Basic Research On Soft Three-Dimensional Polymer Chains Toward Efficient Capture Of Circulating Tumor Cells

Cancer is a kind of serious disease, and every year about 8.2 million people die of cancer. In the field of detection of tumors, traditional gold standard method is “biopsy”, while the detection of circulating tumor cells(CTC) could serve as “liquid biopsy”. It has many advantages, such as small invasiveness, painlessness, high-efficiency, etc. There have been a variety of platforms to capture or enrich CTC. The nanostructure platform is one of the most effective ways to solve this problem. Among the various nanostructure platforms, the organic nanostructures present a much higher efficiency of detection CTC obviously. Based on the previous experiences, we fabricate a soft 3D capture system, which is composed of an electrically conducting polymer substrate and substrate-initiated low density polymer chains with cancer-targeted antibodies but dominated by zwitterionic groups.To fabricate the soft 3D platform with the capacity of selective capturing CTC, we first co-deposited electrochemically the atom transfer radical polymerization(ATRP) initiator and zwitterionic phosphorylcholine group functionalized ethylenedioxythiophene(EDOT) as a conducting polymer thin film onto the conductive substrate. Then the deposited ATRP initiator initialized the copolymerization of biotin functionalized(MBiotin) and zwitterionic phosphorylcholine(MPC) bearing methacryloyl monomers to form polymer chains growing from substrates. The soft 3D platform obtained by this method presents a strong resistance toward nonspecific protein/cell binding due to the dominance of zwitterionic groups on substrates and along chains. Moreover, the biotin groups along polymer chains could strongly complex with streptavidin molecules, and streptavidin molecules could further interact with biotinylated antibody to ensure the potential cancer-targeted capture function.The bromoisobutyryl bearing EDOT molecule(EDOT-Br) was synthesized by the reaction of bromoisobutyryl bromine with hydroxyl functionalized EDOT. The phosphorylcholine functionalized EDOT(EDOT-PC) was prepared by two steps. EDOT-OH was first reacted with 2-chloro-2-oxo-1,3,2-dioxaphospholane; the dioxaphospholane ring was subsequently opened in the presence of trimethylamine to yield the phosphorylcholine functional group. The chemical structure of EDOT-PC and EDOT-Br were confirmed by 1H and 13 C liquid NMR spectra. An inverse micelle electro-polymerization approach was adopted to prepare the copolymer thin film of EDOT-Br and EDOT-PC. In this case, sodiumdioctylsulfosuccinate was used as the inverse micelle surfactant to ensure the solution of EDOT-PC in the organic electrochemistry solvent. With the presence of lithium perchlorate as electrolyte, EDOT-Br and EDOT-PC were co-deposited onto conductive substrates from their acetonitrile solution. The compositions of the copolymers were checked by X-ray photoelectron spectroscopy(XPS). The XPS data confirmed that the EDOT-Br and EDOT-PC co-deposited onto the conductive substrate successfully, and the composition of the EDOT-Br in the thin film could be tuned well by changing the ration of two monomers in the electrolyte solution. The Atomic Force Microscopy results showed that polymer film prepared by cyclic voltammetry procedure has a very low surface roughness. The contact angle of polymer films decreases with the feed composition of EDOT-PC increasing, which further evidenced the XPS analysis.The surface initiated ATRP copolymerization of MPC and M-Biotin still remains as a challenge. We investigated several catalyst systems, and tried to find a good system for the growth of the copolymer chain. Totally, we checked three different catalyst systems including CuBr/bipyridine(bpy), Cu Br2/bpy/ascorbic acid(VC) and CuBr2/ Tris(2-pyridylmethyl)amine(TPMA)/VC. The molecular chain coupling with streptavidin and subsequent with model protein was monitored by a QCM system. The QCM results suggested that the soft 3D platform prepared under the CuBr2/TPMA/VC system has the best performance to conjugate with proteins, which possibly indicates that the polymer chain could get the longest chain length in this case. In addition, as confirmed by the QCM data, all the soft 3D platforms show strong resistance to the nonspecific binding of proteins, further demonstrating the success of our molecular design for the soft 3D platform. Actually, the good nonspecific binding resistance is the precondition for selective capture of targeted rare cells.To optimize the conjugation of the soft 3D platform with proteins, we further tuned the chain growth density(the EDOT-Br composition), the MBiotin composition of chains, and the polymer length of chains(polymerization time). It was found that the platform with 10% EDOT-Br for conducting substrate and 5% MBiotin composition for chains presents an optimized conjugation of proteins. As to the effect of chain length, it was found the conjugation amount increase with the polymerization time, but the incensement slow down much when the polymerization time exceed 6 hours, indicating that 6 hours may be a critical point for the growth kinetics under the adopted catalysis system. In this way, the soft 3D platform with 10% EDOT-Br for conducting substrate, 5% MBiotin composition for chains and 6 hours of polymerization was considered as one of the most suitable CTC capture platform due to its optimized or balanced conjugation of proteins. The optimized platform will be used to capture CTC cells in the near future as a continuous work. The combination of strong resistance to nonspecific binding and high spatial exposition of specific interaction are expected to capture targeted cells with good selectivity and high efficiency.