Empowering Naringin's Anti-Inflammatory Effects through Nanoencapsulation

Abstract

Abundant in citrus fruits, naringin (NAR) is a flavonoid that has a wide spectrum of beneficial health effects, including its anti-inflammatory activity. However, its use in the clinic is limited due to extensive phase I and II first-pass metabolism, which limits its bioavailability. Thus, lipid nanoparticles (LNPs) were used to protect and concentrate NAR in inflamed issues, to enhance its anti-inflammatory effects. To target LNPs to the CD44 receptor, overexpressed in activated macrophages, functionalization with hyaluronic acid (HA) was performed. The formulation with NAR and HA on the surface (NAR@NPs_HA) has a size below 200 nm, a polydispersity around 0.245, a loading capacity of nearly 10%, and a zeta potential of about 10 mV. In vitro studies show the controlled release of NAR along the gastrointestinal tract, high cytocompatibility (L929 and THP-1 cell lines), and low hemolytic activity. It was also shown that the developed LNPs can regulate inflammatory mediators. In fact, NAR@NPs_HA were able to decrease TNF-α and CCL-3 markers expression by 80 and 90% and manage to inhibit the effects of LPS by around 66% for IL-1β and around 45% for IL-6. Overall, the developed LNPs may represent an efficient drug delivery system with an enhanced anti-inflammatory effect.

Keywords: anti-inflammatory activity; hyaluronic acid; lipid nanoparticles; macrophages; naringin.

Figure 1

Particle size determination using NTA: (a) NPs; (b) NAR@NPs, (c) NPs_HA, and (d) NAR@NPs_HA. Average size distribution following 5 measurements. The images correspond to NTA video frames.

Figure 2

Physical stability of developed NPs over time (0, 1, 2, 3, 4, 5, 6, and 9 weeks): (a) size, (b) polydispersity index, (c) zeta potential, and (d) encapsulation efficiency. Values represent mean ± SD (n = 3). Differences between groups were assessed using two-way ANOVA followed by Dunnett test. ^• p < 0.05, ^•• p < 0.01, ^••• p < 0.001 and ^•••• p < 0.0001 relatively to the correspondent week 0.

Figure 3

Transmission electron microscopy photographs of functionalized and non-functionalized lipid nanoparticles. Magnification 80,000×. Scale bar: 100 nm.

Figure 4

Cumulative NAR release for NAR@NPs, NAT@NPs_CTAB, and NAR@NPs_HA. Formulations for cumulative release profiles simulated in three conditions after oral administration. Vertical dashed lines represent mimetic medium changes: (i) gastric media, (ii) intestinal media, and (iii) physiological media. Free NAR was used as control. Values represent mean ± SD (n = 3).

Figure 5

Effect of developed nanoparticles, free HA, and free NAR, at different concentrations after 24 h of incubation, on the viability of (a) fibroblasts (L929 cell line), (b) monocytes (THP-1 cell line), and (c) macrophages (THP-1 cell line). Data are expressed as mean ± SD (n = 3). Differences between groups were assessed using two-way ANOVA followed by Dunnett test. ^• p < 0.05, ^•• p < 0.01, ^••• p < 0.001 and ^•••• p < 0.0001 in comparison to the positive control.

Figure 6

Cell cytotoxicity assessed by LDH assay for developed nanoparticles, free HA, and free NAR, at different concentrations after 24 h of incubation, on the viability of (a) fibroblasts (L929 cell line), (b) monocytes (THP-1 cell line), and (c) macrophages (THP-1 cell line). Data are expressed as mean ± SD (n = 3). Differences between groups were assessed using two-way ANOVA followed by Dunnett test. ^• p < 0.05, ^•• p < 0.01, ^••• p < 0.001 and ^•••• p < 0.0001 in comparison to the positive control.

Figure 7

Hemolysis percentage obtained for the developed nanoparticles. Data are expressed as mean ± SD (n = 3). Dashed line is indicative of a hemolytic rate of 5%. Differences between groups were assessed using two-way ANOVA followed by Dunnett test. ^• p < 0.05 and ^•••• p < 0.0001 in comparison to the free NAR.

Figure 8

(a) Cellular uptake of fluorescence-marked nanoparticles in THP-1 macrophages over time (0.5, 1, 2, 3, 4, and 24 h), at a concentration of 0.25 µg·mL⁻¹. Data are expressed as mean ± SD (n = 3). Differences between groups were assessed using two-way ANOVA followed by Tukey test. ^•• p < 0.01, ^••• p < 0.001, and ^•••• p < 0.0001 in comparison to the NPs. (b) Effect of low temperature and pathway mechanism inhibitors on the uptake of fluorescence-marked nanoparticles by THP-1 macrophages. Data are expressed as mean ± SD (n = 3). Differences between groups were assessed using two-way ANOVA followed by Dunnett test. ^• p < 0.05, ^•• p < 0.01, ^••• p < 0.001 and ^•••• p < 0.0001 in comparison to the control at 37 °C.

Figure 9

ELISA for interleukin secretion: (a) IL-1β, (b) IL-6, (c) IL-8, (d) TNF-α, and (e) CCL-3. Cells were pre-incubated with 0.5 µg·mL⁻¹ of LNPs and controls (free NAR and free HA), for 2 h and then were stimulated to M1 macrophages with LNPs and incubated for 24 h. Data are expressed as mean ± SD (n = 3). Differences between groups were assessed using one-way ANOVA followed by Dunnett test. ^• p < 0.05, ^•• p < 0.01 and ^••••p < 0.0001 in comparison to the LPS.

Figure 10

(a) Reactive oxygen species (ROS) scavenging activity was measured by DCFH-DA assay. Data are expressed as mean ± SD (n = 3). Differences between groups were assessed using one-way ANOVA followed by Dunnett test. ^•••• p < 0.0001 in comparison to the H₂O₂. (b) Confocal microscopy for ROS detection. THP-1 macrophages were treated for 24 h in the absence (control) and in the presence of developed NPs, NAR, or HA. Cells were then stained for ROS (2′,7′-dichlorofluorescein diacetate—DCFH-DA, green), nucleus (Hoechst 33342, blue), and cell membrane (CellMask^TM, red). Scale bar: 20 µm.