| Sonodynamic therapy(SDT)is one of hot topics in the field of tumor treatment because of its non-invasive and controllable spatio-temporal advantages.SDT mainly uses low-intensity ultrasound to activate the sono-sensitive factors(sonosensitizers)enriched in the lesion to produce reactive oxygen species(ROS).ROS triggers a series of oxidation reactions and promotes tumor cells to cause irreversible damage.Sonosensitizers are the key to the anti-tumor efficacy of SDT.Traditional sonosensitizers mainly include porphyrins,porphine compounds,dye molecules,anti-inflammatory drugs,etc.,which often exhibit poor water solubility,low active oxygen production and less accumulation of drug in tumor cell.Therefore,these shortcomings lead to unsatisfactory curative effect of SDT.The introduction of nanomaterials can effectively improve the above disadvantages.A variety of organic/inorganic nano-sonosensitizers have been used in the research of precise diagnosis and SDT.In particular,the attribute modification of organic-inorganic nano-hybrids cleverly combines the advantageous functions of different components,providing novel development opportunities for SDT.SDT triggers different cell death response modes,which are affected by the interaction of ultrasound stimulation parameters and a variety of physical and chemical factors.Ferroptosis is a regulatory cell death mode proposed in recent years.It is attributed to the cell behavior driven by iron ion dependence and lipid peroxides,manifested by mitochondrial shrinkage,increased membrane density and lipid ROS levels.Ferroptosis depends on the high iron ion level of tumor cells,namely "iron pool",which lays a certain sensitive foundation.Once peroxidation stimulates the "iron pool",it will initiate the occurrence of ferroptosis.Fenton Reaction is one of the main mediated way of ferropoptosis.Most ferroptosis inducers emphasize the disorder of iron ion levels through the supplement of extra iron to induce Fenton catalysis,which lead to the accumulation of lipid peroxide overload,break the lipid metabolism of oxygen balance.And the death pathway accompanied by glutathione peroxidase 4(GPX4)removal mechanism disintegrates,causing the tumor cells die.In addition,the occurrence of ferroptosis is also related to the immune response,which has attracted the attention of many scholars in recent years.However,the involvement of heavy metal particles in the inducer cannot circumvent the potential biological toxicity,and the induction efficiency is expected to increase.Therefore,the exploration of the high-efficiency induction pathway of Ferroptosis and its relationship with SDT Fenton catalysis are the key scientific issues of this research.Hemoglobin(Hb)is the main functional protein in red blood cells,being responsible for the transport and carrying of free oxygen.A large number of studies have confirmed that Hb is an effective functional element of artificial oxygen carriers,which can intelligently combine oxygen according to the partial pressure of oxygen to adjust the tumor microenvironment.Among them heme containing iron-like porphyrin structure is the key component of Hb,usually in a divalent state.It avoids iron toxicity,and some literatures show that Hb can induce ferroptosis potential by changing the iron valence state and participating in Fenton under excessive ROS.As a carrier,Hb has dual functions of supplying oxygen and iron.On the one hand,iron ions are taken in a highly biocompatible way to improve treatment safety.On the other hand,its oxygen carrier design can effectively amplify the sensitivity of tumor cells to oxgen-dependnent treatment.In this study,Hb,DVDMS and TiO2 were hybridized by preparing nano-oxygen carrier(HDT),which not only improved the sono-activity of nano-oxygen carrier,but also laid the foundation for a multi-therapeutic synergistic nano-media.Through ultrasound irradiation,the oxygenation of HDT amplifies the generation of ROS,effectively promotes the valence transition of Hb,mediates the ferroptosis pathway.And this strategy has immunoregulatory advantages for anti-tumor effects in vivo and in vitro.HDT has the dual-modality imaging performance of photoacoustic and fluorescence tracing,which provides a valuable research basis for expanding new methods of image-guided SDT tumor treatment.The main research contents and results are summarized as follows:Part 1 Preparation and characterization of nano-oxygen carrierThe inorganic sonosensitizer TiO2 was selected as the nano-carrier,DVDMS and Hb were sequentially conjugated.After the nano-oxygen carrier HDT was constructed by electron affinity.XPS,SDS-PAGE and other methods were used to identify the tight bonding degree between different HDT nanocomponents and the effective load of Hb.Based on HRSEM,HRTEM and other properties characterization,it was found that the diameter of HDT nanoparticles synthesized in this study was relatively uniform,with good dispersion,and a relatively high level of stability over a long period of time.Part 2 Study on the effect of ultrasound combined with HDT on the induction of tumor cell ferroptosisThe 4T1 cell line was selected as the research object.Ultrasound stimulation of HDT by a variety of probes led to the production of intracellular ROS and the accumulation of lipid oxidation products,which provided the basis for the sono-activity of the nano-oxygen carrier.Further studies confirmed that SDT-mediated ROS production can reverse the valence state of Hb iron and promote ferroptosis.The study found:(1)Compared with a single sonosensitive component,HDT can produce a larger amount of hydroxyl radicals and singlet oxygen in a low dose range under ultrasound.And its sonochemical effect is significantly improved.(2)The potential toxicity of the nano-oxygen carrier is extremely low,and the improvement of hypoxia reduced the pumping of drugs and promoted the uptake of HDT by tumor cells under hypoxic conditions.Thereby oxygen carrier helping to enhance the efficiency of SDT treatment.(3)Ultrasound irradiation stimulated HDT to produce high levels of ROS in vitro,especially to simulate the physiological hypoxic culture of tumor cells.The sono-activity of the combined treatment group effectively regulated the Hb iron valence state,and stimulated the occurrence of ferroptosis via Fenton pathway.(4)The classic lipid-oxygen metabolism imbalance was shown in the process of ferroptosis,by using specific probes to label lipid peroxide levels,intracellular GPX4 enzyme activity and dynamic changes of mitochondrial membrane potential.In addition,the combination therapy of sonodynamics and ferroptosis significantly inhibited the proliferation of tumor cells and resulted in the exposure of the"immune activation signal" CRT protein.The level of intracellular inflammatory factors tends to rise,suggesting that ultrasound combined with HDT has a certain immune activation effect.Part 3 HDT dual-modality imaging potential and its regulation on tumor hypoxic microenvironmentThe tumor-bearing mouse models with different oxygen deficiencies were established.Immunofluorescence was used to detect the improvement of hypoxic tissue by nano-oxygen carrier.By using the MSOT and IVIS systems,the study evaluated the photoacoustic and fluorescence imaging performance of HDT.The study found:(1)After HDT administration,the hypoxic signal of tumor-bearing mice decreased significantly,and the nano-oxygen carrier effectively improved the hypoxic microenvironment of the tumor tissues.(2)By analyzing the HDT photoacoustic tomography signal in vitro and in vivo,HDT presented a higher oxygenation signal,which significantly improved the blood oxygen saturation of the target sites after administration.In addition,ultrasound enhanced the deep tissue penetration of the nano-oxygen carrier,further amplified the oxygen regulation effect of HDT.(3)Ultrasound treatment conquered the fluorescence quenching phenomenon of hybrid nanoparticles to some extent,suggesting good guidance for fluorescence tracing.The dual-modality imaging performance of HDT is extremely valuable for improving the efficiency of comprehensive tumor diagnosis and treatment.Part 4 Study on the anti-tumor effect of ultrasound combined with HDT-mediated ferroptosis4T1 tumor-bearing mice were chosen as the research model,and the tumor inhibition rate was analyzed through the observation and monitoring of solid tumors.Moreover,the immune activation level of tumor tissues was evaluated with flow cytometry.In order to ensure the safety of the therapy,the toxic and side effects of the treatment approach were detected by histochemical analysis,blood biochemistry and other indicators.The study found:(1)The treatment of HDT-SDT initiated ferroptosis and significantly promoted the maturation of dendritic cells,as well as the activation of T lymphocytes.The increase of prostaglandins and other inflammatory factors contributed to the immune regulation of target tissues,which has long-term advantages for the treatment of malignant tumors.(2)SDT combined with ferroptosis therapy has a significant in vivo anti-tumor effect and effectively inhibited lung metastasis of malignant tumors,reaching a tumor suppression rate as high as 94%.(3)There was a large gap in tumor tissue after the combined treatment,and cell proliferation was effectively inhibited.The other main organs of mice had no obvious pathological damage.ConclusionsIn this study,a dual-modal imaging-guided nano-oxygen-carrying sonosensor platform was successfully constructed,which realized the sonodynamic cascade brake ferroptosis combined with anti-tumor strategy based on the amplification of reactive oxygen species.Besides,the therapy overcomes the therapeutic resistance of tumor hypoxia microenvironment.This research innovatively combines ferroptosis induction with sonodynamic Fenton catalysis,and the combined treatment shows excellent anti-tumor and immune response effects,which provides a certain research basis for the exploration of nano-SDT for precise diagnosis and treatment of tumors. |
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