Ultrasound-assisted Oxygen-loaded HPPH Microbubble Sensitization PDT Effect Study

Breast cancer is a high incidence of cancer and mortality for female and is the number one killer of women.The current treatment of breast cancer include surgery,radiotherapy,chemotherapy and endocrine therapy.Photodynamic therapy(PDT),which have the advantages of safety and mall side effects,developed in the 1970 s as tumor treatment.PDT mainly relys on its reactive oxygen species(ROS)to kill the tumor,which in a large extent depends on oxygen content in the tumor tissue.But tumor tissue is a hypoxic microenvironment.In view of tumor tissue hypoxia,different scholars put forward different solutions.For example,oxygen carrying microbubbles as oxygen carrier,can be carried out to the hypoxic tissue oxygen transport.Currently,the ultrasound(US)targeted MB destruction(UTMD)technique can be used for the increased drug and oxygen concentrations at the tumor site-and be expected to achieve accurate,targeted and efficient PDT therapy.Ultrasound-loaded drug microbubbles as ultrasound controlled carrier,is the use of ultrasound to locate and deliver drug to targeted system.That is,under the ultrasonic irradiation,the cavitation effect is enhanced by microbubbles as cavitation nuclei and the energy released by local ultrasound can make the drug released after microbubble disintegration into the tumor tissue,so as to play the role of targeted therapy.Photosensitizer,as one of the key elements of photodynamic therapy,plays an important role in photodynamic therapy.HPPH is 3-(1-hexyloxyethyl-3-de-vinyl pyropheophorbide-a)and its basic structure is chlorin compounds,which Belongs to the second generation photosensitizer.Nowadays,HPPH has a good effect on lung cancer,head and neck cancer and esophageal cancer.It is a kind of potential photosensitizer for PDT.HPPH has been in the late pre-clinical studies in China and in Phase ? clinical in the United States.In this paper,based on the literature search and preliminary research work,aiming at the hypoxia characteristics of tumor tissue,photosensitive HPPH was selected.Ultrasound(US)targeting microbubble(MB)destruction(UTMD)was adopted and oxygen and HPPH were wrapped into lipid microbubbles to synthesize HPPH-oxygen microbubbles(OxyMBs-HPPH),which were transported by ultrasound.The in vitro cytotoxicity of ultrasound combination with OxyMBs-HPPH mediated PDT was evaluated by establishing hypoxia cell systems of two breast cancer.The mouse 4T1 breast cancer model was established,and the antitumor effect of ultrasound combination with OxyMBs-HPPH-mediated PDT in vivo was studied.The results obtained were as follows:1.OxyMBs-HPPH was prepared by the factors of film composition,oxygen perfluoropropane ratio and dosage.The particle size was about 1007.23 ± 53.93 nm.The entrapment efficiency of HPPH was about 82.23%± 4.12%and about had 85%oxygen loading.2.The spectral characteristics of HPPH in OxyMBs-HPPH were analyzed.It was found that there were no significant changes in the peak positions of the UV spectra and the fluorescence spectra in the excitation and emission compared with HPPH.Under the same light dose and the concentration of photosensitizer in HPPH and OxyMBs-HPPH,The latter had higher singlet oxygen yield.3.The hypoxia model of two kinds of breast cancer cells was established by cell survival analysis and HIF-la expression.The conditions were as follows:1 ?mol/L Na2S2O4 and in hypoxia box(95%N2:5%CO2)for 2 h.4.Flow cytometry was used to determine the accumulation of HPPH at 2 h achieved the maximum after ultrasonic treatment with OxyMBs-HPPH by flow cytometry.5.MTT assay was used to detect the killing effect of different treatment groups on breast cancer cells.The results showed that ultrasound combined with OxyMBs-HPPH mediated photodynamic therapy was more effective than OxyMBs-HPPH mediated photodynamic therapy or HPPH mediated photodynamic therapy for breast cancer.At the same time,PI staining and flow cytometry were used to analyze cell membrane and scanning electron microscopy was used to analyze cell morphology.We found that combination treatment had the most serious injury in the cell membrane and cell morphology.Annexin V/PI double staining combined with flow cytometry detection of apoptosis found that ultrasound combined with OxyMBs-HPPH mediated photodynamic therapy had the largest proportion of apoptosis and necrosis.Finally,the levels of ROS and mitochondrial membrane potential(MTR)were analyzed.The results showed that ultrasound combined with OxyMBs-HPPH mediated photodynamic therapy resulted in the highest intracellular ROS production and the most severe mitochondrial membrane potential.6.The 4T1 BALB/c tumor bearing mice model was established.The results showed that the combination of ultrasound and OxyMBs-HPPH mediated photodynamic therapy had a stronger inhibitory effect on the growth of mouse breast cancer no matter in tumor volume or in tumor weight than OxyMBs-HPPH mediated photodynamic therapy and HPPH mediated photodynamic therapy.At the same time,H&E staining results showed that the nuclear loss of combination treatment,the space widening of tumor tissue and the destruction of tumor tissues were the most serious.Pulmonary metastasis results showed that ultrasound combined with OxyMBs-HPPH mediated photodynamic therapy had the lowest degree of metastasis.Analysis of the safety of mice found that this approach is relatively safe.