Apoptotic Action Mechanism And Cytotoxicity Of Modified Montmorillonite And The Study On The Transfection Mediated By Modified Calcium Phosphate

Part I Apoptotic action mechanism and cytotoxicity of oligomeric poly(styrene-co-acrylonitrile) modified montmorilloniteTo evaluate the cytotoxic effect and the mechanism of the apoptotic action of oligomeric poly (styrene-co-acrylonitrile)-modified montmorillonite (PSAN-MMT), the present study is focused on the involvement of anti-proliferation, LDH release, apoptosis level, caspase-3 activity, transcriptional activity of apoptosis-related gene, and apoptosis-related protein and mRNA level.Non-cytotoxic montmorillonite (MMT) incubated cells were used as control. PSAN-MMT and MMT induced anti-proliferation and upregulated LDH release in a dose-dependent manner, and the effect was obvious at the concentration of 1 g/L while this effect was weaker for PSAN-MMT compared to MMT. Lower level of apoptosis was detected in 1 g/L PSAN-MMT treated cells than in 1 g/L MMT cells, and caspase-3 activity in PSAN-MMT treated cells was lower than one of cells treated in MTT. In PSAN-MMT treated cells, p53-dependent transcriptional activity and p53 expression level were lower as compared to MMT treated cells.In addition, mRNA levels of p53 and p21 upregulated by PSAN-MMT were much lower compared with MMT, which was consistent with the results for the apoptotic rate of the cells exposed to PSAN-MMT and MMT, respectively. In conclusion, PSAN-MMT (at the concentration of less than 1g/L) is non-cytotoxic like MMT. We speculated that the mechanism of PSAN-MMT and MMT-induced apoptosis seemed to follow a transcription-dependent route by activating proapoptotic genes such as p53 and p21.It could be expected that the modified MMT may be a promising safety controlled-release carrier for large molecules drug and for gene delivery. Part II The study on the transfection mediated by nucleoprotamine modified calcium phosphateTo evaluate whether nucleoprotamine modified calcium phosphate could enhance the transfection efficiency and the calcium phosphate particles grain diameter, this research use pEGFP-N1 green fluorescent protein as a indicator to study the change in transfection efficiency. Atomic force microscope was used to evaluate the morphological change and grain diameter of the calcium phosphate particles, and MTT assay was induced to detect the cell viability inhibit effect. Coprecipitated superphosphate was used as control.In 293 FT,HEK 293 and NIH 3T3 cells, the transfection efficiency of nucleoprotamine modified calcium phosphate was obviously higher than coprecipitated superphosphate. Moreover, the transfect efficiency of nucleoprotamine modified calcium phosphate could maintain at least 7 days, which indicated that nucleoprotamine may increase the stability of calcium phosphate.Atomic force microscope image showed that the grain diameter of nucleoprotamine modified calcium phosphate was much smaller than coprecipitated superphosphate. Two phosphates made no difference as cell viability inhibition assay was induced. It could be expected that nucleoprotamine modified calcium phosphate could be a better transfect agent as compared to coprecipitated superphosphate.