Preliminary Experimental Study Of Magnetic Particle Imaging Combined With Annexin Ⅴ Targeted MPI-tailored Tracer In Tumor Apoptosis Imaging

Non-invasive,accurate and timely evaluation of tumors response to treatment has important clinical significance for optimizing treatment plan,standardizing medical management,evaluating prognosis and developing new drugs.At present,commonly used non-surgical treatment strategies for tumors,such as radiotherapy,chemotherapy,hyperthermia and photodynamic therapy,mainly aim at achieving successful treatment by significantly inducing iatrogenic apoptosis of tumor cells[1-4].Therefore,it is urgent to effectively monitor the state of apoptosis during tumor treatment.Although mature methods such as flow cytometry,TUNEL detection or Cleaved Caspase staining have been used to detect apoptosis in cultured cells and sliced tissues,the research on non-invasive apoptosis imaging and quantitative detection of apoptosis in vivo is still in the exploratory stage.We hope to have a powerful imaging method to detect and monitor the process of apoptosis of tumor cells in vivo.As a non-invasive imaging strategy,molecular imaging is an attractive monitoring method,which can obtain more valuable pathological information intuitively and non-invasively at the molecular level[5].Magnetic particle imaging(MPI),as a new molecular imaging technology in recent years,uses the nonlinear magnetization response principle of magnetic nanoparticles to quantitatively obtain the information such as the concentration,molecular markers,temperature and viscosity of magnetic nanoparticles in living animals[6-8].This new non-invasive imaging technology in vivo has potential high sensitivity(reaching nanomole level,and 1000 times higher than magnetic resonance imaging),high speed(millisecond of tomographic scanning speed),large field of view(depth field of view is greater than 5cm,exceeding the optical limit of 2cm),high resolution(sub-millimeter level,and 3 times higher than radionuclide imaging),quantitative information(used to quantify the number of SPIO labeled cells in the imaging area)and no ionizing radiation[9-11].These advantages make it possible to provide an unprecedented imaging tool for visualization and quantification of apoptosis in vivo.Therefore,in order to verify the above hypothesis and study the application value of MPI in apoptosis imaging,we have carried out the following three experimental schemes:Part I: Preparation and characterization of bi-modality MPI-tailored apoptosis-targeted magnetic nanoparticleObjective:To prepare magnetic nanoparticles with tailored MPI response,which can specifically target apoptotic cells and conform to certain physical and chemical characteristics,and thus provide basic imaging tracers for subsequent verification of MPI apoptosis imaging.Methods:In this study,phosphatidylserine,exposed outside the apoptotic cell membrane in the early stage of apoptosis was selected as imaging target,Annexin Ⅴ with high affinity and specific binding to apoptotic cell was used as imaging tracer ligand,and superparamagnetic iron oxide nanoparticles(SPIONs),Vivo Trax,which had been commercialized for MPI imaging research was used as its basic imaging signal source.Alexa Flour 647(AF647)was used as the fluorescent signal source.MPI tracer,AF647-Anx V-SPIO was synthesized by chemical crosslinking method.It was purified and characterized,including(1)physical property characterization: Dynamic Light Scattering(DLS)technology was used to detect the hydration particle size and stability of tracer particle size;The Zeta potential of tracer was detected by laser Doppler electrophoresis;The morphology and distribution were observed by Transmission Electron Microscope(TEM).(2)chemical property characterization:Inductively Coupled Plasma Optical Emission Spectrometer(ICP-OES)was used to detect the iron content of tracer,and BCA protein assay was used to detect and calculate the binding rate of Annexin Ⅴ protein with SPIONs.(3)tracer imaging performance: fluorescence characteristics of tracer were detected by fluorescence spectrophotometer;The MPI signal changes before and after tracer synthesis were detected by MPI scanner and the correlation between MPI signal and tracer concentration was analyzed.(4)Detection of biosafety performance(cellular level):The cytotoxicity of tracers with different concentrations on normal cells(human umbilical vein vascular endothelial cells)and tumor cells(EL-4 lymphoma cells)was detected by CCK8(Cell Counting Kit-8).Results:The related characterization results of the synthesized AF647-Anx V-SPIO are as follows:(1)physical property characterization: AF647-Anx V-SPl O is dark brown in appearance,easily soluble in water,and free of precipitation and impurities;Under transmission electron microscope,it is a circularly or oval-like regular high electron density shadow with an average particle size of 6.8±4.7nm;The results of malvinnano-particle size and Zeta potential analyzer show that the average hydration particle size(Z-Ave)of AF647-Anx V-SPIO is about 87 nm,while the hydration particle size of SPIO before attachment to Annexin Ⅴ is about 67 nm.the hydration particle size of the synthesized tracer increases,which accords with the size and characteristics of attached biomolecular materials.The hydrated particle size of AF647-Anx V-SPIO has little change after 72 hours,which is about 80 nm,indicating that it has good stability.AF647-Anx V-SPIO has a small polydispersity index and PDI is less than 0.3,indicating that the particle size distribution is relatively uniform;The Zeta potential test results show that AF647-Anx V-SPIO has high surface negative charge,and its Zeta potential is-21.4 ± 6.11 mv,which is beneficial to the dispersion of tracers in solution and prevents mutual aggregation.(2)Chemical property characterization: Fluorescence spectrophotometer detection results: AF647-Anx V-SPIO had the same characteristic peak as AF647,indicating that the tracer labeling was successful,and the fluorescence intensity of AF647-Anx V-SPIO was significantly higher than that of the ultrafiltrate(about 20times),so it was assumed that most AF647-Anx V proteins were bound to SPIO;Quantitative detection results of BCA protein showed that the cross-linking rate of Anx V protein and SIPO was about 91.8%,that is,most Annexin Ⅴ was intersecting with SPIO.The results of Inductively Coupled Plasma Emission Spectrometer showed that the iron concentration of AF647-Anx V-SPIO was about 4.75 mg/m L.(3)Imaging performance: AF647-Anx V-SPIO has the same characteristic peak as AF647,and has AF647 fluorescence imaging characteristics.The MPI imaging performance of AF647-Anx V-SPIO is basically consistent with that of Vivo Trax and has good MPI response.Moreover,the concentration of AF647-Anx V-SPIO had a significant linear correlation with MPI signal,and the fitting coefficient R2 was 0.998.(4)Biosafety: Assay of cytotoxicity of AF647-Anx V-SPIO to HUVECs and EL-4by CCK-8 method: The cell survival rate of AF647-Anx V-SPIO with different concentrations(0-250 g/m L)was over 90% after 24、48 hours of co-incubation with HUVECs and EL-4 cells,and AF647-Anx V-SPIO showed relatively low cytotoxicity.Conclusion:In this study,Annexin Ⅴ,an apoptosis-targeted ligand,was successfully combined with MPI-tailored SPIO(Vivo Trax)to prepare AF647-Anx V-SPIO tracer with good water-soluble,uniform particle size distribution,stable,high surface negative potential,AF647 fluorescence and MPI imaging performance,and low cytotoxicity,which can be used in subsequent experimental studies.Part II: The experimental study of apoptosis-targeted tracer AF647-Anx V-SPIO in vitroObjective:To evaluate the ability of AF647-Anx-SPIO as MPI tracer to target tumor apoptotic cells and the ability of MPI in apoptosis imaging,so as to lay a foundation for MPI apoptosis imaging in vivo.Methods:Etoposide,a commonly used chemotherapy drug,was used to induce mouse T lymphoma EL-4 cells,and the apoptosis model in vitro was established.The ability of the synthesized tracer AF647-Anx V-SPIO to target apoptotic cells was quantitatively and qualitatively detected by in vitro flow cytometry and fluorescence confocal microscope.The ability of MPI to image apoptotic cells in vitro was detected by using different treatment groups of cells,and the ability to quantify apoptotic cells was detected by MPI imaging of apoptotic cells with different number gradients.Results:Flow cytometry verified that the apoptosis model of EL-4 lymphoma incubated with 20 μM etoposide for 14 h was successfully established,and the apoptosis rate was about 67.21%.AF647-Anx V-SPIO has a good ability of targeting apoptotic cells in vitro.MPI imaging experiment in vitro verified that MPI has a good imaging performance on imaging apoptotic cells in vitro,which shows that the treatment experimental group is obviously different from other control groups in MPI images.The MPI signal intensity increases with the increase of apoptotic cells,and there is a good linear correlation between them,and the fitting coefficient R2 is 0.9991.Conclusion:AF647-Anx V-SPIO has a good ability to target apoptotic cells in vitro.At the same time,MPI,as a new imaging technology,has a good ability to visualize and quantify apoptosis cells in vitro,and has the potential to be further applied to imaging apoptosis in vivo.Part III: The experimental study of apoptosis-targeted tracer AF647-Anx V-SPIO in vivo and ex vivoObjective:The biosafety of the tracer AF647-Anx V-SPIO in mice was evaluated systematically.To evaluate the application potential of MPI in tumor apoptosis imaging at animal level,we constructed tumor apoptosis model induced by chemotherapeutic agents in mice with lymphoma.Methods:The tracer AF647-Anx V-SPIO was injected into normal nude mice by tail vein at different concentrations(control group,50,100,and 200 μg/g).The biological safety of the tracer was detected by observing the weight,skin,mouth,nose and eyes and other vital signs of nude mice in each group,as well as blood routine examination,liver and kidney function,and the HE staining of major organs.The mouse subcutaneous tumor apoptosis model was induced by etoposide combined with cyclophosphamide,and confirmed by TUNEL apoptosis staining of tumor tissue.The IVIS imaging of apoptotic model mice injected with AF647-Anx V-SPIO preliminarily confirmed the best imaging time of MPI,and further verified by ICP-OES.In this study,MPI imaging of tumor-bearing mice in vivo,MPI imaging of tumors and main organs ex vivo were performed to detect the MPI ability of apoptosis imaging and the distribution of AF647-Anx V-SPIO.Results:The mice of different treatment groups showed good biosafety in the series of vital signs and the results of HE section staining of the main organs.TUNEL staining of tumor tissue confirmed that the mouse subcutaneous tumor apoptosis model induced by etoposide combined with cyclophosphamide was successfully established,and the tumor apoptosis rate was about 16.6%.According to IVIS observation,the aggregation of tracer can be observed at the tumor site of the treatment group after injecting AF647-Anx V-APIO for 2 hours.After injecting tracers for 4 hours,the fluorescence signal intensity of the treatment group all increased,while no obvious fluorescence signal was detected at the tumor site of the untreated group.The optimal imaging time is set to 4h.and the ICP-OES testing was also further confirmed this result.While in vivo MPI imaging,no obvious MPI signal was detected in tumor sites of both two groups,but MPI imaging of tumor ex vivo can observe the highlight signal of mice in Etoposide + CTX treatment group which is obviously different from other treatment groups or the control group.The difference of MPI signal between groups reflects the difference of apoptosis rate in tumor tissues,and the MPI detection results are consistent with TUNEL staining results of tumor tissues.MPI imaging of major organs showed that the tracer was mainly distributed in liver(92±10%),followed by spleen(5±1%)and tumor(1.6±3%),and almost no tracer signal was detected in kidney,intestine and heart.Conclusion:For the first time,MPI was used to image apoptosis at cell and animal levels.Although in vivo MPI imaging has not achieved the expected results,we have obtained a high contrast image through MPI imaging of tumor ex vivo.The difference of MPI signals between the different treatment groups also reflects the difference of apoptosis rate in tumor tissues,which proves the apoptotic targeting of AF647-Anx V-SPIO,and the ability of MPI to apoptotic imaging.It is believed that with the continuous optimization of the performance of MPI devices and the development of more MPI-tailored nanoparticles,MPI will be developed as a promising diagnostic tool for apoptotic imaging in vivo,which is different from the existing medical imaging models.Our preliminary study reaffirms that tracers with excellent MPI characteristics are important for the development of potential preclinical imaging modalities and for the identification of new possible clinical applications.