Cytotoxicity Of Calcium Rectorite Nanoparticles Before And After Organic Modification And The Related Application

Rectorite (REC) is a regular layered silicate clay mineral which has been found to be useful in the field of food and medicine. However, the toxicity research of REC is still limited. Paclitaxel (PTX) has been found to be a potent anti-neoplastic agent to efficiently treat cancer, but with severe limitations and deficiencies. In this paper, we discussed the cytotoxicity and toxicology of REC nanoparticles before and after organic modification, and the influence of REC on the antitumor activity of PTX incorporated nanofibrous mats as a drug delivery system.The work in this paper consists of two parts:Part I Cytotoxicity of Calcium Rectorite Nanoparticles before and after Organic ModificationObjective: The goal of the present study was to investigate the cytotoxicity of REC and organically modified rectorite (OREC) in human normal hepatic cells Chang liver and human hepatoma cells HepG2, the modes of cell death (apoptosis or necrosis), and the underlying mechanisms. Our results would provide potential implications for the safety application of REC and OREC in the future.Methods: OREC was prepared by cation exchange of REC galleries with hexadecyl trimethylammonium bromide. The size distributions and ?-potential of REC and OREC were determined. The characterization of REC and OREC were determined by thermogravimetric analysis, Fourier transform infrared, transmission electron microscope and small-angle X-ray diffraction. The uptake of REC and OREC by Chang liver cells was detected by energy-dispersive X-ray spectroscopy. Chang liver and HepG2 cells were treated with 0,1,2.5,5,7.5, and 10 ?g/mL REC or OREC for 6,24, and 48 h, and then the cytotoxicity was determined by MTT assay. Chang liver cells were treated with 0,1,2.5,5,7.5, and 10 ?g/mL REC or OREC for 24 h, then the modes of cell death were examined by flow cytometry. To investigate the underlying mechanisms of REC- and OREC-induced apoptosis in Chang liver cells, the protein levels of Bax, Bcl-2, and procaspase-3 were measured by Western blot after the cells were exposed to 0,1,2.5,5,7.5, and 10 ?g/mL REC or OREC for 6 and 24 h. After treated with 5 ?g/mL REC or OREC for 24 h, HepG2 cells were observed by transmission electron microscop and the autophagy related protein was examined by Western blot.Results:The interlayer spacing of OREC was enlarged after organic modification. After treatment with REC or OREC for 24 h at 1 and 5 ?g/mL, both of the two nanoparticles were taken up by Chang liver cells. REC and OREC induced cytotoxicity in Chang liver and HepG2 cells at almost all doses (1,2.5,5,7.5, and 10 ?g/mL) after 6,24, and 48 h of treatment (P< 0.05 or P< 0.01). Compared with REC, OREC was more cytotoxic. However, there was no difference in the cytotoxicity of REC and OREC between the two cell lines. After treatment with REC or OREC at 7.5 and 10 ?g/mL for 24 h, the apoptotic and necrotic percentages of Chang liver cells were increased (P< 0.05 or P< 0.01). The levels of apoptosis-related proteins Bax, Bcl-2, and pro-caspase-3 were all decreased in Chang liver cells after 24 h of exposure to REC or OREC at 5,7.5,10 ?g/mL. There was no change in the relative ratio of Bax/Bcl-2 after treatment, indicating that REC or OREC-induced apoptosis was not associated with Bax-related mitochondria-mediated apoptotic pathway. Autophagosome was observed in HepG2 cells after treated with 5 ?g/mL REC or OREC for 24 h and the ratio of LC3-?/LC3-? was increased.Conclusion: Our results suggested that the physical and chemical properties of REC were improved after organic modification. But the cytotoxicity was upregulated at the same time. Besides, both of REC and OREC induced apoptosis and autophagy, and therefore led to cell death. So caution should be taken when OREC was used in the field of food and medical.Part II Effect of Rectorite on the Antitumor Activity of Paclitaxel Incorporated Nanofibrous MatsObjective: PTX-loaded CA nanofibrous mats (CA/PTX), as well as REC and PTX incorporated CA nanofibrous mats (CA/PTX/REC) were electrospun and characterized to realize good incorporation and controlled release of PTX. We are aim to produce the nanofibrous mats as drug carriers for slow drug release system to treat gastric cancer.Methods:The morphologies of the nanofibrous mats were observed by field-emission scanning electron microscope. The Brunauer-Emmett-Teller specific surface areas of the fibrous mats were characterized by nitrogen adsorption. The composition of the samples was examined by energy-dispersive X-ray spectroscopy. The interaction between PTX, REC and CA was investigated by Fourier transform infrared spectra and the wide angle X-ray diffraction. The thermal properties of the nanofibrous mats were determined by differential scanning calorimetry and Thermogravimetric analysis. The release of PTX was determined in 40 mL phosphate buffered saline (PBS, pH 7.4) and performed by high performance liquid chromatography. Human gastric adenocarcinoma SGC7901 and human gastric epithelium GES-1 cells were planted on the electrospun nanofibrous mats, and then the cytotoxicity was determined by LIVE/DEAD Viability/Cytotoxicity kit. And ultrastructural cell morphology was examined by scanning electron microscopy.Results: The mats were characterized to imply that PTX and REC were successfully incorporated into the composite nanofibrous mats without impacting the thermal properties of CA. The morphology of the composite nanofibrous mats maintained perfect fiber shape and 3D structure before and after cell culture while the added REC increased the BET specific surface area of the mats. PTX was released from CA/PTX and CA/PTX/REC mats at a relative stable rate. And the release rate of PTX from CA/PTX mats was slower than that from CA/PTX/REC mats. Moreover, the presence of REC reduced the viability of both SGC7901 and GES-1 cells on composite nanofibrous mats. But the mats induced more cytotoxicity in GES-1 cells than in SGC7901 cells. Furthermore, all mats provided idea scaffold for both GES-1 and SGC7901 cells to spread and spit.Conclusion:These results suggested that both CA and CA/REC composite nanofibrous mats were ideal scaffold for cells to adhere and spread. PTX could be released by CA/PTX and CA/PTX/REC at a stable and slow speed. Both CA/PTX and CA/PTX/REC induced cytotoxicity with a synergistic effect by REC. CA/PTX, CA/REC and CA/PTX/REC may have enormous potential values in the area of control released drug delivery system but targeted therapy is needed for further investigations.