The Study Of Nanoparticles Based On Porous Structure Zif-8 For Bimodal PA/NIR-Ⅱ FL Imaging Guided Photodynamic Therapy

Objectives:1.To investigate the physical and chemical properties of ZIF-8-IR820-MnPc-HA nanoparticles and verify their photodynamic effects.2.To further investigate the photoacoustic/near-infrared Ⅱ fluorescence bimodal imaging effect and biological safety of ZIF-8-IR820-MnPc-HA nanoparticles in vitro and in vivo.Methods:1.Preparation and Characterization of ZIF-8-IR820-MnPc-HA:ZIF-8-IR820-MnPc-HA nanoparticles（ZIMH NPs）were synthesized by one-step method,and the whole process was protected from light.The physical and chemical properties of ZIF-8-IR820-MnPc-HA nanoparticles were characterized by dynamic light scattering（DLS）,transmission electron microscopy（TEM）,ultraviolet spectroscopy（UV）and thermogravimetric analysis（TGA）.And the photodynamic effects of ZIMH NPs were verified by NIR laser.2.In vitro experiments:The cytotoxicity study of ZIF-8-IR820-MnPc-HA was carried out by CCK-8 method after co-incubation with 4T1 cells;verify that nanoparticles can generate singlet oxygen under 808 nm laser irradiation at the cellular level;the photodynamic therapy effect of ZIMH NPs at the cellular level was analyzed by living/dead cell assay;the photoacoustic imaging system scans ZIMH solution with different concentration gradient to observe the imaging effect;ZIF-8-IR820-MnPc-HA solutions with different concentration gradients were scanned by near-infrared Ⅱ fluorescence imaging system,and the feasibility of ZIF-8-IR820-MnPc-HA solutions as contrast agents for optical imaging was primarily verified respectively.3.In vivo experiments:A subcutaneous tumor in nude mice was established.After ZIF-8-IR820-MnPc-HA nanoparticles were intratumorally injected into each mouse,photoacoustic imaging and near-infrared Ⅱ fluorescence imaging scanning were respectively performed in different time points to dynamically observe the enrichment of nanoparticles in local tumors in real time.After the injection of the nanomaterials,nude mice were sacrificed at different times.And the vital organs such as heart,liver,spleen,lung and kidney were harvested,as well as blood was collected for further pathological tissue and blood biochemical analysis,to study the biological safety.Results:1.Characterization of nanoparticles:Both TEM and DLS results showed that after the surface of ZIF-8-IR820-MnPc（ZIM）was coated with HA,the size of the nanoparticles become larger and the morphology got irregular,which may be due to the expansion of hyaluronic acid.The absorption curves of the raw materials and the staged products were measured by ultraviolet spectrophotometer.The results showed that the absorption peaks of ZIF-8-IR820-MnPc overlapped with that of IR820 and MnPc,initially confirming the successful synthesis of ZIM.TGA curves verified that HA was successfully coated on ZIM surface with an effective loading of about 10%.DPBF and SOSG,two reactive oxygen species（ROS）trapping,were used to carry out that a certain amount of singlet oxygen could be produced by ZIMH at the solution level.2.In vitro experiments:The results of CCK-8 cytotoxicity experiments showed that the cell survival rate remained above 90%after the nanomaterials were incubated with cells for 24 hours with a concentration of 50μg/m L,proving that ZIMH nanoparticles had good biocompatibility.Then ZIMH NPs labeled with DCFH-DA were co-incubated with4T1 cells and irradiated with an 808 laser,and bright green fluorescence could be observed,which proved the production of singlet oxygen in cells.Live/dead cell staining analysis also confirmed that photodynamic therapy after co-incubation with nanoparticles had a significant effect.Both photoacoustic and near-infrared Ⅱ fluorescence scanning results showed that as concentration gradient of ZIF-8-IR820-MnPc-HA solution become higher,the respective signal got stronger.3.In vivo experiments:ZIF-8-IR820-MnPc-HA NPs were intratumorally injected to experimental mice at a dose of 5mg/m L.The photoacoustic imaging scan results were consistent with that of the fluorescence imaging scan.It is found that after the injection,the average signal at the tumor site reached peak at 1.0h post injection,then the signal gradually decreased,and the signal basically disappears until 12 hours.What’s more,there was no obvious difference in indicators of biosafety,including H&E staining of the main organs and blood biochemical analysis.Conclusion:In this study,we reported a kind of ZIF-8-IR820-Mn PC-HA nanoparticles with excellent photodynamic effect,which exhibited good photoacoustic/NIR-Ⅱ fluorescent dual-modality imaging capability.Interestingly,the HA coating on the surface of ZIF-8-IR820-MnPc could act as a sponge,enhancing the production of ¹O₂ and promoting the therapeutic effect at cellular level to some extent.Through the preliminary study of photodynamic effect,we expect that the above research results can provide assistance for the integration process of diagnosis,treatment of ZIF-8-IR820-MnPc-HA and realize clinical transformation as soon as possible.