FITC/¹⁷⁷Lu Labeling Of Aminopeptidase N Targeting Peptide And Properties In Vivo And In Vitro

Objective:Studies have shown that aminopeptidase N?CD13?,as a malignant cell surface marker,is overexpressed on many tumor cells and neovascular cell membranes,and the tripeptide structure Asn-Gly-Arg?NGR?can specifically bind to CD13 and is a targeting ligand for the APN/CD13receptor.In this study,we plan to design and synthesize NGR polypeptide with tumor neovascularization effects,use FITC for fluorescent labeling,and study the uptake of NGR polypeptide derivatives of different structures by target tumor cells.Linking the chelating agent DOTA and using the radionuclide ¹⁷⁷Lu to label the derivatives,and tested the in vitro stability of the radiolabeled compounds,then it was subjected to in vitro cell experiments with CD13 receptor positive expression cells to analyze its specific uptake.Establish a tumor animal model,use MircoPET imaging to explore the metabolism and tumor uptake of ⁶⁸Ga-labeled NGR in tumor-bearing mice,and explore the biodistribution of ¹⁷⁷Lu-labeled NGR polypeptide in tumor-bearing mice.The design and construction of a highly-targeted peptide structure and the study of ¹⁷⁷Lu integrated radionuclide labeling studies will ultimately provide the research basis for obtaining radioactive peptide drugs with targeted therapeutic effects on tumor neovascularization.Method:?1?Synthesis of NGR fragments containing a targeting structure that specifically binds to the CD13 receptor by peptide solid-phase synthesis,labeling linear NGR?zNGR?and cyclic NGR?cNGR?with the fluorescent agent FITC,and cyclic NGRs?cNGR and ky-cNGR?were labeled with the chelating agent p-NCS-Bn-DOTA,then the two cyclic NGRs were purified and characterized using HPLC and mass spectrometry.?2?Using confocal imaging for qualitative experiments of cell uptake fluorescence and flow cytometry for quantitative experiments of cell uptake fluorescence,to study the uptake ability and specific targeting ability of zNGR,cNGR,ky-cNGR peptides in lung cancer cells,respectively.?3?Labeling two cyclic NGR polypeptides with different chain lengths using ¹⁷⁷LuCl3.The reaction conditions were:PH=5-5.5,temperature 80°C,time 30 minutes,and the radiochemical purity of each labeled product was measured by thin layer chromatography?TLC?.The stability of the prodrugs ¹⁷⁷Lu-DOTA-cNGR and ¹⁷⁷Lu-DOTA-ky-cNGR in fetal bovine serum and physiological saline at 37°C was measured in vitro.?4?Human small cell lung cancer cells NCI-H1688,human lung adenocarcinoma cell A549,human fibrosarcoma cell line HT-1080,and normal human umbilical vein endothelial cells HUVEC were tested for their ability to uptake ¹⁷⁷Lu-DOTA-cNGR and ¹⁷⁷Lu-DOTA-ky-cNGR,then evaluate the in vitro properties of related NGR peptide derivatives.?5?Use SPF BALB/c female nude mice to establish tumor-bearing mouse tumor models.DOTA-cNGR was labeled with ⁶⁸Ga and injected into the NCI-H1688 and A549 tumor-bearing mice along the tail vein and imaged with MiroPET one hour later.Inject ¹⁷⁷Lu-DOTA-cNGR and ¹⁷⁷Lu-DOTA-ky-cNGR along the tail vein into HT1080,A549 and NCI-H1688 tumor-bearing mice?n=2?,then biodistribution were performed after 24 hours to compare the differences between the two probes in vivo,and the application value of¹⁷⁷Lu-DOTA-cNGR and ¹⁷⁷Lu-DOTA-ky-cNGR for the diagnosis and treatment of tumor targeted imaging was evaluated.Results:?1?FITC-cNGR,FITC-zNGR,DOTA-cNGR,and DOTA-ky-cNGR were successfully synthesized and analyzed using high-performance liquid chromatography and mass spectrometry.?2?Both FITC-cNGR?FITC-zNGR can be combined with NCI-H1688 cells for 1 hour.The FITC signal is basically the same as the DAPI?nuclear staining?signal,which indicates that the NGR peptide can be taken up by the cells.After pretreatment with the corresponding cNGR or zNGR peptide for 30 minutes and performing blocking experiments,the former significantly reduced the uptake of FITC-cNGR by the cells,while the latter was still highly uptaked by the cells.It can be seen that NCI-H1688 cells have certain specificity for the uptake of cNGR.?3?When the added peptide concentration was fixed at 5 nM,the uptake of FITC-cNGR by NCI-H69 cells in 4 minutes was measured by flow cytometry in 15 minutes.As time increases,the cell uptake rate increases accordingly.And When the incubation time is 15 minutes,as the concentration of cNGR peptide increases,the cell uptake rate will also increase accordingly.?4?The labeling rates of¹⁷⁷Lu-DOTA-cNGR and¹⁷⁷Lu-DOTA-ky-cNGR are more than 95%.The radiochemical purity of the former in FBS and saline remains about 70%after 24 hours,while the latter is still about 90%.?5?¹⁷⁷Lu-DOTA-cNGR and ¹⁷⁷Lu-DOTA-ky-cNGR have different uptake rates for each cell type,the former uptake rate:HUVEC>HT-1080>A549>NCI-H1688;the uptake rate of the latter:A549>NCI-H1688>HT-1080>HUVEC.?6?According to MicroPET imaging results,68Ga-DOTA-cNGR showed high liver and kidney uptake and low tumor uptake in both NCI-H1688 and A549 tumor-bearing mice.?7?The biological distribution of ¹⁷⁷Lu-DOTA-cNGR and ¹⁷⁷Lu-DOTA-ky-cNGR in HT1080,A549,and NCI-H1688 tumor-bearing mice showed that both probes have high uptake of liver,spleen,kidney,and bone,and low tumor uptake,which is not significantly different from the results of MicroPET imaging,and¹⁷⁷Lu-DOTA-ky-cNGR has a longer retention time in the tissues of the body than ¹⁷⁷Lu-DOTA-cNGR,and tumor uptake is higher.Conclusion:In this study,we successfully labeled multiple NGR peptides with FITC and ¹⁷⁷Lu.By increasing the length of the terminal chain of the NGR polypeptide,although the specific in vitro binding capacity of the polypeptide against some target tumors is affected,it effectively extends the circulation time of the radiolabeled cNGR polypeptide derivative in the body,which increases the aggregation effect in tumors.The results of the radionuclide¹⁷⁷Lu-labeled NGR peptide research carried out in this project are expected to provide new design ideas and basic parameters for targeted therapeutic drugs for tumors.