Encapsulation of the dual FLAP/mPEGS-1 inhibitor BRP-187 into acetalated dextran and PLGA nanoparticles improves its cellular bioactivity

Abstract

Background: Dual inhibitors of the 5-lipoxygenase-activating protein (FLAP) and the microsomal prostaglandin E₂ synthase-1 (mPGES-1) may exert better anti-inflammatory efficacy and lower risks of adverse effects versus non-steroidal anti-inflammatory drugs. Despite these advantages, many dual FLAP/mPGES-1 inhibitors are acidic lipophilic molecules with low solubility and strong tendency for plasma protein binding that limit their bioavailability and bioactivity. Here, we present the encapsulation of the dual FLAP/mPGES-1 inhibitor BRP-187 into the biocompatible polymers acetalated dextran (Acdex) and poly(lactic-co-glycolic acid) (PLGA) via nanoprecipitation.

Results: The nanoparticles containing BRP-187 were prepared by the nanoprecipitation method and analyzed by dynamic light scattering regarding their hydrodynamic diameter, by scanning electron microscopy for morphology properties, and by UV-VIS spectroscopy for determination of the encapsulation efficiency of the drug. Moreover, we designed fluorescent BRP-187 particles, which showed high cellular uptake by leukocytes, as analyzed by flow cytometry. Finally, BRP-187 nanoparticles were tested in human polymorphonuclear leukocytes and macrophages to determine drug uptake, cytotoxicity, and efficiency to inhibit FLAP and mPGES-1.

Conclusion: Our results demonstrate that encapsulation of BRP-187 into Acdex and PLGA is feasible, and both PLGA- and Acdex-based particles loaded with BRP-187 are more efficient in suppressing 5-lipoxygenase product formation and prostaglandin E₂ biosynthesis in intact cells as compared to the free compound, particularly after prolonged preincubation periods.

Keywords: Acetalated dextran; BRP-187; Dual inhibitor; FLAP inhibitor; Leukotriene biosynthesis; MPGES-1; Nanoparticles; PLGA.

Fig. 1

SEM images of NPs: Acdex (a), Acdex[BRP-187] (b), PLGA (c), PLGA[BRP-187] (d)

Fig. 2

Degradation of NPs at physiological (7.4) and acidic (4.8) pH values: Acdex and Acdex[BRP-187] (a, b); and PLGA and PLGA[BRP-187] (c, d) measured by DLS (n = 1)

Fig. 3

PMNL (1 × 10⁶) were incubated with 0.5 mg mL⁻¹ PLGA-DY635[BRP-187] or Acdex-RhoB[BRP-187] NPs for the indicated time points at 37 °C. a Percentage of PMNL gated as positive due to the fluorescence signal of the dye-labeled NPs in the cell. b, c Mean fluorescence intensity (MFI) of NPs in the cell, measured by flow cytometry. For statistics analysis, a multiple t-test was used; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); n = 4

Fig. 4

Confocal laser scanning microscopy of PMNL after isolation (a, g) and incubation at 37 °C with NPs for the indicated timepoints (b–f and h–l). Micrographs in the upper rows display the individual fluorescence channel while images in the bottom rows display the additional overlay of the transmitted light channel

Fig. 5

M1 macrophages (a) or M2 macrophages (b) were incubated with control (0.1% DMSO), free BRP-187 (10 µM), NP without drug, BRP-187 (10 µM) encapsulated into NP, or positive control (staurosporin 3 µM) for 24 h. Then, the release of LDH was analyzed using the Promega Cytotox 96^® assay. Values are normalized to the DMSO control (100%) and given as percentage of cell viability; data are means + S.E.M., n = 3

Fig. 6

PMNL (5 × 10⁶) were preincubated with control (0.1% DMSO), BRP-187, Acdex, PLGA, Acdex[BRP-187] and PLGA[BRP-187] NPs for the indicated time points at 37 °C. The cells were then stimulated with 2.5 µM A23187 for 10 min, and 5-LO product formation was analyzed. Values are given as ng of 5-LO products (sum of the trans-isomers of leukotriene B₄ (LTB₄), LTB₄ and 5-hydroxyeicosatetraenoic acid (5-HETE)). For statistical analysis, one-way ANOVA and Tukey’s multi comparison test was performed. p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); n = 4

Fig. 7

Determination of PGE₂ formation in human macrophages: M1 macrophages (2 × 10⁶) were preincubated with BRP-187 or with BRP-187 encapsulated into Acdex or PLGA NPs for the indicated timepoints at 37 °C. Cells were then exposed to E. coli (O6:K2:H1), MOI = 50. After 90 min at 37 °C the reaction was stopped and PGE₂ was analyzed after solid phase extraction (SPE) by UPLC-MS/MS. Values are given as pg of PGE₂ per 2 × 10⁶ M1. For statistical analysis one-way ANOVA (p < 0.0001) and a Tukey‘s multi comparison test was performed. p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); n = 3–4