The Study Of IR-780 Photothermal Therapy And Sonodynamic Therapy On Breast Cancer

Backgroud Malignant tumors are still the main cause of death in the world top diseases.The major treatments are surgery, radiotherapy and chemotherapy. Althoughcombination therapy of cancer treatment has the advantages over a single treatment,while sometimes it will increase the side effects which patients have to give up thetreatment. So looking for new methods of treatment of the malignant tumor whichhave good selectivity and small side effects are still today’s hot spot in medicalresearch. Photothermal therapy(PTT) is a mothod that light is converted into energy toreach a critical heating treatment temperature and maintain for a period of time toachieve the purpose of the treatment of tumor. Photothermal therapy(PTT)graduallygets the affirmation of the clinical research, because of its small side effects as wellas the advantages of high selectivity. IR-780 iodide, a lipophilic cation heptamethinedye with peak absorption at 780 nm, has emerged as a potential fluorescent probefor in vivo tumor imaging due to its high fluorescence intensity, excellent stabilityand preferential accumulation in the tumor. In addition, it can be used to PTT due toits strong absorption in near-infrared light area.Sonodynamic therapy(SDT) is a new method of neoplastic treatment in recent years. Japanese scientist Yumita first put forward the concept of the sonodynamic therapy(SDT), He proved for the first time that the low-frequency ultrasound could activate photosensitizer to suppress tumor growth, and the processes were similar to laser activation process. Sonodynamic therapy(SDT) is that sonosensitizers selectively gathered in tumor tissue, sonosensitizer is activated upon ultrasound irradation to produce a series of chemical reactions to achieve the purpose of inhibiting tumor growth. SDT includes three parts:sonosensitizer; ultrasound; oxygen molecules. Sonodynamic therapy (SDT) is based on photodynamic therapy(PDT), the current sonosensitizers most are photosensitizers, sonosensitizer is one of the key factors of the success in SDT treatment. Although the mechanism of SDT has not been fully elucidated, while ultrasound can activate the sonosensitizer to produce a series of chemical reactions to induce tumor cell necrosis or apoptosis.SDT has two unique features which give it a decided advantage over PDT. (i) US (a mechanical wave) can easily penetrate human tissue and get into a deep cancer, (ii) US can precisely focus on a specific tumor site and effectively activate the sonosensitizer. It has been demonstrated that SDT can accurately kill the cancer cells with a great many fewer damage to normal cells. Many photosensitizers can be used as sonosensitizers for SDT, as porphyrin and porphyrin derivatives, et, these agents not only show good therapeutic effect in the PDT as well as SDT. Considering the photosensitizer and sonosensitizer have long absorption wavelength and can be activated under light, We assumed that the IR-780 can be activiate by ultrasound for SDT experiments.Part One the study of IR-780 photothermal therapy on breast cancerObjectiveTo study the effect of IR-780 combined with laser on breast cancer of the mice.Materials and methodsExperimental materialsThe IR-780 iodide, dimethyl phosphite (DMSO). Weighed a certain amount of IR-780 dissolved in DMSO, made its concentration at 32 mg/ml, then dissolved in basic medium at the final concentration of 800μg/ml for experiments. Single step apoptosis detection kit (TUNEL).Laser Machine; Vevo2100 High frenquency Ultrasound Imaging Systeml; Inverted Fluorescence Microscope.The Paraffin Section Machine. Methods4T1 breast cancer cells were inoculated in the right subcutaneous abdomen of Balb/c mice to establish tumor models. Experimental mice were randomly divided into four groups (each group five mice):(1) Contral group (0.1 ml saline was injected); (2) Laser+Saline group (laser 2.2 W/cm2,4 min); (3) IR-780 group (80 μg IR-780 was injected); (4) IR-780+Laser group (80 μg IR-780 was injected, then irradated with laser,2.2 W/cm2,4 min). Then the tumor size was measured and recorded. And the ultrasonic echo of tumor tissue in each group was studied with Vevo 2100 high frequency system at 0 h,12 h and 60 h after injection of drug in tumor respectively. Tumor tissues in each group were detected with TUNEL to monitor apoptotic cells after 60 h of treatment. ResultsThe volume of IR-780 plus laser decreased significantly compared with IR-780 group (P<0.01). The ultrasonic echo of IR-780+Laser tumor tissue at 12 h reduced compared with 0 h (P<0.05), and ultrasonic echo tumor tissue at 60 h apparently reduced compared with 0 h (P<0.01). while other three groups were observed no significant change in tissue echo except slightly increased tumor size. TUNEL experiment presented abundant apoptosis in the group of IR-780+Laser compared with IR-780 group (P<0.01), and other three groups had no difference.ConclusionThe obvious killing effect was observed in PTT with IR-780 on breast cancer tumor, presented as change of ultrasonic echo of tumor tissue in high frequency, increased apoptosis in the TUNEL experiment.Part Two Inhibition effect of sonodynamic therapy(SDT) with IR-780 on breast tumor cells and its machanismsObjectiveTo study the inhibition effect of sonodynamic therapy(SDT) with IR-780 on breat tumor cells and its machanisms.Materials and methodsExperimental materialsThe IR-780 iodide, dimethyl phosphite (DMSO). Weighed a certain amount of IR-780 dissolved in DMSO, made its concentration at 10 mM, then dissolved in FBS at the final concentration of 100 μM for experiments. Cell Counting Kit-8(CCK-8), Annexin V-FITC Apoptosis Kit, MitoSOXTM Red mitochondrial superoxide indicator,5-(and-6)-chloromethy-2’,7’-dichlorodihydroflurescein diacetate, acetyl ester(CM-H2DCFDA), Hydroxyphenyl fluorescein solution (HPF).BD FACSCantoⅡ flow cytometry, Laica TCS SP5 laser confocal microscopy, sonicator device, SynergyTM4 multimode plate reader, BD Accuri C6 flow cytometry.MothodsTo test 4T1 tumor cells uptake the IR-780 iodide at different concentrations of IR-780 iodide for different incubation time, flow cytometry experiments were conducted. To test the effect of SDT with IR-780 on viability of tumor cells through CCK-8 testing, different ultrasonic irradiation time(0 s,20 s,40 s) and different IR-780 iodide concentration were used. In order to detect apoptosis necrosis of SDT with IR-780, flow cytometry experiments were examined. Reactive oxygen species as 1O2, H2O2 and ·OH were detected to explore the mechanism SDT with IR-780.Results1. Results were demonstrated that there were obvious dose-and time-dependence for cellular uptake of IR-780 iodide at 4 μM,10 μM,16 μM at 1 h, 2 h,3 h. Also, stronger fluorescence intensities might be found in these cells with longer incubation time at the same dye dose.2. Under the condition without US, treatment of tumor cells had minor inhibition effect on cell viability. Only US irradiation for 20 s had not obvious effect on cell viability, but longer US irradiation for 40 s could observe some inhibitive effect on tumor cell viability. It was notable that significant growth inhibition effects on cell viabilities could be seen when IR-780 iodide plus US was used. The higher concentrations of the IR-780 iodide or longer US duration were used, the better inhibitory effect on tumor cell growth would be.3. The results demonstrated that IR-780 plus US induced significant tumor cell apoptosis/necrosis and there were in positive correlation with IR-780 concentration.4. The tumor cells treated with IR-780 plus US caused significant increased level of 102. cells treated with IR-780 plus US had a significant increase of H2O2. There were higher fluorescence intensities compared with other groups (P< 0.01). No significant increase of OH levels was found in the tumor cells treated with IR-780 plus US. ConclusionIR-780 can be efficiently uptaken by 4T1 cells. IR-780 plus US could inhibit the activity of 4T1 cells and induce tumor cells apoptosis/necrosis. At the same time can obviously increase 102 and H2O2 in tumor cells.Part Three Inhibition Effect of Sonodynamic Therapy with IR-780 on 4T1 Transplatation TumorObjectiveTo study Inhibition effect of SDT with IR-780 on 4T1 transplatation tumor and further explore its mechanisms.Materials and methodsExperimental materialsThe IR-780 iodide, dimethyl phosphite (DMSO). Weighed a certain amount of IR-780 dissolved in DMSO, made its concentration at 32 mg/ml, then dissolved in basic medium at the final concentration of 800μg/ml for experiments. Single step apoptosis detection kit (TUNEL), Eosin and Hematoxylinstaining staining solution. MothodsThe IR-780 fluorescent intensity in the tumors of mice were detected at different time points through the near infared imaging system by intra-tumor injection of IR-780 solution, then determined the best ultrasonic irradiation time. 4T1 transplanted tumor mice were divided into four groups:(1) PBS group; (2) ultrasound group; (3) IR-780; (4) IR-780 plus ultrasound group. To explore the inhibition effects of SDT with IR-780, changes of the tumor volume and weight were observed after US treatment. Tumor tissue were stained by H&E to observe histomorphology and TUNEL experiment were conducted to detect the tissue apoptosis.Results1. Fluorescence signals increased gradually as IR-780 diffusing in the tumor tissue, reaching a maximum tumor coverage area after 1 h. A longer time (>6 h) would result in the permeability of IR-780 into the peripheral normal tissues.The tumor localization of IR-780 was still dominant even if 48 h later(P<0.01).2. mice received with IR-780 plus US showed a significant tumor growth inhibition (P< 0.01). By contrast, there were not significant inhibition effects on tumor growth for IR-780 group or only US group (P> 0.05).3. A significantly increased degree of cell necrosis could be observed in the tumors receiving IR-780 and US. TUNEL-positive cells were significantly more in the tumors treated using SDT with IR-780, compared with other groups.ConclusionIR-780 as a sonosensitizer agent for SDT treatment achieved obvious effects. H&E staining and TUNEL experiment showed significantly increased necrosis and apoptosis after ultrasonic irradiation in the IR-780 plus US group compared with other groups.