Self-assembled Bio-nanoprobes For Molecular Image-guided Enhanced Photodynamic Therapy Of Hypoxic Malignant Tumors

Objective:The purpose of this study is to address the limitations of photodynamic therapy（PDT）in clinical applications,particularly in hypoxic tumor environments,visualization,and light penetration depth.To achieve this,a novel self-assembled fluorescent nanoprobe is designed,which utilizes molecular imaging technology for visualization guidance and enables enhanced photodynamic therapy for hypoxic tumors.Methods:Firstly,a rational synthesis route was designed,and finally compound I-BODIPY 4（B4）was synthesized,followed by a series of characterization experiments.Subsequently,CAT and B4,the two functional moieties,were self-assembled in an ordered manner under various supramolecular interactions to form a water-soluble and biocompatible fluorescent nanoprobe（CAT-I-BODIPY NPs）.The optimal assembly conditions were screened based on parameters such as morphology,particle size,and spectral properties,and the assembly kinetics and stability of the sensor in aqueous solution were investigated.Next,the amount of oxygen produced and consumed by CAT-I-BODIPY NPs in hydrogen peroxide（H₂O₂）solutions of diverse concentrations were measured to simulate the oxygen production and consumption in tumor tissues.Cell experiments were carried out using the nanoprobe,including dark toxicity assay,uptake assay,singlet oxygen（¹O₂）production assay,phototoxicity assay,and PDT assay.Finally,the nanoprobe was used in in vivo experiments on nude mice,including the establishment of subcutaneous tumor models,small animal imaging,PDT experiments in subcutaneous tumors,and in vivo biosafety evaluation.Results:In this study,a B4 molecule was synthesized that had both NIR fluorescence and PS functionalities.Through a self-assembly strategy,B4 was combined with CAT to construct a spherical fluorescent nanoprobe CAT-I-BODIPY NPs with a diameter of approximately 100 nm.After a series of in vitro and in vivo experiments,the nanoprobe exhibited decent biocompatibility and cell uptake performance,and was able to efficiently accumulate in hypoxic tumor cells.Under the guidance of fluorescence imaging（FI）technology,the localization of hypoxic tumors and monitoring of the probe accumulation time were achieved.When the probe reached optimal accumulation in the tumor,B4 was stimulated by NIR laser from outside the body,resulting in the in-situ release of ¹O₂ in the tumor site.At the same time,CAT catalyzed the production of O₂ from H₂O₂ within the tumor,not only alleviating tumor hypoxia but also facilitating the production of ¹O₂,enhancing the tumor killing ability,thus achieving enhanced photodynamic therapy.Conclusion:Based on the analysis of in vitro and in vivo experimental results,this study successfully developed a novel nanoscale probe,CAT-I-BODIPY NPs,which achieved enhanced PDT with in situ oxygen production at hypoxic tumor sites under FI guidance.In summary,this study provides a new strategy for the diagnosis and treatment of hypoxic tumors,and CAT-I-BODIPY NPs have the potential to become a novel nanoscale probe with potential application value in hypoxic tumor imaging guidance.