In Vitro Delivery of Brassica oleracea L. (Broccoli)- Derived Nanovesicles (BDNVs) to Lung Epithelial Cell Lines as Potential approach for the treatment of Chronic Respiratory Diseases

In this in vitro study, we test our hypothesis that Broccoli-derived nanovesicles (BDNVs), combining the anti-oxidant properties of their components and the advantages of their structure, can influence the metabolic activity of different cancer cell lines. BDNVs were isolated from homogenized fresh broccoli (Brassica oleracea L.) using a sucrose gradient ultracentrifugation method and were characterized in terms of physical properties, such as particle size, morphology, and surface charge by transmission electron microscopy (TEM), Cryo transmission electron microscopy (Cryo-TEM), Nanoparticle Tracking Analysis (NTA) and laser doppler electrophoresis (LDE). Glucosinolates (GSLs) content was assessed by RPLC–ESI–MS analysis and Sulforaphane (SFN) content was assessed by LC-MS/MS. Four therapeutically potent molecule were identified by Global Natural Product Social Molecular Networking (GNPS) library searching. Four different human cancer cell lines (colorectal adenocarcinoma Caco-2, lung adenocarcinoma NCI-H441, A549 and neuroblastoma SHSY5Y) as well as two non cancer cell lines (differentiated THP-1, diff.THP-1 and immortalized human alveolar type I-like cell, hAELVi) were evaluated for metabolic activity by the MTT assay. Cellular uptake of BDNVs by cancer cell lines (Caco-2, NCI-H441 and SHSY5Y) was evaluated by fluorescence as well as confocal microscopy whereas cellular uptake by non-cancer cell lines (diff. THP-1, hAELVi) was evaluated by confocal microscopy. Anti-oxidant activity was evaluated by a fluorimetric assay detecting intracellular reactive oxygen species (ROS) considering Caco-2, NCI-H441 and SHSY5Y cells. Anti-inflammatory activity was evaluated as a means of cytokine gene expression (TNF, IL-6, IL-8 and IL-10) by RT-qPCR as well as a means of measurement of cytokine release (TNF, IL-6 and IL-10) by Cytokine Cytometric Bead Array (CBA) method. Suitability of the freeze-drying method for the storage of BDNVs was also analyzed and long term stability study was assessed for this condition along with conventional -80ᵒC and 4ᵒC storage conditions. Co-culture model of hAELVi and diff.THP-1 was used for assessing the epithelial tightness and barrier integrity after treated with BDNVs. Three bands were obtained with average size measured by TEM based size distribution analysis of 52 nm (Band 1), 70 nm (Band 2), & 82 nm (Band 3) as well as by NTA based size distribution analysis of 170 nm (B 1), 173 nm (B 2), & 179 nm (B 3) and by Cryo-TEM size distribution analysis of 100 nm (B 1), 101 nm (B 2), & 113 nm (B 3). Glucobrassicin, glucoraphanin and neoglucobrassicin were found mostly concentrated in Band 1. Sulforaphane was also found mostly concentrated in Band 1. BDNVs affected the metabolic activity of different cancer cell lines in a dose dependent manner compared with untreated cells. Overall, Band 2 and 3 were more toxic than Band 1 irrespective of the cell lines. The metabolic activity of non cancer cell lines (diff. THP-1, hAELVi) were less affected as compared to cancer cell lines. BDNVs were taken up by cells in a dose- and time-dependent manner. Pre-incubation of cells with BDNVs resulted in a significant decrease in ROS production in Caco-2 and NCI-H441 stimulated with hydrogen peroxide and SHSY5Y treated with 6-hydroxydopamine, with all three Bands. BDNVs were also able to reduce the pro inflammatory cytokine gene expression (TNF) as well as pro inflammatory cytokine release (TNF and IL-6). Long term stability study revealed that freeze drying or lyophilization can be a suitable alternative storage condition for the storage of BDNVs. Trans-Epithelial Electrical Resistance (TEER) value measurement revealed that BDNVs treatment had no negative effect on the barrier integrity of co-culture model (hAELVi and diff. THP-1 cells). Our findings open to the possibility to find a novel “green” approach for the treatment of chronic diseases including cancer, focused on using vesicles from broccoli, although a more in-depth characterization of bioactive molecules is warranted.