The Sonodynamic Effects Of Chlorin E6on S180Sarcoma In Mice

Malignant tumor is the leading cause of death in the world. Therapeutic means include operation, radiotherapy, chemotherapy and targeted therapy. At the time of diagnosis, most of patients are in advanced or metastatic stage, which lose the chance of surgery. The chemotherapy and radiotherapy destroy not only tumor but also normal tissue, which induce side effect. Although targeted therapy has a low toxicity, its efficiency depend on gene states. So seeking new treatment for malignant tumor is a great challenge in cancer research.Sonodynamic therapy (SDT) is a promising new approach for cancer therapy developed from photodynamic therapy (PDT). Sonodynamic therapy functions by activating a tumor-localizing sonosensitizing agent with ultrasound, which generates singlet oxygen, The singlet oxygen can destruct tumor. Compared with PDT (activating a tumor-localizing photosensitizer with light) ultrasound can penetrate tissues and reach deep-seated tumors with ability to focus the ultrasound energy on a small volume, which makes sonodynamic therapy become a highly potential approach for tumor therapy. The main problem is that the accumulation of porphyrin and its derivatives is not highly selective in tumor and does not clear from normal tissue, which induces higher phototoxic side effect. Seeking new sonosensitizer which have selective accumulation in tumor tissue and determining the best SDT modes is a new chanlleage in front of us.Chlorin e6is the degradation products of chlorophy II which is similar to Hematoporphyrin (HPD) in structure. Previous studies showed that Chlorin e6can selectively accumulated in tumor tissue, rapidly clear from normal tissue and exerted antitumor effect with PDT. However SDT with Chlorin e6is limited, Our previous studies have shown that Chlorin e6can selectively accumulated in lung cancer and breast cancer cells and exerted antitumor effect with SDT. Unitil now, there is still lack evidence for the antitumor effect of Chlorin e6induce SDT in whole animol. The first purpose of this study is to determine whether Chlorin e6can accumulate selectively in the S180sarcoma of mice by laser scan confocal microscope (LSCM); the second purpose of this study is to assess its antitumor effects of SDT.PartⅠ The accumulation of Chlorin e6in tumor and muscle in the S180sarcoma miceObjectiveTo explore the accumulation of Chlorin e6in the S180sarcoma of mice and the optimal timing of ultrasound.MethodsFrozen sections of tumor and muscle tissue were made from mice sacrificed at3h,6h,12h,18h,24h and72h after intraperitoneal injection of Chlorin e6(10mg/kg). Fluorescence images were attained after intraperitoneal injection of Chlorin e6(10mg/kg) with laser scan confocal microscope (LSCM). Results were analyzed by repeated measures analysis of variance.ResultsAfter intraperitoneal injection of Chlorin e6, the concentration of Chlorin e6in tumor was significantly higher than that in normal muscle tissue within24h, which reached to a peak at18h. At the same time, the difference of Chlorin e6concentration between tumor and muscle tissue was greatest, which showed Chlorin e6in tumor was6.14times that in muscle, This results suggested that it was the optimal timing for SDT. During18-24h after injection, fluorescence decreased rapidly and reached to normal at24h, fluorescence was undetectable at72h. The results suggested that Chlorin e6can clear fastly, which induce less toxicity in normal tissue.ConclusionChlorin e6can selectively accumulated in tumor tissue and can be cleared quickly from normal tissue. Tumor should be treated at18h after Chlorin e6injection in SDT. PartⅡ The sonodynamic effects of Chlorin e6on S180sarcoma in miceObjectiveTo explore the antitumor effect of Chlorin e6with SDT.Methods1、The antitumor effect of ultrasound aloneThe tumor-bearing mice were divided into4groups (5mice for one group):the control group, ultrasound alone (the intensity of ultrasound was separated to0.4W/cm2,0.8W/cm2and1.6W/cm2). The1.0MHz ultrasound irradiation was treated for180s2、The antitumor effect of Chlorin e6aloneThe tumor-bearing mice were divided into4groups (5mice for one group):the control group, Chlorin e6alone (the dose of Chlorin e6was separated to10mg/kg,20mg/kg and40mg/kg). 3、The antitumor effect of Chlorin e6with ultrasoundThe tumor-bearing mice were divided into4groups (5mice for one group):the control group, ultrasound alone (1.6W/cm2), Chlorin e6alone(40mg/kg), Chlorin e6+ultrasound (1.6W/cm2ultrasound+40mg/kg Chlorin e6), The1.0MHz ultrasound irradiation was treated for180s at18h after intraperitoneal injection of Chlorin e6.4、The relationship of the antitumor effect and the intensity of ultrasound in SDT with Chlorin e6The tumor-bearing mice were divided into4groups (5mice for one group):the control group, the dose of Chlorin e6was set to40mg/kg, the intensity of ultrasound was separated to0.4W/cm2,0.8W/cm2and1.6W/cm2. The1.0MHz ultrasound irradiation was treated for180s at18h after intraperitoneal injection of Chlorin e6.5、The relationship of the antitumor effect and the dose of Chlorin e6in SDT with Chlorin e6The tumor-bearing mice were divided into4groups (5mice for one group):the control group, the intensity of ultrasound was set to1.6W/cm2, the dose of Chlorin e6was separated to10mg/kg,20mg/kg and40mg/kg; The1.0MHz ultrasound irradiation was treated for180s at18h after intraperitoneal injection of Chlorin e6.The tumor size was recorded every2days and the tumor was weighted on the15th day after treatment. Data are presented as mean±S.E.M, experiments between two groups were analyzed by t test. Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparision test.Results1、The antitumor effect of ultrasound alone or Chlorin e6aloneCompared to control group, ultrasound (0.4～1.6W/cm2) alone or Chlorin e6(10～40mg/kg) alone had no significant antitumor effects (P>0.05) 2、The antitumor effect of Chlorin e6with ultrasoundIn the control group, the tumor sizes increased over the time period. It was observed that ultrasound (1.6W/cm2) alone or Chlorin e6(40mg/kg) alone had no significant antitumor effects (P>0.05). In the Chlorin e6with ultrasound treatment group (sono1.6W/cm2+Chlorin e640mg/kg), the tumor growth was significantly delayed compared to other groups. The combination of ultrasound with Chlorin e6(SDT) exerted significant antitumor effects (P<0.05)3、The relationship of the antitumor effect and the intensity of ultrasound in SDT with Chlorin e6Compared to control group, the Chlorin e6with SDT treatment groups (the dose of Chlorin e6was set to40mg/kg, the intensity of ultrasound was separated to0.4W/cm2,0.8W/cm2and1.6W/cm2) exerted significant antitumor effects (P<0.05) This antitumor effects was intensity-dependent for ultrasound. The1.6W/cm2ultrasound may be the optimal intensity in SDT.4、The relationship of the antitumor effect and the dose of Chlorin e6in SDT with Chlorin e6Compared to control group, the Chlorin e6with SDT treatment groups (the intensity of ultrasound was set to1.6W/cm2, the dose of Chlorin e6was separated to10mg/kg,20mg/kg and40mg/kg) exerted significant antitumor effects (P<0.05). This antitumor effects was dose-dependent for Chlorin e6. The40mg/kg Chlorin e6may be the optimal dose in SDT.ConclusionBoth ultrasound (0.4～1.6W/cm2) or Chlorin e6(10～40mg/kg) alone had no significant antitumor effects. However, the combination of ultrasound with Chlorin e6(SDT) exerted significant antitumor effects. This antitumor effects was intensity-dependent for ultrasound and dose-dependent for Chlorin e6. The adverse reaction such as photosensitive dermatitis was not observed.