Macrophage folate receptor-targeted antiretroviral therapy facilitates drug entry, retention, antiretroviral activities and biodistribution for reduction of human immunodeficiency virus infections

Abstract

Macrophages serve as vehicles for the carriage and delivery of polymer-coated nanoformulated antiretroviral therapy (nanoART). Although superior to native drug, high drug concentrations are required for viral inhibition. Herein, folate-modified ritonavir-boosted atazanavir (ATV/r)-encased polymers facilitated macrophage receptor targeting for optimizing drug dosing. Folate coating of nanoART ATV/r significantly enhanced cell uptake, retention and antiretroviral activities without altering cell viability. Enhanced retentions of folate-coated nanoART within recycling endosomes provided a stable subcellular drug depot. Importantly, up to a five-fold enhanced plasma and tissue drug levels followed folate-coated formulation injection in mice. Folate polymer encased ATV/r improves nanoART pharmacokinetics bringing the technology one step closer to human use.

From the clinical editor: This team of authors describes a novel method for macrophage folate receptor-targeted antiretroviral therapy. Atazanvir entry, retention, and antiretroviral activities were superior using the presented method, and so was its biodistribution, enabling a more efficient way to address human immunodeficiency virus infections, with a hoped for clinical application in the near future.

Keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ATV; ATV/r; BSA; CHO; Chinese hamster ovary; FA; FOLR; Folate; Human immunodeficiency virus; LAMP; MDM; MTT; Macrophages; NanoART; PBS; PDI; PFA; Poloxamer 407; RME; RT; RTV; Targeted drug delivery; atazanavir; bovine serum albumin; folate receptor; folic acid; lysosomal associated membrane protein; monocyte-derived macrophages; nanoART; nanoformulated antiretroviral therapeutics; paraformaldehyde; phosphate buffered saline; polydispersity index; receptor-mediated endocytosis; reverse transcriptase; ritonavir; ritonavir-boosted atazanavir.

Figure 1. Synthesis of folate conjugated poloxamer 407 (FA-P407)

Schematic representation of the modifications for (A) poloxamer 407 and (B) folate and subsequent reaction to yield (C) FA-P407. (D) Scanning electron micrographs of FA-P407-ATV and P407-ATV.

Figure 2. Characterization of folate receptor (FOLR) on MDM

(A) Western blot for FOLR1 and FOLR2. (B) Confocal image demonstrating FOLR2 expression on MDMs. FOLR2 (green) and nuclei (blue). Scale bar = 10 μm. Characterization of purified FA-P407-ATV. (C) ¹H NMR spectra of nanosuspension before purification (inserts show peaks for P407 and ATV in supernatant and FA-P407-ATV pellet after purification) (D) Folic acid quantification in the supernatant and FA-P407-ATV pellet before and after purification. (E) MDM uptake of purified FA-P407-ATV compared to unpurified starting material and non-targeted P407-ATV. Data are expressed as mean ± SEM, n = 3.

Figure 3. Uptake, retention, release and antiretroviral efficacy of FA-P407-ATV in MDM

(A) Time course for MDM uptake of ATV nanoformualtions. Macrophages were administered with 100 μM FA-P407-ATV containing either 0%, 20%, or 40% FA-P407 for 8 hours. Timecourse of (B) MDM retention and (C) release of nanoART into medium over 15 days following an 8 hour loading with 100 μM nanoART. (D) Inhibition of MDM uptake of 100 μM FA-P407-ATV by pre-incubation with 1.5–50 mM free FA for 45 minutes. Data are expressed as mean ± SEM, n = 3. (E) Antiretroviral efficacy of FA-P407-ATV in MDM infected with HIV-1_ADA for 1, 5, 10 and 15 days after drug treatment. Level of infection was determined by RT activity in culture supernatant fluids. The left logarithmic y-axis shows RT levels of HIV-1 infected cells without drug. The right linear y-axis shows data for control (uninfected), FA-P407-ATV and P407-ATV treated cells. Data are expressed as mean ± SEM, n = 3. Statistical differences were determined using Student’s t-test; *P < 0.05 considered significant. (F) HIV-1 p24 (brown) staining for MDM challenged with HIV-1_ADA at 1, 5, 10 and 15 days after FA-P407-ATV treatment. Images are representations of 3 identically treated wells.

Figure 4. Subcellular localization of FA-P407-ATV and P407-ATV

MDM were cultured for 7 days on LabTek CC2 chamber slides and treated with 100 μM CF633-labeled P407-ATV or FA-P407-ATV for 8 hours. (A) The cells were stained with LAMP1, Rab7, Rab11, or Rab14 primary antibodies and AlexaFluor 488-labeled secondary antibodies to visualize the corresponding cell compartments using confocal microscopy. Nanoparticles are shown in red, cell compartments in green, and nuclei in blue. Quantitation of subcellular compartment co-localization of FA-P407-ATV and P407-ATV. (B) Ratio of nanoART co-localized with cell compartments (intensity) to total intensity of the compartment in the cell (n=40) was determined by Zeiss LSM 510 Image browser version 4.2. (C) Overlap coefficients for each of the cell compartments with CF633-FA-P407-ATV. Statistical analysis was performed by 2 way ANOVA followed by Bonferroni’s Multiple Comparison; *P < 0.05 considered significant (**P < 0.01, ***P < 0.001).

Figure 5. Comparison of pharmacokinetics and biodistribution of FA-P407-nanoART and P407-nanoART in mice

Balb/cJ mice were administered 50 mg/kg (each drug) FA-P407-ATV/r or P407-ATV/r by intramuscular injection. (A) Plasma and (B, C) tissues were collected at indicated days and ATV levels determined by UPLC-MS/MS. Data are means ± SEM.