP within MDMs (left y-axis) and release of drug to surrounding media (right y-axis) over 15 days were determined by high-performance liquid chromatography. (E) Flow cytometry data and (F) high-performance liquid chromatography data of MDMs exposed to fluorescent RTV-NPs demonstrate that treating MDMs with the clathrin inhibitor Dyna significantly reduces uptake. All data represent the mean ± standard error of the mean for n = 3. ART: Antiretroviral therapy; DOTAP: Dioleoyl trimethylammonium propane; Dyna: Dynasore; Indo: Indomethacin; NP: Nanoparticle; RTV: Ritovanir.

Figure 2. Proteomic analyses of RTV-NP locale

(A) Intracellular RTV-NP were identified within distinct membrane-bound compartments by transmission electron microscopy (magnification 15,000×). (B) The subcellular localization process. RTV-NP were labeled with Brilliant Blue-250 and exposed to MDM. The cells were lysed and subcellular compartments separated by centrifugation on a sucrose gradient. Blue bands represent compartments that contain RTV-NP. These bands were collected, and the proteins separated by electrophoresis. Following in-gel trypsin digest, the proteins were identified using liquid chromatography/mass spectrometry. (C) Subcellular distribution of the identified proteins. A total of 38 endosomal proteins were identified. Each protein was included in each category in which it was identified up to this point in time. Proteomic analysis indicated that RTV-NP distribution was primarily with RE and EE compartments. EE: Early endosomes; LE: Late endosomes; MDM: Monocyte-derived macrophage; NP: Nanoparticle; RE: Recycling endosomes; RTV: Ritovanir; SE: Sorting endosomes.

Figure 3. Immunohistological identification of nanoparticle subcellular localization

(A–H) Confocal microscopy confirmed distribution of RTV-NP within endocytic compartments. Note that RTV-NP are (A–F) red and (G–H) green, and yellow signifies marker-particle overlap in all panels. (I) Pearson’s colocalization coefficients indicate RTV-NPs are preferentially distributed to Rab11 and Rab14 recycling endosomes compared with early endosomes, Rab8 or Rab7 endosomes, and lysosomes. Analysis of distribution of RTV-NP within acidified (degrading) compartments, identified by pHrodo-dextran beads, revealed minimal overlap indicating RTV-NP likely bypass degradation within the cell and are primarily recycled for release. High RTV-NP colocalization with transferrin also indicates that particles are most likely recycled. Measure bars equal 1 μm. Graphical data represent the mean ± standard error of the mean for n = 3. NP: Nanoparticle; RTV: Ritonavir.

Figure 4. Validation of nanoparticle subcellular localization

(A & B) Disruption of endocytic recycling with siRNA (Rab8, 11 and 14) as well as disruption of cell secretion with brefeldin A resulted in knockout of the associated protein and caused RTV-NPs to be redistributed within monocyte-derived macrophages. (A & B) In each case, siRNA treatment resulted in aggregation of RTV-NPs at the perinuclear region within large vacuoles. (C) siRNA silencing of specific proteins was confirmed by Western blot. High-performance liquid chromatography quantitation of RTV-NP in (D) cells and (E) culture fluids demonstrated that disruption of endocytic recycling and inhibition of secretion significantly increased cellular retention of RTV-NPs and reduced release. Upper p-value signifies difference from control cells and lower p-value signifies difference from cells treated with scrambled siRNA. Measure bars equal 1 μm. Graphical data represent the mean ± standard error of the mean for n = 3. NP: Nanoparticle; RTV: Ritonavir.

Figure 5. Ritonavir nanoparticles are transported during endocytic sorting

Since RTV-NPs were labeled with lipophillic dyes (DiD or DiO), which bind to the polymer coat but not the drug crystal itself, we tested whether the endocytic distribution of drug matched that of labeled polymer. (A) Treatment of MDM with RTV-NP and subsequent immune isolation of subcellular compartments and HPLC analysis of drug content. (B) Image of magnetic beads along with immune isolated endosomal compartments prior to HPLC analysis; the white matter on top of the bead pellet in the Rab11 tube was presumably RTV-NP filled endosomes. (C) HPLC analyses of immune isolated compartments confirmed a greater amount of RTV present in Rab11 endosomes than in either EEA1 or LAMP1. Graphical data represent the mean ± standard error of the mean for n = 3. ^†Significantly (p < 0.01) different from control. ^‡Significantly (p < 0.01) different from Rab11. Ab: Antibody; HPLC: High-performance liquid chromatography; MDM: Monocyte-derived macrophage; NP: Nanoparticle; RTV: Ritovanir.

Figure 6. Ritovanir nanoparticles are released intact and retain their antiretroviral efficacy

The data indicated that RTV-NPs were not being degraded but were recycled through the cell, suggesting that intact RTV-NPs should be released. Scanning electron microscopy (magnification 15,000×) of native RTV-NPs (A) and RTV-NPs released from cells into the surrounding medium (B). RTV-NPs were separated from dissolved drug by ultracentrifugation; the percentage of total drug in both particulate and dissolved form is shown. Total drug concentration was 40 μg/ml (C). Monocyte-derived macrophages were treated with either free RTV, native RTV-NP or released RTV-NP and subsequently challenged with HIV. Treatment of monocyte-derived macrophages with released RTV-NP reduced viral infection to similar levels as the native (non-endocytosed) particles as seen by p24 staining and formation of multinucleated giant cells (D), measurement of RT activity (E), and density of p24 staining (F). For both RT activity and p24 density measurements all data represent the mean ± standard error of the mean for n = 4. NP: Nanoparticle; NS: Nonsignificant; RT: Retroviral; RTV: Ritovanir.

Figure 7. Intracellular pathways of ritonavir nanoparticles

RTV-NPs (shown in blue) enter MDM via clathrin-coated pits and are then transported to the early endosome (EE) compartment. From the EE compartment, the particles can have three different fates: fast recycling via Rab4⁺ or 14⁺ endosomes [69,70]; trafficking to late endosome, regulated in part by ESCRT machinery [71] for eventual release as a secretory lysosome [72]; or for most of the particles, transport to the recycling endosome (RE) compartment where they will be stored for long periods and slowly recycled via Rab11⁺ endosomes [–75]. BFA: Brefeldin A; ESCRT: Endosomal sorting complex required for transport; NP: Nanoparticle; RTV: Ritonavir.