A polymeric material with CTCs capture, its preparation and application

Abstract

This invention relates to a polymer material for CTC (cytotoxic tumor cells) capture, its preparation, and its application. The CTC-capturing polymer material P4, P5, or P6 is composed of a network backbone P3 and CTC functional monomers (methacrylated bishistidine; methacrylated histidine; acrylated 3-aminophenylboronic acid), and its preparation polymerization process involves the random copolymerization of the network backbone P3 and the CTC functional monomers. This invention also relates to the preparation of a smart responsive hydrogel. Using CTCs as a template, the cellular microstructure is introduced onto the surface of a hydrogel polymer material carrying functional groups to form a cell imprint, obtaining a dual smart responsive hydrogel biomaterial for cancer diagnosis carrying both functional groups and molecular imprints. This material possesses efficient enrichment and non-destructive release capabilities, and can be applied in cancer diagnosis and treatment, including CTC enrichment, non-destructive release for downstream molecular biological analysis of diseases, and assessment of cancer progression.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the screening of anti-adhesion and anti-swelling gel frameworks, methods for preparing functional monomers and cell-imprinted microstructures of smart responsive hydrogels, and the clinical applications of smart responsive hydrogel biomaterials for cancer diagnosis. Background Technology

[0002] The low survival rate of liver cancer is due to the high cost of screening and the dulled liver pain response, meaning most patients are diagnosed at an advanced stage, missing the optimal treatment window. Alpha-fetoprotein (AFP), a serum biomarker, is currently the primary clinical test for liver cancer screening, diagnosis, recurrence risk assessment, and monitoring treatment efficacy. However, a significant number of patients with low AFP levels are positive, greatly reducing the sensitivity of AFP testing. Therefore, finding more precise and sensitive biomarkers is crucial for the prevention and treatment of liver cancer.

[0003] Circulating tumor cells (CTCs) in peripheral blood are important biomarkers for liquid biopsies because they carry genetic and epigenetic information about tumor tissue. As an effective approach to tumor diagnosis and treatment, the development of CTC capture materials has profound clinical significance for early tumor diagnosis and postoperative assessment. However, due to the extremely low number of CTCs in blood and their susceptibility to damage during capture, precise capture and non-destructive release of CTCs present significant challenges. Existing CTC capture materials can be broadly categorized into two types based on the type of capture unit: those based on natural antibodies and those based on artificial antibodies. While natural antibody-based capture materials offer significant advantages in specificity, those based on artificial antibodies allow for more flexible, personalized, and diversified functional designs, better addressing many current challenges in CTC capture. Furthermore, materials based on artificial antibodies also offer advantages in terms of material stability and manufacturing costs. Compared to blood cells, CTCs possess larger size, mechanical plasticity, and dielectric migration properties. These characteristics allow for the separation of CTCs using various strategies, such as membrane filtration, density gradient stratification, inertial focusing, and dielectric migration. This invention develops a biocompatible gel framework with significant anti-adhesion and anti-swelling properties based on cellular micromorphology design. Furthermore, by copolymerizing multi-hydrogen-bonded artificial antibody functional monomers, smart responsive polymer surface hydrogels with varying degrees of polymerization, enrichment capabilities, and ease of processing are prepared. These smart responsive hydrogels have shown excellent performance in the separation and enrichment of cellular cytokines (CTCs), primarily exhibiting highly selective capture and non-destructive release. Therefore, they hold promise for widespread application in CTC enrichment and identification, and consequently, in clinical cancer diagnosis and treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing intelligent responsive biomaterials for cancer diagnosis, which possess the ability to efficiently enrich and release without damage. By preparing a class of functional monomers with selective recognition capabilities of multiple hydrogen bonds and using azobisisobutyronitrile (AIBN)-initiated copolymerization, a biomaterial for cancer diagnosis and treatment is prepared by combining cell microstructure within a gel framework exhibiting good biocompatibility, non-swelling, anti-adhesion, and plasticity.

[0005] The objective of this invention is achieved through the following approach:

[0006] First, the purpose of this invention is to provide a class of smart responsive polymer cancer biodiagnostic materials having a hydrogel polymer network backbone of P1, P2 and P3 and hydrogel functional polymers targeting CTCs of P4, P5 and P6.

[0007] The specific description is as follows: The hydrogel polymer P1 or P2 is composed of a network backbone (P1: acrylic acid; P2: acrylamide) and a crosslinking agent (N,N-methylenebisacrylamide, MBA), wherein x and 1-x are the molar percentages of the network backbone and the crosslinking agent, and 1-x = 0.01~0.5. The network backbone P3 (polyethylene glycol dimethacrylate, PEGDMA) has the same mass as acrylic acid and acrylamide, and n = 13.

[0008] The hydrogel functional polymers P4, P5, or P6 in the cancer biodiagnostic material that target CTCs are composed of a network backbone P3 and functional monomers (P4: methacrylamide bishistidine; P5: methacrylamide histidine; P6: acrylamide 3-aminophenylboronic acid), where y and 1-y are the molar percentages of P3 and the functional monomers, and y = 0.01 to 0.5, n = 1 to 15.

[0009] The molecular structures of P1, P2, P3, P4, P5, and P6 are shown below (the polymerization process is random copolymerization):

[0010]

[0011] Secondly, the preparation method of the intelligent responsive hydrogel of the present invention: using the atom transfer radical polymerization reaction mechanism, through azobisisobutyronitrile (AIBN) initiation, the functional monomers with high specificity, affinity and multi-hydrogen bond selective recognition ability of CTCs are randomly copolymerized onto the surface of the biocompatible, non-swelling, anti-adhesion and plastic gel network backbone P3, to obtain a hydrogel cancer diagnostic biomaterial carrying functional groups.

[0012] Finally, the preparation method of the intelligent responsive hydrogel of the present invention is as follows: using CTCs as templates, the cell microstructure is introduced into the surface of a hydrogel polymer material carrying functional groups to form a cell imprint, thereby obtaining a dual intelligent responsive hydrogel cancer diagnostic biomaterial carrying both functional groups and molecular imprints. The intelligent responsive hydrogel biomaterial of the present invention can be applied in the fields of CTC enrichment and non-destructive release for downstream molecular biological analysis of diseases in cancer diagnosis and treatment, as well as in assessing cancer progression. Attached Figure Description

[0013] Figure 1 Evaluation of CTC enrichment efficiency and anti-interference ability of P4+ blood samples with smart responsive hydrogel. Figure 2 Receiver operating characteristic (ROC) curve of the clinical liver cancer differentiation ability of intelligent responsive hydrogel P4+.

[0014] Figure 3 Selection of gel skeleton for smart responsive hydrogels.

[0015] Figure 4 Surface potential of HH@SiO2 material.

[0016] Figure 5 Affinity test of dual histidine functional monomers with cell membrane proteins.

[0017] Figure 6 1. Testing of the internal water content of intelligent responsive hydrogels.

[0018] Figure 7 Preparation and characterization of the microstructure of intelligent responsive hydrogel cell imprints.

[0019] Figure 8 The anti-protein adsorption capacity of the intelligent responsive hydrogel backbone.

[0020] Figure 9 Processability of smart responsive hydrogels.

[0021] Figure 10 Smart responsive hydrogel CTCs capture and re-release cycle availability. Detailed Implementation

[0022] (I) Taking P4+ hydrogel as an example, the experiment on the enrichment efficiency and anti-interference ability of CTCs is described: 1. Statistical method of the number of white blood cells (WBCs) in blood samples.

[0023] Obtain 1 ml of whole blood (from a healthy individual undergoing a routine blood test, using purple blood collection tubes, collected immediately before use) from the Second Affiliated Hospital of Dalian Medical University. Take 1 μL of this sample (to minimize error, the whole blood sample can be serially diluted with phosphate buffered saline (PBS = 10 mM, pH = 7.4, the same below)). Then, add 10 μL of phycoerythrin (PE) anti-human CD45 (leukocyte common antigen, LCA) antibody (Enabling Legendary Discovery, 368510) to 1 ml of PBS solution and vortex for 3 minutes. Afterward, add 500 μL of the above WBC solution to 1 μL of blood, carefully mix by pipetting, and stain at room temperature for 30 minutes. Subsequently, the cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the unbound antibodies were washed with 1 mL of cell culture medium [89% Dulbecco's modified eagle medium, DMEM (Gibco), 10% Fetal bovine serum, FBS (ExCell Bio), and 1% penicillin-streptomycin, 100X (Beyotime; the cell culture medium composition is the same as described here], and centrifuged again. This washing and centrifugation process was repeated 3 times. Finally, the cell pellet was resuspended in 1 mL of cell culture medium, and 10 μL was pipetted onto a hemocytometer. The pellet was placed on an inverted research fluorescence microscope (Nikon Ti-s, Japan), the PE channel was selected (emission wavelength: 575 nm), and the number of cells was observed and recorded using the hemocytometer. This number was recorded as N0. Therefore, the number of WBCs in 1 mL of cell culture medium was N0 / 4 × 10⁻⁶. 4 The number of WBCs in 1 ml of whole blood is N0 / 4 × 10⁻⁶. 7 .

[0024] 2. The capture process of CTCs and WBCs by P4+ hydrogel.

[0025] With 10 5 The experimental procedure is described using SMMC-7721 cells as an example:

[0026] When SMMC-7721 cells in 100mm diameter and 100mm height cell culture dishes reach 90% cell density (cell coverage), remove them from the cell culture incubator (37℃, 5% CO2 air). Wash three times with 3 ml of preheated PBS solution (PBS ~10mM, pH ~7.4) to remove cell culture medium from the cell surface. Then, add 1 ml of preheated trypsin solution [Trypsin EDTA solution: 0.25% Trypsin & 0.02% EDTA; pH ~8.0 (Viva Cell)] and digest for 1-2 minutes until the cells separate and become rounded. Discard the 1 ml trypsin solution and immediately add 6 ml of preheated 37℃ cell culture medium. Use a pipette to aspirate the cell culture medium and blow away any cells adhering to the bottom of the cell culture dish. Subsequently, 1 mL of solution from 6 mL of cell culture medium containing SMMC-7721 cells was transferred to a 1.5 mL sterile EP tube, labeled A. Then, 100 μL of solution from A was transferred to a new 1.5 mL sterile EP tube, labeled B. 900 μL of the aforementioned cell culture medium was added to B, and the mixture was thoroughly mixed using a pipette. 10 μL of this mixture was then seeded onto a hemocytometer and placed under an inverted research fluorescence microscope. The cell count was observed and recorded using a cell counter; this count is denoted as N1. Therefore, the number of SMMC-7721 cells in 1 mL of cell culture medium in tube B is N1 / 4 × 10⁻⁶. 4 10 5 The volume of each SMMC-7721 cell was 40 / N1 mL.

[0027] 40 / N 1 mL of SMMC-7721 cells were mixed with 1 mL of whole blood sample and seeded onto the surface of a P4 hydrogel (area: 14 cm²). 2(Height: 0.7 cm) Place the P4 hydrogel in a cell culture incubator. After 1 hour, slowly rinse the surface of the P4 hydrogel with 1 mL of PBS solution to remove red blood cells, unbound SMMC-7721 cells, and WBCs. Then, add 300 μL of pre-warmed (37°C) trypsin solution (0.25% Trypsin & 0.02% EDTA; pH ~8.0 (VivaCell)) and digest for 1–2 minutes. Immediately add 1 mL of pre-warmed (37°C) cell culture medium and use a pipette to remove cells adhering to the P4 hydrogel surface. Collect the solution in a 1.5 mL EP tube and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, leaving the cell pellet. During this period, 10 μL of fluorescein isothiocyanate isomer (FITC) anti-human CD326 (epithelial cell adhesion molecule, EpCAM) antibody (Enabling Legendary Discovery, 369814) and 10 μL of PE anti-human CD45 antibody were added to 1 mL of PBS solution and vortexed for 3 minutes to obtain a fluorescent antibody mixture. Then, 500 μL of the above fluorescent antibody mixture was added to an EP tube containing cell pellet and stained at room temperature for 30 minutes. Subsequently, the cells were washed with 1 mL of PBS solution and centrifuged, and the washing and centrifugation process was repeated 3 times. Then, the cell pellet was resuspended with 1 mL of cell culture medium, and 10 μL was pipetted and seeded onto a hemocytometer. The plate was placed under an inverted research fluorescence microscope, and the number of SMMC-7721 cells was observed and recorded using FITC (emission wavelength: 525 nm). This number was recorded as N2. The capture rate of SMMC-7721 cells by the P4 hydrogel was N² / 40 × 100%. Finally, 10 μL of the cell suspension was added to 1 mL of cell culture medium, mixed thoroughly, and then 10 μL was pipette-loaded onto a hemocytometer. The microscope was placed under an inverted fluorescence microscope, the PE channel (emission wavelength: 575 nm) was selected, and the cell count was observed and recorded using a cell counter; this count was recorded as N³. The capture efficiency for WBCs was N³ / 10N₀ × 100%. Each experimental data was tested in triplicate.

[0028] 3. Numerical calculations and result analysis in specific experimental operations.

[0029] The number of cells was counted using a cell counter on a hemocytometer as follows:

[0030] N0 = 1451, 1673, 1543; N1 = 181, 175, 195; N2 = 37, 38, 37; N3 = 3250, 3547, 3628. Therefore, the P4+ hydrogel has a specific effect on 10... 5 The capture efficiency of SMMC-7721 cells was calculated using the formula N2 / 40×100% as 92.5%, 95%, and 92.5%, respectively. The capture efficiency of WBCs in 1 ml of whole blood was calculated using the formula N3 / 10N0×100% as 22.4%, 21.2%, and 23.51%, respectively.

[0031] from Figure 1 As can be seen, this smart responsive hydrogel exhibits excellent capture ability for SMMC-7721 cells of different densities, and it is expected to be developed for the selective enrichment of CTCs.

[0032] (II) Multi-indicator combined diagnosis gradually improves the efficiency of cancer diagnosis.

[0033] Based on the background survey, the researchers enrolled 70 clinical samples with AFP levels less than 20 ng / mL, including 29 patients with liver cancer, 18 patients with cirrhosis, and 23 healthy volunteers.

[0034] The experimental procedure is described using one patient and P4+ hydrogel as an example:

[0035] A 5 ml whole blood sample (purple blood collection tube, collected fresh) was obtained from the Second Affiliated Hospital of Dalian Medical University. The blood was centrifuged at 450 g for 5 minutes to remove serum, and plasma was collected. Since the volume of 5 ml blood was much larger than the capacity of the hydrogel, the collected plasma was used to lyse red blood cells using erythrocyte lysis buffer (0.8 g ammonium chloride (NH4Cl), 37 mg ethylenediaminetetraacetic acid (EDTA), 84 mg sodium bicarbonate (NaHCO3), and 100 ml deionized water). Ten times the volume of the erythrocyte lysis buffer was added to the plasma, gently mixed by pipetting, and lysed for 5 minutes. The plasma was centrifuged at 450 g for 5 minutes at 4°C, the red supernatant was discarded, and ten times the volume of the remaining cells in PBS solution was added to resuspend the pellet. The pellet was centrifuged at 450 g for 3 minutes, and the supernatant was discarded. The pellet was resuspended in 1 mL of cell culture medium [89% Dulbecco's modified eagle medium, DMEM (Gibco), 10% Fetal bovine serum, FBS (ExCell Bio), and 1% penicillin-streptomycin, 100X (Beyotime; the cell culture medium composition is the same as described here], and evenly inoculated (dropped onto the gel surface) onto the surface of the P4+ hydrogel (area: 14 cm²). 2After placing the cells (height: 0.7 cm) in a cell culture incubator for 1 hour, the surface of the P4 hydrogel was slowly rinsed with 1 mL of PBS solution to remove residual red blood cells and unbound cells. During this time, 10 μL of fluorescein isothiocyanate isomer (FITC) anti-human CD326 (epithelial cell adhesion molecule, EpCAM) antibody (Enabling Legendary Discovery, 369814) and 0.4 μL of 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) were added. TMAdd D1306 to 1 mL of PBS solution and vortex for 3 minutes. Evenly drop the solution onto the surface of the P4+ hydrogel and stain at room temperature for 30 minutes. Then, slowly rinse the P4+ hydrogel surface with 1 mL of PBS solution, repeating the rinsing operation 3 times. Invert the P4+ hydrogel surface downwards (so that the upper surface of the rinsed P4+ hydrogel surface is facing down) and place it under an inverted research fluorescence microscope (Nikon Ti-s, Japan). Select the FITC and DAPI channels (emission wavelength: 416 nm) to observe and record the number of circulating tumor cells. In addition, the solution used to rinse the surface of the P4+ hydrogel was collected, centrifuged at 1000 rpm for 3 minutes, and stained with 500 μL of tetramethylindo(di)-carbocyanines(Cy5) anti-rabbit CD56 (Neural cell adhesion molecule, NCAM) antibody (Beijing Bio-Sens Biotechnology Co., Ltd., 0805R) at room temperature for 30 minutes. The solution was immediately transferred to a flow cytometer (SONY SH800S, Japan) and the percentage of natural killer (NK) cells was detected using the Cy5 channel (emission wavelength: 670 nm). The percentage of cells exhibiting red fluorescence in the solution was determined by the flow cytometer. Here, AFP (provided by the hospital), CTCs (the polymer material used to capture CTCs in this study), and NK (detected by flow cytometry) can be considered as three different classification indicators for liver cancer (the specific data is too large to be provided in the text, but the data's authenticity and reliability are guaranteed). The data from the three indicators were input into the Support Vector Machine (SVM) in the deep learning model for data integration and analysis. Data from 70 samples (29 liver cancer patients, 18 cirrhosis patients, and 23 healthy volunteers) were fed into the model system, and the cancer discrimination ability was determined using a binary classification model – ROC curve. The area under the ROC curve (AUC) values ​​(AFP: 0.633; AFP+NK: 0.837; AFP+CTCs: 0.889; AFP+CTCs+NK: 0.99) demonstrate that the combined use of the three indicators significantly improves the discrimination ability against liver cancer (see...). Figure 2 ).

[0036] Raw materials used in the examples:

[0037] Acrylic acid (AA), acrylamide (AAm), azobisisobutyronitrile (AIBN), N,N-methylenebisacrylamide (MBA), polyethylene glycol dimethacrylate (PEGDMA), 3-aminophenylboronic acid (PBA), and histidine (His) were purchased from Sigma-Aldrich Corp. (St. Louis, Missouri, USA). Bis-histidine was obtained from Shanghai Qiangyao Biotechnology Co., Ltd. Methacrylamide chloride, methanol, sodium hydroxide, and hydrochloric acid were purchased from Thermo Fisher Scientific. Other solvents and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The water used in this study was ultrapure water obtained from the Milli-Q system. 1 Hspectra was detected at AVANCE III HD 700MHz.

[0038] Example 1

[0039] P1, P2, and P3 hydrogel polymer network frameworks were prepared using atom transfer radical polymerization.

[0040]

[0041] (1) Synthesis of polyacrylic acid [PAA, P1]: The polymerization method used was free radical random copolymerization. The initiator used was azobisisobutyronitrile (AIBN, CAS No. 78-67-1, Sigma-Aldrich Corp.), the monomer used was acrylic acid (AA, CAS No. 79-10-7, Sigma-Aldrich Corp.), and the crosslinking agent used was N,N-methylenebisacrylamide (MBA, CAS No. 110-26-9, Sigma-Aldrich Corp.). Taking 1-x = 0.015 as an example, the specific synthesis steps are as follows: AA (9.0 mL, 130.0 mmol) and MBA (300.0 mg, 2.0 mmol) were added sequentially to a 50 mL EP tube at a molar ratio of 65:1. At the same time, 30 mL of ultrapure water was added as a solvent, and the mixture was dissolved by sonication and shaken to mix. Subsequently, initiator AIBN (118.1 mg, 0.7 mmol) was added, and the mixture was shaken and mixed for 1 hour. The mixture was then poured into a 50 mL glass sample vial, sealed, and placed in an oven. For the first 12 hours, the temperature was maintained at 30°C. Afterward, the temperature was increased to 80°C, maintained for 6 hours, and then decreased to 30°C, continuing this process for 2 days (including the initial 18 hours). The heating and cooling rates were both 5°C / hour. The glass vial containing P1 was carefully broken, and P1 was removed and immersed in ultrapure water for dialysis purification for 7 days to remove unreacted monomers. The immersion solution was changed every half day during this period. The successful synthesis of P1 was confirmed by elemental analysis using Fourier-transform infrared spectroscopy (FT-IR) and energy dispersive spectroscopy (EDS) (structure shown in the figure above, 1-x = 0.015). Number-average molecular weight = 20000 Da, polydispersity (PDI) = 1.3. FT-IR: 3446 cm⁻¹ -1 (hydroxyl acrylic acid), 1725cm -1 (N,N-methylenebisacrylamide carbonyl); EDS: 58.69% (C), 14.87% (N), 26.31% (O).

[0042] (2) Synthesis of polyacrylamide [PAAm, P2]: The polymerization method used was free radical random copolymerization. The initiator used was azobisisobutyronitrile (AIBN, CAS No. 78-67-1, Sigma-Aldrich Corp.), the monomer used was acrylamide (AAm, CAS No. 79-06-1, Sigma-Aldrich Corp.), and the crosslinking agent used was N,N-methylenebisacrylamide (MBA, CAS No. 110-26-9, Sigma-Aldrich Corp.). Taking 1-x = 0.015 as an example, the specific synthesis steps are as follows: AAm (9.2 g, 130.0 mmol) and MBA (300.0 mg, 2.0 mmol) were added sequentially to a 50 mL EP tube at a molar ratio of 65:1. At the same time, 30 mL of ultrapure water was added as a solvent, and the mixture was dissolved by sonication and shaken to mix. Subsequently, initiator AIBN (118.1 mg, 0.7 mmol) was added, and the mixture was shaken and mixed for 1 hour. The mixture was then poured into a 50 mL glass sample vial, sealed, and placed in an oven. For the first 12 hours, the temperature was maintained at 30°C. Afterward, the temperature was increased to 80°C, maintained for 6 hours, and then decreased to 30°C, continuing this process for 2 days (including the initial 18 hours). The heating and cooling rates were both 5°C / hour. The glass vial containing P2 was carefully broken, and P2 was removed and immersed in ultrapure water for dialysis purification for 7 days to remove unreacted monomers. The immersion solution was changed every half day during this period. The successful synthesis of P2 was confirmed by elemental analysis using Fourier-transform infrared spectroscopy (FT-IR) and energy dispersive spectroscopy (EDS) (structure shown in the figure above, 1-x = 0.015). Number-average molecular weight = 640,000 Da, polydispersity (PDI) = 2.1. FT-IR: 3415 cm⁻¹ -1 (Acrylamide free amino group), 1725cm -1 (N,N-methylenebisacrylamide carbonyl); EDS: 56.54% (C), 19.68% (N), 22.96% (O).

[0043] (3) Synthesis of polyethylene glycol dimethacrylate [PP, P3]: The polymerization method used was free radical random copolymerization. The initiator used was azobisisobutyronitrile (AIBN, CAS No. 78-67-1, Sigma-Aldrich Corp.), and the monomer used was polyethylene glycol dimethacrylate (PEGDMA, n=13, CAS No. 25852-47-5, Sigma-Aldrich Corp.). The same monomer mass as P1 was used. The specific synthesis steps were as follows: PEGDMA (9.0 mL, 13.32 mmol) was added to a 50 mL EP tube, and 30 mL of ultrapure water was added as a solvent. The mixture was dissolved by sonication and then shaken to mix. Subsequently, initiator AIBN (118.1 mg, 0.7 mmol) was added, and the mixture was shaken and mixed for 1 hour. The mixture was then poured into a 50 mL glass sample vial, sealed, and placed in an oven. For the first 12 hours, the temperature was maintained at 30°C. Afterward, the temperature was increased to 80°C, maintained for 6 hours, and then decreased to 30°C, continuing this process for 2 days (including the initial 18 hours). The heating and cooling rates were both 5°C / hour. The glass vial containing P3 was carefully broken, and P3 was removed and immersed in ultrapure water for 7 days for dialyzing to remove unreacted monomers. The immersion solution was changed every half day during this period. The successful synthesis of P3 was confirmed by elemental analysis using Fourier-transform infrared spectroscopy (FT-IR) and energy dispersive spectroscopy (EDS) (structure shown in the figure above, n = 13, degree of polymerization 19). The number-average molecular weight was 16000 Da, and the polydispersity index (PDI) was 0.3. FT-IR: 3059, 2341, 1754cm -1 (Polyethylene glycol dimethacrylate carbonyl and backbone); EDS: 89.64% (C), 10.27% (O).

[0044] Example 2

[0045] Selection of gel skeleton for smart responsive hydrogels

[0046] Gels P1, P2, and P3, each weighing 1 g, were immersed in 10 mL of phosphate-buffered saline (PBS = 10 mM, pH = 7.4) at 0.2, 0.5, 1, 4, 24, 48, 72, 96, and 120 hours, respectively. After removing the gels, the surface liquid was blotted dry with filter paper, and the gels were weighed. The weight at 0 hours was recorded as W0, and the weights at the other times were recorded as W. x Using the formula: SR(%) = (W) x Calculate the swelling ratio and plot it using (-W0)×100 / W0. Figure 3 As can be seen, P1 and P2 have different degrees of swelling, which is not conducive to the preservation of the microstructure of cell imprints, while the swelling and deformation of P3 is negligible and can be selected as a substrate for the preparation of smart responsive gels.

[0047] Example 3

[0048] Structure and Synthesis Methods of Functional Monomers

[0049] To prepare the aforementioned smart responsive hydrogel, a series of functional monomers need to be synthesized, and the specific synthetic methods are as follows:

[0050]

[0051] (1) Synthesis of methacrylamide dihistidine.

[0052] Sodium hydroxide (2.4 g, 0.06 mol) was added to 50 mL of an ultrapure aqueous solution containing 100 mg (0.34 mmol) of dihistidine (two histidines covalently linked by peptide bonds). The mixture was stirred at ambient temperature for 5 hours. Then, methacryloyl chloride (660 μL, 6.8 mmol) was added to the mixture, and stirring was continued at ambient temperature for 24 hours. After evaporating the solvent, 50 mL of methanol was added, and the mixture was sonicated for 20 minutes. The mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected, discarding the precipitate. The supernatant was then freeze-dried to obtain a white powder product (106.97 mg, yield: 86.77%). 1 ¹H NMR (700 MHz, deuterated dimethyl sulfoxide (DMSO-d6)): δ (ppm): 8.18 (d, J = 7.0 Hz, 2H, NH), 7.48 (s, 1H, CH), 7.44 (s, 1H, CH), 6.75 (s, 1H, CH), 6.64 (s, 1H, CH), 5.82 (s, 2H, CH), 5.37 (s, 2H, CH), 4.09 (dd, J = 11.9, 6.6 Hz, 1H, CH₂), 3.45–3.42 (m, 1H, CH₂), 2.62 (dd, J = 14.3, 7.1 Hz, 2H, CH₂), 1.52 (s, 3H, CH₃); MADLI-MS: m / z calcd for C 27 H 39 N7O 13 :360.1546; found:361.1542.

[0053] (2) Synthesis of methacrylated histidine.

[0054] Sodium hydroxide (16.0 g, 0.4 mol) was added to 50 mL of an ultrapure aqueous solution containing histidine (15.5 g, 100 mmol). The mixture was stirred at ambient temperature for 5 hours. Then, methacryloyl chloride (5 mL, 51.5 mmol) was added to the mixture, and stirring was continued at ambient temperature for 24 hours. After evaporating the solvent, 50 mL of methanol was added, and the mixture was sonicated for 20 minutes. The mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected, discarding the precipitate. The supernatant was then freeze-dried to obtain a white powder product (2.08 g, yield: 93.27%). 1 H NMR (700MHz, heavy water (D2O)): δ (ppm): 8.66 (m, 2H, NH), 7.46 (m, 2H, CH), 5.68 (m, 1H, CH), 5.47(m,1H,CH),4.65(m,1H,CH),3.47(m,2H,CH2),1.96(m,3H,CH3);MADLI-MS:m / z calcd for C 10 H 13 N3O3:223.2285; found:224.1015.

[0055] (3) Synthesis of acrylated 3-aminophenylboronic acid.

[0056] Sodium bicarbonate (16.8 g, 25.0 mmol) was added to 50 mL of an ultrapure water and tetrahydrofuran (2:1 v / v) solution containing 3-aminophenylboronic acid (1.5 g, 1.0 mmol). The mixture was stirred at ambient temperature for 5 hours. Then, acryloyl chloride (203 μL, 2.5 mmol) was added to the mixture, and stirring was continued at ambient temperature for 24 hours. After evaporating the solvent, 50 mL of methanol was added, and the mixture was sonicated for 20 minutes. The mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and the precipitate was discarded. The supernatant was then freeze-dried to obtain a white powder product (1.92 g, yield: 91.87%). 1 ¹H NMR (700MHz, deuterated dimethyl sulfoxide (DMSO-d6)): δ (ppm): 7.88 (s, 4H, -ph), 6.40 (qd, J = 17.0, 5.9Hz, 2H, CH), 5.76 (dd, J = 9.8, 1.7Hz, 1H, CH), 1.34 (d, J = 45.9Hz, 2H, -OH); MADLI-MS: m / z calcd for C9H 10 BNO3:190.9916; found:191.0847.

[0057] Example 4

[0058] Hydrogel functional polymers targeting CTCs, namely P4, P5 and P6, were prepared by atom transfer radical polymerization.

[0059]

[0060] (1) Synthesis of poly(polyethylene glycol dimethacrylate-co-methpropenylbishistidine) [Poly(PP-co-acrylamidobishistidine), PP-co-AHH, P4]: The polymerization method (random copolymerization) and initiator used were the same as in Example 1. The monomer used was methpropenylbishistidine (AHH) (successful synthesis was characterized by NMR and mass spectrometry). The crosslinking agent used was polyethylene glycol dimethacrylate (PEGDMA, n=13, CAS No.25852-47-5, Sigma-AldrichCorp). Taking y = 0.4 as an example, the specific synthesis steps are as follows: PEGDMA (13.5 mL, 20.0 mmol) and AHH (10.8 g, 30.0 mmol, added in two equal portions) are added sequentially to a 50 mL EP tube at a molar ratio of 2:3. Simultaneously, 45 mL of ultrapure water is added as a solvent. The mixture is sonicated and shaken to dissolve and homogenize. Then, the initiator AIBN (168.8 mg, 1.0 mmol) is added, and the mixture is shaken and homogenized for 1 hour. The mixture is then poured into a 50 mL glass sample vial, sealed, and placed in an oven. Initially, the temperature is maintained at 30°C for the first 12 hours. Afterward, the temperature is increased to 80°C, maintained for 6 hours, and then decreased to 30°C, continuing this process for 2 days (including the initial 18 hours). The heating and cooling rates are both 5°C / hour. The glass vial containing P4 is carefully broken, and P4 is removed and immersed in 10 mL of ultrapure water for dialyzing and purification for 7 days to remove unreacted monomers. The immersion solution is changed every half day during this period. The successful synthesis of P4 was confirmed by elemental analysis using infrared spectroscopy and scanning electron microscopy (structure shown in the figure above, y = 0.4). Number-average molecular weight = 18700 Da, polydispersity (PDI) = 0.9. FT-IR: 2869 cm⁻¹ -1 (PEGDMA ester group), 1455, 1405, 1328 cm -1 (AHH imidazole ring); EDS: 46.62% (C), 23.63% (N), 29.51% (O).

[0061] (2) Synthesis of poly(polyethylene glycol dimethacrylate-co-methacrylamidohistidine) [Poly(PP-co-acrylamidohistidine), PP-co-AH, P5]: The polymerization method (random copolymerization) and initiator used were the same as in Example 1. The monomer used was methacrylated histidine (AH) (successful synthesis was characterized by NMR and mass spectrometry). The crosslinking agent used was polyethylene glycol dimethacrylate (PEGDMA, n=13, CAS No.25852-47-5, Sigma-Aldrich Corp.). Taking y = 0.4 as an example, the specific synthesis steps are as follows: PEGDMA (13.5 mL, 20.0 mmol) and AH (6.69 g, 30.0 mmol, added in two equal portions) are added sequentially to a 50 mL EP tube at a molar ratio of 2:3. Simultaneously, 45 mL of ultrapure water is added as a solvent. The mixture is sonicated and shaken to dissolve and mix thoroughly. Then, the initiator AIBN (168.8 mg, 1.0 mmol) is added, and the mixture is shaken and mixed for 1 hour. The mixture is then poured into a 50 mL glass sample vial, sealed, and placed in an oven. Initially, the temperature is maintained at 30°C for the first 12 hours. Afterward, the temperature is increased to 80°C, maintained for 6 hours, and then decreased to 30°C, continuing this process for 2 days (including the initial 18 hours). The heating and cooling rates are both 5°C / hour. The glass vial containing P5 is carefully broken, and P5 is removed and immersed in 10 mL of ultrapure water for dialyzing and purification for 7 days to remove unreacted monomers. The immersion solution is changed every half day during this period. The successful synthesis of P5 was confirmed by elemental analysis using infrared spectroscopy and scanning electron microscopy (structure shown in the figure above, y = 0.4). Number-average molecular weight = 16400 Da, polydispersity (PDI) = 1.1. FT-IR: 2853 cm⁻¹ -1 (PEGDMA ester group), 1680, 1593cm -1 (AH imidazole ring); EDS: 63.12% (C), 13.63% (N), 22.51% (O).

[0062] (3) Synthesis of poly(polyethylene glycol dimethacrylate-co-acrylamide 3-aminophenylboronic acid) [Poly(PP-co-3-(acrylamido)phenylboronic acid, PP-co-APBA, P6]: The polymerization method and initiator used were the same as in Example 1. The monomer used was acrylamide 3-aminophenylboronic acid (APBA) (NMR and mass spectrometry characterization confirmed successful synthesis). The crosslinking agent used was polyethylene glycol dimethacrylate (PEGDMA, n=13, CAS 13). No. 25852-47-5, Sigma-Aldrich Corp. Taking y = 0.4 as an example, the specific synthesis steps are as follows: PEGDMA (13.5 mL, 20.0 mmol) and APBA (10.8 g, 30.0 mmol, added in two equal portions) are added sequentially to a 50 mL EP tube at a molar ratio of 2:3. Simultaneously, 45 mL of ultrapure water is added as a solvent. The mixture is sonicated and shaken to dissolve and homogenize. Then, the initiator AIBN (168.8 mg, 1.0 mmol) is added, and the mixture is shaken to homogenize for 1 hour. The mixture is then poured into a 50 mL glass sample vial, sealed, and placed in an oven. The initial temperature was maintained at 30°C for 12 hours, then increased to 80°C, maintained for 6 hours, and then decreased to 30°C, continuing this process for 2 days (including the initial 18 hours). The heating and cooling rates were both 5°C / hour. The glass vial containing P6 was carefully broken, and the P6 was removed and immersed in 10 mL of ultrapure water for 7 days of dialyzing to remove unreacted monomers. The immersion solution was changed every half day during this period. Characterization by infrared spectroscopy and scanning electron microscopy (SEM) elemental analysis confirmed the successful synthesis of P6 (structure shown in the figure above, y = 0.4). Number-average molecular weight = 17000 Da, polydispersity (PDI) = 1.4. FT-IR: 2769 cm⁻¹ -1 (PEGDMA ester group), 1586, 1500cm -1 (Benzene ring); EDS: 52.81% (C), 11.13% (N), 25.63% (O), 9.56% (B).

[0063] Example 5

[0064] Because the structure of the dihistidine molecule is sensitive to pH, a material with dihistidine molecules grafted onto silica microspheres was designed and synthesized. The following is a detailed description of the preparation process of HH@SiO2 material.

[0065]

[0066] First, acidification is used to expose hydroxyl groups on the surface of silica microspheres. Then, silane coupling agents are grafted onto the surface of the silica microspheres using these hydroxyl groups. Subsequently, a ring-opening reaction between the epoxy groups of the silane coupling agent and the terminal amino groups of the dihistidine is performed to modify the surface of the silica microspheres with dihistidine. The specific reaction steps are as follows:

[0067] 10.0g of silica gel (diameter 10μm, inner pore diameter...) The silica gel was suspended in 50 mL of nitric acid (HNO3, 50% by mass) for 10 hours to generate sufficient hydroxyl groups on the silica gel surface. The hydroxylated silica gel was then separated by centrifugation at 7000 rpm for 5 minutes, washed five times with ultrapure water, and dried in an oven at 80 °C. Next, 5 g of the hydroxylated silica gel was added to 30 mL of anhydrous toluene containing 5 mL of 3-glycidoxypropyltrimethoxysilane (GLYMO). The reaction mixture was stirred at 60 °C for 48 hours. The resulting silica gel (denoted as GLYMO@SiO2) was separated by centrifugation at 7000 rpm for 5 minutes and washed three times sequentially with toluene and ethanol, respectively, by repeating the dispersion / precipitation cycle to remove residual GLYMO. The GLYMO@SiO2 was then dried in an oven at 80 °C. Next, 500 mg of GLYMO@SiO2 and 400 mg of bishistidine were added to 30 mL of ultrapure water. The mixture was stirred at 37 °C for 72 hours. Then, after removing residual chemicals by repeated dispersion / precipitation cycles of washing with ultrapure water and ethanol, respectively, HH@SiO2 was obtained by freeze drying. The successful synthesis of HH@SiO2 was confirmed by thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) energy dispersive spectroscopy (EDS).

[0068] Example 6

[0069] The surface potential of HH@SiO2 material was determined using a Malvern nanoparticle size analyzer.

[0070] Eight 20 mg portions of HH@SiO2 material obtained in Example 5 were weighed into 4 mL EP tubes, labeled A1, A2, A3, A4, A5, A6, A7, and A8. 2 mL of ultrapure water was added to each of the eight EP tubes. A1-A5 were adjusted to pH solutions 2, 3, 4, 5, and 6 using concentrated hydrochloric acid (12 mol / L), while A6-A8 were adjusted to pH solutions 7, 8, and 9 using sodium hydroxide (2 mol / L). After thorough mixing, the solutions were sequentially added to potentiometric cuvettes and placed in a Malvern nanolaser particle size analyzer (Zetasizer Nano). The surface potentials of A1, A2, A3, A4, A5, A6, A7, and A8 were measured at room temperature for 60 cycles. The measurements were repeated three times, and the average values ​​were calculated and plotted. Figure 4As can be seen, HH@SiO2 materials carry a positive charge in acidic environments (pH 2-7) and a negative charge in alkaline environments (pH 8-9). Combined with the fact that sialic acid, which is abnormally expressed on the surface of CTCs compared to other cells, carries a negative charge, the dual histidine charge-switching characteristics can be used to achieve the dynamic release of CTCs by smart responsive hydrogel materials.

[0071] Example 7

[0072] Biolayer interferometry (BLI) curves showing high affinity binding of dual histidine functional monomers to cell membrane suspensions.

[0073] Because circulating tumor cells (CTCs) are covered with a large amount of glycocalyx on their surface, they can be captured by recognizing and enriching target molecules on the cell surface. Based on this idea, we extracted SMMC-7721 cell membrane proteins and used biolayer interferometry to evaluate the affinity of histidine dinucleotides for SMMC-7721 cell membrane proteins.

[0074] First, SMMC-7721 cell membrane proteins were extracted according to the instructions of the Thermo Scientific Mem-PER Plus (89842) membrane protein extraction kit, yielding a white lyophilized powder. Second, 1 mg of the lyophilized powder was dissolved in 2 mL of ultrapure water to prepare a protein solution of 0.5 mg / mL. The bicinchoninic acid (BCA) concentration was verified to be 0.508 mg / mL using a Beyotime (P0010) assay kit, indicating that the lyophilized powder was free of impurities and consisted entirely of protein.

[0075] Finally, 1 mg of histidine was dissolved in 200 μL of ultrapure water and grafted onto the surface of the amine reactive 2ndgeneration (AR2G) biosensor probe (Sartorius, 2211021111) of the Molecular Interaction Instrument (ForteBio Octet, Octet K2, USA) to conduct adsorption experiments on membrane proteins at different concentration gradients (3.31, 6.25, 12.5, 25, 50, 100 μg / mL). The specific procedure is as follows: Before testing, 30 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (3-[(Ethylimino)methylidene]amino-N,N-dimethylpropan-1-amine, EDC, CAS No. 1892-57-5, Sigma-Aldrich Corp) and 18 mg of N-hydroxysulfosuccinimide (N-Hydroxysuccinimide, NHS, CAS No. 6066-82-6, Sigma-Aldrich Corp) were dissolved in 400 μL of ultrapure water and placed in a 96-well plate with 200 μL of solution in each well. The two biosensor probes were placed in the wells containing the solution and the carboxyl groups on the surface of the biosensor probes were activated under room temperature and light-protected conditions. Two hours later, the solution in the wells was discarded. 2 mg of dihistidine was dissolved in 400 μL of ultrapure water and added to the wells. Two biosensor probes were placed in the wells containing the solution, and the reaction was allowed to proceed for 24 hours at room temperature in the dark. Using the amino group of dihistidine, a amide reaction was performed to graft dihistidine onto the surface of the carboxyl-terminated biosensor probe. The sensor surface was then carefully washed with ultrapure water to remove unreacted sample. Subsequently, the extracted membrane protein was prepared into a 1 mg / mL protein solution with ultrapure water, and then serially diluted with ultrapure water to obtain protein solutions of different concentration gradients (3.31, 6.25, 12.5, 25, 50, 100 μg / mL). Then, the dihistidine-modified biosensor probe of the experimental group (one of the biosensor probes) was exposed (immersed) in protein solutions of different concentration gradients (3.31, 6.25, 12.5, 25, 50, 100 μg / mL). Binding, dissociation, and interaction analyses were performed at 37°C. Simultaneously, a control group (another biosensor probe) with a bishistidine-modified biosensor probe was exposed to ultrapure water. Binding, dissociation, and interaction analyses were performed at 37°C. Data were analyzed by ForteBio (Data Analysis 11.0). Figure 5 The results show that the effect of dihistidine on the SMMC-7721 cell membrane protein solution is concentration-dependent, indicating that dihistidine has the ability to bind to SMMC-7721 cells and is expected to be developed for the efficient enrichment of CTCs.

[0076] Example 8

[0077] The water content inside the prepared smart responsive hydrogel was determined using low-field nuclear magnetic resonance (LF-NMR).

[0078] In the T2 relaxation of LF-NMR, relaxation peaks at different positions represent different states of binding of H nuclei, thus characterizing their different mobility. H nuclei with a higher degree of binding have poorer mobility and shorter relaxation times; conversely, relatively free H nuclei with a lower degree of binding have better mobility, higher degrees of freedom, and longer relaxation times. Based on this property, we can determine the state of water molecules inside the gel, and further, we can use peak area to indicate the water content. The specific operation is as follows: First, calibrate the LF-NMR (Suzhou Newmai Technology). Select the instrument default for the magnet probe; select Q-FID for the sequence name, with default parameters; place the standard sample (standard oil sample); click "single sampling," wait 10 seconds, and click "stop sampling"; click "find center frequency (SF+01)" until the FID curve no longer shows regular oscillations; click "pulse width," and the software automatically finds 90° and 180° pulse widths until a peak and a trough appear, indicating that the pulse width has been correctly found. Then, 1g of P1-P6 gel (5cm in diameter and 5cm in height) was sequentially placed into the magnetic field cavity. The CPMG sequence was selected, the sequence name was entered, appropriate parameters were set, and sampling was clicked to determine the T2 relaxation time. The states of different water molecules were then obtained through inversion. For example... Figure 6 As shown, hydrogels P1, P2, P3, P4, P5, and P6 were immersed in 10 mL of PBS solution (PBS = 10 mM, pH = 7.4, the same below) for 7 days, and then removed and placed in a magnetic field cavity for testing. The T2 component peaks located at 0.1–3 ms, 70–200 ms, and 800–2200 ms correspond to bound water, fixed water, and free water, respectively. Sharp and strong peaks were observed in P1 and P2 at 1649–2078 ms and 1250–1649 ms, with signal intensities (744 and 741) significantly greater than those at 71–89 ms (74) and 81–103 ms (39). This indicates that a large amount of free water exists inside the P1 and P2 hydrogels, with water in the dominant form, which is consistent with the significant swelling of P1 and P2 in Example 2. In contrast, the signals corresponding to free water and fixed water in P3-P6 are significantly weaker, especially the free water intensity in P4-P6, which confirms its low swelling properties.

[0079] Example 9

[0080] Preparation and characterization of microstructure of P4+, P5+, and P6+ cell imprints using smart responsive hydrogels: Taking P4+ hydrogel as an example, the formation process of cell imprints is described:

[0081] First 4×106 The previous night, SMMC-7721 cells were placed in a 100mm diameter, 100mm high cell culture dish and 10mL of cell culture medium was added [volume concentration: 89% Dulbecco's modified eagle medium, DMEM (Gibco), 10% Fetal bovine serum, FBS (ExCell Bio), and 1% penicillin-streptomycin, 100X (Beyotime; the cell culture medium composition described below is the same as described here]] and cultured until the morning of the following day (12 hours in total). The culture medium was removed, and the cells were washed three times with PBS solution (PBS = 10mM, pH = 7.4, the same below). 2mL of pre-prepared, membrane-passed (0.22μm) paraformaldehyde solution (mass concentration: 4%) was added, and the cells were fixed at 37℃ for 5 minutes. The fixative was removed, and the cells were washed three times with PBS solution (PBS = 10mM, pH = 7.4, the same below). Next, PEGDMA (13.5 mL, 20.0 mmol) and AHH (10.8 g, 30.0 mmol, added in two equal portions) were added sequentially to a 50 mL EP tube at a molar ratio of 2:3. 45 mL of ultrapure water was added as a solvent, and the mixture was sonicated and vortexed. Then, the initiator AIBN (168.8 mg, 1.0 mmol) was added, and the mixture was vortexed for 1 hour. 5 mL of the solution was then transferred to a cell culture dish containing immobilized cells and shaken at 300 rpm for 5 hours to allow the functional monomers in the solution to seek target molecules on the cell surface. The culture dish was then transferred to an oven and kept at 30 °C for the first 12 hours. The temperature was then increased to 80 °C, held for 6 hours, and then decreased to 30 °C, continuing this process for 2 days (including the initial 18 hours) at a rate of 5 °C / hour. This yielded a hydrogel P4-cell with cell imprints and functional groups. The P4-cell hydrogel was rinsed with ultrapure water. Ultrapure water was slowly added from the edge of the cell culture dish to detach the P4-cell hydrogel from the dish. The P4-cell hydrogel was then removed and immersed in 10 mL of ultrapure water for dialyzing purification for 7 days, changing the immersion solution every half day. Afterward, the gel was removed and immersed in 10 mL of trypsin EDTA solution (0.25% Trypsin & 0.02% EDTA; pH ~8.0 (VivaCell)). After 4 hours, the gel was rinsed three times with 5 mL of phosphate-buffered saline (PBS = 10 mM, pH = 7.4) and stored in 10 mL of PBS solution (PBS = 10 mM, pH = 7.4). This process was to remove the cell template, unreacted monomers, and AIBN residues, thereby preparing a smart responsive molecularly imprinted hydrogel carrying functional monomers and cell micromorphology. The hydrogel was observed under an optical microscope. Figure 7 The white arrows shown indicate the microscopic morphology of the cellular imprint.

[0082] Application Examples

[0083] Example 10: Anti-protein adsorption properties of the gel backbone of a smart responsive hydrogel

[0084] First, 3 ml blood samples (from healthy individuals undergoing physical examinations) were obtained from the Second Affiliated Hospital of Dalian Medical University. Based on cell density differences (erythrocytes and granulocytes: 1.090 g / mL; lymphocytes and monocytes: 1.075–1.090 g / mL; platelets: 1.030–1.035 g / mL), cells were separated from the blood by centrifugation to distribute them according to their respective density gradients. The remaining solution was collected, and the protein content was determined using a BCA assay kit. 4 mg of protein was taken and diluted in 13 ml of ultrapure water (initial protein concentration: 0.3 mg / mL), and aliquoted into 12 2 ml glass vials (1 ml solution per vial). P1, P2, and P3 were cut into 1 cm × 1 cm × 1 cm cubes (3 aliquots, 70 mg each) and added to the aforementioned glass vials. Three blank control groups (without any added material) were also included. Four hours later, the solution in the glass vial was aspirated, and the absorbance (OD) was measured using a nanodrop microspectrophotometer. The protein content was then calculated using a standard curve. x1 (corresponding to P1), A x2 (corresponding to P2), A x3 (corresponding to P3) and A con (Corresponding to the blank control group), calculate the adsorption contents of proteins P1, P2, and P3. The specific calculation process is as follows: First, prepare a standard curve by preparing bovine serum albumin (BSA) solutions of 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL and measuring their OD values ​​as 0.0195, 0.0425, 0.0835, 0.158, 0.2445, 0.3085, and 0.39. Based on the OD values ​​and concentrations, plot the standard curve in Excel to obtain the equation y = 0.7741x + 0.0039; R 2=0.999. Then, after soaking for 4 hours, 100 μL of solution was taken from each of the 12 glass vials, and 2 μL was taken to measure the OD value. The OD values ​​of the control group were 0.222, 0.209, and 0.213; the OD values ​​of P1 were 0.211, 0.1995, and 0.202; the OD values ​​of P2 were 0.219, 0.206, and 0.209; and the OD values ​​of P3 were 0.219, 0.208, and 0.211. The corresponding protein concentrations were as follows: control group: 0.2817, 0.2649, 0.2701 mg / mL; P1 group: 0.2675, 0.2526, 0.2559 mg / mL; P2 group: 0.2778, 0.2610, 0.2649 mg / mL; P3 group: 0.27787, 0.2636, 0.2675 mg / mL. The protein concentrations adsorbed by gels P1, P2, and P3 were obtained by subtracting the protein concentrations of the sample groups from the protein concentrations of the control group (corresponding sequentially). Since each gel had a volume of 1 mL, the adsorbed protein amounts were: P1 - 14.21, 12.27, 14.21 μg; P2 - 3.87, 3.87, 5.16 μg; P3 - 0.38, 0.13, 0.26 μg. The corresponding mass adsorption percentages were: P1 - 0.203, 0.175, 0.203 μg / mg; P2 - 0.055, 0.055, 0.073 μg / mg; P3 - 0.005, 0.002, 0.004 μg / mg. Figure 8 As can be seen, the P3 hydrogel framework has excellent anti-protein adsorption ability.

[0085] Example 11: Processability of Smart Responsive Hydrogel P4

[0086] P4 gel (10 cm in diameter and 10 cm in height) was fixed on the probe of a texture analyzer (Shanghai Baosheng, TA.XTC-20). A certain force was applied axially to the cylinder using the texture analyzer, and the probe moved downwards at a constant speed to apply pressure. When the pressure reached a set value, the probe moved upwards, and the change in the cylinder height before and after compression was observed and measured under a microscope to reflect the mechanical properties of the hydrogel. Under a 15% strain condition (quantitatively describing the degree of hydrogel stretching and compression, with compression equal to 15% of the gel height, i.e., the gel height is compressed from 10 cm to 8.5 cm), the gel was compressed to a fixed position at a speed of 1 mm / s, and then compressed for 2 seconds before rebounding. The damage to the smart responsive hydrogel caused by compression and rebound during 50 cycles was tested. The experimental results showed that P4 did not suffer damage after 50 cycles, with only a 7% decrease in mechanical properties (change in gel height from the initial gel height to the gel height after 50 cycles). This indicates that P4 has excellent plasticity (see...). Figure 9 It is suitable for processing and preparing into biomedical materials.

[0087] Example 12: CTCs capture and re-release cycle availability of smart responsive hydrogel P4+

[0088] 10 5 A 1 ml blood sample (from a healthy patient undergoing a physical examination) with an SMMC-7721 cell doping density was dropped onto a P4+ hydrogel (area: 14 cm²). 2 Cells were cultured on the surface of a hydrogel (height: 0.7 cm) for 30 minutes. Unbound cells were then washed with PBS solution (PBS = 10 mM, pH = 7.4, the same below), and the capture rate was calculated by observing the number of CTCs in the PBS. Next, 0.2 mL of pre-warmed (37°C) trypsin solution [Trypsin EDTA solution: 0.1–0.5% Trypsin & 0.005%–0.05% EDTA; pH ~8.0 (Viva Cell)] was added, and digestion was performed for 1 minute. Immediately afterward, 1 mL of pre-warmed (37°C) cell culture medium was added, and cells adhering to the hydrogel surface were removed by pipetting. The solution was collected and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, leaving the cell pellet. The cells were resuspended in 100 μL of cell culture medium, and the cell count was calculated using a hemocytometer to determine the release rate. This process was repeated 10 times, with each single experiment consisting of 3 replicates. Taking one cycle as an example, the specific experimental data is described as follows: First, the capture rate was calculated. The obtained PBS solution was centrifuged at 1000 rpm for 3 minutes, and 100 μL of cell culture medium [volume concentration: 89% Dulbecco's modified eaglemedium, DMEM (Gibco), 10% Fetal bovine serum, FBS (ExCell Bio), and 1% penicillin-streptomycin, 100X (Beyotime; the cell culture medium composition below is the same as here] was added to resuspend the cells. 10 μL of the medium was taken, and the cell count was calculated using a hemocytometer (N1 = 24, 26, 27). Therefore, the cell count in 100 μL of cell culture medium was 6 × 10⁶ cells / mL. 3 6.5×10 3 6.75×10 3 Dividing the individual cell counts by the total number of seeded cells yields the percentages of uncaptured cells as 6%, 6.5%, and 6.75%, respectively. Therefore, the P4+ hydrogel is effective against 10... 5The capture rates of SMMC-7721 cells were 94.0%, 93.5%, and 93.3%. Then, the release rate was calculated, yielding 94,000, 93,500, and 93,250 cells captured on the P4+ hydrogel surface, respectively. Next, the number of cells released from the gel surface was calculated using a hemocytometer (N² = 353, 360, 347), resulting in 88,250, 90,000, and 86,750 cells released, leading to release rates of 93.88%, 96.26%, and 93.03%. Figure 10 As can be seen, this smart responsive hydrogel has good capture efficiency and reusability, which can reduce detection costs and is expected to be developed for the selective enrichment of CTCs, promoting the popularization of cancer detection.

Claims

1. A polymeric material captured by CTCs, characterized in that: The polymer material is composed of a PEGDMA network backbone and CTCs binding units. The polymerization reaction process of PEGDMA and CTCs binding units is random copolymerization. The number average molecular weight of the polymer material is: P4=5000~20000 Da, P5=5000~20000 Da, P6=5000~20000 Da. Its molecular structure is shown below. ； Where y = 0.01~0.5, n = 1~15 integers; the CTCs binding unit is one or more of methacrylamide bishistidine AHH, methacrylamide histidine AH, and acrylamide 3-aminophenylboronic acid APBA.

2. The polymer material with CTCs captured according to claim 1, characterized in that: y=0.1~0.4, n=3~13; the number-average molecular weight range is: P4=8000~18700 Da, P5=7000~16400 Da, P6=7000~17000 Da.

3. A method for preparing a polymer material with CTCs captured according to claim 1, characterized in that: The specific process is as follows: (1) Synthesis of poly(polyethylene glycol dimethacrylate) co P4: In a 10-200 mL beaker, add 5-50 mL of PEGDMA and 5-100 g of AHH sequentially, with a molar ratio of PEGDMA to AHH of 0.5:1 to 3:

1. Simultaneously add 10-200 mL of ultrapure water as a solvent, sonicate to dissolve and mix thoroughly. Then, add 100-1000 g of... After mixing mg of the initiator azobisisobutyronitrile (AIBN) by shaking for 30–2880 minutes, the mixture is poured into 10–100 mL glass sample vials, the vials are sealed, and placed in an oven. Initially, the temperature is maintained at 10–50 °C for 3–20 hours. Then, the temperature is increased to 60–150 °C, maintained for 2–10 hours, and then decreased to 10–50 °C. This process, including the initial and maintenance times of 5–30 hours, is continued for a total of 2–9 days, with a heating / cooling rate of 1–10 °C / hour. The glass vial containing P4 is carefully broken, and P4 is removed and immersed in 5–50 mL of ultrapure water for dialyzing purification for 1–14 days to remove unreacted monomers. The immersion solution is changed every half day to one day during this period to obtain gel P4. Or, (2) synthesize poly(polyethylene glycol dimethacrylate- co P5: In a 10-200 mL beaker, add 5-50 mL of PEGDMA and 1-500 g of AH sequentially, with a molar ratio of PEGDMA to AH of 0.5:1 to 3:

1. Simultaneously add 10-200 mL of ultrapure water as a solvent, sonicate to dissolve and mix thoroughly. Then, add 100-1000 g of... After mixing mg of the initiator azobisisobutyronitrile (AIBN) by shaking for 30–2880 minutes, the mixture is poured into 10–100 mL glass sample vials, the vials are sealed, and placed in an oven. Initially, the temperature is maintained at 10–50 °C for 3–20 hours. Then, the temperature is increased to 60–150 °C, maintained for 2–10 hours, and then decreased to 10–50 °C. This process, including the initial and maintenance times of 5–30 hours, is continued for a total of 2–9 days, with a heating / cooling rate of 1–10 °C / hour. The glass vial containing P5 is carefully broken, and P5 is removed and immersed in 5–50 mL of ultrapure water for dialyzing purification for 1–14 days to remove unreacted monomers. The immersion solution is changed every half day to one day during this period to obtain gel P5. Or, (3) synthesize poly(polyethylene glycol dimethacrylate- co (Acryloylated 3-aminophenylboronic acid) P6: In a 10-200 mL beaker, add 5-50 mL of PEGDMA and 5-100 g of APBA sequentially, with a molar ratio of PEGDMA to APBA of 0.5:1 to 3:

1. Simultaneously add 10-200 mL of ultrapure water as a solvent, sonicate to dissolve, and shake to mix thoroughly. Then, add 100-1000 g of... After mixing mg of the initiator azobisisobutyronitrile (AIBN) by shaking for 30–2880 minutes, the mixture is poured into 10–100 mL glass sample vials, the vials are sealed, and placed in an oven. Initially, the temperature is maintained at 10–50 °C for 3–20 hours. Then, the temperature is increased to 60–150 °C, maintained for 2–10 hours, and then decreased to 10–50 °C. This process, including the initial and maintenance times of 5–30 hours, is continued for a total of 2–9 days, with a heating / cooling rate of 1–10 °C / hour. The glass vial containing P6 is carefully broken, and P6 is removed and immersed in 5–50 mL of ultrapure water for dialyzing purification for 1–14 days to remove unreacted monomers. The immersion solution is changed every half day to one day during this period to obtain gel P6.

4. A method for preparing a polymer material with CTCs captured according to claim 1, characterized in that: The specific process of cell imprinting on the material surface is as follows: (1) Preparation of smart responsive molecularly imprinted hydrogel P4+: First, 10 3 ~10 6 Tumor cells, specifically liver cancer, lung cancer, breast cancer, cervical cancer, prostate cancer, colon cancer, or ovarian cancer cells, are placed in a cell culture dish with a diameter of 30-150 mm and a height of 30-150 mm. 1-30 mL of cell culture medium is added, and the cells are cultured for 2-48 hours. The culture medium is removed, and the cells are washed 1-10 times with PBS. 0.1-5 mL of a pre-prepared and membrane-filtered paraformaldehyde solution is added. The membrane is 0.1-0.8 μm thick, and the paraformaldehyde solution has a mass concentration of 0.5-10%. o C~37 o Fix cells at 1-15 min, remove the fixative, and wash 1-10 times with PBS. Then, add 5-50 mL of PEGDMA and 5-100 g of AHH to a 10-200 mL beaker, with a molar ratio of PEGDMA to AHH of 0.5:1-3:

1. Add 10-200 mL of ultrapure water as a solvent, sonicate to dissolve, and vortex to mix. Next, add 100-1000 mg of the initiator azobisisobutyronitrile (AIBN), vortex to mix for 30-28 minutes, and then transfer 1-200 mL of the solution to a cell culture dish containing fixed cells. Incubate at 300°C on a shaker. Shake at 0.5-10 rpm for 0.5-10 hours to allow the functional monomers in the solution to seek target molecules on the cell surface. Then, transfer the culture dish to an oven and maintain the temperature at 10-50°C for the first 3-20 hours. Afterward, gradually increase the temperature to 60-150°C, maintain for 2-10 hours, and then decrease to 10-50°C. Including the initial and maintenance times of 5-30 hours, continue this process for a total of 2-9 days, with a heating / cooling rate of 1-10°C / hour. This yields P4-cell hydrogels with cell imprints and functional groups. Rinse the P4-cell hydrogels with ultrapure water, slowly adding ultrapure water from the edge to detach the P4-cell hydrogel from the culture dish. Remove the P4-cell hydrogel and dialysis it in 1-50 mL of ultrapure water for 1-14 days, changing the dialysis solution every half day to one day. Afterward, remove the gel and dialysis it in 1-50 mL of pH 10 solution. A ~8.0% trypsin solution, wherein the trypsin solution consists of 0.1~0.5% trypsin and 0.005%~0.05% ethylenediaminetetraacetic acid; after 0.1~10 hours, the gel is rinsed 1~10 times with 0.5~10 mL of phosphate buffer solution, wherein the phosphate buffer solution is PBS = 10 mM, pH=7.4, and is stored in 1~100 mL of PBS solution; The above process is to remove cell templates, unreacted monomers and AIBN residues, thereby preparing a smart responsive molecularly imprinted hydrogel P4+ carrying functional monomers and cell micromorphology. Or, (2) Preparation of smart responsive molecularly imprinted hydrogel P5+: First, 10 3 ~10 6 Tumor cells, specifically liver cancer, lung cancer, breast cancer, cervical cancer, prostate cancer, colon cancer, or ovarian cancer cells, are placed in cell culture dishes with a diameter of 30-150 mm and a height of 30-150 mm. 1-30 mL of cell culture medium is added, and the cells are cultured for 2-48 hours. The culture medium is removed, and the cells are washed 1-10 times with PBS. 0.1-5 mL of a pre-prepared and membrane-filtered paraformaldehyde solution is added. The membrane is 0.1-0.8 μm thick, and the paraformaldehyde solution has a mass concentration of 0.5-10%. o C~37 o Fix cells at 1-15 min, remove the fixative, and wash 1-10 times with PBS. Then, add 5-50 mL of PEGDMA and 1-500 g of AH to a 10-200 mL beaker (PEGDMA to AH molar ratio of 0.5:1-3:1), along with 10-200 mL of ultrapure water as the solvent. Sonicate to dissolve and mix thoroughly. Next, add 100-1000 mg of the initiator azobisisobutyronitrile (AIBN), and mix thoroughly for 30-28 minutes. Then, take 1-200 mL of the solution and add it to a cell culture dish containing fixed cells. Incubate at 300°C on a shaker. Shake at 0.5-10 rpm for 0.5-10 hours to allow the functional monomers in the solution to seek target molecules on the cell surface. Then, transfer the culture dish to an oven and maintain the temperature at 10-50°C for the first 3-20 hours. Afterward, increase the temperature to 60-150°C, maintain for 2-10 hours, and then decrease to 10-50°C. Including the initial and maintenance times of 5-30 hours, continue this process for a total of 2-9 days, with a heating / cooling rate of 1-10°C / hour. This yields P5-cell hydrogels with cell imprints and functional groups. Rinse the P5-cell hydrogels with ultrapure water, slowly adding ultrapure water from the edge to detach the P5-cell hydrogel from the culture dish. Remove the P5-cell hydrogel and dialysis it in 1-50 mL of ultrapure water for 1-14 days, changing the dialysis solution every half day to one day. Afterward, remove the gel and dialysis it in 1-50 mL of pH 10 solution. A ~8.0% trypsin solution, wherein the trypsin solution consists of 0.1~0.5% trypsin and 0.005%~0.05% ethylenediaminetetraacetic acid; after 0.1~10 hours, the gel is rinsed 1~10 times with 0.5~10 mL of phosphate buffer solution, wherein the phosphate buffer solution is PBS=10 mM, pH=7.4, and stored in 1~100 mL of PBS solution; The above process is to remove cell templates, unreacted monomers and AIBN residues, thereby preparing a smart responsive molecularly imprinted hydrogel P5+ carrying functional monomers and cell micromorphology. Or, (3) Prepare smart responsive molecularly imprinted hydrogel P6+: First, 10 3 ~10 6 Tumor cells, specifically liver cancer, lung cancer, breast cancer, cervical cancer, prostate cancer, colon cancer, or ovarian cancer cells, are placed in cell culture dishes with a diameter of 30-150 mm and a height of 30-150 mm. 1-30 mL of cell culture medium is added, and the cells are cultured for 2-48 hours. The culture medium is removed, and the cells are washed 1-10 times with PBS. 0.1-5 mL of a pre-prepared and membrane-filtered paraformaldehyde solution is added. The membrane is 0.1-0.8 μm thick, and the paraformaldehyde solution has a mass concentration of 0.5-10%. o C~37 o Fix cells at 1-15 min, remove the fixative, and wash 1-10 times with PBS. Then, add 5-50 mL of PEGDMA and 5-100 g of APBA (molar ratio of PEGDMA to APBA 0.5:1-3:1) to a 10-200 mL beaker, along with 10-200 mL of ultrapure water as the solvent. Sonicate to dissolve and mix thoroughly. Next, add 100-1000 mg of the initiator azobisisobutyronitrile (AIBN), and mix thoroughly for 30-28 minutes. Then, take 1-200 mL of the solution and add it to a cell culture dish containing fixed cells. Incubate at 300°C on a shaker. Shake at 0.5-10 rpm for 0.5-10 hours to allow the functional monomers in the solution to seek target molecules on the cell surface. Then, transfer the culture dish to an oven and maintain the temperature at 10-50°C for the first 3-20 hours. Afterward, increase the temperature to 60-150°C, maintain for 2-10 hours, and then decrease to 10-50°C. Including the initial and maintenance times of 5-30 hours, continue this process for a total of 2-9 days, with a heating / cooling rate of 1-10°C / hour. This yields P6-cell hydrogels with cell imprints and functional groups. Rinse the P6-cell hydrogels with ultrapure water, slowly adding ultrapure water from the edge to detach the P6-cell hydrogel from the culture dish. Remove the P6-cell hydrogel and dialysis it in 1-50 mL of ultrapure water for 1-14 days, changing the dialysis solution every half day to one day. Afterward, remove the gel and dialysis it in 1-50 mL of pH 10 solution. A ~8.0% trypsin solution, wherein the trypsin solution consists of 0.1~0.5% trypsin and 0.005%~0.05% ethylenediaminetetraacetic acid; after 0.1~10 hours, the gel is rinsed 1~10 times with 0.5~10 mL of phosphate buffer solution, wherein the phosphate buffer solution is PBS=10 mM, pH=7.4, and stored in 1~100 mL of PBS solution; The above process is to remove cell templates, unreacted monomers, and AIBN residues, thereby preparing a smart responsive molecularly imprinted hydrogel P6+ carrying functional monomers and cell micromorphology.

5. A method for preparing a polymer material captured by CTCs according to claim 1, wherein the synthetic route of the CTCs binding unit is shown below: ； The specific preparation process is as follows: (1) Synthesis of methacrylyl dihistidine: Add 1-10 g of sodium hydroxide to 1-100 mL of an ultrapure aqueous solution containing 10-500 mg of bishistidine. Stir the mixture at 15-60 °C for 1-24 hours. Then add 0.01-1 mL of methacryloyl chloride to the mixture and continue stirring at 15-60 °C for 1-3 days. After evaporating the solvent, add 10-100 mL of methanol, sonicate to dissolve for 10-60 minutes, centrifuge at 2000-8000 rpm for 5-30 minutes, collect the supernatant, and discard the precipitate. Then, freeze-dry the supernatant to obtain a white powder product. (2) Synthesis of methacrylated histidine: Add 1-20 g of sodium hydroxide to 1-100 mL of an ultrapure aqueous solution containing 1-50 g of histidine. Stir the mixture at 15-60 °C for 1-24 hours. Then add 1-10 mL of methacryloyl chloride to the mixture and continue stirring at 15-60 °C for 1-3 days. After evaporating the solvent, add 10-100 mL of methanol, sonicate to dissolve for 10-60 minutes, centrifuge at 2000-8000 rpm for 5-30 minutes, collect the supernatant, and discard the precipitate. Then, freeze-dry the supernatant to obtain a white powder product. (3) Synthesis of acrylated 3-aminophenylboronic acid: Add 1-30 g of sodium bicarbonate to 1-100 mL of an ultrapure water and tetrahydrofuran solution containing 1-50 g of 3-aminophenylboronic acid, wherein the volume ratio of ultrapure water to tetrahydrofuran is 0.05:1-10:

1. Stir the mixture at 15-60°C for 1-24 hours. Then add 0.01-1 mL of acryloyl chloride to the mixture and continue stirring at 15-60°C for 1-3 days. After evaporating the solvent, add 10-100 mL of methanol, sonicate to dissolve for 10-60 minutes, centrifuge at 2000-8000 rpm for 5-30 minutes, collect the supernatant, and discard the precipitate. Then, freeze-dry the supernatant to obtain a white powder product.

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