Potential of Newly Synthesized Sea Buckthorn Phytocarriers as Anti-Inflammatory Active Agents

Abstract

Background: Phytocarriers are advanced drug delivery systems that use biocompatible and biodegradable materials to enhance the efficacy, stability, and bioavailability of natural products. The sea buckthorn (Hippophae rhamnoides L.) berry extract is rich in essential fatty acids and antioxidants, including vitamin C, vitamin E, and anthocyanins, which contribute to its wide-ranging health benefits. In this study, we assessed the morphology, intracellular delivery, and anti-inflammatory effect of sodium cholate (NaC) and sodium deoxycholate (NaDC)-based phytocarriers loaded with ethanolic extract from sea buckthorn berries (sea buckthorn carrier nanostructures, further defined as phytocarriers). Methods: Negative and electron cryo-microscopy were used to analyze hollow and loaded nanocarriers. The cyto-compatibility of nanocarriers was assessed by endpoint (LDH and MTS) and real-time cell assays, on both human fibroblasts (HS27) and human normal monocytes (SC). The anti-inflammatory effect of hollow and loaded nanocarriers was tested by multiplexing. Results: The negative and electron cryo-microscopy analyses showed that NaC-based phytocarriers were spherical, whilst NaDC-based phytocarriers were predominantly polymorphic. Moreover, the NaDC-based phytocarriers frequently formed large lipid networks or "plaques". Although 24 h cytotoxicity testing showed both types of nanocarriers are biocompatible with human fibroblasts and monocytes, based on a long-term real-time assay, NaDC delayed fibroblast proliferation. NaC sea buckthorn phytocarriers did not impair fibroblast proliferation in the long term and they were uptaken by cells, as shown by hyperspectral microscopy. NaC nanocarriers and NaC sea buckthorn phytocarriers induced an anti-inflammatory effect, lowering IL-8 cytokine production in normal human monocytes as soon as 4 h of treatment lapsed. Conclusions: NaC-derived phytocarriers loaded with sea buckthorn alcoholic extract are a cell-compatible delivery system with anti-inflammatory properties.

Keywords: IL-8; anti-inflammatory effect; nanocarriers; phytocarriers; sea buckthorn ethanolic extract.

Figure 1

TEM analysis of phytocarriers. NaC hollow nanocarriers were relatively small and highly homogenous in size and shape in both NS-TEM (a) and cryo-TEM (b). By contrast, NaDC hollow nanocarriers were frequently aggregated and appeared polymorphic in NS-TEM (c), whereas individual particles were more electron-dense (darker) and with a comparatively broader size distribution in cryo-TEM (d). NaC phytocarriers were also smaller and more homogenous in both size and shape (e,f), compared to their NaDC counterparts (g,h). The scale bar is 200 nm for all NS-TEM images and 50 nm for all cryo-TEM images.

Figure 2

Viability and proliferation rates of cells treated with NaC and NaDC nanocarriers and phytosomes, assessed by endpoint assay (upper panel) and real-time assay (lower panel). (a) MTS bars and LDH points represent the average of triplicates ± S.D. (b) Real-time impedance graph points represent the average of triplicates ± S.D. Points were registered every 15 min. (c) Doubling times were calculated for 1/100 dilution, and the bars represent the average of triplicates ± S.D. One-way ANOVA, with Dunnet post hoc analysis, was used for the statistical analysis. **** p < 0.0001).

Figure 3

Phase-contrast and dark-field fluorescence hyperspectral imaging of phytocarriers. (a) Mean spectral analysis of phytocarriers obtained via hyperspectral analysis; (b) phagocytosis abilities of normal human macrophages investigated by phase-contrast microscopy (left, 40×) and fluorescence microscopy (60×), using fluorescently labeled E coli particles; (c) digitally highlighted overlayed HSI libraries of NaC phytocarriers and nanocarriers onto macrophage cell cultures (60×), which demonstrated their presence inside the cells. The focal plane was chosen based on the nucleus focal plane.

Figure 4

Dose-dependent pattern inhibition of IL-8 at 4 h (a) and of IL-6 and IL-8 at 20 h (b). One-way ANOVA, Dunnett’s post hoc analysis; * p < 0.05, *** p < 0.001, and **** p < 0.0001.