Synthesis Of Re-engineering PAMAM Dendrimers With Targeting Antibodies For Their Anti-cancer And Anti-adhesion Studies

OBJECTIVE:The high death rate resulting from cancer metastasis after surgical removal of tumor is a serious social and scientific problem that needs urgent solution. The root cause of the problem is the residual dormant circulating tumor cells (CTC), on which the anti-cancer drugs have no effects. Dendritic polymers have been widely applied in biomedical fields because of their unique properties of good biocompatibility, degradability, size effect and multivalent complexing effects. In this work, we aimed at preparing the dendrimer-single/dual antibody conjugates with high specificity which used biosafely nanoscale G6 PAMAM dendrimers as the platforms for multivalently coupling more than one targeting antibodies against the biomarkers of human colon cancer cell lines and further explored their biostability, anti-cancer and anti-adhesion capability in vitro. The design theory aimed to provide theory reference for targeting CTCs, and inhibit their activity.METHOD:Completely carboxylated G6 dendrimers (CC G6) were firstly synthesized by reacting primary PAMAM with succinic anhydride. By taking control of the molar ratio of CC G6 and aEpCAM or aSialyl Lewis X, G6 PAMAM-5aEpCAM (G6-5A), G6 PAMAM-5aSialyl Lewis X (G6-5S) and G6 PAMAM-5aEpCAM-5aSialyl Lewis X (G6-5A-5S) conjugates were respectively synthesized. The structural and morphological analysis of G6 dendrimer conjugates were confirmed by 1H NMR, FTIR, UV-Vis and DLS. The stability of three conjugates under different temperature, pH and vibration velocity was respectively determined by using ultramicro UV/Vis spectrophotometer according to the ultraviloent absorption values at 220 nm. The inhibition effects of these conjugates on the growth of human colon cancer cell lines LoVo and SW480 were evaluated by MTT assay. Their cell cycle distributions were analyzed by flow cytometry. The capability of intervening the colon cancer cell lines adhered to fibronectin (Fn)-coated substrates or human umbilical vein endothelial cells (HUVECs) were also investigated by MTT and fluorescence-photographed methods.RESULTS:The successfully synthesized G6-5A、G6-5S and G6-5A-5S conjugates with precise structure and controllable size could be kept stable in PBS solution (pH 7.4) for at least 12 h at 37℃ and two weeks at 4 ℃, and hardly affected by shaking velocity even at 210 rpm. Compared with CC G6, these three conjugates had concentration-dependent inhibitory effects on two colon cancer cells and arrested the cell proliferation in G0/G1 phase. The adhesion assay in vitro showed that the adherence of colon cancer cells to Fn-coated substrates or HUVECs could be hindered by these conjugates at different degrees.CONCLUSION:These three conjugates not only could down-regulate the activities of two colon cancer cells in vitro by inhibiting the cell proliferation in G0/G1 phase, but also intervene the adherence of cancer cell lines to Fn-coated substrates or HUVECs. In summary, these studies will provide the important theoretical foundation for the related research on preventing cancer metastasis in vitro and in vivo.