A Novel Sensitizer DVDMS Acoustic-dynamic Anti-tumor Experimental Study

Background and Objective:Sonodynamic therapy(SDT)is an emerging anti-cancer method that was presented in 1989 by Umemuras,SDT involved the synergistic effects of low-intensity ultrasound and a sonosensitizer.The low tissue attenuation coefficient and nonradiative nature characterized by dominant tissue penetration,non-invasion and regional focusing of ultrasound contributed SDT to treat deep sited tumors.Ultrasound and sonosensitizer are the vital element in SDT,the physical and chemical properties of sonosensitizers directly determine the therapeutic efficacy of SDT.DVDMS is a porphyrin dimmer connected by an ether bond,its strong antitumor photosensitivity has been previously demonstrated in many kinds of tumors,but the sonosensitivity of DVDMS hasn't been fully studied.Abundant researches were needed to confirm DVDMS-SDT anticancer effect.Nanotechnology has been widely used in the early diagnosis and treatment of cancer,it achieved the non-invasive targeted delivery of drugs to overcome the low selectivity and systemic side effects of traditional chemotherapy drugs.Recently,nanotechnology has gradually applied to the SDT field.The nano-modification of traditional sensitizers achieved target drug delivery,controlled release,multimodal imaging,and enhanced anti-tumor effect of SDT.In this paper,we focus on the wide anti-tumor study of DVDMS-SDT,the following research content were mainly studied focusing on DVDMS.(1)The interaction between DVDMS and protein molecules and the damage effect of DVDMS-SDT on protein and nucleic acid molecules were studied,which provided the basis for in vivo DVDMS transportation,metabolism,distribution and the in vivo.(2)Investigate the cytotoxicity induced by DVDMS-SDT on various tumor cells like ECA-109,SGC-7901,MDA-MB-231,U373,CT26 and C6,mitochondrial injury,DNA oxidative damage and apoptosis were detected by morphological observation,biochemical analysis and molecular measurements.The relationship between ROS and apoptosis was further obtained by applying NAC inhibition studies.(3)Explore the combined anti-cancer effect of micro-bubbles and DVDMS-SDT both in vitro and in vivo,moreover,the potential mechanism was evaluated from membrane permeability and intracellular DVDMS concentration.(4)In order to improve DVDMS bioavailability,iRGD target DVDMS-liposome(iRGD-Lipo-DVDMS)were designed and synthesized,physical and chemical properties,and metabolic characteristics were firstly analyzed.(5)The anti-cancer effect of iRGD-Lipo-DVDMS-SDT was next analyzed on glioma.Focus ultrasound and microbubbles were used to open blood brain barrier(BBB)and the killing effect of iRGD-Lipo-DVDMS-SDT on glioma were evaluated both in vivo and in vitro.The available findings may provide valuable information about DVDMS in SDT anticancer field,which promoted DVDMS-SDT mediated cancer therapy to clinical application.Results and main conclusion:1.Interaction analysis between DVDMS and BSA and the damage of DVDMS-SDT to BSA and DNA.Fluorescence spectroscopy revealed a strong interaction between DVDMS and BSA molecules in aqueous solution,affecting DVDMS targeting distribution and metabolism.After co-incubation of DVDMS and BSA,DVDMS absorption spectrum hasn't show obvious changes but the fluorescence emission spectroscopy moved from 620nm to 641nm along with a significant enhancement of DVDMS fluorescence intensity.The characteristic absorption of BSA(280nm)and DNA(260nm)decreased and BSA fluorescence intensity raised,at the same time gel electrophoresis experiment showed DNA degradation after DVDMS-SDT treatment,which indicated seriouse damage of BSA and DNA structure.2.DVDMS-SDT triggers mitochondrial dependent apoptosis in ECA-109 cells via ROS production.DVDMS-SDT showed a general anti-tumor effect on various malignant cells including ECA-109,SGC-7901 and MDA-MB-231,but no cytotoxicity on normal cells(HEK-293)using the same SDT parameter.The sensitive ECA-109 cells were chosen for further studies.Compared with Hp,DVDMS mediated SDT showed stronger cytotoxicity on ECA-109 cells.Abundant of intracellular ROS was found in SDT groups,and the cytotoxicity induced by SDT was effectively remitted by ROS scavenger.DVDMS mainly located in mitochondria of ECA-109 cells,and after SDT treatment the mitochondria were serious damaged.The release of Cyt C,enhancive apoptosis rate and activated apoptosis protein were detected in SDT group.In addition,relatively serious cell damages were observed under SEM after DVDMS-SDT treatment.3.DVDMS-SDT induces tumor cell apoptosis through DNA oxidative damage by generating ROS.Previous studies have demonstrated that DVDMS-SDT injury DNA structure,which indicating DNA may be a potential damage target in DVDMS-SDT anti-tumor treatment.We used U373 cells(P53 mutant)to analyze the relationship between DNA damage and cell apoptosis after DVDMS-SDT treatment.MTT and PI assay showed significant cytotoxicity induced by DVDMS-SDT treatment,microscope observation found serious cells injury and condensed chromatin and cell death.Abundant ROS were detected after DVDMS-SDT and ROS scavenger NAC can significantly rescues cytotoxicity caused by SDT.DNA fragment analysis,single cell gel electrophoresis and ?H2AX expression proved DNA double-strand breaks,Annexin V staining manifest cell apoptosis in DVDMS-SDT group and NAC also rescue DNA damage and cell apoptosis.4.Microbubbles enhance the antitumor effects of DVDMS-SDT both in vitro and in vivo.Microbubbles can significantly enhance the cytotoxicity and necrocytosis rate induced by SDT treatment.The increase membrane permeability and much higher uptake of DVDMS were founded in SDT combined with microbubbles group.For the in vivo experiments,tumor weight and size in SDT combined with microbubbles group were much smaller than other treatment groups with pimping difference of mice weight.In addition,microbubbles notably improved tumor tissue destruction caused by ultrasound and SDT treatment.5.iRGD-Lipo-DVDMS synthesis and characterization.Lipo-DVDMS and iRGD-Lipo-DVDMS size were about 120nm,and TEM image showed round shaped with a smooth surface and uniform particle size.Compare with free DVDMS,Lipo-DVDMS exerts longer serum half-life and higher level in C6 cells.iRGD-Lipo-DVDMS can be selectivity uptake by C6 cells,which were overexpress?v?3 protein,similarly,the target liposome can preferentially accumulated in C6 tumor tissues in vivo,which proved the well target function of iRGD-Lipo-DVDMS.6.Anti-glioma effect of iRGD-Lipo-DVDMS-SDT both in vitro and in vivo.We mainly foused on the anti-cancer effect of iRGD-Lipo-DVDMS-SDT on glioma in this part.Fluorescence macromolecule marker and Evans Blue leakage test demonstrated that appropriate ultrasound parameters combined microbubble treatment can open BBB safely and reversible.MTT assay showed that iRGD-Lipo-DVDMS-SDT could effectively kill C6 cells and C6 tumor sphere.After iRGD-Lipo-DVDMS-SDT treatment,large amounts of reactive oxygen species were detected in C6 cells and SDT also induced cell apoptosis.For the in vivo test,iRGD-Lipo-DVDMS-SDT significantly prolonged the survival of C6 tumor-bearing mice and relieved the rate of weight loss in tumor-bearing mice.All the results indicated that iRGD-Lipo-DVDMS-SDT can be used as an alternative treatment for glioma.