Two-dimensional (2D) monolayer cell cultures are the most common in vitro models for mechanistic studies on the toxicity of engineered nanoparticles (NPs). However, 2D cell cultures may not accurately model the functions of three-dimensional (3D) tissues that have extensive cell-cell and cell-matrix interactions, and thus may lead to wrong experimental results. Hence, to obtain more adequate and detailed information about ZnO NP-tissue interactions, we here assess the toxicity and inflammatory effects of different-sized ZnO NPs at various concentrations on Caco-2 cells which are threedimensionally cultured in agarose gels, measuring ROS expression, pro-inflammatory cytokines, cell proliferation and death. The results showed that the significant effects of cell dimensionality to cellular responses such as inflammatory response, cytotoxicity and different modes of cell death in response to ZnO NPs treatment. Moreover, ZnO NPs induced the toxicity of 2D and 3D cells in different sizedependent manners. This study revealed that the nanotoxicity conclusions obtained from the 2D cell model might overestimate the toxic effects of ZnO NPs. There is a more realistic mechanism study of nanoparticle-induced toxicity in the 3D cell model, which acts as an intermediate stage bridging in vitro 2D and in vivo models.

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Introduction: The leaves of Morus alba L is a traditional Chinese medicine widely applied in lung diseases. Moracin N (MAN), a secondary metabolite extracted form the leaves of Morus alba L, is a potent anticancer agent. But its molecular mechanism remains unveiled. Objective: In this study, we aimed to examine the effect of MAN on human lung cancer and reveal the underlying molecular mechanism. Methods: MTT assay was conducted to measure cell viability. Annexin V-FITC/PI staining was used to detect cell apoptosis. Confocal microscope was performed to determine the formation of autophagosomes and autolysosomes. Flow cytometry was performed to quantify cell death. Western blotting was used to determine the related-signaling pathway. Results: In the present study, we demonstrated for the first time that MAN inhibitd cell proliferation and induced cell apoptosis in human non-small-cell lung carcinoma (NSCLC) cells. We found that MAN treatment dysregulated mitochondrial function and led to mitochondrial apoptosis in A549 and PC9 cells. Meanwhile, MAN enhanced autophagy flux by the increase of autophagosome formation, the fusion of autophagsomes and lysosomes and lysosomal function. Moreover, mTOR signaling pathway, a classical pathway regualting autophagy, was inhibited by MAN in a time-and dose-dependent mannner, resulting in autophagy induction. Interestingly, autophagy inhibition by CQ or Atg5 knockdown attenuated cell apoptosis by MAN, indicating that autophagy serves as cell death. Furthermore, autophagy-mediated cell death by MAN can be blocked by reactive oxygen species (ROS) scavenger NAC, indicating that ROS accumulation is the inducing factor of apoptosis and autophagy. In summary, we revealed the molecular

Abstract:The main purpose of this study was to optimize the preparation of lysozyme nanoliposomes using response surface methodology and measure their stability. The stabilities of lysozyme nanoliposomes in simulated gastrointestinal fluid (SGF), simulated intestinal fluid (SIF), as well as pH, temperature and sonication treatment time were evaluated. Reverse-phase evaporation method is an easy, speedy, and beneficial approach for nanoliposomes' preparation and optimization. The optimal preparative conditions were as follows: phosphatidylcholine-to-cholesterol ratio of 3.86, lysozyme concentration of 1.96 mg/mL, magnetic stirring time of 40.61 min, and ultrasound time of 14.15 min. At the optimal point, encapsulation efficiency and particle size were found to be 75.36%˘3.20% and 245.6 nm˘5.2 nm, respectively. The lysozyme nanoliposomes demonstrated certain stability in SGF and SIF at a temperature of 37˝C for 4 h, and short sonication handling times were required to attain nano-scaled liposomes. Under conditions of high temperature, acidity and alkalinity, lysozyme nanoliposomes are unstable.

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