Anti-tumor Effect Of Sodium Porphyrin-mediated Multiple Sonodynamic Therapy On Mouse Sarcoma S180

Background and purposeThe tumor has become a major danger to human health with the rapid development of the society, serious environmental damage and pollution, shrouded in a pollution haze. Although traditional therapy such as surgery, radiotherapy and chemotherapy can largely improve patient's symptoms, the side effects of high toxic, poor safety and heavy wound still exists. Thus, explore targeted, safe and effective methods and techniques for clinical treatment is the biomedical major issues currently.Japanese scholar proposed that using low-intensity ultrasound activate photosensitizer namely Sonodynamic therapy (SDT) on the basis of photodynamic therapy (PDT), in 1989. SDT of cancer is based on preferential uptake and/or retention of sonosensitizer in tumor tissues and subsequent activation of the drug by ultrasound irradiation, specially causing tumor killing. Low-intensity ultrasound can avoid local high temperature damage and systemic inflammatory response by comparison to high-intensity ultrasound. Sonosensitizer has the characteristics of preferentially uptake in tumor tissues, little side effect to normal tissues and spontaneous fluorescence, which is better to in the process of treatment location, imaging and monitor. Ultrasonic equipment is comparatively cheap and easily operation. The use of ultrasound has the advantages of being noninvasive and convenient while also possessing deep-penetration properties. Therefore SDT have the advantages of potential targeting, safe, effective, etc.Owing to the rapid expansion of knowledge on the fundamental mechanisms underlying SDT, investigators have recognized that the sonosensitizer is one of the most essential factors in SDT. The present study selected a new porphyrin dimers sodium salt (sinoporphyrin sodium, referred to DVDMS) as sonosensitizer by Professor Qicheng Fang from the Chinese Academy of Medical Sciences. DVDMS has 98.5% chemical purity, is highly soluble in water, results in relatively short-time skin sensitivity, and generates high singlet oxygen production. What's more, DVDMS was granted intellectual property rights in China, quality can be controlled and industrialized production.The SDT research hotspots focus on ultrasonic equipment and sensitizer optimization at the present stage. However few people research the effect of handling mode on the therapeutic effect. The study includes studying the chemical and physical aspects and the effect of treatment about DVDMS. There has been no report on the effect of ultrasound handling mode on the therapeutic effect. Here, we focused our investigation on the anti-tumor effects of the new sensitizer DVDMS combined with multiple ultrasounds. The findings may have significant implications for the treatment of cancer.Methods and ResultsPart 1 Cytotoxicity studies of DVDMS in normal cell and tumor cellCytotoxicity and cellular uptake of DVDMS was measured by flow cytometry, Xenogen IVIS spectrum system and MTT assay. The results were as follows:?the fluorescence signal of DVDMS elevated gradually and reached a maximum at approximately 3h. ?the florescence intensity of DVDMS was significantly higher in the S180 cells, suggesting that DVDMS, compared withthe other three sensitizers, accumulated to a much larger extent in cancer cells. ?A significant loss in cell viability was observed in tumor cell when the tumor cell and normal cell were incubated in same drug concentration.Part 2 In vitro studies-the proliferation and antiangiogenesis effect of DVDMS-SDT was investigated in S180 cell lineMTT assay, colony formation assay and flow cytometry combined with AnnexinV-PE/7-AAD displayed that DVDMS could be effectively activated by sound and the cytotoxicity was much higher in tumor cells than normal cells under the same conditions. DVDMS-SDT repressed tumor growth and that the effect was strengthened with the increase in ultrasound irradiation time. This phenomenon may represent the primary mechanism underlying the arrest of cancer cell proliferation. Besides, endothelial cell capillary-like tube formation assay indicated that tube formation was markedly inhibited by multiple ultrasonic treatments. The data revealed that DVDMS-mediated SDT play an important role in inhibiting tumor angiogenesis.Part 3 Pharmacoskinetic study of DVDMS in S180 tumor bearing miceThe DVDMS biodistribution and concentrations S180 tumor bearing mice were detected by Xenogen IVIS spectrum system. The results showed that DVDMS in tumors reached a peak at 2 h post-injection, DVDMS concentrations at 6 h and 24 h of DVDMS were about 98.77% and 70.37% of the peak, respectively. This study utilizes three times of ultrasound exposure at 2,6 and 24 h after DVDMS injection to maximumly excite DVDMS in the tumor tissue, producing much higher anti-tumor efficiency.Part 4 In vivo studies-the anti-tumor effect of DVDMS combined with multiple focused ultrasound treatments was studiedS180 tumor-bearing mice models were established to evaluate DVDMS combined with multiple focused ultrasound effects of DVDMS through tumor weight, tumor volume, histopathological changes and immunohistochemical staining. The results revealed that PDT with DVDMS markedly prolonged the survival of the tumor-bearing mice and inhibited tumor growth and lung metastasis, consistent with in.yitro findings. TUNEL assay indicated that DVDMS-mediated SDT could also induce apoptosis in vivo, thereby contributing to tumor growth suppression DVDMS-SDT decreased VEGF expression in a treatment time-dependent manner; furthermore, its combination with DVDMS further decreased VEGF expression. In the acute toxicity test, mortality, clinical signs, and body weight of the mice were measured; no obvious changes were observed. Based on this result, the No Observed Adverse Effect Level (NOAEL) of DVDMS-mediated SDT could be 2 mg/kg.ConclusionIn conclusion, the multiple ultrasound strategy is a relatively safe, repeatable, easily accessible, inexpensive, non-invasive, and non-toxic form of treatment that can be focused on a specific region deep in the tissue. Importantly, the combination of multiple ultrasounds with the new sensitizer DVDMS showed efficient anticancer effects in mouse sarcoma 180 cells in vivo and in vitro, including apoptosis induction, proliferation inhibition and suppression of tumor microvasculature formation, with no visible side effects. These results indicate that DVDMS combined with multiple ultrasounds may represent a new promising strategy against solid tumors.