| Background:CD 13(aminopeptidase N,APN)is a type of Ⅱ transmembrane glycoprotein compring of 967 amino acids,which is overexpressed in the endothelium of angiogenic blood vessels and some tumor cells,promotes angiogenesis,participates in tumor invasion,metastasis,and progression,and is an ideal target for tumor therapy.In vitro phage display technology found that peptides containing Asn-Gly-Arg(NGR)sequences could specifically recognize CD 13 and dispay specific tumor targeting,making them an important vector for CD 13 targeting diagnosis and treatment.Labeling polypeptides containing NGR sequences with radionuclides,fluorescent dyes and magnetic particles can provide a variety of molecular probes for the diagnosis and efficacy evaluation of tumors and other diseases associated with angiogenesis through different in vitro imaging techniques.Objective:This study was designed to construct and synthesize a cyclic dimer polypeptide molecule probe labeled with NGR motif by positron radionuclide 68Ga,and to evaluate its targeting to CD 13 receptor in vitro and in vivo,and to investigate the feasibility of its application in evaluating the expression level of CD 13 receptor in vivo and PET imaging of CD 13 positive tumors.Methods:DOTA-c(NGR)2 was constructed,synthesized,and labeled with 68Ga to prepare 68Ga-DOTA-c(NGR)2 probe.Reverse high performance liquid chromatography(RP-HPLC)analyzer was used to identify and analyze the probe.The in vitro stability of the probe was investigated by measuring the radiological purity of the probe at different time points.Two ovarian cancer cell lines with different CD 13 expression levels,ES2 and SKOV3,were selected for cell binding assay and cell binding inhibition assay to investigate the specific binding ability of 68Ga-DOTA-c(NGR)2 to CD 13 positive tumor cells in vitro to determine its CD 13 receptor targeting in vitro.In this study,68Ga-DOTA-c(NGR)2 was used as imaging agent to detect CD 13 expression in ES2 and SKOV3 nude mice with different levels of CD 13 expression by MicroPET imaging.The tumor targeting ability of the probe in nude mice with tumor bearing was investigated,and the feasibility of using the probe to evaluate CD 13 expression level in tumor was evaluated.Results:DOTA-c(NGR)2 could be easily labeled by 68Ga,and the labeling rate was(98.01%±1.44)%,without further purification.68Ga-DOTA-c(NGR)2 showed good stability in vitro.The results of biological distribution in mice showed that 68Ga-DOTA-c(NGR)2 was mainly metabolized from the kidney and cleared quickly from the blood.The pharmacokinetics of 68Ga-DOTA-c(NGR)2 was consistent with single-compartment model.The uptake of 68Ga-DOTA-c(NGR)2 in ES2 and SKOV3 cells increased gradually,and the upktake in ES2 cells was higher than that in SKOV3 at all time points.The binding of 68Ga-DOTA-c(NGR)2 to ES2 can be blocked by excessive unlabeled DOTA-c(NGR)2.MicroPET imaging of tumor-bearing nude mice showed different degrees of radioactive accumulation at tumor sites in both ES2 and SKOV3 tumor-bearing nude mice 15 min after injection of 68Ga-DOTA-c(NGR)2.High focal accumulation occurred in the tumor site at 1 h and 1.5 h post-injection.ROI quantitative analysis showed that the uptake values of 68Ga-DOTA-c(NGR)2 in ES2 tumors were(0.62±0.09)%ID/g,at 1 h,(0.53 ±0.08)%ID/g,at 1.5 h,and the T/B ratios were 10.30±0.26 and 8.04±1.75,respectively,while the uptake values of SKOV3 tumors at the same time point were(0.32±0.03)%ID/g and(0.24±0.05)%ID/g,respectively,and the T/B ratios were 3.99±0.18 and 4.24±0.73,respectively.All the above differences were statistically significant(P<0.05).Radiation uptake of ES2 at tumor sites was significantly reduced by simultaneous injection of excessive nonlabelled DOTA-c(NGR)2.Immumohistochemical staining confirmed that the density of neovasculature and the expression of CD 13 on neovascular endothelial cell surface and tumor cells in ES2 tissue.was significantly higher than that in SKOV3 tissue.Conclusions:68Ga-DOTA-c(NGR)2 is easy to synthesize,stable in vitro,and has excellent biological distribution and metabolic properties.It can specifically bind to CD 13 receptor in vivo and in vitro and may be a potential PET imaging probe for non-invasive detection of neovasculature and CD 13 positive tumors in vivo. |
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