Synthesis Of Quaternary Ammonium Modified Porphyrin Photosensitizer And Its Targeting Properties To Mitochondria

Photodynamic therapy（PDT）is a new non-invasive method for the treatment of tumor diseases.Different from radiotherapy,chemotherapy and surgery,photodynamic therapy has the advantages of low drug resistance,good selectivity,small trauma,good repeatability and good biocompatibility.The treatment principle is:through the injection of photosensitizer drugs into the human body,some photosensitizers are enriched to the tumor site;The tumor was irradiated by near infrared excitation,and the photosensitizer was excited by light to produce strong oxidizing reactive oxygen species（mainly singlet oxygen ¹O₂）;Biological macromolecules（such as protein,nucleic acid,etc.）will react with reactive oxygen species to inhibit the growth of tumor tissue.Mitochondria is a kind of organelle which exists in most cells and is coated by two layers of membrane.It is the main place for cells to carry out aerobic respiration and produce energy.Therefore,it is called"energy factory"in biological system.In addition,mitochondria also play a mediating role in apoptosis.Therefore,the development of photosensitizer drugs targeting mitochondria is of great significance for photodynamic therapy of tumors.The results show that the transmembrane potential of mitochondria is negative,and the positively charged lipophilic photosensitizer is easy to bind to the mitochondrial membrane and enter the interior of mitochondria.At present,many studies connect the positively charged pyridine group with photosensitizer drugs in the form of covalent bond,so that the cells can absorb photosensitizer drugs through endocytosis and target the mitochondria in the tumor.However,photosensitizer drugs containing pyridine cationic groups have high toxicity and strong hydrophobicity,which are easy to combine with negatively charged proteins in the blood,and then recognized by the reticuloendothelial system or macrophage system in the body.As a result,they are cleared before reaching the tumor site,and can not reach the effective drug dose at the tumor site.In order to develop photosensitizers with mitochondrial targeting function,reduce the impact on the body and improve its effective dose at the tumor site,three quaternary ammonium modified porphyrin photosensitizers were designed and synthesized and encapsulated by liposomes.The specific research contents and results are as follows:（1）Quaternary ammonium salt modified porphyrin photosensitizers QP1,QP2and QP3 were designed and synthesized,and their structures were characterized by ¹H NMR,IR and MS.（2）The production and photostability of ¹O₂ of QP1,QP2 and QP3 under 650nm（33 W/cm²）light were studied by UV absorption spectrum.The results showed that the three photosensitizers could effectively produce ¹O₂ under 650 nm（33 W/cm²）light,and the photostability was good.Transmission electron microscopy（TEM）was used to observe the morphological characteristics of QP1,QP2 and QP3 in water.The results showed that the shape of QP1 was mostly irregular and large-size block nanoparticles,while photosensitizers QP2 and QP3 could form nanoparticles with uniform particle size in water,and the particle size decreased with the increase of the amount of quaternary ammonium groups.The measured surface potentials of QP1,QP2 and QP3were 6.75±1.19 m V,19.13±2.13 m V and 27.75±1.79 m V respectively.The surface potential test results provided a feasible basis for the three photosensitizers to target mitochondria.（3）It was found that QP1,QP2 and QP3 could be encapsulated by liposomes to form spherical nanoparticles QP1@DSPE-m PEG₂₀₀₀、QP2@DSPE-m PEG₂₀₀₀ and QP3@DSPE-m PEG₂₀₀₀ with average particle sizes of 98 nm,96 nm and 67 nm,and QP3@DSPE-m PEG₂₀₀₀ compared with QP1@DSPE-m PEG₂₀₀₀、QP2@DSPE-m PEG₂₀₀₀,its particle size is relatively uniform,and the three nanoparticles have good stability in PBS solution and culture medium.The QP1@DSPE-m PEG₂₀₀₀、QP2@DSPE-m PEG₂₀₀₀ and QP3@DSPE-m PEG₂₀₀₀ surface potentials of were-17.25±1.34 m V,-15.20±1.76 m V and-11.75±2.09 m V.The results of surface potential test showed that the liposomes successfully encapsulated three quaternary ammonium salt modified porphyrin photosensitizers.（4）Cell experiment results show that,QP1@DSPE-m PEG₂₀₀₀、QP2@DSPE-m PEG₂₀₀₀ and QP3@DSPE-m PEG₂₀₀₀ can be absorbed by mouse 4T1 cells to achieve mitochondrial targeting.In addition,QP3@DSPE-m PEG₂₀₀₀has a stronger ability to target mitochondria than QP1@DSPE-m PEG₂₀₀₀ and QP2@DSPE-m PEG₂₀₀₀.After the cells were irradiated with 650 nm（33 W/cm²）laser,a large amount of ¹O₂ was produced in the cells,QP3@DSPE-m PEG₂₀₀₀ was more phototoxic to 4T1 cells than QP1@DSPE-m PEG₂₀₀₀ and QP2@DSPE-m PEG₂₀₀₀。...