Tumor-targeting Theranostics Based On A Fluorescent Coumarin Probe And RhPd-H Nanoparticles

One characteristic that distinguishes solid tumor cells from normal cells is disrupted metabolism.Tumor cells and their microenvironment differ from normal tissue cells in that they have a high metabolic rate,little oxygen,and a low p H.We completed the following two tasks for this paper:We used rhodium-palladium-hydrogen alloy nanoparticles（RhPd-H nanopaticle,RhPd-H NP）with in situ NIR photocontrolled hydrogen release by the material in tumor sites because the balance of reactive oxygen species（ROS）is more fragile in tumor cells than in normal cells.A novel approach for the safe and effective treatment of breast cancer may involve the controlled release of hydrogen under the influence of near-infrared light.Contrarily,we created a fluorescent coumarin probe to detect endogenous alkaline phosphatase（ALP）in tumor cells based on the high expression characteristics of metabolic enzymes in tumor cells.This probe can measure the level of intracellular ALP and reflect tumor metabolism by specifically binding to ALP and generating ratiometric fluorescence,which has good diagnostic and prognostic value for evaluation.Part Ⅰ:In situ NIR-controlled hydrogen release of RhPd-H material for hydrothermal therapy of breast cancerBackground and purpose:Conventional radiotherapy has a propensity to cause skin damage and radiation pneumonia,while conventional chemotherapy in the non-surgical treatment of breast cancer primarily kills rapidly proliferating cells.It also has varying degrees of killing effects on the blood cells and digestive tract epithelium.The population has little benefit from targeted drug therapy.New breast cancer treatments must therefore be created immediately.According to recent research,tumor cells’redox balance is more prone to being upset,making them more susceptible to dying from an excess of ROS.With its anti-inflammatory and anti-tumor properties,hydrogen（H2）,a reducing agent with high biosafety,can successfully upset the ROS balance in solid tumor cells.We therefore created a combined hydrogen-thermal therapeutic approach based on rhodium-palladium metal alloy hydrogen storage nanoparticles（RhPd-H nanopaticle,RhPd-H NP）,which can achieve near-infrared phototactic hydrogen gas release and redox reactions with intracellular ROS to specifically kill tumor cells by taking advantage of the differences in ROS levels and sensitivity between tumor cells and normal cells.Methods:1.Hydrogen storage nanomaterial RhPd-H nanoparticles were prepared by the solvent thermal method,and the products were characterized by XRD and XPS to verify the stable storage of hydrogen atoms in nanoparticles.2.The ability of RhPd-H to release H2 under 808 nm laser irradiation was evaluated by UV-Vis and methylene blue（MB）.The photothermal conversion ability of RhPd-H nanoparticles was studied in animals and in the lab using a thermocouple and a thermal imager.The photoacoustic imaging ability of RhPd-H was also studied in animals.3.To investigate whether RhPd-H can be taken up by tumor cells by transmission electron microscopy and inductively coupled plasma mass spectrometry（ICP-MS）characterization,and to investigate the safety of RhPd-H and the killing of tumor cells and normal cells by cytotoxicity experiments.4.Establish a 4T1 mammary carcinoma tumor-bearing mouse model to investigate the in vivo therapeutic effect of the RhPd-H-based hydrogen-thermal treatment strategy and to demonstrate the biosafety of RhPd-H by blood biochemical assay and tissue organ section staining.5.The mechanism of intracellular action of RhPd-H material was probed by the ROS probe to further explore the clinical application potential of this hydrogen-thermal therapeutic strategy for tumor treatment.Results:1.The RhPd-H material formed by H2 stored in a rhodium-palladium metal alloy structure as a prototype has a stable structure and highly NIR light-controllable release properties.2.The H₂release profile of RhPd-H was calculated by MB at the characteristic absorption peak of 664 nm,and it was found that RhPd-H could continuously and stably release H₂under 808 nm NIR light irradiation,and RhPd-H warmed up more than 60°C for 5 mins under 1 W/cm²power density irradiation.Photoacoustic imaging also showed that RhPd-H could build up at the tumor site in mice with tumors for about 6 hours and stay stable for more than 24 hours.This showed that RhPd-H could build up at the tumor site.3.The results of electron microscopy and ICP showed that RhPd-H could be effectively taken up by the tumor cells,and RhPd-H could reduce the MB in the cells and kill the cells effectively with the help of NIR controlled release hydrogen.4.The results of the RhPd-H-based hydrogen-heat treatment strategy for 4T1tumor-bearing mice showed a significant reduction in tumor size and no significant systemic toxic effects.Tumor cells in immunohistochemical sections of tumors showed a significant decrease in Ki67 cell proliferation markers of tumor cells,indicating that tumor cell growth was inhibited.H&E staining of other tissues and organs and blood biochemical results showed that RhPd-H had no systemic toxicity.5.The results of in vivo experiments confirmed that the RhPd-H-based hydrothermal treatment strategy not only has significant tumor suppressive effects,but also protects normal cells from high temperature damage,which is a safe and efficient synergistic treatment mode.The mechanism may be further explored that H₂kills cells by disrupting the balance of reactive oxygen species in tumor cells and leads to an abnormal elevation of ROS.It was also found that the amount of ALP in the treated tumor dropped by a lot.This suggests that enzyme activity is related to how fast a tumor is growing.Conclusions:1.RhPd-H is a hydrogen storage nanoparticle with good warming ability that can controllably regulate the release of H₂by NIR and exert the bio-reduction of H₂.Also,RhPd-H has the potential for photoacoustic imaging.2.RhPd-H can accumulate at the tumor site and enter the cell interior,thus effectively killing tumor cells.However,this process has little effect on normal cells,which reflects its good biosafety.3.The results of in vivo experiments confirmed that the RhPd-H-based hydrogen-thermal treatment strategy not only has an obvious tumor suppression effect but can also protect normal cells from high temperature damage,which is a safe and efficient synergistic treatment mode.If more research is done,it may be found that H₂kills cells by upsetting the balance of reactive oxygen species（ROS）in tumor cells and causing ROS levels to rise in a strange way.In summary,we have prepared multifunctional RhPd-H nanoparticles with stable,high hydrogen storage,photocontrolled hydrogen release,and photothermal conversion properties to achieve efficient tumor-targeted hydrogen delivery and photothermal conversion,activate ROS to selectively kill tumor cells,and establish a new model of hydrogen-thermal synergistic tumor suppression with potential applications in the field of tumor therapy.PartⅡ:Ratiometric detection and imaging of endogenous alkaline phosphatase activity by fluorescein-coumarin-based fluorescence probeBackground and purpose:ALP is an enzyme widely present in various human tissues,and it plays an important role in the regulation of many cellular functions.In contrast,the levels of ALP are significantly different in tumor cells and normal cells,while the levels vary greatly within different tumors.The detection of intracellular ALP levels can help distinguish tumor cells from non-tumor cells and enable the diagnosis of tumors.Coumarin fluorescent dyes have good two-photon properties and large Stokes shifts,so fluorescent probes developed with coumarin and its derivatives as the master are well suited for detection in the biomedical field.Based on the above research background,we designed a ratiometric fluorescent probe（FCP）based on organic small molecules for the specific detection of ALP based on the hydroxyl group electron giving group protected by a phosphate group in fluorescent coumarin,which demonstrated strong anti-interference ability and could effectively distinguish the activity of different cellular endogenous ALP.Methods:1.Fluorescein-coumarin（FC）and fluorescent probe（FCP）probes were synthesized and characterized by electrospray ionization mass spectrometry（ESI-MS）and nuclear magnetic resonance（NMR）,and the mechanism of specific binding of FCP probes to ALP was initially investigated to provide a theoretical basis for further A theoretical basis for further detection was provided.2.To investigate the spectral changes of the FCP ratio fluorescent probe after the reaction with ALP in an in vitro environment using UV-Vis spectrophotometry（UV-Vis）,and to obtain the relationship between the bimodal ratio and ALP concentration before and after the FCP reaction.ALP was also found in samples of human serum,which was used to confirm the level at which it could be found.3.The stability of FCP’s ability to detect ALP was evaluated under different p H and reaction time conditions,and mixed with non-target analytes to simulate the specificity and anti-interference ability of FCP detection under different interference environments.4.Study the principle and mechanism of FCP ratiometric fluorescence detection by theoretical computational analysis,and elucidate the source of the fluorescence signal and clarify the response changes in the detection process.5.FCP will be used to detect ALP in malignant tumor cells and analyze its diagnostic ability,as well as to demonstrate the biosafety of the FCP probe through cytotoxicity testing.Results:1.ESI-MS and NMR results showed that the FCP molecule has a phosphate group on it,which can react with ALP specific binding and generate FC.2.The UV-Vis results showed that the reaction of FCP with ALP produced an absorption peak at 450 nm,accompanied by a color change of the solution.Under 320nm excitation light,the fluorescent coumarin structure had a fluorescence emission peak at 465 nm,which continuously diminished with the increase of ALP concentration,while a new fluorescence emission peak appeared at 530 nm.The fluorescence intensity ratio I₅₃₀/I₄₆₅was linearly related to the ALP concentration within a certain range,with an LOD of 0.006 m U/m L.3.As the p H of the reaction system increased,I530/I465 increased continuously and decreased after reaching the peak,thus obtaining the optimum p H of 8.0 for the assay;by comparing I₅₃₀/I₄₆₅with different reaction times,it was found that FCP and ALP could react rapidly and reach stability after 15 min.The results of mixed detection with different substances showed that the detection values of FCP and ALP reactions far exceeded the control interfering substances,which proved their anti-interference ability.4.The results of DFT/TDDFT calculations show that the FCP detects fluorescent signals from-conjugated fluorescent coumarin groups,and molecular docking calculations prove that FCP,FC,and ALP can be firmly bound.5.There was no significant toxicity after 24 hours of incubation with a high-concentration FCP probe（50μM）.The intracellular fluorescence signal was significantly attenuated after early removal of ALP from the Hep G2 hepatocellular carcinoma cell line and the normal human hepatocyte line LO2 with ALP scavenger.Conclusions:1.FCP,is a ratiometric fluorescent probe that specifically binds ALP.2.FCP has good adaptability;it adapts to a wide range of p H values,has a short detection time,and can avoid the interference of other substances.3.The FCP probe can distinguish the difference in ALP activity in different cells,which provides a theoretical basis and experimental foundation for clinical diagnosis.PartⅡ:In situ NIR-controlled hydrogen release of RhPd-H material for hydrothermal therapy of breast cancer.