Construction Of A Novel Three Target Rare Earth Doped Down Conversion Near Infrared Micro Nano Composite Probe And Its Differential Tracing Of Lung Cancer Cells

Lung cancer is common in malignant tumors,and is the most malignant tumor with high incidence rate and mortality.At the early stage,lung cancer usually appears as small nodules in CT(computed tomography).Adenocarcinoma,squamous cell carcinoma and small cell cancer are the most common pathological types in lung cancer,and their incidence rate is also increasing rapidly.At present,in addition to surgical biopsy,there is no specific screening method for the nodules of early lung cancer,which can only be judged by clinicians’ experience in CT imaging.So,it is better to have probes that can recognize early lung cancer cells for early identification clinically.Compared with the current CT,MRI(magnetic resonance imaging),color Doppler ultrasound,PET-CT(positron emission tomography computed tomography)and other scanning imaging technology,the imaging technology of NIRF(near-infrared fluorescence)has the advantages of high feedback speed,high imaging resolution,high sensitivity,low cost and no radiation,and can be used in surgery,which is incomparable to the current technology.NIRF can be used to guide the scope of operation in clinic,can accurately locate the lesion and assist surgical resection,and can be used to identify pulmonary nodules and pulmonary vessels.Compared with the organic dyes of ICG(indocyanine green),rare earth conversion micro nanocrystals are the latest light-emitting basic materials,which have good light stability,low biological toxicity,high chemical stability,better tissue compatibility,and have deeper light penetration depth and less radiation damage,and are more suitable for biological tissues.The most common types of lung cancer are non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).The most common types of NSCLC are adenocarcinoma and squamous cell carcinoma.In this study,three down conversion rare earth materials,erbium(Er),neodymium(Nd)and holmium(Ho),combined with folate,SCC(squamous cell carcinoma)antibody and ProGRP(pro gastrin releasing peptide)antibody,which as markers of lung adenocarcinoma,squamous cell carcinoma and small cell carcinoma,were selected as targets to construct rare earth doped down conversion micro nano materials.They have excellent near-infrared imaging properties,and can generate different wavelengths by reflection.Different objects can be identified by NIRF.In this study,we intend to use rare earth doped down conversion micro nano materials combined with target to construct rare earth doped down conversion micro nano crystals,and prepare a new three target rare earth doped down conversion near infrared micro nano composite probe,so as to identify different lung cancer cells and explore their differential tracing ability.The research content is divided into the following three parts,and the fourth part is exploratory verification experiment:Part one:preparation of three target rare earth doped down conversion micro nano composite probeObjective:To prepare a novel three target rare earth doped down conversion micro nano composite probe.Methods:GD-OA(Gadolinium-Oleic acid)and Na-TFA-OA(Sodiumtrifluoroaceticacid-Oleic acid)was synthesized for the rare earth down conversion shell precursor material.Then synthesis of core-shell structure high efficiency quenched neodymium,erbium and holmium doped down conversion fluorescent nanocrystals.And then through reaction of EDC-NHS(1-Ethyl-three-(3’dimethylaminopropyl)carbodiimide-N-Hydroxysuccinimide),the target folate,SCC antibody and pro-GRP antibodies were grafted on different down conversion micro nano crystals for surface modification,will be used to identify of different lung cancer cells.Finally,three target rare earth doped down conversion micro nano composite probes were prepared,and their properties were tested.Results:Down conversion fluorescent micro nano crystals with water-soluble core-shell structure and high efficiency anti quenching were successfully prepared of NaErF4@NaGdF4,NaYF4:Yb,Ho@NaGdF4 and NaYF4:Nd@NaGdF4.Under electron microscope,the micro nano particles showed hexagonal phase.The water-soluble core-shell structure high efficiency quenched down conversion fluorescence micro nano crystals were successfully prepared,and the micro nano probes NaErF4@NaGdF4-folic acid,NaYF4:Yb,Ho@NaGdF4-SCC antibodies,NaYF4:Nd@NaGdF4-ProGRP antibodies were obtained by grafting the target.Their sizes are about 29 nm,72 nm and 44 nm,respectively.The test shows that the probe has good uniformity.The crystal of NaErF4@NaGdF4 and NaYF4:Yb,Ho@NaGdF4 has luminescence peaks in the visible region.The crystal of NaErF4@NaGdF4,NaYF4:Yb,Ho@NaGdF4 and NaYF4:Nd@NaGdF4 has a luminescence peak in the near-infrared region.The NIR emission peaks of the three probes are 1525 nm,1150 nm and 1060 nm,respectively.The green and red emission of the crystal NaErF4@NaGdF4 is a two photon process,while the NIR emission is a single photon process.The emission of green,red and NIR of the crystal NaYF4:Yb,Ho@NaGdF4 is a single photon process.The NIR emission of the crystal NaYF4:Nd@NaGdF4 is a single photon process.Conclusion:Down conversion fluorescent micro nano crystals were successfully prepared,and a new type of three target rare earth doped micro nano composite probe has been successfully prepared.The results show that the composite probe has good stability and luminescence characteristics.Part two:cell culture,cytotoxicity test and recognition imaging of three target rare earth doped down conversion micro nano composite probeObjective:Lung adenocarcinoma cell line NCI-H522,lung squamous cell carcinoma cell line NCI-H1703 and small cell lung cancer cell line HTB-119 were cultured in vitro.The cytotoxicity of NCI-H522,NCI-H1703 and HTB-119 were tested and identified by using composite probe,and near infrared imaging was performed.Methods:Human lung adenocarcinoma cell line NCI-H522,human lung squamous cell carcinoma cell line NCI-H1703 and human small cell lung cancer cell line HTB-119 were resuscitated,cultured,subcultured and cryopreserved in the laboratory.Then the CCK-8(cell counting Kit)cytotoxicity test was carried out by using the three target rare earth doped down conversion micro nano composite probe to understand the cytotoxicity of the composite probe on lung cancer cells.Finally,the composite probe was used to perform the near-infrared imaging of lung cancer cells under the near-infrared fluorescence microscope to explore the recognition accuracy.Results:Human lung adenocarcinoma cell line NCI-H522,human lung squamous cell carcinoma cell line NCI-H1703 and human small cell lung cancer cell line HTB-119 which purchased from ATCC can be successful resuscitated,cultured,subcultured and cryopreserved by the experiment.The cells survived well and could meet the experimental requirements.The survival rate of lung adenocarcinoma cells treated with NaErF4@NaGdF4-folate probe was more than 70%,the survival rate of lung squamous cell carcinoma cells treated with NaYF4:Yb,Ho@NaGdF4-SCC antibody probe was more than 70%,and the survival rate of small cell lung cancer cells treated with NaYF4:Nd@NaGdF4-ProGRP antibody probe was more than 60%.The cell proliferation toxicity of the composite probe to lung adenocarcinoma cells,lung squamous cell carcinoma cells and small cell lung cancer cells was within the controllable range,and there was no obvious toxic reaction.The final dosage of the composite probe was 40 μmol/ml.Compared with DAPI,the recognition imaging of three lung cancer cells with the composite probe can accurately overlap,indicating that the composite probe can carry out accurate near-infrared recognition.Conclusion:Lung cancer cells can be resuscitated,cultured,subcultured and cryopreserved.In this experiment,the proliferative toxicity of the micro nano probe NaErF4@NaGdF4-folic acid probe,NaYF4:Yb,Ho@NaGdF4-SCC antibody probe and NaYF4:Nd@NaGdF4-ProGRP antibody probe to lung adenocarcinoma cells,lung squamous cell carcinoma cells and small cell lung cancer cells was within the controllable range,and lung cancer cells could be accurately near-infrared identified.Part three:construction of subcutaneous tumor of lung cancer nude mice and trace of the three target rare earth doped down conversion micro nano composite probeObjective:The subcutaneous tumor of lung cancer in nude mice was constructed and traced by three target rare earth doped down conversion micro nano composite probe.Methods:Lung adenocarcinoma cell line NCI-H522,lung squamous cell carcinoma cell line NCI-H1703 and small cell lung cancer cell line HTB-119 were subcutaneously injected into the right forearm of B ALB/c nude mice to establish three kinds of subcutaneous tumor models.Three target rare earth doped down conversion micro nano composite probe was then injected into the models’ tail vein different time for near infrared tracing of subcutaneous tumor.The accuracy of the composite probe recognition was evaluated by pathological sections.The changes of blood routine,liver and kidney function,respiration and heart rate were observed by the composite probe to subcutaneous tumor models.Results:The subcutaneous tumor model of lung cancer in nude mice was established by culturing lung adenocarcinoma cell NCI-H522,lung squamous cell carcinoma cell NCI-H1703 and small cell lung cancer cell HTB-119.The mortality of nude mice was about 10%,and the existence of subcutaneous tumor was confirmed by CT.When the three target rare earth doped down conversion micro nano composite probe was injected into the tail vein for 10 minutes,the near infrared signal intensity is the highest,the subcutaneous tumor model of lung cancer in nude mice could be identified by near infrared spectroscopy.Under the guidance of fluorescence imaging region,the lesions and surrounding tissues of the subcutaneous tumor were excised.Pathological sections confirmed that the subcutaneous tumor tissue cells were cultured cancer cells.The probe identified that the tissue around the tumor was normal tissue cells,and there was no cancer cell infiltration.The accuracy of the composite probe recognition and the accuracy of the recognition range were confirmed by pathology.The results showed that the main blood routine,liver and kidney function indexes of the composite probe had no significant change before injection,20 minutes and 60 minutes after injection,P values were greater than 0.05.And the respiration and heart rate had no significant changes,P values were greater than 0.05.Conclusion:The subcutaneous tumor models of lung adenocarcinoma cell NCI-H522,lung squamous cell carcinoma cell NCI-H1703 and small cell lung cancer cell HTB-119 were successfully established in nude mice.The new three target rare earth doped down conversion micro nano composite probe can realize differential identification and tracing of subcutaneous tumor model in nude mice,and has less biological toxicity.Part four(additional experiments):exploratory study of three target rare earth doped down conversion micro nano composite probe for lung carcinoma in situ in nude mice(single cancer validation test)Objective:This extended experiment is an extension of the study.It is an exploratory experiment on the success rate for the follow-up of in situ cancer tracing.Human lung squamous cell carcinoma cells were used to construct the lung squamous cell carcinoma in situ model in nude mice,and to understand the tracer effect of three target rare earth doped down conversion micro nano composite probe on lung squamous cell carcinoma in situ in nude mice.Methods:Human lung squamous cell carcinoma cell line NCI-H1703 was selected and injected into the lung of nude mice through skin in the front chest of nude mice to construct the nude mouse carcinoma in situ model of lung squamous cell carcinoma.The nude mouse carcinoma in situ model was confirmed by CT imaging.Then,the three target rare earth doped down conversion micro nano composite probe was injected to trace the cancer model in situ by near-infrared to understand the tracing performance of the composite probe.In addition,the near-infrared imaging of other organs was carried out to understand whether there was wrong recognition.Results:After 30 nude mice were injected with lung squamous cell carcinoma cell line NCI-H1703,16 survived,and 7 of them were confirmed by CT to construct a successful carcinoma in situ model of lung squamous cell carcinoma in nude mice.The success rate of carcinoma in situ model was 23.3%.The lesion of lung squamous cell carcinoma was located in the lung parenchyma of nude mice throughing anatomy.When the three target rare earth doped down conversion micro nano composite probe was injected for 10 minutes,there was obvious near-infrared imaging in the lung of nude mice in situ cancer model,no near-infrared imaging in other parts,and the signal-to-noise ratio between tumor tissue and normal tissue was 5.3.The pathological section of the lesion confirmed that it was lung squamous cell carcinoma cells,the surrounding lung tissue was normal tissue,and there was no cancer cell infiltration.In situ cancer model,there was obvious near-infrared enrichment signal in the lung,but there was no near-infrared enrichment signal in the spleen,heart,kidney,intestine and liver of nude mice.It shows that the three target rare earth doped down conversion micro nano composite probe can accurately identify the lesions of lung squamous cell carcinoma,and has no effect on other organs of nude mice.Conclusion:The research shows that the three target rare earth doped down conversion micro nano composite probe can accurately identify the in situ cancer model of lung squamous cell carcinoma in nude mice,and has good performance without identification deviation for other organs in nude mice.The experimental data laid an experimental foundation for the follow-up planning experiments of other carcinoma in situ,metastatic carcinoma and benign nodule.