Ag₂S Quantum Dots-mediated Self-assembled Molecular Imaging Probe For Imaging-guided Synergistic Treatment Of Colorectal Cancer

Objective:Colorectal cancer has the third-highest incidence and second-highest mortality rate worldwide,and its prevalence is expected to increase alarmingly in the coming years.Currently,clinical research is focused on combining photodynamic therapy（PDT）with other therapies such as chemotherapy and photothermal therapy（PTT）,as this is more effective than monotherapy.However,the hypoxic tumor microenvironment（TME）and drug leakage,as well as the optimal time window for phototherapy,have largely limited the anticancer efficacy of phototherapy in combination with chemotherapy.Molecular imaging（MI）,as a non-invasive imaging technology,can visualize biological processes and disease development at the molecular level,with advantages such as high sensitivity and high resolution.Among them,fluorescence imaging technology,as a promising molecular imaging technology,has been widely used in the biomedical field.Based on fluorescence imaging technology,fluorescence imaging-guided phototherapy is an emerging technique.By using fluorescence imaging technology to monitor the growth of tumors and the release of drugs in real time,phototherapy can be performed when the drug reaches the optimal concentration,thus improving the treatment efficacy.Therefore,we proposed a"bottom-up"protein self-assembly strategy,which assembles silver sulfide quantum dots（Ag₂S QDs）with good fluorescence imaging and photo-thermal effects,the photosensitizer chlorin e6（Ce6）,the catalytic oxygen producer catalase（CAT）,and the first-line chemotherapeutic agent oxaliplatin（Oxa）for colon cancer through multivalent electrostatic interactions.Ag₂S@CAT-Ce6@Oxa NPs as molecular imaging probes were assembled to achieve TME p H-stimulated responsive disassembly and in vivo PDT/PTT synergistic chemotherapy guided by near-infrared quantum dots（QDs）imaging.Methods:（1）Firstly,functional units of Ag₂S QDs were synthesized and their surfaces were polyamine-modified.The photosensitizer Ce6 was covalently modified with CAT to obtain CAT-Ce6,which has multivalent negative charges on the surface.Finally,Ag₂S@CAT-Ce6@Oxa NPs were co-assembled with Oxa under constantly optimized conditions.Various characterizations and stability investigations were performed on the resulting nanoparticles.（2）In vitro experiments were conducted to evaluate the fluorescence spectra,photodynamic therapeutic properties,photothermal properties,and controlled release of drug Oxa at the TME before and after disassembly of Ag₂S@CAT-Ce6@Oxa.（3）Furthermore,cellular uptake assay,cytotoxicity assay,ROS generation measurement,and dissolved oxygen measurement were carried out to assess the system’s cellular uptake effect,the killing effect of Ag₂S@CAT-Ce6@Oxa NPs on tumor cells,and the possible mechanism underlying the enhanced synergistic PDT/PTT/chemotherapy effect.（4）Finally,a colon cancer model was constructed to validate in vivo the efficacy of Ag₂S@CAT-Ce6@Oxa NPs NIR imaging-guided synergistic PDT/PTT/chemotherapy and the assessment of biosafety.Results:（1）The assemblies Ag₂S@CAT-Ce6@Oxa NPs were successfully constructed in a physiological environment（p H=7.4）,showing homogeneous spherical size nanostructures under transmission electron microscopy and the results of assembly theory simulations supported the formation of the assemblies,while the unassembly was achieved under simulated TME（p H=6.5）.（2）In vitro tests showed that the fluorescence intensity of Ag₂S@CAT-Ce6@Oxa NPs remained unchanged before and after the unassembly,with excitation at 475 nm and emission at 800 nm.The fluorescence intensity of Ag₂S@CAT-Ce6@Oxa NPs remained unchanged before and after disassembly,with excitation at 475 nm and emission at 800 nm.In vitro,they also exhibited good photodynamic therapeutic properties,photothermal properties and controlled release of drugs.（3）Compared with other experimental groups,Ag₂S@CAT-Ce6@Oxa NPs showed the strongest tumor suppression rate in vivo and also maintained good biosafety.Conclusions:（1）An excellent NIR quantum dot-mediated protein nanosystem,Ag₂S@CAT-Ce6@Oxa,with regular morphology and good stability,was successfully constructed in this paper,which can be successfully disassembled at TME（p H=6.5）.（2）Ag₂S@CAT-Ce6@Oxa nanosystem has good near-infrared imaging PDT/PTT/chemotherapy effects in vitro.（3）Ag₂S@CAT-Ce6@Oxa nanosystem has achieved enhanced near-infrared imaging PDT/PTT/chemotherapy effects and excellent biocompatibility in vivo.