Green Synthesized Liquid-like Dynamic Polymer Chains with Decreased Nonspecific Adhesivity for High-Purity Capture of Circulating Tumor Cells

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLES3 Feb 2024Green Synthesized Liquid-like Dynamic Polymer Chains with Decreased Nonspecific Adhesivity for High-Purity Capture of Circulating Tumor Cells Feng Wu†, Xiaofeng Chen†, Shuli Wang, Ruimin Zhou, Chunyan Wang, Lejian Yu, Jing Zheng, Chaoyong Yang and Xu Hou Feng Wu† State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 College of Physical Science and Technology, Xiamen University, Xiamen, Fujian 361005 College of Physics and New Energy, Xuzhou University of Technology, Xuzhou, Jiangsu 221018 , Xiaofeng Chen† State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Shuli Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 Fujian Engineering Research Center for Solid-State Lighting, Department of Electronic Science, School of Electronic Science and Engineering, Xiamen University, Xiamen, Fujian 361005 Institute of Artificial Intelligence, Xiamen University, Xiamen, Fujian 361005 , Ruimin Zhou College of Physical Science and Technology, Xiamen University, Xiamen, Fujian 361005 , Chunyan Wang State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Lejian Yu State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Jing Zheng State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Chaoyong Yang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province (IKKEM), Xiamen, Fujian 361102 and Xu Hou *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 College of Physical Science and Technology, Xiamen University, Xiamen, Fujian 361005 Institute of Artificial Intelligence, Xiamen University, Xiamen, Fujian 361005 Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province (IKKEM), Xiamen, Fujian 361102 State Key Laboratory of Bio-Fibers and Eco-Textiles, Qingdao University, Qingdao, Shandong 266071 https://doi.org/10.31635/ccschem.023.202302731 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The capture of circulating tumor cells (CTCs) is of great significance in reducing cancer mortality and complications. However, the nonspecific binding of proteins and white blood cells (WBCs) weakens the targeting capabilities of the capture surfaces, which critically hampers the efficiency and purity of the captured CTCs. Herein, we propose a liquid-like interface design strategy that consists of liquid-like polymer chains and anti-EpCAM modification processes for high-purity and high-efficiency capture of CTCs. The dynamic flexible feature of the liquid-like chains endows the modified surfaces with excellent antiadhesion property for proteins and blood cells. The liquid-like surfaces can capture the target CTCs and show high cell viability due to the environment-friendly surface modification processes. When liquid-like surface designs were introduced in the deterministic lateral displacement (DLD)-patterned microfluidic chip, the nonspecific adhesion rate of WBCs was reduced by more than fivefold compared to that in the DLD chip without liquid-like interface design, while maintaining comparable capture efficiency. Overall, this strategy provides a novel perspective on surface design for achieving high purity and efficient capture of CTCs. Download figure Download PowerPoint Introduction Metastasis is the primary factor in fatalities from cancer. The majority of cancer patients receive a cancer metastasis diagnosis and get a dismal prognosis. Circulating tumor cells (CTCs), shed from solid tumors into the vasculature, have been studied as a key class of cancer biomarkers for a deeper understanding of cancer metastasis. Therefore, the capture of CTCs from blood samples facilitates monitoring of cancer progression and treatment response and is of great significance in reducing cancer mortality and complications.1–4 The existing technologies for separating CTCs from the peripheral blood can be classified into two categories: (1) physical force-based separation methods5–9 that depend on the size, deformability, density, and inertial forces and (2) affinity-based separation methods10–15 that depend on the specific interaction between the biomarkers on the CTC membrane and the recognition ligands of aptamer/antibody/peptide on the capture interface. In recent years, affinity-based microfluidic technologies, which utilize the synergistic effect of both physical forces and affinity for sensitive and specific capture of CTCs, have aroused extensive research interest.16–20 For example, Nagrath et al. reported a microfluidic device with specific antibody-modified micropost arrays, where the micropost arrays increase the contact chance of CTCs with the target antibody through improving the mixing efficiency, and the antibody can specifically capture the target CTCs.18 However, the increased mixing efficiency also led to the increase of the frequency of nontargeted cells, such as white blood cells (WBCs), which nonspecifically adsorb on the microchannel surfaces and further decrease the purity of the captured CTCs and reduce the accuracy of molecular analysis. Therefore, it is extremely urgent to find new CTC capture technologies with high efficiency and purity. Aiming at the above-mentioned problem, a series of microfluidic-based CTC capture devices were developed by integrating solid nanointerface design strategies with deterministic lateral displacement (DLD) patterned structures.21,22 In DLD-patterned microfluidic devices, the large-sized CTCs show continuous and frequent collisions with immune-modified patterned structures, while small-sized blood cells migrate in the original flow direction, which minimizes their interaction with the patterned structures and further results in the decreased nonspecific WBC adsorption and high purity CTC capture. By modifying the structures with enhanced interface affinity nanointerface, the capture efficiency of the microfluidic devices was significantly improved compared to that without nanointerface designs.16 In addition, through high-affinity modifications, such as multivalent nanointerface designs, the capture efficiency of CTCs was further improved. However, some proteins such as fibrinogen (Fn) can adsorb on the solid surfaces of the microfluidic devices, which may promote eukaryotic cell adhesion and further reduce the purity of the captured CTCs.23 Therefore, it is urgently needed to develop novel approaches to further minimize the nonspecific interaction and maximize the specific binding. Inspired by the blood cell-resistant capability of the leukocyte membrane, leukocyte membrane-cloaked nanoparticles were reported to decrease their nonspecific adsorption to blood cells.24,25 Recently, we proposed a bioinspired fluidic multivalent nanointerface design strategy by modifying the DLD-patterned microfluidic chip with aptamer-functionalized leukocyte membrane nanovesicles by biotin-streptavidin interaction for high-performance isolation of CTCs with minimized background blood cell adsorption.17 Therefore, soft and flexible interfaces provide a novel design strategy for the high-efficient and purity capture of CTCs for further clinical application of cell-based liquid biopsy.26 Liquid-like surfaces are a kind of surfaces that are modified by flexible polymer brushes, typically polydimethylsiloxane (PDMS) or perfluoropolyether brushes/film, with an extremely low glass transition temperature, and their chemical bonds have a very low rotational conformation transition energy barrier, generally comparable to the thermal motion energy.27–31 Therefore, the surface molecules are highly mobile at room temperature, and the molecular chains have a high dynamic characteristic similar to that of the fluids.32,33 In recent years, liquid-like surfaces have shown great potential applications in areas such as hydro/oleophobic,27 lossless/directional liquid transport,34 antifouling,35 antiicing,36 and condensation heat transfer37 due to their unique interfacial physicochemical properties. However, the applications of liquid-like surfaces for CTC isolation and detection were rarely investigated.38–40 The authors assumed that this resulted from the currently reported preparation processes for liquid-like surfaces that usually use cytotoxic organic solvents, such as toluene, acetone, tetrahydrofuran (THF), etc. When liquid-like molecules are modified on the surfaces of polymers such as PDMS, the residual organic solvents in the networks of polymers lead to cytotoxicity, which results in the death of the target cells and influences the downstream biomedical analysis. Thus, the development of a green and biocompatible preparation method that utilizes nontoxic solvents for liquid-like surfaces seems to be urgently required for the exploration of liquid-like surfaces in the application of the isolation CTCs. The highly flexible liquid-like molecule chains hold the potential of the reduction of the nonspecific adsorption of proteins through the weak interactions between the surface and the proteins (such as hydrogen bond, ionic bond, and ligand/receptor interactions) and the nonspecific adhesion of WBCs, which is important for the CTC isolation with high purity. Herein, we propose a green liquid-like interface design strategy for antibiofouling and high-purity capture of CTCs in affinity-modified DLD-patterned microfluidic devices. Flexible PDMS brushes are modified on the selected substrate through an ethanol solution-based preparation process, and further functionalized by biotinylated anti-EpCAM for specific capture of CTCs. In addition, due to the dynamic flexible feature of the liquid-like chains, the nonspecific adsorption of proteins and WBCs is significantly decreased on the liquid-like surfaces compared to that on solid surfaces. Moreover, by integrating the green liquid-like surface designs with the DLD patterns, high-efficiency and high-purity capture of CTC was achieved, with nonspecific WBC adsorption decreased more than fivefold compared to traditional solid surface designs. Hence, our designed liquid-like interface is an attractive candidate for highly efficient and specific capture of CTCs in clinical settings. Experimental Methods Materials The quartz crystals were obtained from Jiaxing Crystal Electronics Co. (China). Bis(3-aminopropyl) terminated PDMS (μPDMS) with a molecular weight of 3000, N,N'-disuccinimidyl carbonate (DSC), and 3-aminopropyl triethoxysilane (APTES) were purchased from Aladdin (China). Streptavidin (SA), and acid orange was purchased from Sigma-Aldrich (United States). Biotinylated anti-EpCAM and 6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific Inc. (United States). Fluorescein isothiocyanate isomer (FITC)-labeled antihuman fibrinogen was purchased from Bioss (China). Ethanol, toluene, n-hexane, N,N-dimethylformamide (DMF), THF, and dimethyl sulfoxide (DMSO) were used as received. Deionized water with a resistivity of 18.2 MΩ cm was obtained from Milli-Q system. Preparation of liquid-like polymer chains on the substrates Ethanol solution of DSC (1 μg mL−1) and ethanol solution of μPDMS (1 mg mL−1) were prepared for the grafting of liquid-like polymer chains. The amino-functionalized substrates were then dipped in ethanol solution of DSC for 60 min at room temperature, and rinsed in ethanol and washed rapidly. The sample was then dipped in the ethanol solution of μPDMS for another 60 min at room temperature, rinsed in ethanol, and then dried by compressed air. The above two processes were repeated for several cycles to obtain longer μPDMS polymer brushes. Next, the samples were immersed in the phosphate-buffered saline (PBS) solution of SA (10 μg mL−1) at room temperature for 1 h. After the samples were ultrasonically washed with PBS and dried by nitrogen gas, the samples were immersed in PBS solution of anti-EpCAM (20 μg mL−1) at 4 °C overnight. The preparation of liquid-like interface modified microfluidic inner surface was similar to that described above. CTC viability SW480 colorectal tumor cells were employed as representative CTCs for the cell counting kit-8 (CCK-8) assay to confirm whether liquid-like interface was toxic to CTCs. SW480 cells were cultured on the pure PDMS and liquid-like interface for 24 h, respectively. The medium was removed, and the percentage of the survival of CTCs was quantified by CCK-8 assay. To investigate the apoptosis of CTCs on each surface, a cell permeable acridine orange (AO) in combination with a plasma membrane-impermeable DNA-binding dye propidium iodide (PI) was applied. AO and PI excite green and red fluorescence respectively when they are intercalated into DNA. Only AO but not PI can cross the plasma membrane of a normal cell. In brief, CTCs were cultured onto the samples for 1 h. Subsequently, the cells were stained with a 1:1 mixture of AO (100 mg mL−1) and PI (100 mg mL−1) at 37 °C for 5 min, and then inspected in a fluorescence microscope immediately. Characterization of the liquid-like interface The self-assembly process of the liquid-like polymer was monitored using quartz crystal microbalance with dissipation (QCM-D) (Q-Sense AB, Sweden). The QCM-D has the ability of simultaneously measuring the normalized resonant frequency and energy dissipation shifts. First, the gold-coated crystals were modified with amine groups. Second, the ethanol solution of DSC was injected into the measurement cell at a proper flow rate using a peristaltic pump. Then, the ethanol solution of μPDMS was injected into the measurement cell. All the experiments were conducted at room temperature. The acid orange II (AO II) colorimetric method was used to determine the density of amine groups. In detail, the samples were immersed in an aqueous solution of AO II (pH 4.0) for 4 h. Then the samples were washed with aqueous solution of HCl (pH 4.0) to remove unreacted AO II. Afterward, the AO II on the sample was eluted with 200 μL of aqueous solution of NaOH (pH 11.0), and the amount of AO II was measured by the fluorescence signal with excitation at 485 nm and emission at 520 nm using a Molecular Devices SpectraMax ID5. The density of amine groups was calculated based on a standard curve. The chemical compositions of the liquid-like polymer layer were analyzed by X-ray photoelectron spectroscopy (XPS). The instrument (PHI Quantum 2000 Scanning ESCA Microprobe, Physical Electronics, Eden Prairie, Minnesota, United States) was equipped with a monochromatic Al Kα (1486.6 eV) X-ray source operated at 15 kV and 35 W at a pressure of 5 × 10−7 Pa. The carbon peak (284.4 eV) was designated as the reference for charge calibration. The thickness of the grafted liquid-like polymer layer was measured by a spectroscopic ellipsometer M-2000U. The surface topologies of the grafted liquid-like polymer layer were analyzed by an atomic force microscope (Cypher S, Asylum Research-Oxford Instruments, Santa Barbara, California, United States) using the tapping mode in air. Sliding angle measurements were carried out using OCA20 equipment (Data Physics, Filderstadt, Germany) under ambient conditions. The surface was tilted with respect to the horizontal plane until the liquid droplet started to slide along the surface. Adsorption of protein on the surfaces The samples were covered with 50 μL of fresh human platelet poor plasma extracted from fresh venous blood obtained from a healthy adult volunteer for 1 h and then rinsed three times with PBS to remove the nonadhered Fn. Subsequently, the samples were blocked with 1 wt % bovine serum albumin (BSA) in PBS at 37 °C for 30 min and then rinsed with PBS. Finally, FITC-labeled antihuman fibrinogen was added and incubated at 37 °C for 1 h. After rinsing with PBS again, the stained samples were observed under an inverted fluorescence microscope (TH4-200, Olympus, Tokyo, Japan). Quantitation of the protein adsorption was measured using a QCM-D. After construction the liquid-like interface on sensors, a PBS solution (pH 7.4) of BSA (50 μg mL−1) and Fn (1 μg mL−1) was introduced to the axial flow sample chamber and kept at a constant flow rate of 1 mL h−1 for 60 min in sequence. Adhesion of WBCs on the surfaces Fresh whole blood of a volunteer was added to the sample surfaces for 1 h. Then, the adherent blood cells were fixed with 2.5% glutaraldehyde solution at 4 °C for 2 h. Next, the morphology of the adhered blood cells was examined using by a scanning electron microscope (SEM, 2400 s, Hitachi, Tokyo, Japan). Adhesion of CTCs on the surfaces SW480 colorectal tumor cells were cultured on the samples for 1 h. After washing, SW480 cells were fixed by 4% polyformaldehyde, and then blocked by goat serum, followed by immunofluorescence staining with anti-panCK. The immunofluorescence results were scanned by a fluorescence microscope (Nikon, Tokyo, Japan). Capture of CTCs in the DLD patterned microfluidic chips 100 LNCap prostate and SW480 colorectal cells were suspended in 1 mL of whole blood obtained from healthy donors, respectively. All the blood samples were injected into the chip at a flow rate range from 0.5 to 3 mL h−1. After capture and washing, cells on the chip were fixed by 4% polyformaldehyde and then blocked by goat serum, followed by immunofluorescence staining with DAPI and anti-panCK. The immunofluorescence results were scanned by a fluorescence microscope (Nikon, Japan). Results and Discussion Design and working principle of liquid-like interface The schematic illustration of the working mechanism of liquid-like surface design for reduced nonspecific adhesion of proteins and WBCs and specific capture of CTCs is shown in Figure 1a. The liquid-like surfaces are composed of PDMS polymer brushes and terminal-modified biotinylated anti-EpCAM. The PDMS polymer brushes grafted on the surfaces are highly mobile at room temperature because of their highly melted state resulting from their extremely low glass transition temperature, and they act as a liquid-like slippery layer.37 When the proteins contact the liquid-like surfaces, it is difficult for them to nonspecifically adhere on the surfaces due to the highly mobile polymer chains, which further reduce the adhesion of WBCs. Though at a highly mobile state, the terminal biotinylated anti-EpCAM on the liquid-like molecule chains offer the affinity sites for the target CTCs and can specifically capture them from the biological fluids. Figure 1 | Design and working principle of the liquid-like interfaces. (a) Schematic diagram showing the "liquid-like" interface for decreased nonspecific adsorption of proteins and WBCs and specific capture of CTCs. (b) Schematic of the green and environmentally friendly design processes of liquid-like interface. (c) Biocompatibility of the liquid-like interfaces. Using ethanol as the solvent for liquid-like interface design, the PDMS film does not swell and shows low cytotoxicity. Using toluene as the solvent, the PDMS swells and shows high cytotoxicity. AO in combination with a plasma membrane, impermeable DNA-binding dye PI was applied to detect the vitality of CTCs. Only AO but not PI can pass through the plasma membrane of normal CTCs. Normal CTCs emit green fluorescence (AO), and dead CTCs emit red fluorescence (PI). Download figure Download PowerPoint The green and noncytotoxic liquid-like interface modification process by a covalent layer-by-layer method is illustrated in Figure 1b. To fulfill the requirements of nontoxic organic solvents in the whole process, we use ethanol and deionized water as the solvent in the whole solution-based modification process, and chose the ethanol soluble monomers DSC and μPDMS to graft the polymer brushes. First, the substrates were grafted a monolayer of amino groups by oxygen plasma treatment and immersed in the ethanol solution of APTES. Second, the amino modified surface was immersed in the ethanol solution of DSC and μPDMS subsequently to obtain liquid-like polymer chains via the ring-opening reaction between succinimide groups and the amino groups.41 Third, the above process was repeated for several cycles to obtain longer liquid-like μPDMS polymer brushes. Fourth, the amino-group terminated μPDMS brushes were further modified by the SA and anti-EpCAM to endow the liquid-like surfaces with specific binding capability to CTCs. Compared to traditional cytotoxic organic solvent-based preparation processes, the liquid-like surfaces prepared through our green synthesis processes here are more suitable to modify a broad range of materials (such as glass, silicon, PDMS, etc.) and show significant high cell viability. Due to the ethanol solution-based modification processes, the PDMS (mostly used material in microfluidic devices) film was not swelled and maintained its flatness after the modification processes. In contrast, PDMS film was swelled and deformed when immersed in solvents such as toluene, acetone, THF, etc. (Figure 1c and Supporting Information Figure S1), which indicate that a significant amount of residual toxic solvent is left in the PDMS networks. The residual toxic solvent in the PDMS networks can be released onto the surface and into the cell culture medium, which brings and results in the of CTCs as low as However, the of CTCs on the liquid-like surfaces prepared through green synthesis processes to Supporting Information Figure the high viability of the surfaces and the of our which is for the cell culture and analysis. Supporting Information Figure shows that the CTC viability of the liquid-like interface was than the PDMS as polymer employed for applications in the biomedical that the liquid-like interface is and cytotoxicity. Characterization of liquid-like surface To the reaction in our green liquid-like interface design processes, the self-assembly processes of DSC and μPDMS polymer chains were by quartz crystal microbalance with dissipation mode which can detect at the surface in a sensitive and shown in Figure the grafting of the DSC on the surface was in a and the grafting density of DSC on the surface was increased to while the grafting density on the surface was increased to after grafting of μPDMS chains on the DSC surface. The amino density of the surfaces also provides an method for the modification The surface amino density decreased from the to after DSC modification with a reaction of min, and then to a density the original state after μPDMS modification with a reaction of min (Figure a The surface chemical after each modification process were also by and the of of the modification of DSC and μPDMS chains (Figure Figure 2 | of the liquid-like interface. (a) the self-assembly process by measuring the grafting density of DSC and μPDMS using QCM-D. (b) The amount of amine groups was by the acid orange (c) The of of of measurement and after modification of DSC and of liquid-like polymer as a of the of modification Sliding of a droplet of blood (10 on the liquid-like interface with of modification The surface of the liquid-like interface as a of the of modification Sliding angle of with surface on the liquid-like interface with modification Sliding of μL of blood on the solid interface and liquid-like interface with a angle of Download figure Download PowerPoint The thickness of the modified flexible polymer brushes is increased with the of the of the layer-by-layer modification shown in Figure the thickness of the layer from to nm when the modification from 1 to The thickness increase is for the of the liquid-like The liquid-like property of the surfaces is by measuring the angle of the of ethanol, and organic solvents such as n-hexane, toluene, and on the modified surfaces. shown in Figure the angle of the blood droplet (10 from to when the modification increased to the of the of the liquid-like However, it was increased to a when the modification was further due to the increase in the of the modified surface (Figure and Supporting Information Figure Therefore, cycles of modification is proper for the liquid-like surface Figure shows the angle of the on the liquid-like surface with modification Due to the high of the angle of blood is than water and organic the blood droplet (20 ethanol and droplet onto the liquid-like surface without on the liquid-like surface and on the solid surface at a angle of (Figure and Supporting Information Figure The above results indicate the of the polymer and liquid-like property of the modified surface. nonspecific adhesion of proteins and WBCs, and the specific adhesion of CTCs In addition, the of the polymer chains also endows the liquid-like surface with reduced nonspecific adhesion of protein and WBCs. The of plasma protein on the surface has a significant on the blood protein adsorption is the that the such as cell we blood BSA and Fn as the representative proteins to the antiadhesion of the liquid-like interfaces. The BSA adsorption when the blood samples contact with the capture surface. Then the BSA is by Fn with surface affinity based on the The Fn was with to their adhesion on the surfaces by the fluorescence shown in Figure

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