| Objective:The aim of this study was to explore the preparation method of99Tcm-labeled peptide PI(CASPSGALRSC) with high affinity to human breast cancer cell line MDA-MB-231, conjugated with hydrazinonictinamide(HYNIC) and to evaluate its biodistribution in vivo in normal mice and tumor bearing nude mice, and imaging in tumor bearing nude mice, as to seek the feasibility of PI as targeting tumor vector in the diagnosis and treatment of breast cancer.Methods:PI(CASPSGALRSC) with high affinity to human breast cancer cell line MDA-MB-231, conjugating the bifunctional chelator-HYNIC was synthesiz-ed. The peptide was labeled with99Tcm through an indirect method using tricine/TPPTS as co-ligands. then the preparation of [99Tcm-(HYNIC-PI)(tricine)(TPPTS)] was completed. The radiolabeling efficiency, radiochemical purity analysis and stability of the labeled mixture was demonstrated by HPLC testing and whatman3MM paper chromategraphic system; study of the specificity uptake in cell in vitro:setting human lung cancer cell A549as a negative control group, observed the bioactivity of labeled peptide, which specifically conjugated and entered MDA-MB-231cell lines; biodistribution in normal mice were studied. There were40adult KM mice, which were divided to eight groups by random.1.85MBq99Tcm-(HYNIC-PI)(tricine)(TPPTS) was injected into each mouse via a tail vein. At1,5,10,30,60,120,240,360min after injection, whole blood samples were collected and the mice were sacrificed by cervical dislocation. The heart, liver, lung, kidney, spleen, stomach, bone, muscle, and intestines were harvested, weighed and counted on a γ counter. The percentage of the injected radioactive dose per gram of tissue wet weight (ID%/g) was calculated after correction by reference source; biodistribution in tumor bearing nude mice:Balb/c nude mice bearing human breast cancer MDA-MB-231xenografts were studied.25tumor bearing mice were divided to five groups.1.85MBq99Tcm-(HYNIC-PI)(tricine)(TPPTS) was injected into each mouse via a tail vein. At30,60,120,240,360min after injection, the mice were sacrificed by cervical dislocation. The heart, liver, lung, kidney, spleen, stomach, bone, muscle, intestines, blood, and tumor were harvested, weighed and counted on a γ counter. We calculated the percentage of the injected radioactive dose per gram of tissue wet weight (ID%/g) after correction by reference source and tumor/non-tumor ratio (T/NT) at various time; SPECT imaging in tumor bearing nude mice:7.4MBq the labeled peptide was injected into the caudal vein of the bearing human breast cancer cell MDA-MB-231mice. Then static acquisitions were performed at30,60,120,240,360min with a gammar camera fitted with a low-energy high resolution collimator using a128X128matrix with a20%energy window set at140keV.Results:Analyzed by HPLC and whatman3MM paper chromategraphic system, the radiochemical purity and the radiolabeling yield of99Tcm-(HYNIC-PI)(tricine)(TPPTS) were over97%; After conserved at room temperature(25℃), and incubated with physiological saline and1%BSA at37℃for24h, the radiochemical purities were over95%; the cysteine replacement rate was beneath1%; Cells binding experiment in vitro proved the labeled peptide specifically conjugated the MDA-MB-231cells membrane, barely conjugated the A549lung cancer cells.The biodistribution in normal and bearing tumor mice showed it excreted mainly through kidney and quickly removed from blood, tumor uptake reached the highest radioactive at0.5h postinjection for (2.48±0.24)%ID/g, at this time the tumor/blood, tumor/muscle ratio for respectively (2.85±0.41),(7.49±1.83), but the tumor intake at4h remained at (1.38±0.29)%ID/g, tumor/blood, tumor/muscle ratio for respectively (5.85±1.81) and (9.67±2.88). all above indicated that the labeled peptide had higher intake in cancer and longer retention time; Compared with the radioactive of liver and lungs at same time, the difference of tumor tissue radioactive was statistically significant (p<0.05), excluding the kidney radioactive; Imaging of tumor bearing nude mice was clearly visible after the injection30min, and imaging at60min is much better.Conclusion:99Tcm-(HYNIC-PI)(tricine)(TPPTS) can be easily prepared and had high radiolabeling yield and well stability. The study of imaging and biodistribution in vivo showed that99Tcm-(HYNIC-PI)(tricine)(TPPTS) remains fine biological activity, higher tumor uptake and longer retention time. It is well worth researching as a promising targeting tumor vector in the treatment and diagnosis of breast cancer. |
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