In Vitro Study Of Photodynamic Effect Of Hemoporfin On Vascular Endothelial Cells

Photodynamic Therapy(PDT)is a drug-device combination modality.Its basic principle is that photosensitive drugs are enriched in target tissues after administration,when irradiated by light of specific wavelength and with the participation of oxygen molecules,it can produce singlet oxygen and other reactive oxygen species(ROS),which are cytotoxic and can cause damage to organelles and ultimately lead to the apoptosis and necrosis of target cells,so as to achieve the effect of treating diseases.As the three basic elements of PDT,the interaction of light,photosensitizer and oxygen molecule plays a decisive role in the clinical therapeutic effect of PDT.In the early stage,PDT was mainly used for the treatment of malignant tumors,and then gradually applied to the treatment of precancerous lesions and benign diseases,such as actinic keratosis,macular degeneration,port wine stains and condyloma acuminatum.Port wine stains(PWS)is a common congenital cutaneous vascular disease with an incidence rate of 0.3%-0.5%,and its pathogenesis is still unclear.PWS can occur on the face,hands,legs and other parts of the body.It usually exists since the birth of a baby and cannot disappear with age.China pioneered photodynamic therapy of PWS based on photosensitizer Hemoporfin.Hemoporfin~?is a new second-generation photosensitizer independently developed by China.It belongs to ClassⅠinnovative drugs,with more simple composition,relatively stable structure,less damage to normal skin,short retention time in vivo,fast metabolism and absorption,and the time of avoiding light in clinical use can be shortened to one to two weeks.At present,clinical treatment of PWS is mainly based on the combination of Hemoporfin combined with laser and LED light source of 532 nm.Although certain curative effects have been achieved,the photosensitizer dose,light dose and other important parameters in the treatment process are dependent on empirical dose.The treatment mechanisms at the cellular levels are not fully understood and still need to be further studied.Therefore,this study attempted to explore the photodynamic effect of Hemoporfin on Human vascular endothelial cells in vitro to provide theoretical foundation and guidance for optimizing clinical treatment.In this study,the optical properties of Hemoporfin,i.e.absorption and fluorescence spectra,were utilized as research tools.First,the fluorescence intensity of Hemoporfin photosensitizer in skin microvascular endothelial cells(HDMEC)was measured by using a microplate reader,and the concentration of Hemoporfin photosensitizer in vascular endothelial cells(HDMEC)was semi-quantitatively studied.High Performance Liquid Chromatograph(HPLC)was used to separate and extract the photosensitizer,and the concentration of photosensitizer in vascular endothelial cells was quantitatively studied.The subcellular localization of photosensitizer was observed by fluorescence markers of organelles and confocal laser microscopy.Finally,the photodynamic killing effect on the vascular endothelial cells in vitro was carried out to analyze the dose-effect relationship.