Glioprotective Effect of Chitosan-Coated Rosmarinic Acid Nanoemulsions Against Lipopolysaccharide-Induced Inflammation and Oxidative Stress in Rat Astrocyte Primary Cultures.

Rosmarinic acid (RA) is a natural polyphenolic compound with a well-documented neuroprotective effect mainly associated with its anti-inflammatory and antioxidant activities. Recently, our research group developed and optimized chitosan-coated RA nanoemulsions (RA CNE) intended to be used for nasal delivery as a new potential neuroprotective therapy. In this sense, the present study aimed to evaluate the protective and/or therapeutic potential of RA CNE in inflammation/oxidative stress induced by LPS (1 µg mL-1) in rat astrocyte primary cultures. In summary, pre-treatment with RA CNE before exposure to LPS (protective protocol) reduced significantly the LPS-induced alterations in astrocyte cell viability, proliferation, and cell death by necrosis, which was not observed in therapeutic protocol. RA CNE protective protocol also enhanced anti-oxidative status by ~ 50% by decreasing oxygen reactive species production and nitric oxide levels and preventing total thiol content decrease. Finally, our results demonstrate the protective effect of RA CNE in migratory activation and GFAP expression of reactive astrocytes. Overall, our findings indicate for the first time the RA CNE glioprotective potential, associated with an increase in cell viability and proliferation, a preventive effect on cellular death by necrosis, migratory ability and hypertrophic reactive astrocytes, and the reparation of astrocyte redox state.

Incorporation of 3,3'-Diindolylmethane into Nanocapsules Improves Its Photostability, Radical Scavenging Capacity, and Cytotoxicity Against Glioma Cells.

3,3'-Diindolylmethane (DIM) is a phytochemical that presents health benefits (antitumor, antioxidant, and anti-inflammatory effects). However, it is water insoluble and thermo- and photolabile, restraining its pharmaceutical applications. As a strategy to overcome such limitations, this study aimed the development and characterization of DIM-loaded nanocapsules (NCs) prepared with different compositions as well as the in vitro assessment of scavenging activity and cytotoxicity. The formulations were obtained using the interfacial deposition of preformed polymer method and were composed by Eudragit® RS100 or ethylcellulose as polymeric wall and primula or apricot oil as the core. All the formulations had adequate physicochemical characteristics: nanometric size (around 190 nm), low polydispersity index (< 0.2), pH value at acid range, high values of zeta potential, drug content, and encapsulation efficiency (~ 100%). Besides, nanoencapsulation protected DIM against UVC-induced degradation and increased the scavenging activity assessed by the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and 1-1-diphenyl-2-picrylhydrazyl methods. The developed DIM-loaded nanocapsules were further evaluated regarding the in vitro release profile and cytotoxicity against a human glioblastoma cell line (U87 cells). The results demonstrated that the nanoencapsulation promoted a sustained release of the bioactive compound (in the range of 58-78% after 84 h) in comparison to its free form (86% after 12 h), as well as provided a superior cytotoxic effect against the U87 cells in the highest concentrations. Therefore, our results suggest that nanoencapsulation could be a promising approach to overcome the DIM physicochemical limitations and potentialize its biological properties.

Hydrogels containing soybean isoflavone aglycones-rich fraction-loaded nanoemulsions for wound healing treatment - in vitro and in vivo studies.

Soybean isoflavone aglycones have been investigated as potential wound healing compounds for topical application. The aim of this study was to evaluate the wound healing properties of a soybean isoflavone aglycones-rich fraction (IAF) when incorporated into lipid nanoemulsions dispersed in acrylic-acid hydrogels. Formulations exhibited a mean droplet size in the sub 200 nm range, negative ζ-potential (-60 mV), and displayed non-Newtonian pseudoplastic behavior. The addition of a gelling agent decreased the IAF release from formulations and improved the retention of these compounds in intact porcine ear skin when compared with a control propylene glycol solution. No IAF were detected in receptor fluid of Franz-type diffusion cells. However, increasing amounts of IAF were noticed in both skin layers and the receptor fluid when the tissue was partially injured (tape stripping), or when the epidermis was completely removed. In vitro studies showed that IAF elicits an increased proliferation and migration of keratinocytes (HaCaT cell line). Subsequently, the healing effect of the formulations was evaluated in a model of dorsal wounds in rats, by assessing the size of the lesions, histology, inflammatory markers, and antioxidant activity. Overall findings demonstrated the potential of IAF-loaded formulations to promote wound healing by increasing angiogenesis by ∼200 %, reducing the lipid oxidation (TBARS) by ∼52 % and the inflammation (TNFα) by ∼35 %, while increasing re-epithelialization by ∼500 %, visualized by the epithelium thickness.

Development, physico-chemical characterization and in-vitro studies of hydrogels containing rosmarinic acid-loaded nanoemulsion for topical application.

OBJECTIVES: Natural sources with antioxidant activity, such as rosmarinic acid (RA), have been considered as an interesting approach for the development of new anti-ageing skin products. In this context, this study aimed to develop hydrogels containing RA-loaded nanoemulsions and evaluate the effect of the addition of Tween® 80 (a nonionic cosurfactant) in formulations intended to be used for topical application. METHODS: Physico-chemical characterization, in-vitro release and skin retention/permeation from hydrogels of RA-loaded nanoemulsions (containing or not Tween® 80) were evaluated. The RA-loaded nanoemulsion safety profiles were also investigated in keratinocytes (HaCaT cells). KEY FINDINGS: It was observed that all formulations presented adequate physico-chemical characterization for topical application. Furthermore, the results also demonstrated that the presence of Tween® 80 decreased the droplet size and polydispersity index of nanoemulsions and hydrogels. An extended RA release was noted for the hydrogels. However, when comparing the hydrogels, a positive effect of the presence of Tween® 80 on RA retention/permeation in total skin was seen. The RA-loaded nanoemulsion safety profiles demonstrated a good tolerability (3.125-100 µm) in HaCaT cells. CONCLUSIONS: The overall results demonstrated that the formulations developed in this study can be considered as a suitable carrier for RA in a topical application targeting new anti-ageing skin care products.

Box-Behnken design optimization of mucoadhesive chitosan-coated nanoemulsions for rosmarinic acid nasal delivery-In vitro studies.

Mucoadhesive chitosan-coated nanoemulsions for rosmarinic acid (RA) nasal delivery were optimized. The optimum ratio between the formulation components that led to minimum droplet size and PDI, and maximal ζ-potential and RA content was obtained using Box-Behnken design (BBD). Optimized conditions were 8.5% oil phase (w/v), 3:10 lecithin to oil phase ratio (w/w), and 0.1% chitosan final concentration (w/v). Physicochemical characterization, mucoadhesion measurement, in vitro release and permeation/retention were performed. Optimized chitosan-coated RA nanoemulsions presented adequate physicochemical characteristics, high mucoadhesive potential, prolonged drug release, and long-lasting permeation time with a higher RA penetration/retention through porcine nasal mucosa. Cell viability and death by necrosis in fibroblasts cells were also evaluated to investigate the formulations safety. Formulations did not induce cytotoxicity following 24 h (3.125-50 µM) or 48 h (3.125-25 µM) of treatments. Overall results demonstrated that optimized chitosan-coated nanoemulsion showed to be a suitable carrier for RA nasal delivery aiming neuroprotective therapies.