| The lymphatics serve as one of the most common route for the dissemination of many solid tumors. However, the diagnosis and therapy for the lymphatic metastasis often have bad effects. It is well known that polymer prodrugs deliver to the lymphatic system when injected subcutaneously. However, the polymer prodrug could not actively targeted the lymphatic system with metastatic tumor. In addition, how to release of the antitumor drugs from polymer carrier is still unsolved. In the present study, the strategies for solving aboved two problems are based on the ideas of modification of folic acid, polyethylene glycol carrier and release by enzymatic catalysis. It aims to develop a folate-polyethylene glycol conjugate as carrier of radionuclide and antitumor drug applied for the lymphatic tumor.The main content for this study is including as following: first, The Folate-PEG conjugate was prepared and determined. Second, Folate-PEG-DTPA was synthesized and biological evaluated for the lymphoscintigraphy with metastatic tumor. Third, the folate-polyethylene glycol-doxorubicin conjugates were prepared and studied for antitumor effect.The first step for the study was to prepare H2N-PEG-NH2 by chrorination, azide and catalytic hydrogenation reactions, the birth rates were 75%, 87% and 88%. Using cation exchange resin as a separate medium and acetic buffer as the eluent, H2N-PEG-NH2 was purified from HO-PEG-OH and HO-PEG-NH2. Finally, the purity of H2N-PEG-NH2 was over 95%. The product structure was characterized by HPLC, IR and HNMR.Catalyzed by dicyclohexylcarbodiimide, folic acid was reacted with H2N-PEG-NH2 to yield Folate-PEG. After purification by anion-exchange chromatography, Folate-PEG was obtained from H2N-PEG-NH2, Folate-PEG-Folate and folic acid. The purity of Folate-PEG was about 95%. In vitro and in vivo test confirmed Folate-PEG kept the activity of combining with the folate receptor.Folate-PEG-DTPA was synthesized as carrier of radionuclide applied for the lymphatic tumor. In vivo competitive binding test showed that the uptake of [125I] folic acid was inhibited by Folate-PEG-DTPA and 50% inhibitory concentration was 4.37pmol/L. In comparison with folic acid, the relative affinity of Folate-PEG-DTPA was 0.18 for human folate receptor. The results showed that Folate-PEG-DTPA could compete with folic acid for folate receptor.Ethylenediamine was a bifunctional molecule that linked isothiocyanate fluorescein to Folate-PEG-DTPA. In the cultured tumor cell, uptake of fluorescein labeled Folate-PEG-DTPA was found to increase significantly compared with that of untargeded MPEG-DTPA. The competition with free folic acid blocked the cell uptake of Folate-PEG-DTPA. The results conformed that Folate-PEG-DTPA could enter into KB cells through for the folate receptor endicytosis pathway in vitro.Folate-PEG-DTPA was labeled with 99mTc by a stannous chroride reduction method. The better condition was selected, and the radiolabled yield of [99mTc] Folate-PEG -DTPA was in excess of 96%. After 24h at room temperature, the radiolabled yield of [99mTc] Folate-PEG-DTPA was not less than 96%. The data showed [99mTc] Folate-PEG-DTPA had a good stability. After subcutaneous injection, [99mTc] Folate-PEG-DTPA exhibited an initial increase and successively decline of accumulation in poplitael nodes in normal Wistar rats. Except for kidney, uptake of other tissues was rather low. In conclusion, [99mTc] Folate-PEG-DTPA may have a potential as a lymphatic tumor-targeted radiopharmaceutical.Adopting solid-phase synthesis, Folate-Cys and Gly-Phe-Leu-Gly were prepared. Catalyzed by EDC and NHS, two of folate-polyethylene glycol-doxorubicin conjugates were obtained. Both of the aboved conjugates were purified, and the purity were over 95%.The cytotoxicities of folate-polyethylene glycol-doxorubicin conjugates to KB were compared by MTT assay. The IC50 of DOX, Folate-PEG-Gly-Phe-Leu-Gly-DOX and Folate-PEG-SAD-DOX was 0.16, 0.79 and 3.92μmol/L, respectively. At the range of 7.42×10-1～1.16×10-5mol/L, the IC50 of the methoxylene glycol-doxorubicin conjugates could not detemined. The results indicated that modification of folic acid and release of enzymatic catalysis resulted in the marked cytotoxicity of Folate-PEG-Gly-Phe-Leu-Gly-DOX. Fluorescein-tracing method was adopted for determining the lymphatic targeting of Folate-polyethylene glycol-doxorubicin conjugates. The dates showed that both Folate-PEG-SAD-DOX and Folate-PEG-Gly-Phe-Leu-Gly-DOX exhibited the biggest accumulation in the poplineal nodes that that of other tissues in normal animals, after subcutaneous injection. Kidney as main excretion organ, the accumulation was bigger than that of other tissues, but lower than that of the lymph nodes.
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