Breast Cancer Cell Membrane Biomimetic Contrast Agent For Dualmodal Imaging Guided Phototherapy

PART ? PREPARATION AND CHARACTERIZATION OF BREAST CANCER CELL MEMBRANE BIOMIMETIC CONTRAST AGENTObjective To prepare a novel breast cancer cell membrane biomimetic contrast agent(MPFTNPs),and detect its material characterization,photothermal performance and photodynamic performance.The specific homologous targeting ability of MPFTNPs to homologous tumors and the photothermal and photodynamic performance were evaluated in vitro.Methods The PpIX@Fe~? TA(PFTNPs)was prepared by a solvent exchange method.The biomimetic contrast agent(MPFTNPs)was prepared by modifying the cell membrane of breast cancer on the surface of the PFTNPs under sonication.The morphological characteristics(transmission electron microscope and scanning electron microscope),particle size potential characteristics,PpIX encapsulation efficiency,UV absorption spectrum,membrane protein and other material characterization were detected.The photothermal characteristics of PFTNPs and MPFTNPs in different pH values under 808 nm laser irradiation were detected.The production of reactive oxygen species(ROS)was detected under 660 nm laser irradiation.The phagocytosis of MPFTNPs by different tumor cell lines was observed by flow cytometry and laser confocal microscopy.Results PFTNPs was successfully prepared by a solvent exchange method.Then,the biomimetic contrast agent(MPFTNPs)was prepared by modifying the cell membrane of breast cancer on the surface of the PFTNPs under sonication.Transmission electron microscope(TEM)showed that the prepared MPFTNPs was uniform with a classical putamen structure.Scanning electron microscopy(SEM)showed that MPFTNPs had good dispersion with a typical spherical structure.The particle size of MPFTNPs was 295.3 nm(PDI:0.311),and the Zeta potential was-15.4 mV.The UV absorption spectrum showed that MPFTNPs retained the absorption peak of PpIX,and its encapsulation efficiency was 91%.SDS-PAGE results showed that MPFTNPs retained the surface membrane protein of cell membrane.In vitro photothermal experiments showed that MPFTNPs had high photothermal conversion efficiency.The photothermal ability of MPFTNPs was higher than PFTNPs in acidic environment.MPFTNPs had good biocompatibility and can target to homologous tumor cells efficiently.Conclusions MPFTNPs were successfully prepared with uniform size distribution,regular morphology,excellent PpIX encapsulation rate,and considerable membrane protein.PDT can be carried out under the excitation of 660 nm laser,and PTT can be carried out under the excitation of 808 nm laser.After the decoration of breast cancer membrane,the photothermal capability of PFTNPs was significantly enhanced.The MPFTNPs could achieve both PDT and PTT,which is expected to provide a new biomimetic contrast agent for breast cancer therapy.PART ? BREAST CANCER CELL MEMBRANE BIOMIMETIC CONTRAST AGENT FOR PHOTOACOUSTIC IMAGING/MRI DUALMODAL IMAGINGObjective To observe the effect of MPFTNPs for photoacoustic imaging(PAI)and magnetic resonance imaging(MRI)both in vitro and in vivo.Methods MR imaging model was established by using 50-hole centrifuge tube box.MR imaging system was used to image MPFTNPs of different concentrations and analyze the corresponding signal intensity.A gel model was established to detect photoacoustic imaging of different concentrations of MPFTNPs and analyze the corresponding signal intensity.Furthermore,MDA-MB-231 cells were injected subcutaneously into the nude mice to establish the subcutaneous breast cancer transplantation model for in vivo photoacoustic imaging/MRI dual-modal imaging experiments.Photoacoustic imaging and T1-weighted MRI were performed at different time points(0,2,6 and 24 h)after injection of MPFTNPs via tail vein of the tumor-bearing mice.Results In vitro imaging results showed that MPFTNPs as dual-modal imaging contrast agents could achieve photoacoustic imaging and T1-weighted MRI imaging.The photoacoustic imaging signal and T1-weighted MRI were positively correlated with MPFTNPs concentration.After intravenous injection with MPFTNPs via tail vein,T1-weighted MRI and PAI signal of tumor site gradually increased,reaching its peak in 24 h.Conclusions The prepared MPFTNPs nanoprobe could achieve PAI and T1-weighted MRI dual-modal imaging in vitro.The experimental results of tumor bearing nude mice showed that MPFTNPs can be effectively enriched in tumor site to achieve PAI and MRI dual-modal imaging.Therefore,MPFTNPs have shown a broad clinical application prospect in the field of breast cancer dual-modal imaging.PART ? THE EXPERIMENTAL STUDY OF BREAST CANCER MEMBRANE BIOMIMETIC CONTRAST AGENT FOR COMBINED PHOTOTHERAPYObjective To study the PTT and PDT therapeutic effect on breast cancer in vitro and in vivo.The biocompatibility of MPFTNPs was also evaluated.Methods CCK-8 method was used to detect the killing effect of MPFTNPs on MDA-MB-231 cells and evaluate the biocompatibility of MPFTNPs.The killing effects of PFTNPs and MPFTNPs at different concentrations on MDA-MB-231 tumor cells were detected under 808 nm laser irradiation.A CCK-8 assay,flow cytometry and laser confocal microscopy were used to evaluate the synergistic killing effect between photothermal effect(PTT)and photodynamic effect(PDT)on MDA-MB-231 cells.24 h after injection of MPFTNPs,808 nm laser was used to irradiate the tumor site,and an infrared camera was used to monitor the temperature change during the treatment process,and corresponding thermal imaging pictures were recorded.MDA-MB-231 tumor-bearing mice model was established.When the tumor volume grew to 60-70 mm3,the tumor bearing nude mice were randomly divided into 6 groups:(1)control group;(2)laser only group;(3)MPFTNPs group;(4)PDT only group;(5)PTT only group;(6)PDT/PTT combined therapy group.808 nm laser(2.0 W/cm2,5 min)and 660 nm laser(100 m W/cm2,30 min)were carried out in group(2)and group(6),respectively.660 nm laser(100 m W/cm2,30 min)was carried out in group(4).Group(5)was irradiated with 808 nm laser(2.0 W/cm2,5 min).In the process of PTT,the temperature change of tumor site was detected and recorded by an infrared camera,and the corresponding temperature rise curve was drawn and corresponding thermal images were recorded.The diameter of tumor and the weight of tumor bearing nude mice were measured every two days.24 h after different treatments,one mice in each group was randomly killed and the tumor was taken out.After H&E,PCNA and TUNEL staining,the proliferation and apoptosis rate of tumor cells were counted by optical microscope and analyzed quantitatively.In order to evaluate the biological safety of MPFTNPs,one mice in each group of tumor-bearing nude mice was killed randomly at 24 h after treatment,and the main organs(including heart,liver,spleens,lungs,kidneys and brain)were taken out for H&E staining,and the morphological changes were observed.In addition,different doses of MPFTNPs(0,10,20 and 40 mg/kg)were injected into the tail vein of healthy Kunming mice.After 30 d,the blood was collected for blood routine and biochemical analysis.The main organs(including heart,liver,spleens,lungs,kidneys,and brain)were taken out for H&E staining,and the morphological changes were observed.Results MDA-MB-231 cells were killed by PTT under 808 nm laser irradiation,and the cells were killed by PDT under 660 nm laser irradiation.The combination of PDT and PTT showed a maxium killing capability for combating tumors.The tumor-site temperature gradually increased after 808 nm laser irradiation.In vivo thermal imaging(PTI)showed that the temperature of tumor site increased significantly in MPFTNPs treated group with a temperature up to 56.1 ?.As contrasts,the temperature of PFTNPs treated group reached 48.5 ? and that of control group was only 42.3 ?.The tumor volume increased 3.98 times in the PTT group and 5.20 times in the PDT group,respectively,and the tumor growth rate was slower than that in the control group(tumor volume increased 10.76 times),indicating that both PTT and PDT can inhibit tumor growth to a certain extent.In the PDT and PDT combined group,the tumor growth was completely inhibited.The results of H&E staining of tumor tissue showed that the morphology of tumor cells in PDT/PTT combined treatment groups was significantly damaged,and nuclear pyknosis,fragmentation and dissolution were seen;PCNA and TUNEL staining showed that the proliferation index of cells in the PDT/PTT combined treatment group was lower than the other groups,while the apoptosis index in the PDT/PTT combined treatment group was higher than the other groups(*** P < 0.001).The H&E staining results of the main organs(including heart,liver,spleens,lungs,kidneys and brain)of the tumor bearing nude mice showed no obvious damage.The H&E staining results of the main organs(including heart,liver,spleens,lungs,kidneys and brain)of Kunming mice also showed no obvious damage.The blood routine and biochemical results of Kunming mice were no significant difference among different groups.Results MPFTNPs could inhibit tumor growth completely through the mechanism of PDT and PTT in vitro and in vivo.In addition,it showed good biocompatibility in short-term(1 day)and relatively long-term(30days),which showed broad clinical application prospects.