NanoART synthesis, characterization, uptake, release and toxicology for human monocyte-macrophage drug delivery

Abstract

Background: Factors limiting the efficacy of conventional antiretroviral therapy for HIV-1 infection include treatment adherence, pharmacokinetics and penetration into viral sanctuaries. These affect the rate of viral mutation and drug resistance. In attempts to bypass such limitations, nanoparticles containing ritonavir, indinavir and efavirenz (described as nanoART) were manufactured to assess macrophage-based drug delivery.

Methods: NanoART were made by high-pressure homogenization of crystalline drug with various surfactants. Size, charge and shape of the nanoparticles were assessed. Monocyte-derived macrophage nanoART uptake, drug release, migration and cytotoxicity were determined. Drug levels were measured by reverse-phase high-performance liquid chromatography.

Results: Efficient monocyte-derived macrophage cytoplasmic vesicle uptake in less than 30 min based on size, charge and coating was observed. Antiretroviral drugs were released over 14 days and showed dose-dependent reduction in progeny virion production and HIV-1 p24 antigen. Cytotoxicities resulting from nanoART carriage were limited.

Conclusion: These results support the continued development of macrophage-mediated nanoART carriage for HIV-1 disease.

Figure 1. Nanoparticle morphology

Scanning-electron microscopy analysis (magnification: 15,000×) of nanoformulations shown include (A) IDV-1, (B) IDV-4, (D) RTV-1, (E) RTV-4, (G) EFV-1 and (H) EFV-3 on top of a 0.2 μm polycarbonate filtration membrane. Scale bar = 2.0 μm. (A) IDV-1 and (B) IDV-4 showed ellipsoid structures with sizes of approximately 1 μm; (D) RTV-1 and (E) RTV-4 showed rod structures with sizes of approximately 550 nm; (G) EFV-1 showed cuboidal structures with sizes of approximately 600 nm while (H) EFV-3 showed spherical structures with sizes of approximately 300 nm. Transmission-electron microscopy (magnification: 15,000×) demonstrated uptake of nanoART into monocyte-derived macrophages exposed to (C) IDV-4, (F) RTV-4 and (I) EFV-3. Within the cells, each type of nanoART is readily identifiable by shape and an example has been outlined in blue for (B & C) IDV, yellow for (E & F) RTV and red for (H & I) EFV. EFV: Efavirenz; IDV: Indinavir; RTV: Ritonavir.

Figure 2. Uptake of indinavir nanoART into monocyte-derived macrophages

Data represent the means ± standard deviation for n = 3 determinations/time point. (A) Levels of IDV from cell lysates of cultured monocyte-derived macrophages treated with 100 μM nanoART and collected at specified times were assayed by reverse-phase high-performance liquid chromatography. The effect of particle size, charge and surfactant on uptake of nanoART was determined. (B) Fluorescent microscopy (time in min) of monocy-derived macrophages (green) labeled with Vybrant DiO cell-labeling solution and cocultured with 100 μM IDV nanoparticles (red) labeled with rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt confirmed intracellular localization of nanoART and demonstrated the effect of surfactant coating on nanoparticle uptake. IDV: Indinavir.

Figure 3. Uptake and release of indinavir, ritonavir and efavirenz nanoART from monocyte-derived macrophages

Data represent the means ± standard deviation for n = 3 determinations/time point. (Uptake) Levels of IDV, RTV or EFV from cell lysates of cultured monocyte-derived macrophages treated with 100 μM nanoART and collected at specified times were assayed by reverse-phase high-performance liquid chromatography. (Release) Levels of IDV, RTV or EFV were assayed over 15 days by reverse-phase high-performance liquid chromatography from cell lysates of cultured monocyte-derived macrophages treated with nanoART (cells) and extracellular media (media) at specified times. EFV: Efavirenz; IDV: Indinavir; RTV: Ritonavir.

Figure 4. Effect of nanoART loading on monocyte and macrophage viability and blood–brain barrier migration

Data represent the means ± standard deviation for n = 3 determinations/treatment. (A) Monocytes and (B) monocyte-derived macrophages were loaded with 0.1 mM IDV-4 or RTV-4 and cell viability assessed 24 h later by the alamarBlue™ assay. At 0.1 mM concentration, IDV-4 did not significantly alter monocyte or macrophage viability, but RTV-4 decreased monocyte viability by approximately 20% (p < 0.05). At 0.1 mM concentration, both IDV-4 and RTV-4 increased monocyte migration across in vitro blood–brain barrier model (C), but the increase was not statistically significant. Control represents monocytes or monocyte-derived macrophages not loaded with nanoparticles. IDV: Indinavir; RTV: Ritonavir.

Figure 5. NanoART effects progeny virion release from virus-infected monocyte-derived macrophages

Comparison of antiretroviral effects of (A) IDV-1 versus IDV-4, (B) RTV-1 versus RTV-4 and (C) EFV-1 versus EFV-3 over 15 days in monocyte-derived macrophages pretreated with 1, 10 or 100 μM of nanoART, as measured by RT activity and normalized to infected control cells. RT activities as measured by ³H-TTP incorporation for infected monocyte-derived macrophages treated with (D) IDV-1, RTV-1 and EFV-1, and (E) IDV-4, RTV-4 and EFV-3. Data represent the least squared means for n = 3 determinations/treatment. EFV: Efavirenz; IDV: Indinavir; RT: Reverse transcriptase; RTV: Ritonavir.

Figure 6. NanoART effects on HIV-1 p24 antigen expression

Comparison of antiretroviral effect of IDV-1 to ID-4, RTV-1 to RTV-4, EFV-1 to EFV-3 over 15 days in monocyte-derived macrophages pretreated with 100 μM of nanoART. A total of 10 days after each viral challenge cells were immunostained for HIV-1p24 antigen (brown). Three photos at a magnification of 4× from each well were quantified by densitometry using Image-Pro Plus, v. 4.0. Expression of p24 was quantified by determining the positive area (index) as a percentage of the total image area per microscopy field. Data represent least squares means of n = 3 determinations/time point. Cells treated with IDV-1, RTV-1 or EFV-1 showed progressive loss of antiretroviral protection and increased HIV p24 expression over time, while cells treated with IDV-4, RTV-4 or EFV-3 showed complete or near complete suppression of viral p24 production. EFV: Efavirenz; IDV: Indinavir; RTV: Ritonavir.