Preparation Of TCMePyP And Study On Killing Effect On SH-SY5Y Neuroblastoma Cells

Objective:H₂TPyP?5,10,15,20-4?4-pyridine?porphyrin?were modifie d to prepare and obtain the TCMePyP.The physical and chemical properties of TCMePyP were studied.Killing effect and mechanism of photodynamic therapy?PDT?on neuroblastoma SH-SY5Y cells were studied.The killing effect of Q[7]-TCMePyP and Q[7]-anchored PAA loaded TCMePyP?Q[7]-PAA-TCMePyP?withPDT on SH-SY5Y cells was studied.Methods:TCMePyP was synthesized to study its nuclear magnetic resonance?¹H NMR?and dynamic laser scattering.Ultraviolet visible light absorption spectra and fluorescence spectra of H₂TPyP and TCMePyP were measured.Singlet oxygen was detected by SOSG?Singlet Oxygen Sensor Green?.The survival rate of SH-SY5Y neuroblastoma cells was detected by MTT colorimetric assay.The localization of TCMePyP cells and Detection of changes in intracellular ROS during PDT was observed Under the laser confocal microscope.Flow cytometry was used to detect the apoptosis induced by TCMePyP-PDT.The effect of TCMePyP-PDT on migration of SH-SY5Y cells was detected by scratch test.The killing effect of TCMePyP?Q[7]-TCMePyP and Q[7]-anchored PAA loaded TCMePyP with PDT on SH-SY5Y cells was detected by MTT assay.The fluorescence variation of TCMePyP?Q[7]-TCMePyP and Q[7]-anchored PAA loaded TCMePyP in SH-SY5Y cells was observed under confocal laser scanning microscope.Results:The structure of the compound was characterized by nuclear magnetic resonance?¹H NMR?of TCMePyP.The dynamic laser scattering spectra showed that single peak distribution in solution was found,and the diameter of hydrodynamics was significantly reduced.UV-visible absorption of 10?mol/L TCMePyP was significantly increased.Fluorescence intensity was significantly increased with?ex=515nm.The yield of single state oxygen reached the highest when the 525nm laser irradiated 11min.In the MTT assay,the 525nm laser and 660nm laser had no significant Photo toxicity?P>0.05?,in the light density?10.48 mW/cm2?and the light time from 1min to 9min.TCMePyP?2.5?mol/L to 80?mol/L?had no significant dark toxicity?P>0.05?.Under the irradiation of 525nm laser,the cell survival rate was significantly inhibited in 10?mol/L and50?mol/L of TCMePyP.Under the inverted microscope,with the extension of light time,the cell morphology changed obviously,and the necrotic cells increased.The apoptosis rate of cells increased significantly?P<0.001?under PDT after 24 hours detected with Flow cytometry.Under the confocal laser scanning microscope,TCMePyP was located in the cytoplasm of SH-SY5Y cells and located in nucleus after radiation.Under the laser confocal microscope,the detection of?ROS?was significantly increased in radiation time 3min and 5min.Scratch assay showed that TCMePyP-PDT significantly inhibited the migration ability of SH-SY5Y cells.In the concentration of 5?mol/L,TCMePyP?Q[7]-TCMePyP and Q[7]-PAA-TCMePyP,with the prolongation of the time of illumination,the cell survival rate gradually decreased,and the killing effect in Q[7]-PAA-TCMePyP group was remarkable?P<0.05?.Q[7]-PAA-TCMePyPgroup was more remarkable than group TCMePyP and group Q[7]-TCMePyP in each concentration of 1?mol/L?5?mol/Land10?mol/L under 2min laser irradiation?P<0.05?.Under the confocal laser scanning microscope,the concentration of 5?mol,Q[7]-TCMePyP and Q[7]-PAA-TCMePyP enhance the fluorescence intensity in SH-SY5Y cells.Conclusion:Compared with H₂TPyP,the water solubility of TCMePyP is obviously improved,that the absorption in visible region is enhanced and emission of fluorescence is more stronger.High singlet oxygen yield under radiation,low dark toxicity,significant light phototoxicity,specific uptake and cellular localization of SH-SY5Y cells were confirmed in Experiments.The experiment was found that adding Q[7]and Q[7]-anchored PAA nanoparticles to enhance the killing effect of new water-soluble porphyrin compounds?TCMePyP?.Cucurbit[7]uril and cucurbit Cucurbit[7]uril-anchored PAA are expected to be new carriers for photodynamic drugs.