Research On Engineering Bionanomaterials That Can Specifically Restrain Circulating Tumor Cells

The root cause of cancer metastasis can be traced down to the existence of circulating tumor cells(CTCs)in blood.CTCs are cancer cells detached from primary tumors with the capability to enter the bloodstream,adhere to vascular endothelium once activated,and initiate the activation-adhesion-extravasation cascades to form the micrometastatic foci at distant metastatic secondary organs.The traditional"anticancer drugs" or target chemotherapeutics that were originally designed to kill highly proliferating carcinoma cells were ineffective in reversing the CTCs-derived cancer metastasis.The "anticancer drugs" can’t be used in the asymptomatic cancer survivors after surgical removal of primary tumor owing to their serious toxic side effects and ineffective effects on targeting the low proliferating(or dormant)CTCs.Current cancer bionanotechnology approaches that typically combine a post-metastatic chemotherapeutic with a single antibody-coated nanomaterial have not shown effectiveness in preventing and treating cancer metastasis because they focus only on chemotherapeutic delivery to metastatic patients or in vitro CTCs capture for cancer diagnosis.Besides,current technologies have limited specificity and reduced capability in recognizing and capturing CTCs in vivo for cancer metastasis prevention because of the diversity of CTCs surface biomarkers and the variability of the property and abundance of CTCs epigenome and protein.On the basis of having a good understanding of the safe and effective supplementary therapy for cancer metastasis and the current bionanotechnology,we hypothesized that multivalent conjugation of two distinct antibodies against a typical CTC to nanomaterials could significantly enhance the specificity and efficiency of the conjugate to capture the rare CTCs,and further result in down-regulation of the activity of the captured CTCs.In the present study,we took the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)and N-hydroxysuccinimide(NHS)catalytic method for surface functionalization of the PAMAM dendrimers with the single and/or dual targeting antibodies.The single and dual antibody-coated PAMAM dendrimer conjugates with or without fluorescence labeling were designated based on the reaction molar ratio of nanomaterial to antibody.The molecular entity and physiochemical characterization of conjugates were documented by using FTIR,NMR,dynamic light scattering(DLS),UV,fluorescent imaging,scanning electron microscope(SEM),atomic force microscope(AFM),X-ray photoelectron spectroscopy(XPS),and other sophisticated means.The specificity and efficiency of PAMAM-single and dual antibody conjugates in capturing the target CTCs in various conditions were qualitatively and quantitatively determined both by microscopic and flow cytometric analyses.The comparison between single and dual antibody conjugates in capturing CTCs was also made.Cell viability,cell cycle,cell morphology,cell apoptosis and cellular mitochondrial membrane potential(MMP)measurements were,respectively,performed to demonstrate the capability of the conjugates in down-regulating the activity of captured cells.In vitro substrate and endothelium adhesion assays were also carried out to evaluate the possibility of the conjugates in interfering the hetero-adhesion between CTCs and vasular endothelium.The results showed that the surface-functionalized PAMAM dendrimers could be sequentially coated with two targeting antibodies for surface biomarkers(EpCAM and Slex)of human colorectal(or hepatocellular)CTCs to specifically capture and down-regulate the CTCs ex vivo and in vivo.Dual antibody-coated PAMAM dendrimers exhibited a significantly enhanced specificity and efficiency in capturing CTCs in the presence of interfering blood cells,and in both clinical patient bloods and nude mice administered with the fluorescence-labeled CTCs whatever in static in vitro or in dynamic in vivo conditions in comparison with their single antibody-coated counterparts.The capturing effect of conjugates was further confirmed by demonstrating the down-regulation of the captured CTCs.The related adhesion assays in vitro displayed that dual antibody conjugates more effectively interfered the hetero-adhesion of CTCs to fibronectin(Fn)-coated substrates and human umbilical vein endothelial cells(HUVECs)as compared to their single antibody counterparts.In conclusion,this study provides the first conceptual evidence that two antibodies can be biocompatibly conjugated to a nanomaterial to capture,restrain CTCs and inhibite their hetero-adhesion to vessel walls in vivo with the enhanced specificity for the effective prevention of cancer metastasis.