Construction Of PH-Responsive Dual-Modality Imaging Probe For Radiotherapy Sensitization In Tumor

Background:Cancer remains one of the significant threats to human life and health.Radiotherapy（RT）is an efficient local treatment method.However,the efficacy is restricted by many factors,such as the unclear boundary of tumour tissue,insensitivity of tumour cells to radiation,damage of surrounding tissue caused by high-dose radiation,and the high cost of real-time monitoring.Improving the sensitivity of tumours to radiotherapy and guiding the treatment process through imaging methods are crucial for improving treatment efficacy and reducing side effects on normal tissues.So far,many radiosensitizing nanoplatforms have been developed and successfully applied in cancer treatment research.A single nano platform for radiotherapy is challenging to meet the individualized treatment of tumours,and it is impossible to observe the process of radiosensitization in real time.Therefore,developing a functional nano platform that integrates imaging and treatment is urgent.The second near infrared region（NIR-II,950-1700 nm）fluorescence（FL）imaging and photoacoustic（PA）imaging are emerging biomedical molecular imaging methods.The optical imaging in the NIR-II has the characteristics of low photon scattering and weak tissue absorption,so it has the advantages of high signal-to-noise ratio,high contrast,high resolution and deep penetration depth.NIR-II FL and NIR-II PA can be used for qualitative and quantitative analysis of biological processes in vivo at the tissue and cell levels.NIR-II FL and NIR-II PA have the characteristics of non-invasive,high-throughput,non-radiation and functional imaging.Combining the two imaging methods can locate the tumour more accurately and obtain more abundant tumour area information to guide precise RT and improve the efficacy of cancer treatment.Currently,the monitoring signal of many integrated probes is always in the"on"state,which limits the contrast of the probe imaging signal.Based on this,researchers have constructed a series of stimuli-responsive probes that can respond to external stimuli and produce corresponding structural or physicochemical changes.This series of probes can accurately diagnose and treat lesions and has become a research hotspot in tumour diagnosis and treatment.Therefore,designing and developing an intelligent nano platform for NIR-II FL/PA imaging-guided radiotherapy activated by tumour microenvironment stimulation is significant.Objective:1.The tumour microenviroment（acidic pH）responsive radiosensitizing nanoprobe AuNNPs-Ag₂S Ve was prepared by self-assembly.Through surface modification,it had good dispersibility and stability,and the physical and chemical properties of the probe were explored.2.The biological safety and pH responsiveness of AuNNPs-Ag₂S Ve in vitro were studied,and the changes of micromorphology,particle size,NIR-II FL and PA signals under different pH conditions were observed.The signal changes of NIR-II FL and PA in the micro-acid environment of the tumor were used to locate the tumor and guide the radiotherapy.3.The high permeability and high retention（EPR）effect of solid tumors enables AuNNPs-Ag₂S Ve to accumulate and disassemble at the tumor site,thereby improving its permeability in tumor tissues.4.The use of gold nanoparticles in AuNNPs-Ag₂S Ve under X-ray irradiation can promote interaction with water,thereby increasing ROS production,thereby achieving radiosensitization,reducing side effects,and improving radiotherapy efficacy.Methods:1.AuNNPs and Ag₂S QDs were assembled into nanovesicles AuNNPs-Ag₂S Ve by microemulsion method.First,AuNNPs and Ag₂S QDs were synthesized,and then hydrophilic PEG and pH-responsive PS-P4VP block polymers were grafted onto the surface of Ag₂S QDs and AuNNPs,respectively.Then,the amphiphilic AuNNPs and Ag₂S QDs were self-assembled into pH-responsive nanovesicles AuNNPs-Ag₂S Ve by oil-in-water emulsification method,and their physical and chemical properties were characterized.AuNNPs-Ag₂S Ve was dispersed in a neutral and acidic solution,then use TEM and DLS observed its morphology and particle size to verify its acid responsiveness.Vevo LAZR-X photoacoustic imaging system and In-Vivo Master Laser fluorescence imaging system were used to obtain nanovesicle PA and FL images.AuNNPs-Ag₂S Ve was incubated respectively in ultrapure water,PBS solution,cell culture medium and cell culture medium containing 10%fetal bovine serum for 7 days to evaluate its stability and water solubility.2.The cytotoxicity and radiosensitizing ability of AuNNPs-Ag₂S Ve were verified by CCK-8 experiment,ROS production experiment and methylene blue detection of hydroxyl radical（·OH）production experiment.The permeability of AuNNPs-Ag₂S Ve was studied by constructing human breast cancer cells（MCF-7）multicellular spheres.3.The MCF-7 breast cancer-bearing mouse model was constructed for living animal experiments to study the biosafety of AuNNPs-Ag₂S Ve in vivo,NIR-II PA/FL imaging performance and radiosensitizing effect.Results:1.A novel nano-radiosensitizer AuNNPs-Ag₂S Ve with a particle size of about190 nm was successfully prepared,which has good water solubility,good dispersibility and stable properties.The characterization results of physicochemical properties showed that the particle size of the nanovesicles was about 190 nm,and the maximum absorption peak was red-shifted to the NIR-II region.Under an 808 nm laser irradiation,the FL signal was initially quenched,and the NIR-II PA signal was turned on.In the acidic solution,the nanovesicles AuNNPs-Ag₂S Ve are dissociated into smaller clusters or single particles,and the NIR-II FL signal is gradually enhanced with the decrease of the PA signal.The neutral pH solution had no significant change,indicating good acidic pH response characteristics.2.In vitro cytotoxicity experiments,although the concentration of pH-responsive and non-responsive AuNNPs-Ag₂S Ve is as high as 200μg/m L,the viability of MCF-7 cells is still above 80%,indicating that the nanovesicles have good biosafety.MCF-7 cells incubated with different concentrations of pH-responsive nanovesicles were irradiated with X-rays.The viability of MCF-7 cells decreased significantly with the increase of AuNNPs-Ag₂S Ve concentration.In contrast,the cell viability of the nanovesicle group without X-rays was almost unchanged,indicating that AuNNPs-Ag₂S Ve had an excellent radiosensitizing effect and significant anti-tumour effect,which is expected to be used in highly effective tumor therapy.The results of multicellular spheroid penetration experiments showed that the pH-responsive nanovesicles were disassembled in the acidic microenvironment of the tumour,releasing single AuNNPs and Ag₂S QDs,which promoted the penetration of nanoparticles into the deep part of the tumour,thereby improving the imaging contrast and therapeutic efficacy of deep tissues.3.After the tumour-bearing mice were injected with pH-responsive and non-responsive AuNNPs-Ag₂S Ve through the tail vein,the NIR-II PA signal at the tumour site reached a peak at about 20 h.The difference is that the NIR-II FL signal intensity of the tumour site of the mice injected with the pH-responsive nanovesicle group gradually increased.In contrast,the tumour site of the mice in the non-responsive nanovesicle group had no NIR-II signal the whole time,which verified that the tumour micro-acid environment could activate the NIR-II FL signal of the pH-responsive AuNNPs-Ag₂S Ve.In the treatment experiment of MCF-7tumour-bearing mice,the AuNNPs-Ag₂S Ve+X-ray group had the most potent inhibitory effect on tumour growth,and the histological results showed that the nanovesicles had good biosafety and no obvious damage to other essential organs.Conclusion:In this study,we developed pH-responsive dual-modality imaging radiosensitizing probe AuNNPs-Ag₂S Ve for scientific issues such as how to specifically enhance the radiosensitivity of tumor cells,the monitoring of radiotherapy efficacy needs to be revised,and AuNNPs-Ag₂S Ve can be dissociated in the acidic environment of the tumor,promoting the further penetration of nanoparticles into the tumor,which is helpful to improve the efficacy of RT.In addition,the NIR-II FL/PA imaging signal can be used to locate the tumor precisely to guide RT.Meanwhile,the efficacy of radiosensitization can be evaluated early and the side effects can be reduced.This intelligent nanoprobe with NIR-II optical imaging activated by tumor micro-acid environment stimulation provides a new strategy for the research of nano-medical diagnosis and treatment integration and has great potential in future precision biomedicine.