Synthesis And Preclinical Evaluation Of Folate-PEG-DTPA Derivates For Lymph Metastasized Tumor Diagnosis

Lymph metastasis signifies the malignancy of tumor and could account for most of the death. Diagnosis of metastasized tumor in lymph system could significantly help with tumor staging and the sequential treatment. Interstitial injection of drug plays a critical role in lymph system drug delivery. After subcutaneous injection, polymers and macromolecules could be directed into lymph system for the imaging of lymph nodes. However, undesired retention and phagocytosis by macrophages in normal lymph nodes may result in the less specificity of the imaging agents, which could not differentiate the normal and malignant lymph nodes. We developed a molecular carrier, using polyethylene glycol as the backbone modified with folic acid and multiple diethylene triamine pentaacetic acid. The conjugate could be used for carrying imaging agents (e.g.99mTc, fluorescent probe and near infrared dyes) to the lymph system. It could effectively evade phagocytosis and specifically target tumor metastasized nodes.We prepared and characterized the conjugate Folate-PEG-CKK2-DTPA4, studied its ability to chelate 99mTc and the consequential lymph direction property. In vivo and ex vivo study was carried out to evaluate its tumor targeting ability. We also synthesized another novel conjugate, Folate-PEG-DTPA, and committed the preliminary pilot study.For the preparation of Folate-PEG-CKK2-DTPA4, we first synthesized Folate-EDA (folate-ethylenediamine) and CKK2 (Cystein-Lysine-(Lysine)2) using solid phase peptide synthesis technology. We conjugated Folate-EDA and CKK2 to bifunctional polyethylene glycol and obtained Folate-PEG-CKK2. After the conjugation of p-SCN-Bn-DTPA to Folate-PEG-CKK2, Folate-PEG-CKK2-DTPA4 was obtained with high purity (>98%).'H-NMR result indicated that the number of DTPA per molecular is about 3.7.We labeled Folate-PEG-CKK2-DTPA4 with 99mTc using Tin(?) Chloride Reduction method. Central composite design was carried to obtain the optimal labeling conditions:using a minimal of 520?g Folate-PEG-CKK2-DTPA4 to label 2mCi/100?L of Na99mTcO4,high chemical radio purity could be achieved (>90%) in the presence of 3.5?g SnCl2·2H2O at room temperature, pH=7.4.In order to evaluate its capability for lymph direction, Folate-PEG-CKK2-DTPA4 was labeled with 99mTc and biodistribution study was carried in rats and SPECT imaging study in rabbits. Biodistribution study showed the high distribution in lymph nodes different times after subcutaneous injection and SPECT imaging showed fast clearance in normal nodes. After labeled with FITC, both in vitro cell endocytosis study and in vivo fluorescent imaging study indicated that the conjugate could target folate positive tumor. The safety of Folate-PEG-CKK2-DTPA4 was further confirmed using MTT assay and hemolysis study.The preliminary pilot study of preparation of Folate-PEG-DTPA was carried out in two aspects, the synthesis and purification of Folate-PEG-NH2 and then Folate-PEG-DTPA. After purification in a Index Column 140/500 loaded with DEAE-SepharoseTM gel, Folate-PEG-NH2 could be obtained about 3 gram per batch, with high purity (>95%), yields about 18%. After optimization of the synthesis and purification condition, Folate-PEG-DTPA could be obtained about 0.5 gram per batch, with high purity (>95%), yields about 95% for the final step. We further study the 99mTc labeling condition and preliminarily determine the prescription of the Folate-PEG-DTPA kit:every kit should contain 5mg of Folate-PEG-DTPA,80?g of SnCl2·2H2O and 1ml/10mCi of Na99mTcO4 could be added into the kit to obtain Folate-PEG-DTPA-99mTc with high radio chemical purity (>90%).