Clathrin-mediated endocytic uptake of PUFA enriched self-nanoemulsifying lipidic systems (SNELS) of an anticancer drug against triple negative cancer and DMBA induced preclinical tumor model

Abstract

The current studies envisage unravelling the underlying cellular internalisation mechanism of the systematically developed docetaxel (DTH) polyunsaturated fatty acid (PUFA) enriched self-nanoemulsifying lipidic micellar systems (SNELS). The concentration-, time- and cytotoxicity-related effects of DTH-SNELS on triple negative breast cancer (TNBC) MDA-MB-231 and non-TNBC MCF-7 cell lines were assessed through Presto-blue assay. Subsequently, rhodamine-123 (Rh-123) loaded SNELS were employed for evaluating their internalisation through flow cytometry and fluorescence microscopy, establishing it to be "clathrin-mediated" endocytic pathway. Apoptosis assay (65% cell death) and cell cycle distribution (47% inhibition at G2/M phase) further corroborated the cytotoxicity of DTH-SNELS towards cancerous cells. Biodistribution, histopathology and haematology studies indicated insignificant toxicity of the optimized formulation on vital organs. Preclinical anticancer efficacy studies using 7,12-dimethylbenzantracene (DMBA)-induced model construed significant reduction in breast tumor-volume. Overall, extensive in vitro and in vivo studies indicated the intracellular localization and cytotoxicity, suggesting DTH-SNELS as promising delivery systems for breast tumor therapeutics including TNBC.

Nano-Zn Increased Zn Accumulation and Triglyceride Content by Up-Regulating Lipogenesis in Freshwater Teleost, Yellow Catfish Pelteobagrus fulvidraco

“…The primary intestinal epithelial cells from P. fulvidraco were incubated for 12 h in 40 µM ZnSO 4 or 40 µM nano-Zn containing medium with or without 2 h of pretreatment inhibitors. The inhibitors included sodium azide (NaN 3 , 10 mM) for inhibiting energy-dependent internalization pathways, chlorpromazine (CHL, 10 µg/mL), nystatin (5 µg/mL), and cytochalasin-D (5 µg/mL) for inhibiting clathrin-dependent, caveolae-dependent, and phagocytosis-dependent pathways, respectively [14,38]. The concentrations of inhibitors were selected according to our pilot trials and to other in vitro studies [38,39].…”

Nebulised surface-active hybrid nanoparticles of voriconazole for pulmonary Aspergillosis demonstrate clathrin-mediated cellular uptake, improved antifungal efficacy and lung retention

“…As the positively charged NPs are known to internalize by clathrin-mediated endocytosis (CME), this pathway was considered primarily responsible for the internalization of the positively charged LPH NPs in the current studies, for A549 as well as Calu-3 cells. Electrostatic interactions seemed to be primarily involved in the receptor-independent CME [ 47 , 72 , 73 ]. As NPs usually employ multiple pathways to internalize, macropinocytosis, postulated to be involved in the uptake of both positively and negatively charged NPs, was also construed as one of the likely internalisation pathways of LPH NPs in Calu 3 cells [ 74 ].…”

“…Varied concentrations of formulation ( i.e., 1, 2.5, 5 μg mL −1 ) were incubated at 37 ± 1 °C in pre-cultured lung epithelial cells in 12 well-plates for varying time intervals, i.e. , 0.5, 1, 2, 4 and 6 h, and estimated for mean fluorescence intensity [ 47 ].…”

“…The effect of energy-dependent endocytosis was also observed by placing the cells at 4 °C. Samples were processed, as per the standard protocol, and subjected to flow cytometer [ 47 ].…”

“…The cellular uptake studies were conducted to evaluate the effect of concentration and time on the uptake of Rh-LPHNPs in A549 (5×10 4 cells/well) and Calu 3 (1x 10 5 cells/well) cells using a flow cytometer (Guava ® easy-Cyte HT, M/s Merck, Germany). Varied concentrations of formulation (i.e., 1, 2.5, 5 μg mL −1 ) were incubated at 37 ± 1 °C in pre-cultured lung epithelial cells in 12 wellplates for varying time intervals, i.e., 0.5, 1, 2, 4 and 6 h, and estimated for mean fluorescence intensity [47].…”