Modulation of Inflammatory Mediators by Polymeric Nanoparticles Loaded with Anti-Inflammatory Drugs

Abstract

The first-line treatment of osteoarthritis is based on anti-inflammatory drugs, the most currently used being nonsteroidal anti-inflammatory drugs, selective cyclooxygenase 2 (COX-2) inhibitors and corticoids. Most of them present cytotoxicity and low bioavailability in physiological conditions, making necessary the administration of high drug concentrations causing several side effects. The goal of this work was to encapsulate three hydrophobic anti-inflammatory drugs of different natures (celecoxib, tenoxicam and dexamethasone) into core-shell terpolymer nanoparticles with potential applications in osteoarthritis. Nanoparticles presented hydrodynamic diameters between 110 and 130 nm and almost neutral surface charges (between -1 and -5 mV). Encapsulation efficiencies were highly dependent on the loaded drug and its water solubility, having higher values for celecoxib (39-72%) followed by tenoxicam (20-24%) and dexamethasone (14-26%). Nanoencapsulation reduced celecoxib and dexamethasone cytotoxicity in human articular chondrocytes and murine RAW264.7 macrophages. Moreover, the three loaded systems did not show cytotoxic effects in a wide range of concentrations. Celecoxib and dexamethasone-loaded nanoparticles reduced the release of different inflammatory mediators (NO, TNF-α, IL-1β, IL-6, PGE₂ and IL-10) by lipopolysaccharide (LPS)-stimulated RAW264.7. Tenoxicam-loaded nanoparticles reduced NO and PGE₂ production, although an overexpression of IL-1β, IL-6 and IL-10 was observed. Finally, all nanoparticles proved to be biocompatible in a subcutaneous injection model in rats. These findings suggest that these loaded nanoparticles could be suitable candidates for the treatment of inflammatory processes associated with osteoarthritis due to their demonstrated in vitro activity as regulators of inflammatory mediator production.

Keywords: celecoxib; dexamethasone; inflammatory mediators; nanoparticles; osteoarthritis; tenoxicam.

Figure 1

Physicochemical properties of CLX, TNX and DEX-loaded NPs. (A) NP suspensions obtained by the nanoprecipitation method. Image created using ChemDraw. (B) NP size distribution immediately after synthesis. (C) Hydrodynamic diameter (D_h), polydispersity index (PDI) and zeta potential (ξ) values of NPs. (D) SEM micrographs of CLX-10, TNX-5 and DEX-15 NPs. Scale bar: 300 nm.

Figure 2

Effect of (A) CLX, (B) TNX and (C) DEX NPs on HC-a viability after 24 and 48 h, 7 and 14 days. Mean ± SD values are relative to control cells without NP treatment (CNT) for each time, in which cell viability was taken as 100%. ANOVA of the results was performed with respect to their corresponding CNT at a significance level of p < 0.05 (a, b, c and d correspond to statistical significance for 24 h, 48 h, 7 and 14 days samples, respectively).

Figure 3

(A) Effect of CLX, TNX and DEX NPs on RAW264.7 viability after 24 h. Mean ± SD values are relative to control cells without NP treatment (CNT), in which cell viability was taken as 100%. ANOVA of the results was performed with respect to CNT at a significance level of * p < 0.05. (B) Effect of CLX, TNX and DEX NPs on NO production in LPS-stimulated cells. Mean ± SD values are relative to control LPS+, in which NO production was taken as 100%. CLX-20 NPs at a concentration of 1.00 mg mL⁻¹ were not tested since they showed cytotoxic effects on the AlamarBlue^® viability test. ANOVA of the results was performed with respect to LPS+ at significance levels of * p < 0.05, ** p < 0.005 and *** p < 0.001.

Figure 4

Effect of CLX-10 and DEX-15 NPs (0.50 mg mL⁻¹) on the production of inflammatory mediators that were upregulated in LPS-stimulated RAW264.7 (LPS) using a mouse inflammation antibody array. Mean ± SD values are relative to the positive control of each membrane, which was given an arbitrary value of 1. ANOVA between inflammatory (LPS) and treatment membranes (LPS + CLX-10 and DEX-15) was performed at significance levels of * p < 0.05 and *** p < 0.001.

Figure 5

Effect of CLX-10, TNX-5 and DEX-15 (0.50 mg mL⁻¹) on the release of the inflammatory mediators TNF-α, IL-1β, IL-6, PGE₂ and IL-10 in LPS+ RAW264.7 using ELISA kits. Data are represented as mean ± SD values. ANOVA between each NP formulation and LPS+ (* p < 0.05 and *** p < 0.001) and between NPs (^# p < 0.05 and ^### p < 0.001) were performed.

Figure 6

Representative histological photomicrographs of skin cross-sections of rats subcutaneously injected with CLX-10, TNX-5 and DEX-15 NPs compared to the control group after one week. In the image corresponding to CLX-10-treated rats, some tissue reaction (squared and amplified in Figure S5) was observed, mainly based on macrophage infiltration (H-E).

Figure 7

Representative histological photomicrographs of skin cross-sections of rats subcutaneously injected with CLX-10, TNX-5 and DEX-15-loaded NPs compared to the control group after two weeks. (H-E).