Lipid-protein nanovesicles of membrane from "Brassica oleracea L. var. italica" as carriers of bioactive compounds

Abstract

The use of nanocarriers of bioactive compounds or drugs has become more widespread in recent years due to the advantages they bring to the cosmetic and pharmaceutical industries. The development of new encapsulation systems focuses on obtaining nanocarriers of natural origin, such as membrane vesicles. This PhD thesis proposes developing a new encapsulation system for biotechnological applications based on cell membranes obtained from broccoli plants. Broccoli is a crop of great interest in Spain (the fourth largest producer in the world) and in the Region of Murcia, which contributes 70% of the national export of broccoli. This type of crop generates a large amount of waste and by-products. Due to the worldwide scarcity of resources and the environmental problem of waste management, it is necessary to find applications for them. During the experiments carried out in this thesis, membrane vesicles obtained from both broccoli roots and leaves were used to develop and implement their use as nanocarriers of bioactive compounds such as sulforaphane in skin-related pharmaceutical or cosmetic applications. Physicochemical analyses, proteomic studies, microscopy analyses, stability and in vitro release tests, as well as, cell culture assays were carried out. In the latter, cell permeability, cytotoxicity, gene expression, and inflammatory cytokine production assays were performed to determine the functionality and applicability of broccoli membrane vesicles with encapsulated sulforaphane. From the results obtained, it can be determined that broccoli membrane vesicles have a high encapsulation efficiency and good stability under in vitro conditions and when introduced into a cosmetic formulation for at least one year. Furthermore, related to a crucial aspect in transdermal applications, it was determined that membrane vesicles are able to cross the stratum corneum, reach the inner layers of the skin and release the encapsulated compounds into the cells, resulting in an interaction between plant and human membranes. In cell culture experiments, the application of encapsulated sulforaphane revealed significant anti-proliferative activity in cancer cells and anti-inflammatory activity in macrophages. Enhanced uptake of sulforaphane by melanoma cells was observed when applied in the encapsulated form in membrane vesicles, as well as inhibition of cell proliferation and triggering an increase in gene expression of p53, which encodes for a tumour suppressor protein. On the other hand, regarding the application of sulforaphane encapsulated in broccoli membrane vesicles in human macrophages, the results showed a decrease in the inflammatory interleukins TNF-α, IL-1β, and IL-6. From all the assays performed in the thesis, it was further concluded that broccoli membrane vesicles themselves have potent bioactivity, both anticancer and anti-inflammatory. They were found to contain bioactive compounds such as isothiocyanates and antioxidant proteins, which along with the lipids that make up the structure of the vesicles, will determine the activity of the membrane vesicles. Overall, broccoli membrane vesicles could be incorporated as a complementary or main component of cosmetic formulations or topically applied drugs to treat skin disorders related to inflammation and oxidative stress. They could be used as carriers for other compounds or as bioactive in their own right. The results presented in this thesis leave many lines of research open for further investigation until a system with potential applications in the biotechnology industry is obtained.