Cascade-responsive Ultrasound Molecular Probe-mediated Ferroptosis Potentiated Sonodynamic Therapy For Anaplastic Thyroid Cancer

Anaplastic thyroid carcinoma is highly malignant,invasive,and has rapid clinical progression,poor comprehensive treatment effect and poor prognosis.Therefore,exploring early diagnostic strategies and precise treatment methods has become the key to improve the survival rate of patients with ATC.With the research and development of molecular imaging,the construction of nanosystems integrating diagnostic and therapeutic functions has become a hotspot in the field of tumor therapy research.Ferroptosis,an iron-dependent non-apoptosis-regulated cell death mode with excellent tumor killing effect,has gradually become a new way of anti-tumor therapy.However,the problems of ferroptosis inducer accumulation inside the tumor and poor deep penetration greatly limit its therapeutic effect.Therefore,we constructed a cascade permeable metal polyphenol ultrasonic molecular probe FCIPL,and realized multimodal imaging integrating ultrasound,photoacoustic,MRI and fluorescence imaging in diagnosis.In terms of treatment,ferroptosis in deep tumors was achieved through targeted osmosis and cascaded response drug delivery system,and synergistic sonodynamic therapy was achieved.Objective:To construct a cascade-responsive diagnostic and therapeutic integrated ultrasound molecular probe FCIPL based on a metal-polyphenol network,to examine its characterisation and basic properties,and to validate its therapeutic efficacy of targeted cascade-responsiveness,deep tumor penetration,and ferroptosis-enhancing SDT in vitro and in vivo,respectively.Methods:Self-assembly and vacuum thin-film-acoustic vibration methods were used to prepare FCIPL ultrasound molecular probes,which were based on the core of FCr formed by self-assembly of the ferroptosis inducer curcumin(Cur)with Fe3+,and co-embedded with PFP in IR780-loaded liposomes.In the nanoparticle synthesis part,their characterisation and basic properties were examined,including morphology,size,homogeneity,potential,stability and ADV ability;the encapsulation rate,drug loading rate,structural composition,and glutathione(GSH)response of the nanoparticles were examined.The ability of the nanoparticles to induce the occurrence of ferroptosis and the generation of SDT,including the ROS-generating ability of ·OH and 1O2,were also assessed.In the in vitro experimental part,the safety,uptake capacity,targeting function,cytotoxicity,ferroptosis potentiating SDT effect and mechanism of action of the nanoparticles were investigated by Cell Counting Kit-8(CCK-8)assay,confocal laser microscopy,flow cytometry,and Western-blot assay,using the human ATC BHT101 cell line,and the deep penetration of nanoparticles in tumors was also investigated using 3D tumour ball penetration experiments.In in vivo experiments,the biosafety of the nanoparticles was examined using Kunming mice,and the tumor targeting and synergistic therapeutic effects,mechanism of action,and deep tumor penetration ability of FCIPL nanoparticles were investigated using BALB/c ATC ruffed mice.In addition,we performed relevant validation of the ex vivo and in vivo multimodal imaging capabilities of FCIPL nanoprobes.Results:FCIPL ultrasonic molecular probes were successfully prepared.The nanoparticles were regular spherical with uniform size,hydrated particle size of 218.6±55.77 nm，and a zeta potential of-15.4±7.66 mV,with good dispersion and stability,and were successfully encapsulated with FCr and IR780,with encapsulation rates of 47.06%and 91.86%,respectively.Ultrasonic excitation and high concentration of GSH conditions can promote the disassembly of FCIPL nanoparticles,releasing the ferroptosis inducers Cur and Fe2+,and generating·OH by Fenton reaction with H2O2,as well as excellent SDT effect and generating a large amount of ROS.In vivo and in vitro experiments confirmed the good tumor targeting,cascade response and deep penetration ability of the FCIPL nanoprobe with good biosafety,and also verified the ferroptosis potentiating SDT efficacy of the nanoprobe and its mechanism of action.Under Lifu excitation,the nanoparticles underwent ADV,and UTMD delivered the nanoparticles to deep tumor tissues and released FCr by bursting,and then disassembled and released Cur,an ferroptosis inducer,and Fe2+,a catalyst for the Fenton reaction,which constituted the amplifier of the ferroptosis cascade in the tumor microenvironment,inducing the ferroptosis of ATC tumor cells.At the same time,SDT was stimulated to generate a large amount of ROS,which together effectively inhibited the growth of tumor cells and increased cytotoxicity,demonstrating excellent tumor killing effects,and was able to completely eradicate tumor tissues in ATC-transplanted tumor-bearing nude mice.In terms of imaging,the targeting ability of FCIPL ultrasound molecular probes determines their accumulation at the tumor site,successfully realizing multi-modal imaging integrating ultrasound,photoacoustic,MRI and fluorescence imaging,which provides reference value for early diagnosis and precise treatment of tumors.Conclusion:In this study,a cascade-responsive metal polyphenol ultrasound molecular probe FCIPL was successfully synthesised.In diagnostic aspect,the nanoprobe integrates ultrasound,photoacoustic,fluorescence and MRI imaging,and possesses multimodal imaging function.In terms of treatment,ferroptosis in deep tumors was achieved through targeted osmosis and cascaded response drug delivery system,and potentiating the SDT,which was validated in vitro and in vivo with good efficacy,and it is expected to provide a new idea for the integration of diagnosis and treatment of ATC.