Transmembrane Transport Of Cholesterol Axially Substituted Silicon Phthalocyanine By Fluorescence Correlation Spectroscopy

Cancer is one of the common causes of death in the world,and it is very important to study the therapeutic effect of drugs on the tumor site.One of the points that is closely related to the therapeutic effect is the transmembrane and release of drugs after targeting the cancer tumor region.Compared with traditional treatment methods,phototherapy has high targeting to tumor sites and little damage to normal human tissues,which has attracted much attention and has good application prospects.Phthalocyanine photosensitizers,as second-generation photosensitizers,have attracted much attention due to their good properties.Understanding how organelle-targeted phthalocyanine photosensitizers are transported across the membrane into cells and their intracellular transshipment situation can provides some theoretical support for the design of photosensitizers.In this thesis,high-resolution optical microscopy and single-molecule detection techniques were used to study the kinetic properties of the transmembrane transport and intracellular delivery of cholesterol axially substituted silicon phthalocyanine and its nanoparticles,as well as cholesterol-silicon phthalocyanine-piperazine asymmetric silicon phthalocyanine.And the transmembrane properties of cholesterol axially substituted silicon phthalocyanine mediated by cholesterol groups and the effects of cholesterol groups and piperazine groups and nanocarriers on guiding photosensitizer transmembrane transport and organelle localization were studied.First,we prepared cholesterol axially substituted silicon phthalocyanine(Chol-Pc),and prepared water-soluble nanoparticles(DSPE@Chol-Pc)by loading Chol-Pc with DSPE-PEG2000 to characterize the photophysical and chemical properties of these photosensitizers.Secondly,taking breast cancer MCF-7 cells as the research object,the transmembrane transport and intracellular delivery processes were compared by the combined application of confocal laser microscopy and fluorescence correlation spectroscopy.The results showed that Chol-Pc could enter cells rapidly and be enriched in the Golgi apparatus after 3h.Compared with the more concentrated transmembrane diffusion of Chol-Pc,DSPE@Chol-Pc has a longer time span,which is related to their different transmembrane ways: Chol-Pc flips into cells by targeting cholesterol on the plasma membrane,DSPE@Chol-Pc is that DSPE is compatible with the plasma membrane and stays on the plasma membrane,and then DSPE releases Chol-Pc.Chol-Pc and DSPE@Chol-Pc diffused in the cytoplasm for the same time after entering cells.After treatment with plasma membrane cholesterol by cyclodextrin,the diffusion time of Chol-Pc across the plasma membrane was significantly shortened,indicating that Chol-Pc is highly dependent on the properties of cholesterol in transmembrane transport.Low temperature inhibits the partial transmembrane behavior of Chol-Pc at the plasma membrane,indicating that the turnover of Chol-Pc at the plasma membrane has two situations and one of them is energy-dependent.Transmembrane transport studies on cholesterol-silicon phthalocyanine-piperazine(PIP-Pc-Chol)show that PIP-Pc-Chol has a shorter transmembrane time than Chol-Pc,and is hardly affected by the concentration of cholesterol on the membrane.Further indicating that piperazine group(PIP)plays a dominant role in transmembrane transport and may not be mediated by cholesterol on the plasma membrane.PIP-Pc-Chol is simultaneously localized in three organelles in cells,namely mitochondria,lysosomes and Golgi apparatus,and PIP-Pc-Chol can be seen to be enriched in cells within 0.5h,and it is enriched in the Golgi apparatus over time,indicating that the targeting of organelles after entering the cell,the guidance of cholesterol groups has a higher priority.In this thesis,by studying the transmembrane transport and intracellular delivery of nanophotosensitizers mediated by nanocarriers,cholesterol groups and piperazine groups,the relationship between them in cell transmembrane and organelle targeting was summarized.The research results in this thesis provide a theoretical basis for the subsequent selection of phthalocyanine photosensitizers on tumor cell plasma membranes and organelles targeting substitution groups to improve the effect of drug therapy on tumors.