The Targeted Photosensitizer Of 3PRGD₂-pyro For Photodynamic Therapy In Lung Cancer

Objectives:The targeting and water-soluble photosensitizer 3PRGD₂-Pyro was prepared by modifying pyromellitic acid-a with 3PRGD₂ and characterized.To explore the cytotoxicity and biological distribution of 3PRGD₂-Pyro.To evaluate the therapeutic effect of 3PRGD₂-Pyro on non-small cell lung cancer bearing mice.Methods:Tht synthesis of targeting and water-soluble photosensitizer 3PRGD₂-Pyro.A novel RGD dimer E[PEG₄-c?RGDfK?]₂?3PRGD₂?coupled with carboxyl groups of Pyromellitic acid-a?Pyro?via EDC and NHS was characterized by high performance liquid chromatography-mass spectrometry?HPLC-MS?.Pyro and synthetic photosensitizer 3PRGD₂-Pyro were added into distilled water,saline,buffer solution and fetal bovine serum,respectively.The water-solubility was observed after concussion and centrifugation for several minutes.We use chemical trapping spectrophotometry and electron spin resonance?ESR?technology to determine the singlet oxygen yield of 3PRGD2-Pyro.Non-small cell lung cancer A549 cells were cultured and the cytotoxicity of Pyro and 3PRGD₂-Pyro at different concentrations was measured by MTT.The 3PRGD₂-Pyro?+?group,Pyro?+?group,3PRGD₂-Pyro?-?group and Pyro?-?group were established.To evaluate the laser confocal focusing experiment of 3PRGD₂-Pyro uptake in vitro,A549 cells were cultured at low temperature with 3PRGD₂-Pyro and Pyro respectively,and observed with laser confocal microscopy 2 hours later.At the same time,A549 cells were pre-cultured with3PRGD₂ and then cultured with 3PRGD₂-Pyro to observe the uptake.To construct a tumor bearing rat model of non small cell lung adenocarcinoma?A549?.When the tumor volume of nude mice reached about 200 mm³,the tumor-bearing mice were randomly divided into three groups:3PRGD₂-Pyro group,Block group and Pyro group.The rats were injected with PPRGD2-Pyro group,block group?injected with 3PRGD₂-Pyro+3PRGD₂,in which the molar ratio of 3PRGD₂ to 3PRGD₂-Pyro was 100?and Pyro group with equivalent molar amount.The best time point of photodynamic therapy was determined according to the distribution of photosensitizers in vivo,and the fluorescence intensity of the region of interest?T/N ratio?was quantitatively analyzed in real time.Four hours after injection of photosensitizer,the tumor-bearing mice of3PRGD₂-Pyro group and Block group were sacrificed.The heart,liver,spleen,lung,kidney,stomach,intestine,brain and muscle tissues were taken and fluorescence imaging was performed.The distribution of 3PRGD₂-Pyro in the main organs and tissues of the body was observed and quantitatively analyzed.To construct a tumor bearing rat model of non small cell lung adenocarcinoma?A549?.When the tumor volume of nude mice reached about 80 mm3,the mice were randomly divided into three groups:3PRGD₂-Pyro?+?group,Pyro?+?group,3PRGD₂-Pyro?-?group and Pyro?-?group.Photodynamic therapy or no irradiation were performed to record tumor volume every two days.Tumor tissues were taken from tumor-bearing mice before treatment?0days?,8 days and 14 days after treatment for H&E staining and immunofluorescence staining,and compared with untreated tumors.In addition,H&E staining was performed on the main organs and tissues of tumor-bearing mice in each treatment group and control group to investigate the changes of cell level.Result:The results of high performance liquid chromatography and mass spectrometry showed that 3PRGD₂-Pyro was successfully synthesized and its chemical purity was over 98%.The singlet oxygen yield measurement results show that 3PRGD₂-Pyro has a good and stable singlet oxygen yield under the light condition,and it is linearly correlated with the irradiation time.Through water-solubility test,3PRGD₂-Pyro can be completely dissolved in saline and other solutions,while Pyro has a large number of insoluble substances in the solvent.The water-solubility of photosensitizer can be greatly improved by modifying Pyro with 3PRGD₂.In the cytotoxic MMT test,both3PRGD₂-Pyro?+?group and Pyro?+?group showed cytotoxicity.At the same concentration,the cell survival rate of 3PRGD₂-Pyro?+?group was lower,the cytotoxicity was stronger than that of Pyro?+?group,the semi-inhibitory concentration of 3PRGD₂-Pyro?+?group was 0.1,while that of Pyro?+?group was lower.In addition,compared with the control group,no cytotoxicity,high light toxicity and low dark toxicity were observed in the irradiated 3PRGD₂-Pyro group and Pyro group.In the laser confocal focusing experiment of cell uptake of photosensitizers,photosensitizers uptake on cell membrane could be seen in 3PRGD₂-Pyro group,but no photosensitizers were observed on cell membrane surface in Block group and Pyro group blocked by3PRGD₂ in advance.In vivo distribution of 3PRGD₂-Pyro in tumor-bearing mice,the concentration of photosensitizers in 3PRGD₂-Pyro group was not obvious at any part of the body at 1 h.With the extension of time,photosensitizers gradually concentrated in the tumor.Four hours after injection,3PRGD₂-Pyro was significantly concentrated in axillary tumors of tumor-bearing mice,but less in other parts of the body.After 24hours of injection,a small amount of photosensitizer remained in the tumor-bearing mice,and the photosensitizer was quickly removed from the body by metabolism.There was no apparent accumulation of Pyro in the tumor.After injection of 24h,photosensitizers remained in the mice.At the same time,3PRGD₂-Pyro in Block group was not significantly concentrated in tumor tissues.Compared with unmodified Pyro,the photosensitizer 3PRGD₂-Pyro synthesized by us has very high targeting,and the metabolic clearance of 3PRGD₂-Pyro in tumor-bearing mice is faster,showing good biocompatibility.Photosensitizer 3PRGD₂-Pyro shows potential for practical clinical application.In vitro tissue fluorescence imaging,tumor fluorescence intensity was significantly higher than other tissues,3PRGD₂-Pyro was significantly concentrated in the tumor.However,the fluorescence intensity of tumor in Block group was much lower than that in other organs and tissues,and there was no obvious uptake of 3PRGD₂-Pyro.Delineate the region of interest of the tumor and get the T/M ratio.In the 3PRGD₂-Pyro group,the T/M ratio of tumor to 3PRGD₂-Pyro reached the maximum at 4 h,which indicated that most of the 3PRGD₂-Pyro was distributed in the tumor,while the other parts of the tumor-bearing mice had fewer photosensitizers.3PRGD₂-Pyro could effectively target A549 tumor.For Block group,the ratio of fluorescence intensity to muscle fluorescence intensity did not change with time,and the T/M ratio was lower than that of 3PRGD₂-Pyro group.Pyro was also uptake in tumor,but the T/M ratio was smaller than that of 3PRGD₂-Pyro group,and the targeting ability was not as good as that of 3PRGD₂-Pyro.In addition,we analyzed the fluorescence intensity of organs and tissues in vitro 4 hours after injection of photosensitizer,and drew the fluorescence intensity of each organ.In 3PRGD₂-Pyro group,the fluorescence intensity of tumor was much higher than that of other organs and tissues.3PRGD₂-Pyro showed better therapeutic effect than Pyro.On the second day after treatment,the tumor appeared to be damaged and the color changed inside the tumor.On the fourth day after treatment,only a small proportion of the tumor remained.The tumor disappeared completely on the eighth day after treatment.No recurrence occurred on the fourteenth day after treatment.Histological evaluation with H&E staining revealed that the tumor tissue was destroyed,the number of tumor cells was significantly reduced,and the internal blood vessels were seen to be reduced in the tumor.No histological changes were observed in normal organs in any treatment group.3PRGD₂-Pyro can effectively treat tumors without damaging other normal organ tissues.Its light toxicity is strong and dark toxicity is weak,and it has no obvious side effects on organs and tissues.Immunohistochemical results showed that the expression of Integrin beta 3 was significantly decreased in the 3PRGD₂-Pyro?+?group at different time points?-1,8 and14 days?after photodynamic therapy,while the expression of CD31 was not significantly changed.The expression of Integrin beta 3 in the 3PRGD₂-Pyro photodynamic therapy group was significantly decreased in the A549 lung adenocarcinoma model.Both beta 3 and CD31 decreased significantly.Conclusions:3PRGD₂-Pyro was synthesized successfully.It has good water solubility,stability,high cytotoxicity and low dark toxicity.Compared with unmodified Pyro,the synthesized photosensitizer 3PRGD₂-Pyro has higher targeting ability,and the metabolic clearance of 3PRGD₂-Pyro in tumor-bearing mice is faster,showing good biocompatibility.Photosensitizer 3PRGD₂-Pyro shows potential for practical clinical application.In the lung adenocarcinoma A549 tumor model,3PRGD₂-Pyro has excellent photodynamic therapy effect and can effectively inhibit the growth of the tumor.3PRGD₂-Pyro,as photosensitizer for photodynamic therapy,has no side effects on organ and tissue.