Studies On Photo-sensitive Reactive Oxygen Species(ROS) Responsive Liposomes

With the development of science and technology,researchers have explored many microenvironment specific responsive drug carriers—stimuli-responsive carriers—for tumor microenvironment.Stimuli-responsive carriers include pH-responsive carriers,cytoplasmic glutathione?GSH?-responsive carriers,light-responsive carriers,ultrasound-responsive carriers,magnetic response carriers,thermal-responsive carriers and reactive oxygen species?ROS?responsive carriers.ROS responsive carriers have become the hotspot of drug carrier research in recent years.It is thought that sulfur-containing polymer ROS responsive carriers,selenium-containing polymer ROS responsive carriers,tellurium-polymer ROS responsive carriers and unsaturated phospholipid-containing ROS responsive carriers has appeared in succession.Among the numerous ROS responsive carriers,the unsaturated phospholipid-containing ROS responsive carrier has the most potential for development,and the unsaturated phospholipid can be rapidly oxidized by ¹O₂,thereby realizing rapid drug release.However,relying solely on ROS responsive carriers does not guarantee that the drug is released at specific sites on multiple pathological conditions,whereas photosensitizers can produce ¹O₂ after irradiation,and the unsaturated phospholipid-containing carriers can achieve ¹O₂-based ROS responses.ROS responsive liposomes material DLPC is an unsaturated phospholipid with four unsaturated double bonds and has strong responsive activity to ROS.PdPC?OBu?₈ is a highly effective photosensitizer that can produce large quantities of ¹O₂ in the presence of light,enabling rapid oxidation of unsaturated phospholipids.Photosensitizer can produce ¹O₂under light irradiation with specific wavelength.It not only has the characteristics of oxidized unsaturated phospholipid,but also has the function of photodynamic therapy,which can improve the therapeutic effect of ROS responsive nanoliposomes.Therefore,this study designed and studied ROS responsive carriers containing photosensitizers.we applied 1,2-dilinoleoyl-sn-glycero-3-phosphocholine?DLPC?as a ROS responsivematerial,1,4,8,11,15,18,22,25-octabutoxypalladiumphthalocyanine?PdPC?OBu?₈?as a special substances to produce ¹O₂.We used the construction of photo-sensitive ROS responsive liposomes loaded with PdPC?OBu?₈ and Doxorubicin hydrochloride?DOX??LPD?to explore antitumor activity of LPD in vitro and in vivo.Firstly,we synthesized and identified the photosensitizer PdPC?OBu?₈.The methods for the determination of Doxorubicin hydrochloride?DOX?and PdPC?OBu?₈ were established.The DOX concentration was determined by fluorescence spectrophotometry.When the concentration of DOX was 1-20?g·mL^-1,The linear regression equation was F=8.9337C+0.2412?R2=0.9995?.Then the HPLC method of DOX was explored.The linear range of DOX was 0.2-25?g·mL^-1.The linear regression equation was A=0.4494C+0.0100?R2=1?.The concentration of PdPC?OBu?₈ was determined by UV-visible spectrophotometry.The linear regression equation was A=0.0823C+0.0157?R2=0.9994?when the concentration of PdPC?OBu?₈ was 2-12?g·mL^-1.The HPLC method of PdPC?OBu?₈ was also explored.The linear range of PdPC?OBu?₈ was 0.5-20?g·mL^-1.The linear regression equation was A=0.1002C-0.0054?R2=1?.Secondly,the formulation and characterization of LPD were performed in this paper.The optimum formulation of LPD was DLPC 5%,Cholesterol 40%,DSPE-PEG 2000 5%,and DSPC 50%?molar ratio?,PdPC?OBu?₈ to lipids?m:m?was 1:200.The diameter of LPD was?169.3±1.2?nm and the PDI was 0.198±0.003.The particle size was uniform and the dispersity was good.The zeta potential was?-39.8±0.8?mV.The encapsulation efficiency of PdPC?OBu?₈ in LPD was?84.43±3.12?%and the drug loading was?0.42±0.01?%.The encapsulation efficiency of DOX was?91.03±3.33?%and the drug loading was?9.84±0.40?%.LPD had good storage stability and good serum stability.LPD did not release drug without irradiation.Under light irradiation(730 nm,300 mW·cm^-2,5 min),the drug release rate reached 95.5±2.5%?P<0.05?after 5 min,indicating that LPD had the property of rapid response to ROS.LPD did not produce ¹O₂ before light irradiation,but produced a large amount of ¹O₂ after light irradiation(730 nm,300 mW·cm^-2,3 min).Therefore,LPD could achieve ROS response when exposed to light.Then,breast cancer MCF-7 cells were used as model cells to study the in vitro anti-tumor activity of LPD.Light intensity was not toxic to MCF-7 cells.Liposomes loaded with PdPC?OBu?₈?LP?was slightly toxic to MCF-7 cells before light irradiation,and significantly increased cytotoxicity after light irradiation(730 nm,1200 mW·cm^-2,3 min).LPD could promote the uptake of DOX in MCF-7 cells.DOX was mainly distributed in the lysosome after entering into the cell.The LPD pathway of entering into cells was energy dependent through caveolin mediation.LPD produced a small amount of ROS in living cells and produced a large amount of ROS when irradiated(730 nm,1200 mW·cm^-2,3 min).Finally,we used breast cancer MCF-7 cells as model cells and nude mice as model animals to explore the in vivo antitumor activity of LPD.The method of HPLC-MS analysis of DOX showed good specificity with a linear range of 1-1000 ng·mL^-1.The linear regression equation was A_DOX/A_DNX=0.0003C+0.0116,R2=0.9986,with good matrix effect,recovery rate and accuracy and precision,DOX's HPLC-MS method could be used as a DOX pharmacokinetic assay.The half-life of free DOX was 1.42 h,the half-life of LPD was 7.16h,which was 5.04 times of the half-life of free DOX,with significant difference.The half-life of non-ROS responsive liposomes loaded with PdPC?OBu?₈ and DOX?HPD?was 11.39h,which was 8.02 times of that of free DOX with significant difference.LPD and HPD could extend the half-life of DOX.Compared with free DOX,LPD had the characteristics of small clearance and large AUC.When LPD loaded with Amplex UltraRed?LPU?was not irradiated,the tumor had no fluorescence.When the LPU was irradiated(730 nm,1200mW·cm^-2,5 min),the tumor had obvious fluorescence,indicating that the LPD could generate ROS after being irradiated by light(730 nm,1200 mW·cm^-2,5 min).Free DiR was mainly distributed in the liver,spleen with significant fluorescence,indicating that free DiR was mainly distributed in the liver and spleen.LPD loaded with DiR?LDiR?was mainly distributed in the liver,tumor with significant fluorescence,indicating that LDiR were mainly distributed in the liver and tumor.Therefore,LPD mainly distributed in the liver and tumor after intravenous injection,indicating that LPD have passive targeting.Blank liposomes?BP?and liposomes loaded with PdPC?OBu?₈?LP?had no antitumor activity.Free DOX,liposomes loaded with DOX?LD?,liposomes loaded with DOX in the presence of irradiation(730 nm,1200 mW·cm^-2,5 min)?LD+hv?and LPD had similar anti-tumor activity.LP with irradiation(730 nm,1200 mW·cm^-2,5 min)?LP+hv?has good anti-tumor activity.it indicated that light significantly enhanced the antitumor activity of LP.The most significant antitumor activity of irradiated(730 nm,1200 mW·cm^-2,5 min)photo-sensitive ROS responsive liposomes?LPD+hv?was probably due to the combination of chemotherapy and photodynamic therapy.Non-irradiated HPD had anti-tumor activity.HPD could produce photodynamic therapy in the presence of irradiation(730nm,1200 mW·cm^-2,5 min).As a result,irradiated(730 nm,1200 mW·cm^-2,5 min)HPD had a stronger anti-tumor activity.But LPD had similar antitumor effects with irradiated HPD.So photo-sensitive ROS responsive liposomes with irradiation(730 nm,1200 mW·cm^-2,5 min)?LPD+hv?had the most significant anti-tumor activity.In summary,this study preliminarily demonstrated that LPD not only has ROS response characteristics,but also can enhance cytotoxicity and enhance cellular uptake.What's more,LPD has marked targeting characteristics in animals and has significant antitumor activity in vivo.What is significant is that LPD has the effects of photodynamic therapy and chemotherapy,and it is a photo-sensitive drug delivery with good development prospects.In addition,the preparation process of LPD is simple and has certain guiding significance for the industrialization of nanomedicines.