Research Of Biodistribution And Receptor Imaging Of ^99mTc-GE11 Molecular Probe In Lung Adenocarcinoma-bearing Nude Mice

Background:Currently,the receptor imaging and treatment of lung cancer are favored by more and more people.EGFR targets have attracted much attention,and EGFR is over-expressed in most tumor cells,including non-small cell lung cancer.EGFR plays an important role in cell proliferation and signal transduction,and has become an important target for tumor molecular receptor imaging and targeted therapy research at home and abroad.At present,the receptor imaging and treatment research targeted drug of EGFR are mainly includes EGFR-TKI,monoclonal antibodies and polypeptides.Comparing with EGFR-TKI and monoclonal antibodies,polypeptides has uniqueadvantages such as high affinity for target protein,fast clearance in blood,low immunogenicity,easy synthesis and modification and so on.The new peptide GE11 is a small molecule peptide obtained by phage peptide library screening technology,and it is composed of 12 amino acids.According to the studies,can combined with the specificity of EGFR,and effectively target EGFR.As a vector targeting EGFR receptor,GE11 has been widely used and has a significant effect in the tumor bearing model experiment of NSCLC nude mice.But,there have been no reports of studies on achieving the molecular imaging and treatment objectives by using nuclide to label small molecule polypeptide GE11 at home and abroad.Objectives:In this study,the author try to use ⁹9m9m Tc to label small molecular polypeptide GE11 was used to prepare small molecule nuclide molecular probe of targeting EGFR,identify its physical and chemical properties,and explore its biological distribution and in vivo imaging in nude mice,so as to identify its feasibility as a new imaging agent and provide experimental basis for its clinical application.Methods:1.The preparation of ^99mTc-GE11:commission a company to synthesize GE11 a polypeptide containing BCFR by using chemical method and stannous chloride reduction method to label it with ^99mTc.Then,TCL method was used to determine the marking rate,and the marking conditions were explored to maximize the marking rate.And the100μl mixture was purified by Sephadex G50 column.2.The identification of physical and chemical properties of^99mTc-GE11:measuring it’s stability in vitro,combined quantity with plasma protein and analyzing the composition of polypeptides was single or not,by the experiment of human plasma protein,high pressure liquid chromatography（HPLC）and testing at different time points under normal temperature.And then,analyzing the hydrophilic lipophilicity of ^99mtc-ge11and the stability of its chelation with ^99mTc by lipid water distribution test and cysteine test respectively.3.The identification of the specific binding ability both of ^99mTc-GE11and A545 cells:the specific binding ability both of ^99mTc-ge11 and A549cells was verified by in vitro cell competitive assay and saturation experiment.4.The biological distribution and SPECT imaging of ^99mtc-GE11 in normal nude:100μl fresh ^99mTc-GE11（7.4MBq）was injected into the tail vein,and the nude mice were sacrificed at different time points after injection by cervical dislocation method.Then all tissues and organs were taken for weighing,and their radioactive counts were measured byγ-immune counter.And the percentage injection dose rate（%ID/g）per gram of tissue of the results was calculated after reference source correction.At last,observing the distribution of fresh ⁹9m9m Tc-GE11 in vivo after injection via caudal vein by SPECT.5.The biological distribution in vivo and SPECT imaging of^99mTc-GE11 in human lung adenocarcinoma（A549）nude rat model:constructing human lung adenocarcinoma（A549）nude rat model,and when the mass volume was greater than 1cm³,it could be used to study the distribution of ^99mTc-GE11 in vivo and SPECT imaging.Then detecting the radioactivity of each organ（cpm）by using in vivo distribution experiment,and calculating their%ID/g.Observing the imaging of ^99mTc-GE11 in vivo at different time points by SPECT static imaging.After imaging,the radiometric ratios of tumor sites and contralateral sites were calculated by using the region of interest technique,and the tumor/non-tumor（T/NT）radiometric ratios were semi-quantitatively analyzed.Results:1.GE11 was successfully labeled with ^99mTc,and the labeling rate is（93.12±0.83）%,specific activity is（26.19±0.42）TBq/mmol,and purified radiochemical purity is（93.64±0.56）%.2.The identification of physical and chemical properties of^99mTc-GE11:after being placed at room temperature for 6h or 12h,the radiochemical purity went down 2.14%and 3.11%respectively,and its binding rate with plasma protein was 4.8%.After it incubated with different concentrations of cysteine)for 1h in temperature of 37℃,the content of uncombined ^99mTc has no obvious change.And the fat/water distribution coefficient was lgP=-1.87±0.05.3.The results of Cell saturation curve and competitive experimental:the radioactivity on the surface of A549 cells in the experimental group increased with the increase of the concentration of ^99mTc-GE11,and became saturated gradually.As the concentration of ^99mTc-GE11 increased in the control group,the radioactivity count gradually increased,but there was no saturation trend,and its radioactivity was far lower than that in the experimental group.4.The in vivo biological distribution experiment of ^99mTc-GE11 in normal nude mice:^99mTc-GE11 was rapidly cleared of radioactivity in the blood and kidney,showed low radioactive uptake in lung or bone,muscle and other tissues,and showed persistent low radioactive uptake in the brain.And ^99mTc-GE11 was excreted mainly through the urinary system,followed by the hepatobiliary system.5.the biological distribution in vivo and SPECT imaging of^99mTc-GE11 in human lung adenocarcinoma（A549）nude mice model:^99mTc-GE11 in the lungs characterized by low radioactivity uptake,radioactive cleared quickly in the blood,and kidney tumor tissues can selectively gathered ^99mTc-GE11.At the point of 1.0 h in the injection,the tumor/muscle ratio reached the maximum（12.94±2.65）,and the tumor tissue development was the clearest.Conclusion:1.Radionuclide ^99mTc can label polypeptide GE11 with simple method and high labeling rate.2.Labeling polypeptide ^99mTc-GE11 showed good stability in vitro,low binding rate to plasma protein,and firm chelation of^99mTc with GE11.3.^99mTc-GE11 can bind specifically to A549 cells.4.^99mTc-GE11 was used to treat the tumor at an early time,and has a high radionuclide to nontumor ratio,a fast in vivo clearance rate.Therefore,as a nuclide molecular probe targeting EGFR,^99mTc-GE11 has the potential to become a new targeted imaging agent for the diagnosis and treatment of lung cancer.And that’s also can lay an experimental foundation for us to transform from basic research to clinical application in the future.