Gold nanoparticles (AuNPs) are used enormously in different cancers but very little is known regarding their molecular mechanism and surface charge role in the process of cell death. Here, we elucidate the molecular mechanism by which differentially charged AuNPs induce cytotoxicity in triple negative breast cancer (TNBC) cells. Cytotoxicity assay revealed that both negatively charged (citrate-capped) and positively charged (cysteamine-capped) AuNPs induced cell-death in a dose-dependent manner. We provide first evidence that AuNPs-induced oxidative stress alters Wnt signalling pathway in MDA-MB-231 and MDA-MB-468 cells. Although both differentially charged AuNPs induced cell death, the rate and mechanism involved in the process of cell death were different. Negatively charged AuNPs increased the expression of MKP-1, dephosphorylated and deacetylated histone H3 at Ser10 and K9/K14 residues respectively whereas, positively charged AuNPs decreased the expression of MKP-1, phosphorylated and acetylated histone H3 at Ser 10 and K9/K14 residues respectively. High-resolution transmission electron microscopy (HRTEM) studies revealed that AuNPs were localised in cytoplasm and mitochondria of MDA-MB-231 cells. Interestingly, AuNPs treatment makes MDA-MB-231 cells sensitive to 5-fluorouracil (5-FU) by decreasing the expression of thymidylate synthetase enzyme. This study highlights the role of surface charge (independent of size) in the mechanisms of toxicity and cell death.

A series of stable and ready-to-use bioinks have been developed based on the xeno-free and tunable hydrogel (VitroGel) system. Cell laden scaffold fabrication with optimized polysaccharide-based inks demonstrated that Ink H4 and RGD modified Ink H4-RGD had excellent rheological properties. Both bioinks were printable with 25–40 kPa extrusion pressure, showed 90% cell viability, shear-thinning and rapid shear recovery properties making them feasible for extrusion bioprinting without UV curing or temperature adjustment. Ink H4-RGD showed printability between 20 and 37 °C and the scaffolds remained stable for 15 days at temperature of 37 °C. 3D printed non-small-cell lung cancer (NSCLC) patient derived xenograft cells (PDCs) showed rapid spheroid growth of size around 500 µm in diameter and tumor microenvironment formation within 7 days. IC50 values demonstrated higher resistance of 3D spheroids to docetaxel (DTX), doxorubicin (DOX) and erlotinib compared to 2D monolayers of NSCLC-PDX, wild type triple negative breast cancer (MDA-MB-231 WT) and lung adenocarcinoma (HCC-827) cells. Results of flow property, shape fidelity, scaffold stability and biocompatibility of H4-RGD suggest that this hydrogel could be considered for 3D cell bioprinting and also for in-vitro tumor microenvironment development for high throughput screening of various anti-cancer drugs.

The purpose of this study was to develop folic acid functionalized long-circulating co-encapsulated docetaxel (DTX) and curcumin (CRM) solid lipid nanoparticles (F-DC-SLN) to improve the pharmacokinetic and efficacy of DTX therapy. F-DC-SLN was prepared by hot melt-emulsification method and optimized by face centered-central composite design (FC-CCD). The SLN was characterized in terms of size and size distribution, drug entrapment efficiency and release profile. The cytotoxicity and cell uptake of the SLN formulations were evaluated in MCF-7 and MDA-MB-231 cell lines. The in vivo pharmacokinetic and biodistribution were studied in Wistar rats. F-DC-SLN exhibited 247.5 ± 3.40 nm particle size with 73.88 ± 1.08% entrapment efficiency and zeta potential of 14.53 ± 3.6 mV. Transmission electron microscopy (TEM) revealed spherical morphology of the SLN. Fluorescence microscopy confirmed the targeting efficacy of F-DC-SLN in MCF-7 cells. F-DC-SLN exhibited a significant increase in area under the curve (594.21 ± 64.34 versus 39.05 ± 7.41 μg/mL h) and mean residence time (31.14 ± 19.94 versus 7.24 ± 4.51 h) in comparison to Taxotere®. In addition, decreased DTX accumulation from F-DC-SLN in the heart and kidney in comparison to Taxotere may avoid to toxicity these vital organs. In conclusion, the F-DC-SLN improved the efficacy and pharmacokinetic profile of DTX exhibiting enhanced potential in optimizing breast cancer therapy.

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication that are involved in both normal processes and pathological conditions. Latent membrane protein 1 (LMP1) is a major viral oncogene that is expressed in most Epstein-Barr virus (EBV)-associated cancers and secreted in EVs. LMP1-modified EVs have the ability to influence recipient cell growth, migration, and differentiation and regulate immune cell function. Despite the significance of LMP1-modified EVs in EBV malignancies, very little is understood about how this protein hijacks the host EV pathway for secretion. Using the biotin identification (BioID) method, we identified LMP1-proximal interacting proteins that are known to play roles in EV formation and protein trafficking. Analysis of the identified LMP1-interacting proteins revealed an enrichment in the ESCRT pathway and associated proteins, including CD63, Syntenin-1, Alix, TSG101, Hrs, and charged multivesicular body proteins (CHMPs). LMP1 transcriptionally upregulated and increased the protein expression of EV biogenesis and secretion genes. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following the knockdown of Syntenin-1, Alix, Hrs, and TSG101, with altered endolysosomal trafficking observed when Syntenin-1 and Hrs expression was reduced. Knockdown of specific ESCRT-III subunits (CHMP4B, -5, and -6) impaired LMP1 packaging and secretion into EVs. Finally, we demonstrate that the efficient secretion of LMP1-modified EVs promotes cell attachment, proliferation, and migration and tumor growth. Together, these results begin to shed light on how LMP1 exploits host ESCRT machinery to direct the incorporation of the viral oncoprotein into the EV pathway for secretion to alter the tumor microenvironment. IMPORTANCE LMP1 is a notable viral protein that contributes to the modification of EV content and tumor microenvironment remodeling. LMP1-modified EVs enhance tumor proliferation, migration, and invasion potential and promote radioresistance. Currently, the mechanisms surrounding LMP1 incorporation into the host EV pathways are not well understood. This study revealed that LMP1 utilizes Hrs, Syntenin-1, and specific components of the ESCRT-III complex for release from the cell, enhancement of EV production, and metastatic properties of cancer cells. These findings begin to unravel the mechanism of LMP1 EV trafficking and may provide new targets to control EBV-associated cancers.

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