Development of a macrophage-based nanoparticle platform for antiretroviral drug delivery

Abstract

Complex dosing regimens, costs, side effects, biodistribution limitations, and variable drug pharmacokinetic patterns have affected the long-term efficacy of antiretroviral medicines. To address these problems, a nanoparticle indinavir (NP-IDV) formulation packaged into carrier bone marrow-derived macrophages (BMMs) was developed. Drug distribution and disease outcomes were assessed in immune-competent and human immunodeficiency virus type 1 (HIV-1)-infected humanized immune-deficient mice, respectively. In the former, NP-IDV formulation contained within BMMs was adoptively transferred. After a single administration, single-photon emission computed tomography, histology, and reverse-phase-high-performance liquid chromatography (RP-HPLC) demonstrated robust lung, liver, and spleen BMMs and drug distribution. Tissue and sera IDV levels were greater than or equal to 50 microM for 2 weeks. NP-IDV-BMMs administered to HIV-1-challenged humanized mice revealed reduced numbers of virus-infected cells in plasma, lymph nodes, spleen, liver, and lung, as well as, CD4(+) T-cell protection. We conclude that a single dose of NP-IDV, using BMMs as a carrier, is effective and warrants consideration for human testing.

Figure 1.

NP-IDV synthesis, cell uptake, and release. Images were captured with a Hitachi H7500 Transmission Electron Microscope (TEM) or a Hitachi S3000N variable pressure Scanning Electron Microscope (SEM). (A) SEM analysis (magnification, ×12 000) showed smooth surfaces of NPs with sizes of approximately 1.6 μm. (B) TEM (original magnification, ×20 000) demonstrated uptake of NP-IDV into BMMs (arrowhead). (C) BMM cytoplasm appeared dark by light microscopic examination due to uptake and concentration of NPs after culture in the presence of NP-IDV for 12 hours. (D) Fluorescence microscopy of BMMs cocultured with rDHPE-NP-IDV (red) confirmed intracellular localization of NP-IDV. Ingested NPs appeared as red fluorescent dots and showed intensity located within cytoplasm. (E) Levels of IDV were assayed by HPLC from lysates of cultured BMMs sampled at specified times. (F) After single washout, extracellular (media) and intracellular (BMM) IDV levels were determined. (G) With subsequent media changes, intracellular and extracellular levels of IDV progressively diminished until reaching a nadir at day 6, when IDV levels fell below the limit of detection.

Figure 2.

BMM tissue distribution assessed by SPECT and histologic tests. (A) BMM migration and tissue distribution of ¹¹¹In-labeled BMMs are illustrated by SPECT analysis. Planar presentations of the tomographic images from a representative mouse show that radiolabeled BMMs initially accumulate in the lung (Lun), liver (Liv), and spleen (Spl). (B) To quantify BMM migration, ROIs were circumscribed and radioactive counts determined as a function of time (in days) after adoptive transfer. Data represent mean ± SEM for 4 mice. (C) Histologic analysis of Feridex-labeled BMMs, as determined by Prussian blue staining (blue cells), was consistent with SPECT data. BMMs in liver and lung were less than observed in spleen after day 7. Magnifications are (originals) × 100 and (insets) × 400.

Figure 3.

BMM tissue distribution assessed by MRI tests. MRI tests were used to track BMM migration. Feridex was administered to BMMs in vitro. After Feridex labeling, the BMMs were administered intravenously to immune-competent mice. Signal loss (darkened areas) is shown in the spleen (arrow) over time from 0, 3, 6, and 24 hours (A, B, C, and D, respectively) after adoptive transfer, demonstrating cell migration.

Figure 4.

NP-IDV tissue distribution and pharmacokinetics. (A) Sections of spleen, liver, and lung from mice at day 5 after transfer of rDHPE-NP-IDV-labeled BMMs were stained for CD11b and examined by fluorescence microscopy. Higher magnification inserts demonstrate the presence of rDHPE-NP-IDV (red) colocalized in the cell cytoplasm of CD11b⁺ cells (green). BMMs (yellow) were abundantly present in spleen but were less in liver and lung. (B-E) IDV distribution in targeted tissues and body fluids was assessed in mice treated with a single intravenous dose of (B) IDV sulfate solution, (C) cell-free NP-IDV, or (D-E) NP-IDV-BMMs. In contrast to IDV concentrations in mice treated with NP-IDV-BMMs, nadirs within 6 hours after treatment in mice treated with IDV sulfate solution or NP-IDV, levels were prolonged in tissues and plasma over 14 days in mice treated with NP-IDV-BMMs. Data represent mean ± SEM for 4 mice/group per time point. Magnifications are (originals) × 100 and (insets) × 400.

Figure 5.

Antiretroviral activities of NP-IDV-BMMs in HIV-1-infected hu-PBL-NOD/SCID mice. (A) Serum HIV-1p24 levels (mean ± SEM) from HIV-1-challenged hu-PBL-NOD/SCID mice untreated (control) or treated with NP-IDV-BMMs after days 7 and 14. Spleen sections from (B) untreated or (C) NP-IDV-BMM-treated mice were immunostained for CD3 (pink) and HIV-1p24 (brown). CD3⁺ T cells were observed at comparable frequencies, whereas HIV-1p24-positive cells were vastly diminished in NP-IDV-BMM-treated mice. ^*P < .05 compared with untreated controls. Magnification is (originals) ×200 and (insets) ×400. (D) Distribution of human PBLs (Vim⁺) and HIV-1-infected cells (HIV-1p24⁺) were evaluated in lymph nodes, spleen, liver, and lung from HIV-1-infected mice that were untreated or treated with NP-IDV-BMMs. Magnification is ×200.

Figure 6.

Quantitation of HIV-1p24-expressing cells in NP-IDV-BMM-treated HIV-1-infected hu-PBL-NOD/SCID mice. (A) Numbers of human PBLs in cervical lymph nodes (C-LN), M-LN, spleen, liver, and lung were determined from Vim⁺ immunostained sections. (B) Numbers of HIV-1p24⁺ cells were normalized to total Vim⁺ cells and expressed as the mean percent (± SEM). ^*P < .05 and ^**P < .01 compared with untreated controls.

Figure 7.

T-cell subset analyses in hu-PBL-NOD/SCID mice after NP-IDV-BMM treatment. (A) Flow cytometric histograms of splenocytes from mice 14 days following NP-IDV-BMM treatments were used to determine the numbers of mouse CD45 cells and human reconstituted CD4⁺ and CD8⁺ T cells. (B) The level of human lymphocyte reconstitution in mouse spleens was determined compared with mouse CD45⁺ cells. (C-D) Frequencies of (C) CD4⁺ and (D) CD8⁺ T cells were assessed as percentages of total human T cells. (E) Ratios of CD4⁺/CD8⁺ T cells increased significantly in NP-IDV-BMM-treated group compared with control animals. Data represent mean ± SEM for 4-6 mice/group. ^*P < .05 and ^**P < .01 compared with untreated controls.