ZnPc-loaded Multifunctional Nano-probe Mediated Photoacoustic Imaging And Breast Carcinoma Targeting Study In Vitro

Background:Breast cancer has the highest incidence of cancer in women,and the incidence of breast cancer in China is on the rise.Early diagnosis and early treatment of breast cancer is the key to improve the survival rate of patients,which has become a research hotspot.Photoacoustic imaging has high contrast of optical imaging and good tissue penetration of ultrasonic imaging,and it can collect a variety of physiological and pathological data in real time that including the structure,function,metabolism and molecular information,which is helpful to early diagnosis of breast cancer.At the same time,along with the rapid development of molecular imaging technology,the technique of combining photoacoustic imaging with therapeutic methods is increasing with the use of various multi-function photoacoustic contrast agents.This is of great significance for the integration of breast cancer diagnosis and treatment.At present,the commonly used photoacoustic contrast agents are gold nanorods and indocyanine green.Gold nanorods are expensive.ICG is the only dye molecule currently approved by the FDA for clinical use,but it has a poor stability and rapid clearance in vivo.Therefore,it is of great significance to study the integration of new optoacoustic imaging agents in the diagnosis and treatment of diseases.As a second generation photosensitizer,phthalocyanine zinc(ZnPc)has the advantages of good light stability and excellent photothermal conversion effect.It was more widely used in photodynamic therapy,but it was seldom used in the study of photoacoustic imaging.In this study,PLGA was used as the base material,and RGD was used as a targeted drug,and the multifunctional nanometer probe with photosensitizer ZnPc and docetaxel was prepared by the method of double emulsification and carbodiimide method.The purpose of this study was to investigate the ability of probe to induce apoptosis of breast cancer cells and to broaden the idea of integration of breast cancer diagnosis and treatment.Objective:To investigate the targeting ability and photoacoustic imaging of novel nanoparticle probe loaded with ZnPc and docetaxel.Methods:The double emulsification method(O/W/O)was used to prepare the ZnPc-loaded and docetaxel-loaded PLGA nanoparticles(NPs).The morphologyof the NPs were observed by transmission electron microscope.The NPs size and zeta potential were detected by Dynamic light scattering method(DLS).The high-performance liquid chromatography(HPLC)and UV spectrophotometry were used to detect the encapsulating ratio(ER)and the drug loading(DL)of docetaxel and ZnPc in the NPs,respectively.HPLC and dialysis method were used to study the release characteristics of DTX of the NPs in vitro by laser irradiation.The photoacoustic imaging of NPs with different ZnPc content were observed in vitro.Flow cytometry(FCM)and confocal laser scanning microscopy were used to detect the the ligation efficiency between NPs and RGD.Confocal laser scanning microscopy was used to detect the ability of targeting NPs and non-targeting NPs to target breast cancer cells.In the in-vitro experiments of induce apoptosis of breast cancer cells,FCM determined the breast cancer cell apoptotic rate of the blank nanoparticles group,the pure DTX-loaded nanoparticles group,the non-targeting nanoparticles group,the targeting nanoparticles group,the non-targeting nanoparticles + laser irradiation group,the targeting nanoparticles + laser irradiation group,and evaluated the ability of the nanoprobe to induces the apoptosis of the breast cancer cells in vitro.Results:The probes were loaded with ZnPc and docetaxel efficiently and successfully.The size of NPs was(266.0±65.85)nm and the surface potential was(-29.20±6.27)mV.The encapsulation efficiency and drug loading efficiency of docetaxel was(88.00±0.32)%and(34.92±0.02)?g/mg,respectively.It had(97.25±0.22)%and(30.87±0.11)?g/mg for ZnPc loading in the probes,respectively.HPLC was used to determin the release rate of DTX of NPs in the release medium.After 24.0 h,the release rate of the DTX of the NPs was about 50%,and the release rate was about 68%after 48.0 h,and the release rate was about 77%after 72.0 h.The experimental group with laser irradiation compared with the control group without laser irradiation to show a slow release tendency too.Laser irradiation has no significant effect on the drug release.The in-vitro PAI show that the light acoustic wave peak of NPs appears at 700nm and the NPs showed obvious light acoustic signal,compared with the control group without ZnPc nanoparticles,and the optical sound signal enhanced with the increase of ZnPc content.Confocal laser scanning microscopy observed that the fusion of the green fluorescent of FITC-labeled RGDfk and the red fluorescent of the NPs was yellow fluorescence,which indicated that RGDfk have been successfully ligated to NPs.Flow cytometry detection results showed that the RGDfk modification rate was 89.19%.The in-vitro experimental result of targeting to the MBA-MD-231 cells showed that more nanoparticles adhered to MBA-MD-231 cells in the targeting nanoparticles group than the non-targeting nanoparticles group.In the in-vitro experiments of induce apoptosis of breast cancer cells,FCM determined that the MBA-MD-231 cell apoptosis rate of the blank nanoparticles group,the pure DTX-loaded nanoparticles group,the non-targeting nanoparticles group,the targeting nanoparticles group,the non-targeting nanoparticles + laser irradiation group,the targeting nanoparticles + laser irradiation group were(7.06±2.64)%?(19.31±4.83)%?(17.41±1.78)%?(26.99±5.53)%?(41.54±3.27)%?(62.87±5.95)%,respectively.The targeting nanoparticles group was more likely to induce MBA-MD-231 cell apoptosis than non-targeting nanoparticles group(P<0.05).The apoptosis rate of the targeting nanoparticles + laser irradiation group was higher than that of the other five groups(P<0.05).Conclusions:The new polymeric multifunctional nanoparticles probe was successfully prepared.The novel probe had an ideal size and good photoacoustic signals,which would become an excellent photoacoustic contrast agent.It also have the ability to target breast carcinoma cells and stable external docetaxel release,which can inhibit the proliferation of breast carcinoma cells efficiently.