CN116731234A

CN116731234A - CTCs captured polymer material, preparation and application

Info

Publication number: CN116731234A
Application number: CN202310624147.7A
Authority: CN
Inventors: 卿光焱; 孙文静
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2023-05-30
Filing date: 2023-05-30
Publication date: 2023-09-12

Abstract

The invention relates to a polymer material captured by CTCs, and preparation and application thereof, wherein the polymer material captured by CTCs, P4, P5 or P6 respectively consists of a network skeleton P3 and CTCs functional monomers (methacryloylated bis-histidine; methacryloylated histidine; acrylylated 3-aminophenylboronic acid), and the preparation and polymerization process is random copolymerization of the network skeleton P3 and the CTCs functional monomers. The invention also relates to preparation of the intelligent responsive hydrogel, CTCs are used as templates, and cell microscopic morphology is introduced into the surface of the hydrogel polymer material carrying the functional groups to form cell imprinting, so that the dual intelligent responsive hydrogel cancer diagnosis biological material carrying the functional groups and molecular imprinting is obtained. The material has the capability of efficient enrichment and nondestructive release, and can be applied to the fields of CTCs enrichment in cancer diagnosis and treatment, participation of nondestructive release in downstream disease molecular biological analysis, judgment of cancer progress and the like.

Description

CTCs captured polymer material, preparation and application

Technical Field

The invention relates to the biomedical field, in particular to a method for screening an anti-adhesion and anti-swelling gel skeleton, a method for preparing functional monomers and a cell imprinting microstructure of intelligent response hydrogel, and clinical application of an intelligent response hydrogel cancer diagnosis biological material.

Background

The low survival rate of liver cancer is due to the expensive screening cost and the slow pain of the liver, and most patients are already in the middle and late stages when diagnosed, missing the optimal treatment period. Serum marker Alpha-fetoprotein (AFP) is an existing clinical detection means for screening, diagnosing, assessing recurrence risk and observing therapeutic effect of liver cancer. However, there are many positive cases in patients with low AFP, which greatly reduces the sensitivity of AFP. Therefore, the search of more accurate and sensitive biomarkers has great significance for preventing and treating liver cancer.

Circulating Tumor Cells (CTCs) in peripheral blood are important markers for liquid biopsies because they carry genetic and apparent information about tumor tissue. As one of the effective approaches for tumor diagnosis and treatment, the development of CTCs capturing materials has very profound clinical significance for early diagnosis, postoperative evaluation and the like of tumors. However, since CTCs are very few in number in the blood and are susceptible to damage during capture, accurate capture and non-destructive release of CTCs is extremely challenging. Existing CTCs capture materials can be mainly divided into two main types of capture materials based on natural antibodies and artificial antibodies according to different types of capture units. The capture material based on the natural antibody has remarkable advantages in the aspect of specificity, but the capture material based on the artificial antibody can be used for more flexibly carrying out personalized and diversified functional design, so that a plurality of problems faced by the current CTCs capture can be better solved. In addition, artificial antibody-based materials have certain advantages in terms of material stability and manufacturing costs. CTCs have large size, mechanical plasticity and dielectrophoresis properties compared to blood cells. This feature allows CTCs to be isolated by a variety of separation strategies such as membrane filtration, density gradient layering, inertial focusing, and dielectric migration. The invention develops a gel skeleton which has good biocompatibility, obvious anti-adhesion capability and swelling resistance and is designed based on cell microcosmic morphology, and intelligent responsive polymer surface hydrogel with different polymerization degrees, enrichment capability and easy processing is prepared by copolymerizing multi-hydrogen bond artificial antibody functional monomers. This class of smart responsive hydrogels shows excellent effects on the separation and enrichment of CTCs, mainly in terms of highly selective capture and non-destructive release. Therefore, the method is expected to be widely applied to enrichment and identification of CTCs, and further to clinical cancer diagnosis and treatment.

Disclosure of Invention

The invention aims to provide a preparation method of an intelligent responsive biological material applied to cancer diagnosis, which has the capability of efficient enrichment and nondestructive release. The biological material for cancer diagnosis and treatment is prepared by preparing a functional monomer with multi-hydrogen bond selective recognition capability and combining cell microcosmic morphology in a gel skeleton with good biocompatibility, non-swelling, anti-adhesion and plasticity by using copolymerization reaction initiated by Azodiisobutyronitrile (AIBN).

The invention adopts the following scheme to realize the purposes:

first, the present invention aims to provide a class of intelligent responsive polymer cancer biological diagnostic materials, which have hydrogel polymer network backbones of P1, P2 and P3 and hydrogel functional polymers of P4, P5 and P6 targeting CTCs.

The specific description is as follows: the hydrogel polymer P1 or P2 consists of a network skeleton (P1: acrylic acid; P2: acrylamide) and a cross-linking agent (N, N-methylene bisacrylamide, MBA), wherein x and 1-x are the mole percentages of the substances of the network skeleton and the cross-linking agent, 1-x=0.01-0.5, the mass of the network skeleton P3 (polyethylene glycol dimethacrylate, PEGDMA) is the same as that of the acrylic acid and the acrylamide, and n=13.

The hydrogel functional polymer P4, P5 or P6 of the cancer biological diagnostic material for targeting CTCs is respectively composed of a network skeleton P3 and functional monomers (P4: methacryloyl bis-histidine; P5: methacryloyl histidine; P6: acryloyl 3-aminophenylboronic acid), wherein y and 1-y are the mole percentages of substances of P3 and the functional monomers, y=0.01-0.5, and n=1-15.

The molecular structure of P1, P2, P3, P4, P5 and P6 is schematically shown below (the polymerization process is random copolymerization):

secondly, the preparation method of the intelligent responsive hydrogel comprises the following steps: by utilizing an atom transfer radical polymerization reaction mechanism, the functional monomer with high specificity, affinity and multi-hydrogen bond selective recognition capability with CTCs is randomly copolymerized to the surface of a gel network skeleton P3 with good biocompatibility, no swelling, adhesion resistance and plasticity by initiating with Azodiisobutyronitrile (AIBN) to obtain the hydrogel cancer diagnosis biological material carrying functional groups.

Finally, the preparation method of the intelligent responsive hydrogel comprises the following steps: taking CTCs as templates, introducing the cell microcosmic morphology into the surface of a hydrogel polymer material carrying functional groups to form cell imprinting, and obtaining the dual intelligent responsive hydrogel cancer diagnosis biological material carrying the functional groups and the molecular imprinting. The intelligent responsive hydrogel biomaterial can be applied to the fields of CTCs enrichment in cancer diagnosis and treatment, participation in downstream disease molecular biological analysis by nondestructive release, judgment of cancer progress and the like.

Drawings

FIG. 1, evaluation of CTCs enrichment efficiency and anti-interference ability of intelligent responsive hydrogel P4+ blood samples. Fig. 2, receiver operating profile of smart responsive hydrogel p4+ clinical liver cancer differentiation (Receiver operating characteristic, ROC).

FIG. 3, gel scaffold selection for smart responsive hydrogels.

FIG. 4, HH@SiO ₂ Surface potential of the material.

FIG. 5, affinity test of a bis-histidine functional monomer with cell membrane proteins.

FIG. 6, intelligent responsive hydrogel internal moisture content test.

FIG. 7, preparation and characterization of intelligent responsive hydrogel cell blot microcosmic morphology.

FIG. 8, protein adsorption resistance of smart responsive hydrogel gel scaffold.

Figure 9, processability of smart responsive hydrogels.

Figure 10, smart responsive hydrogel CTCs capture re-release cycle availability.

Detailed Description

Taking a P4+ hydrogel as an example, the experiments of the enrichment efficiency and the anti-interference capability of CTCs are described: 1. method for counting the number of White Blood Cells (WBCs) in a blood sample.

1 ml of whole blood (physical examination of healthy people, purple blood collection tube, on-the-fly) was obtained from a secondary civil hospital affiliated with university of Dalian medical science, from which 1 microliter (for smaller errors, whole blood samples may also be diluted in a gradient with phosphate buffered saline (PBS=10 mM, pH=7.4, supra)). Then, 10. Mu.l of phycoerythrin fluorescein-labeled WBCs common antigen Antibody (P-Phycoerythrin (PE) anti-human CD45 (leukocyte common antigen, LCA) Antibody, enabling Legendary Discovery, 368510) was added to 1 ml of PBS solution, and mixed by shaking for 3 minutes. Thereafter, 500. Mu.l of the above WBCs solution was added to 1. Mu.l of blood, and the mixture was carefully blown and mixed with a pipette, and stained at room temperature for 30 minutes. Subsequently, centrifugation at 1000rpm for 3 minutes, the supernatant was decanted, unbound antibody was washed with 1 ml of cell culture medium [ 89% Dulbecco' smodified eagle medium by volume, DMEM (Gibco), 10% Fetal bovine serum, FBS (ExCell Bio) and 1% penicillin-streptomycin, 100X (Biyun days), the following cell culture media being composed here ], and centrifuged, and the washing centrifugation procedure was repeated 3 times. Finally, the cell pellet was resuspended in 1 ml of cell culture medium, 10. Mu.l was inoculated onto a blood cell counting plate by a pipette, placed on an inverted research fluorescence microscope (Nikon, niKon Ti-s), the PE channel (emission wavelength: 575 nm) was selected, and the number was observed and recorded with the blood cell counting plate, the number being N ₀ . Then, the number of WBCs in 1 ml of cell culture medium was N ₀ /4×10 ⁴ WBCs number N in 1 ml whole blood ₀ /4×10 ⁷ 。

The capture process of CTCs and WBCs by p4+ hydrogels.

At 10 ⁵ Experimental procedure description was performed with respect to the number of SMMC-7721 cells:

SMMC-7721 cells were grown to 90% cell density (cell coverage) in 100mm diameter and 100mm height cell culture dishes from the cell incubator (37 ℃ C., 5% CO by volume) ₂ Air, the same applies below). The cell culture medium on the cell surface was removed by washing 3 times with 3 ml of PBS solution (PBS 10mM, pH 7.4, the same applies below) preheated at 37 ℃. Then, 1 ml of pancreatin solution [ Trypsin EDTA solution:0.25% Trypsin, preheated at 37℃in advance, was added&0.02% EDTA; pH-8.0 (Viva Cell), digesting for 1-2 min until the cells in the Cell culture dish are separated, rounding the Cell shape, pouring out 1 ml of pancreatin solution, immediately adding 6 ml of Cell culture medium preheated at 37 ℃ in advance, sucking the Cell culture medium by a pipette, and blowing off the cells adhered to the bottom of the Cell culture dish. Subsequently, 1 ml of the solution was pipetted from 6 ml of cell culture medium containing SMMC-7721 cells into a 1.5 ml sterile EP tube, labeled a. Then, 100. Mu.l of the solution from A was placed in a new 1.5 ml sterile EP tube, labeled B, 900. Mu.l of the above cell culture medium was added to B, mixed well by a pipette, 10. Mu.l was taken therefrom, inoculated onto a blood cell counting plate, placed on an inverted research fluorescence microscope, and the number was observed and recorded with a cell counter, which number was N ₁ . Then, the number of SMMC-7721 cells in 1 ml cell culture medium in tube B was N ₁ /4×10 ⁴ ，10 ⁵ The volume of the SMMC-7721 cells was 40/N ₁ Milliliters.

Will be 40/N ₁ Ml of SMMC-7721 cells were spiked with 1 ml of whole blood sample and seeded onto the surface of P4 hydrogel (area: 14 cm) ² Height: 0.7 cm), the P4 gel was placed in a cell incubator. After 1 hour, the P4 hydrogel surface was slowly rinsed with 1 ml of PBS to remove red blood cells, unbound SMMC-7721 cells and WBCs. Then, 300. Mu.l of pancreatin solution (Trypsin EDTA solution:0.25% Trypsin) preheated at 37℃was added&0.02% EDTA; pH-8.0 (Viva Cell)), digested for 1-2 min, immediately added 1 ml of fine pre-heated at 37℃Cell culture medium was aspirated with a pipette gun to blow off cells adhering to the surface of the P4 hydrogel. The solution was collected in a 1.5 ml EP tube and centrifuged at 1000rpm for 3 minutes, and the supernatant was decanted, leaving behind a cell pellet. During this period, 10. Mu.l of fluorescein isothiocyanate-labeled epithelial cell adhesion molecule Antibody (Fluorescein isothiocyanate isomer (FITC) anti-human CD326 (epithelial cell adhesion molecule, epCAM) anti-body, enabling Legendary Discovery, 369814) and 10. Mu.l of PE anti-human CD45 anti-body were added to 1 ml of PBS solution, and mixed by shaking for 3 minutes to obtain a fluorescent Antibody mixed solution. Then, 500. Mu.l of the above fluorescent antibody mixed solution was added to the EP tube containing the cell pellet, and stained at room temperature for 30 minutes. Subsequently, the washing and centrifugation process was repeated 3 times by washing with 1 ml of PBS solution and centrifuging. Then, the cell pellet was resuspended in 1 ml of cell culture medium, 10. Mu.l was inoculated onto a blood cell counting plate by a pipette, placed on an inverted research fluorescence microscope, and the number of SMMC-7721 cells, which was N, was observed and recorded by selecting FITC (emission wavelength: 525 nm) ₂ . The capturing rate of the P4 hydrogel to SMMC-7721 cells is N ₂ 40X 100%. Finally, 10. Mu.l of the cell suspension was added to 1 ml of the cell culture medium, mixed uniformly, 10. Mu.l was taken by a pipette, inoculated onto a blood cell counting plate, placed on an inverted research type fluorescence microscope, and a PE channel (emission wavelength: 575 nm) was selected, and the number was observed and recorded by a cell counter, the number being N ₃ . The trapping efficiency of WBCs is N ₃ /10N ₀ X 100%. The above experimental data were tested in triplicate.

3. Numerical calculation and result analysis in specific experimental operations.

From the blood cell counting plate, the number of each cell was counted by a cell counter as follows:

N ₀ ＝1451、1673、1543；N ₁ ＝181、175、195；N ₂ ＝37、38、37；N ₃ =3250, 3547, 3628. Then, P4+ hydrogel pair 10 ⁵ The capture efficiency of the individual SMMC-7721 cells is shown by formula N ₂ Calculated as 92.5%, 95%, 92.5%, the trapping efficiency of WBCs in 1 ml of whole blood was calculated as 92.5%, 95%, 92.5% by the formula N ₃ /10N ₀ The x 100% is calculated as 22.4%, 21.2% and 23.51%.

From FIG. 1, it can be seen that the smart responsive hydrogel exhibits excellent capturing ability to SMMC-7721 cells of different densities, and is expected to be developed for selective enrichment of CTCs.

(II) Multi-index Joint diagnosis gradually improves cancer diagnosis efficiency

Referring to background studies, researchers entered clinical samples with AFP less than 20ng/mL for a total of 70, 29 of the liver cancer patients, 18 of the liver cirrhosis patients and 23 healthy volunteers.

Description of the experimental procedure was performed with 1 patient and p4+ hydrogel as examples:

a sample of 5 ml whole blood (purple blood collection tube, on-line) was obtained from a second civil hospital affiliated to university of major company, and 450g of 5 ml blood was centrifuged for 5 minutes to remove serum and collect plasma. Since the blood volume of 5 ml is much greater than the hydrogel carrying capacity, the collected plasma was lysed with red blood cell lysate (0.8 g ammonium chloride (NH ₄ Cl), 37mg ethylenediamine tetraacetic acid (Ethylenediaminetetraacetic acid, EDTA), 84mg sodium bicarbonate (NaHCO) ₃ ) 100 ml deionized water). Red blood cell lysate was added to the plasma in 10 times the plasma volume, gently swirled and mixed, and lysed for 5 minutes. At 4℃for 5 minutes, 450g was centrifuged, the red supernatant was discarded, 10 times the remaining cell volume of PBS solution was added, the pellet was resuspended, and 450g was centrifuged for 3 minutes, and the supernatant was discarded. The pellet was resuspended in 1 ml of cell culture medium [ volume concentration 89%Dulbecco's modified eagle medium,DMEM (Gibco), 10%Fetal bovine serum,FBS (Excell Bio) and 1% penicillin-streptomycin, 100X (Biyun day), the following cell culture medium was composed as described herein ], and homogeneously inoculated (dropped onto the gel surface) on the surface of the P4+ hydrogel (area: 14cm ² Height: 0.7 cm), the surface of the P4 hydrogel was slowly rinsed with 1 ml of PBS solution after being placed in a cell incubator for 1 hour, and the remaining erythrocytes and unbound cells were removed. During this period, 10. Mu.l of fluorescein isothiocyanate-labeled epithelial cell adhesion molecule Antibody (Fluorescein isothiocyanate isomer (FITC) anti-human CD326 (epithelial cell adhesion molecule, epCAM) anti-body, enabling Legendary Discovery, 369814) and 0.4. Mu.l of 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole (DAPI), invitrogen) ^TM D1306) was added to 1 ml of PBS solution and mixed with shaking for 3 minutes. Evenly dripping the mixture on the surface of the P4+ hydrogel, and dyeing the mixture for 30 minutes at room temperature. Subsequently, the surface of the P4+ hydrogel was slowly rinsed with 1 ml of PBS solution, and the rinsing operation was repeated 3 times. The surface of the P4+ hydrogel was inverted downward (with the upper surface of the rinsed P4+ hydrogel face downward), placed on an inverted research fluorescence microscope (Nikon, niKon Ti-s), and FITC channel and DAPI channel (emission wavelength: 416 nm) were selected, and the number of circulating tumor cells was observed and recorded. In addition, a solution for washing the surface of the P4+ hydrogel was collected, centrifuged at 1000rpm for 3 minutes, and a natural Killer cell glycoprotein Antibody (Tetramethylindo (di) -carbocyanines (Cy 5) anti-rubbit CD56 (Neural cell adhesion molecule, NCAM) Antibody, beijing Boboson Biotechnology Co., ltd., 0805R) was used in 500. Mu.l of tetramethyl indole (di) -carbocyanine label, which was stained at room temperature for 30 minutes, and immediately loaded onto a flow cytometer (Japan, SONY SH 800S), and the natural Killer cell (Nature Killer, NK) ratio was measured by a Cy5 channel (emission wavelength: 670 nm) (flow meter measures the number of cells with red fluorescence in the solution as a percentage of the total cell number). AFP (hospital provided), CTCs (polymeric material captured by CTCs herein) and NK (flow detection) can be considered as three different classification indicators for liver cancer (specific data are too huge, not provided herein, but can guarantee the true reliability of the data). Three index data are input into a support vector machine (Support vector machines, SVM) in a deep learning model for data integration and analysis, and data of 70 samples (29 liver cancer patients, 18 liver cirrhosis patients and 23 healthy volunteers) are put into a model system and the cancer distinguishing capability is judged through a binary classification model-ROC curve. The area under the curve (AUC) values of the ROC curve (AFP: 0.633; AFP+NK;0.837; AFP+CTCs:0.889; AFP+CTCs+NK: 0.99) indicate that the combination of the three indexes can significantly enhance the distinguishing ability of liver cancer (see FIG. 2).

The starting materials used in the examples:

acrylic Acid (AA), acrylamide (AAm), azobisisobutyronitrile (AIBN), N-methylenebisacrylamide(MBA), polyethylene glycol dimethacrylate (PEGDMA), 3-aminophenylboronic acid (PBA) and histidine (His) were purchased from Sigma-Aldrich Corp (St. Louis, mitsui, U.S.A.). The bis-histidine is commercially available from Shanghai Yao Biotechnology Co. Methacryloyl chloride, methanol, sodium hydroxide, hydrochloric acid are commercially available from the company Semerle Feier technologies. Other solvents and reagents were purchased from national pharmaceutical group chemical company, ltd. The water used in this study was ultrapure water obtained from the Milli-Q system. ¹ H spectra were obtained at AVANCE III HD MHz.

Example 1

Preparation of P1, P2 and P3 hydrogel polymer network backbones by atom transfer radical polymerization

(1) Synthesis of Polyacrylic acid (PAA, P1)]: the polymerization process used was free radical random copolymerization, the initiator used was azobisisobutyronitrile (AIBN, CAS No.78-67-1, sigma-Aldrich Corp), the reaction 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 mass molar ratio of 65:1, simultaneously adding 30mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and uniformly mixing. Subsequently, initiator AIBN (118.1 mg,0.7 mmol) was added, after shaking and mixing for 1 hour, poured into a 50 ml glass sample bottle, sealed, placed in an oven, first kept at 30℃for 12 hours, then started to heat up to 80℃and then kept at temperature for 6 hours and then cooled down to 30℃for 2 days (including the preceding 18 hours), and both heating and cooling rates were 5℃per hour. The vial containing P1 was carefully broken, P1 was removed, and the vial was immersed in ultrapure water for dialysis and purification for 7 days to remove unreacted monomers, during which time the immersion solution was changed every half a day. By infrared (Fourier-transform infrared spectroscopy, FT-IR) and scanning electron microscopy (Energy dispersive spectro) Elemental analysis characterization of scopy, EDS) demonstrated successful synthesis of P1 (structure see above, 1-x=0.015). Number average molecular weight=20000 Da, polydispersity (PDI) =1.3. FT-IR:3446cm ^-1 (acrylic hydroxyl group), 1725cm ^-1 (N, N-methylenebisacrylamide carbonyl); EDS:58.69% (C), 14.87% (N), 26.31% (O).

(2) Synthesis of Polyacrylamide, PAAm, P2]: the polymerization process used was free radical random copolymerization, the initiator used was azobisisobutyronitrile (AIBN, CAS No.78-67-1, sigma-Aldrich Corp), the reaction 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 in a mass molar ratio of 65:1, simultaneously adding 30mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and uniformly mixing. Subsequently, initiator AIBN (118.1 mg,0.7 mmol) was added, after shaking and mixing for 1 hour, poured into a 50 ml glass sample bottle, sealed, placed in an oven, first kept at 30℃for 12 hours, then started to heat up to 80℃and then kept at temperature for 6 hours and then cooled down to 30℃for 2 days (including the preceding 18 hours), and both heating and cooling rates were 5℃per hour. The vial containing P2 was carefully broken up, the P2 was removed, and the vial was immersed in ultrapure water for dialysis and purification for 7 days to remove unreacted monomers, during which time the immersion solution was changed every half a day. Successful synthesis of P2 was demonstrated by infrared (Fourier-transform infrared spectroscopy, FT-IR) and scanning electron microscopy (Energy dispersive spectroscopy, EDS) elemental content analysis characterization (structure see above, 1-x=0.015). Number average molecular weight= 640000Da, polydispersity (PDI) =2.1. FT-IR:3415cm ^-1 (acrylamide free amine group), 1725cm ^-1 (N, N-methylenebisacrylamide carbonyl); EDS:56.54% (C), 19.68% (N), 22.96% (O).

(3) Synthesis of polyethylene glycol dimethacrylate [ Polypoly (ethylene glycol) dimethacrylate, PP, P3]: the polymerization method is free radical random copolymerization, and the initiator is Azobisisobutyronitrile (AIBN)CAS No.78-67-1, sigma-Aldrich Corp) the reaction monomer used was polyethylene glycol dimethacrylate (PEGDMA, n=13, CAS No.25852-47-5, sigma-Aldrich Corp). The mass of the reaction monomer is the same as that of P1, and the specific synthesis steps are as follows: PEGDMA (9.0 mL,13.32 mmol) was added to a 50 mL EP tube, while 30mL of ultrapure water was added as a solvent, sonicated and mixed by shaking. Subsequently, initiator AIBN (118.1 mg,0.7 mmol) was added, after shaking and mixing for 1 hour, poured into a 50 ml glass sample bottle, sealed, placed in an oven, first kept at 30℃for 12 hours, then started to heat up to 80℃and then kept at temperature for 6 hours and then cooled down to 30℃for 2 days (including the preceding 18 hours), and both heating and cooling rates were 5℃per hour. The vial containing P3 was carefully broken up, P3 was removed, and the vial was immersed in ultrapure water for dialysis purification for 7 days to remove unreacted monomers, during which time the immersion solution was changed every half a day. Successful synthesis of P3 was demonstrated by infrared (Fourier-transform infrared spectroscopy, FT-IR) and scanning electron microscopy (Energy dispersive spectroscopy, EDS) elemental content analysis characterization (structure see above, n=13, degree of polymerization 19). Number average molecular weight=16000 Da, polydispersity (PDI) =0.3. FT-IR: 3059. 2341, 1754cm ^-1 (polyethylene glycol dimethacrylate carbonyl and backbone); EDS:89.64% (C), 10.27% (O).

Example 2

Gel scaffold selection for smart responsive hydrogels

P1, P2 and P3 gels weighing 1g were immersed in 10 ml of phosphate buffer solution (pbs=10 mm, ph=7.4) and removed at 0.2, 0.5, 1, 4, 24, 48, 72, 96 and 120 hours, respectively, and the filters were dried to suck the surface liquid and weighed. Wherein 0 hour weight is W ₀ Weight of the rest of time is W _x Using the formula: SR (%) = (W) _x -W ₀ )×100/W ₀ The swelling ratio was calculated and plotted. As can be seen from FIG. 3, P1 and P2 swell to different extents, which is detrimental to the maintenance of the microscopic morphology of the cell blot, while P3 swells with negligible deformation, and can be selected for the preparation substrate of the smart responsive gel.

Example 3

Structure and synthesis method of functional monomer

To prepare the intelligent responsive hydrogels described above, a series of functional monomers need to be synthesized, and the specific methods of their synthesis are as follows:

(1) Synthesis of methacryloylated bis-histidine.

Sodium hydroxide (2.4 g,0.06 mol) was added to 50mL of an ultrapure water solution containing bis-histidine (two histidines are covalently linked by a peptide bond) (100 mg,0.34 mmol), the mixture was stirred at ambient temperature for 5 hours, then methacryloyl chloride (660. Mu.L, 6.8 mmol) was added to the mixture, and stirring was continued at ambient temperature for 24 hours. After evaporation of the solvent, 50ml of methanol was added, sonicated for 20 minutes, centrifuged at 8000rpm for 10 minutes, the supernatant was aspirated, and the precipitate was discarded. Thereafter, the supernatant was freeze-dried to obtain a product (106.97 mg, yield: 86.77%) as a white powder. ¹ HNMR (700 MHz, deuterated dimethyl sulfoxide (DMSO-d ₆ ))：δ(ppm)：8.18(d,J＝7.0Hz,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＝11.9,6.6Hz,1H,CH ₂ ),3.45～3.42(m,1H,CH ₂ ),2.62(dd,J＝14.3,7.1Hz,2H,CH ₂ ),1.52(s,3H,CH ₃ )；MADLI-MS:m/z calcd for C ₂₇ H ₃₉ N ₇ O ₁₃ :360.1546；found:361.1542。

(2) Synthesis of methacryloylated histidine.

Sodium hydroxide (16.0 g,0.4 mol) was added to 50mL of an ultrapure water 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 evaporation of the solvent, 50ml of methanol was added, sonicated for 20 minutes, centrifuged at 8000rpm for 10 minutes, the supernatant was aspirated, and the precipitate was discarded. Thereafter, the supernatant was freeze-dried to obtain a product (2.08 g, yield: 93.27%) as a white powder. ¹ H NMR (700 MHz, heavy water (D) ₂ O))：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,CH ₂ ),1.96(m,3H,CH ₃ )；MADLI-MS:m/z calcd for C ₁₀ H ₁₃ N ₃ O ₃ :223.2285；found:224.1015。

(3) Synthesis of acrylated 3-aminophenylboronic acid.

Sodium bicarbonate (16.8 g,25.0 mmol) was added to 50mL of a solution of ultrapure water containing 3-aminophenylboronic acid (1.5 g,1.0 mmol) and tetrahydrofuran (volume ratio 2:1), the mixture was stirred at ambient temperature for 5 hours, then acryloyl chloride (203. Mu.L, 2.5 mmol) was added to the mixture, and stirring was continued at ambient temperature for 24 hours. After evaporation of the solvent, 50ml of methanol was added, sonicated for 20 minutes, centrifuged at 8000rpm for 10 minutes, the supernatant was aspirated, and the precipitate was discarded. Thereafter, the supernatant was freeze-dried to obtain a product (1.92 g, yield: 91.87%) as a white powder. ¹ H NMR (700 MHz, deuterated dimethyl sulfoxide (DMSO-d) ₆ ))：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 C ₉ H ₁₀ BNO ₃ :190.9916；found:191.0847。

Example 4

Hydrogel functional polymers of P4, P5 and P6 targeted CTCs were prepared using atom transfer radical polymerization.

(1) Synthesis of Poly (polyethylene glycol dimethacrylate-co-methylpropenyl bis-histidine) [ Poly (PP-co-acrylic acid amide) PP-co-AHH, P4]: the polymerization method (random copolymerization) and initiator used were the same as in example 1, the reaction monomer used was methylpropenyl bis-histidine (AHH) (nuclear magnetic resonance and mass spectrometry characterization was successful), and 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: in a 50 ml EP tube in sequencePEGDMA (13.5 mL,20.0 mmol) and AHH (10.8 g,30.0mmol, in two aliquots) were added at a molar ratio of 2:3, simultaneously adding 45mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding an initiator AIBN (168.8 mg,1.0 mmol), pouring into a 50 mL glass sample bottle after shaking and mixing uniformly for 1 hour, sealing a bottle mouth, placing in an oven, firstly keeping the temperature at 30 ℃ for 12 hours, then starting to rise to 80 ℃, keeping the temperature for 6 hours and then cooling to 30 ℃ for 2 days (including the previous 18 hours), and keeping the temperature rise and cooling rates at 5 ℃/hour. The vial containing P4 was carefully broken, P4 was removed, and the vial was immersed in 10 ml of ultrapure water for dialysis and purification for 7 days to remove unreacted monomers, during which time the immersion solution was changed every half a day. Successful synthesis of P4 was demonstrated by infrared and scanning electron microscopy spectroscopy elemental content analysis characterization (structure see above, y=0.4). Number average molecular weight=18700 Da, polydispersity (PDI) =0.9. FT-IR:2869cm ^-1 (PEGDMA ester group), 1455, 1405, 1328cm ^-1 (AHH imidazole ring); EDS:46.62% (C), 23.63% (N), 29.51% (O).

(2) Synthesis of Poly (polyethylene glycol dimethacrylate-co-methylpropenyl histidine) [ Poly (PP-co-acrylic amide) PP-co-AH, P5]: the polymerization method (random copolymerization) and initiator used were the same as in example 1, the reaction monomer used was methacrylated histidine (AH) (nuclear magnetic resonance and mass spectrometry characterization synthesis was successful), and the cross-linker 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,30.0mmol, in duplicate) were added sequentially to a 50 mL EP tube at a molar ratio of 2:3, simultaneously adding 45mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding an initiator AIBN (168.8 mg,1.0 mmol), pouring into a 50 mL glass sample bottle after shaking and mixing uniformly for 1 hour, sealing a bottle mouth, placing in an oven, firstly keeping the temperature at 30 ℃ for 12 hours, then starting to rise to 80 ℃, keeping the temperature for 6 hours and then cooling to 30 ℃ for 2 days (including the previous 18 hours), and keeping the temperature rise and cooling rates at 5 ℃/hour. Carefully breaking the glass bottle containing P5, taking out P5, soaking in 10 milli The unreacted monomers were removed by dialysis purification in ultra pure water for 7 days, during which the soaking solution was changed every half a day. Successful synthesis of P5 was demonstrated by infrared and scanning electron microscopy spectroscopy elemental content analysis characterization (structure see above, y=0.4). Number average molecular weight=16400 Da, polydispersity (PDI) =1.1. FT-IR:2853cm ^-1 (PEGDMA ester group), 1680, 1593cm ^-1 (AH imidazole ring); EDS:63.12% (C), 13.63% (N), 22.51% (O).

(3) Synthesis of Poly (polyethylene glycol dimethacrylate-co-acrylated 3-aminophenylboronic acid) [ Poly (PP-co-3- (acrylamido) phenylboronic acid, PP-co-APBA, P6 ]]: the polymerization procedure and initiator used was the same as in example 1, the reaction monomer used was acrylated 3-aminophenylboronic acid (APBA) (nuclear magnetic resonance and mass spectrometry characterization was successful), and the crosslinker 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 APBA (10.8 g,30.0mmol, added in duplicate) were added sequentially to a 50 mL EP tube at a molar ratio of 2:3, simultaneously adding 45mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding an initiator AIBN (168.8 mg,1.0 mmol), pouring into a 50 mL glass sample bottle after shaking and mixing uniformly for 1 hour, sealing a bottle mouth, placing in an oven, firstly keeping the temperature at 30 ℃ for 12 hours, then starting to rise to 80 ℃, keeping the temperature for 6 hours and then cooling to 30 ℃ for 2 days (including the previous 18 hours), and keeping the temperature rise and cooling rates at 5 ℃/hour. The vial containing P6 was carefully broken, P6 was removed, and the vial was immersed in 10 ml of ultrapure water for dialysis and purification for 7 days to remove unreacted monomers, during which time the immersion solution was changed every half a day. Successful synthesis of P6 was demonstrated by infrared and scanning electron microscopy spectroscopy elemental content analysis characterization (structure see above, y=0.4). Number average molecular weight=17000 Da, polydispersity (PDI) =1.4. FT-IR:2769cm ^-1 (PEGDMA ester group), 1586, 1500cm ^-1 (benzene ring); EDS:52.81% (C), 11.13% (N), 25.63% (O), 9.56% (B).

Example 5

Because the molecular structure of the double histidine is sensitive to pH, the synthetic silica microsphere grafting is designedMaterial of branched bis-histidine molecules, HH@SiO below ₂ Detailed description of the material preparation process.

Firstly, exposing hydroxyl on the surface of the silicon dioxide microsphere through acidification treatment, then grafting a silane coupling agent on the surface of the silicon dioxide microsphere through hydroxyl, and then modifying the double histidine on the surface of the silicon dioxide microsphere through ring opening reaction between the epoxy group of the silane coupling agent and the terminal amino group of the double histidine. The specific reaction steps are as follows:

10.0g of silica gel (diameter 10 μm, internal pore size) Suspended in 50mL nitric acid (HNO) ₃ 50% by mass) for 10 hours to generate sufficient hydroxyl groups on the silica gel surface. Subsequently, the hydroxylated silica gel was isolated by centrifugation at 7000rpm for 5 minutes, washed with ultrapure water 5 times, and then dried in an oven at 80 ℃. Thereafter, hydroxylated silica gel (5 g) was added to anhydrous toluene (30 mL) containing 3-glycidoxypropyl trimethoxysilane (GLYMO, 5 mL). The reaction mixture was stirred at 60 ℃ for 48 hours. The obtained silica gel (expressed as GLYMO@SiO ₂ ) Separation was performed by centrifugation at 7000rpm for 5 minutes, and washed three times with toluene and ethanol, respectively, in order by repeating the dispersion/precipitation cycle to remove residual GLYMO, and GLYMO@SiO ₂ Drying in an oven at 80 ℃. Thereafter, GLYMO@SiO ₂ (500 mg) and polyhistidine (400 mg) were added to ultrapure water (30 mL). The mixture solution was stirred at 37 ℃ for 72 hours. Then, after removing the residual chemical substances by repeated dispersion/precipitation cycle continuous washing processes with ultrapure water and ethanol, respectively, sequentially, freeze-drying to obtain HH@SiO ₂ . Characterization by thermogravimetry (Thermogravimetric analysis, TGA) and scanning electron microscope energy spectrum elemental content analysis proves that HH@SiO ₂ Is a successful synthesis of (a).

Example 6

Determination of HH@SiO using a Markov nanosize meter ₂ MaterialSurface potential of (2)

8 parts of 20mg of HH@SiO obtained in example 5 were weighed out separately ₂ Materials in 4 ml EP tubes, designated A1, A2, A3, A4, A5, A6, A7 and A8, 2 ml of ultrapure water was added to each of the 8 EP tubes, A1-A5 was adjusted to pH solutions of 2, 3, 4, 5 and 6 with concentrated hydrochloric acid (molar concentration: 12 mol/L), and A6-A8 was adjusted to pH solutions of 7, 8 and 9 with sodium hydroxide (molar concentration: 2 mol/L). Shaking, mixing, sequentially adding into a potential cup, and placing into a Markov nanometer laser particle sizer (Zetasizer Nano), and measuring the surface potential of each of A1, A2, A3, A4, A5, A6, A7 and A8 in 60 cycles at room temperature. The measurements were repeated 3 times, averaged and plotted. As can be seen from FIG. 4, HH@SiO ₂ The material is positively charged in an acidic environment (pH 2-7), is negatively charged in an alkaline environment (pH 8-9), and combines the property that sialic acid which is abnormally expressed on the surface of CTCs compared with other cells is negatively charged, so that the dynamic release of the intelligent responsive hydrogel material to the CTCs can be realized by utilizing the charge conversion characteristic of the double histidine.

Example 7

Biological layer interferometry (biolayer interferometry, BLI) curve of high affinity binding of a bis-histidine-functional monomer to a cell membrane suspension

Because the surface of the circulating tumor cells is covered with a large amount of sugar moieties, the circulating tumor cells can be captured by recognizing and enriching the target molecules on the cell surface. Based on such a hypothesis, the affinity of the bis-histidine for SMMC-7721 cell membrane proteins was assessed by extracting SMMC-7721 cell membrane proteins using biological layer interferometry techniques.

Firstly, the SMMC-7721 cell membrane protein is extracted by referring to the operation description of the Semer femembrane protein extraction kit (Thermo Scientific Mem-PER Plus, 89842), and finally white freeze-dried powder is obtained. Next, 1 mg of the lyophilized powder was dissolved in 2 ml of ultrapure water to prepare a protein solution of 0.5 mg per ml, and the film protein concentration was confirmed to be specifically 0.508 mg per ml according to the bicinchoninic acid assay (bicinchoninic acid, BCA) kit (bi yun, P0010), which indicates that the obtained lyophilized powder was not doped with other impurities and was a protein sample.

Finally, 1mg of bis-histidine was dissolved in 200. Mu.l of ultra pure water and grafted onto the surface of an amine reactive 2nd generation (AR 2G) biosensor probe (Sartorius, 2211021111) of a molecular interaction instrument (ForteBio Octet Co., octet K2, U.S.A.), adsorption experiments were performed on membrane proteins of different concentration gradients (3.31, 6.25, 12.5, 25, 50, 100. Mu.g/mL). The procedure was as follows, and 30 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (3- [ (Ethylimino) methyl ] amino-N, N-dimethylpropan-1-amine, EDC, CASNo.1892-57-5, sigma-Aldrich Corp) and 18 mg of N-Hydroxysuccinimide (N-hydroxyysuccinide, NHS, CAS No.6066-82-6, sigma-Aldrich Corp) were dissolved in 400. Mu.l of a solution, placed in a 96-well plate, 200. Mu.l of a solution per well, and 2 biosensor probes were placed in wells filled with the solution, respectively, to activate carboxyl groups on the surface of the biosensor probes at room temperature under a dark condition. After 2 hours, the solution in the wells was poured off, 2 mg of bis-histidine was dissolved in 400. Mu.l of ultra pure water, added to the wells, and 2 biosensor probes were placed in the wells containing the solution, respectively, and allowed to stand still at room temperature under dark conditions for 24 hours. The amino group of the double histidine is utilized to graft the double histidine on the surface of the carboxyl end capped biosensor probe through an amide reaction. Thereafter, the sensor surface was carefully cleaned with ultrapure water to remove unreacted sample. Subsequently, the extracted membrane protein was prepared into a 1mg/mL protein solution with ultrapure water, and the protein solution was subjected to gradient dilution with ultrapure water to obtain protein solutions of different concentration gradients (3.31, 6.25, 12.5, 25, 50, 100. Mu.g/mL). The experimental set (1 of which were) of bis-histidine-modified biosensor probes was then exposed (soaked) to protein solutions of different concentration gradients (3.31, 6.25, 12.5, 25, 50, 100 μg/mL). Binding, dissociation and interaction analysis was performed at 37 ℃. Meanwhile, the control group (another 1 biosensor probe) of the bis-histidine-modified biosensor probe was exposed to ultrapure water. Binding, dissociation and interaction analysis was performed at 37 ℃. The data were analyzed by ForteBio (data analysis 11.0). From FIG. 5, it can be seen that the double histidine shows concentration dependence on SMMC-7721 cell membrane protein solution, demonstrating the ability of double histidine to bind to SMMC-7721 cells, and hopefully developed for efficient enrichment of CTCs.

Example 8

The internal water content of the prepared intelligent responsive hydrogel is measured by using low-field nuclear magnetism (LF-NMR).

In the T2 relaxation of LF-NMR, the relaxation peaks at different positions represent different states of the H nuclei bound, thus characterizing the differences in their mobility. The H nucleus with larger constraint degree has poorer mobility and short relaxation time; in contrast, the relatively free H-nuclei, which are less constrained, have good mobility, high degrees of freedom and long relaxation times. From such properties, we can determine the state of water molecules inside the gel, and further can also use the peak area to indicate how much water is contained. The specific operation is as follows: firstly, correcting LF-NMR (Suzhou New Mich technology), and selecting a magnet probe to default; selecting Q-FID by sequence name, default parameters; placing a standard sample (standard oil sample); clicking for single sampling, waiting for 10s, and clicking for stopping sampling; clicking to find the center frequency (SF+01) until the FID curve no longer shows regular oscillation; clicking on the pulse width, the software automatically looks for 90 ° and 180 ° pulse widths until a peak appears and a trough indicates that the pulse width lookup is correct. Then, P1-P6 gel (with the diameter of 5 cm and the height of 5 cm) with the mass of 1g is sequentially placed into a magnetic field cavity, CPMG sequences are selected, the names of the sequences are input, proper parameters are set, T2 relaxation time is measured by clicking sampling, and the states of different water molecules are obtained through inversion. As shown in fig. 6, the P1, P2, P3, P4, P5 and P6 hydrogels were respectively placed in 10 ml of PBS solution (pbs=10 mm, ph=7.4, the same applies below) for 7 days, and then taken out and respectively placed in a magnetic field cavity for testing. The peaks of the T2 component at 0.1-3, 70-200 and 800-2200 ms correspond to bound water, fixed water and free water, respectively. Sharp and intense peaks were observed for P1 and P2 at 1649-2078ms and 1250-1649ms, with signal strengths (744 and 741) significantly greater than 71-89ms (74) and 81-103ms (39). This suggests that there is a significant amount of free water inside the P1 and P2 hydrogels to dominate the water main form, which is consistent with the significant swelling of P1 and P2 in example 2. In contrast, the signals corresponding to free and fixed water in P3-P6 are significantly weaker, and in particular the free water strength of P4-P6 is very low, which also confirms its low swelling.

Example 9

Preparation and characterization of the intelligent responsive hydrogel P4+, P5+ and P6+ cell blot microcosmic morphology taking the P4+ hydrogel as an example, the formation process of the cell blot is described as follows:

first 4×10 ⁶ The following cell culture media, 100X (Biyun days), were placed in 100mm diameter and 100mm height cell culture dishes the evening before each SMMC-7721 cell, 10mL of cell culture media [ 89%Dulbecco's modified eagle medium,DMEM (Gibco) at a volume concentration, 10% Fetal bovine serum, FBS (ExCell Bio) and 1% penicillin-streptomycin, 100X (Biyun days), were added and cultured until the next morning (total 12 h). The medium was removed, washed 3 times with PBS solution (pbs=10 mm, ph=7.4, the same applies hereinafter), added with 2mL of paraformaldehyde solution (mass concentration 4%) pre-prepared and coated with membrane (0.22 μm), fixed at 37 ℃ for 5min, the fixing solution removed, and washed 3 times with PBS solution (pbs=10 mm, ph=7.4, the same applies hereinafter). Thereafter, PEGDMA (13.5 mL,20.0 mmol) and AHH (10.8 g,30.0mmol, in duplicate) were added sequentially to a 50 mL EP tube at a molar ratio of 2:3, simultaneously adding 45mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding an initiator AIBN (168.8 mg,1.0 mmol), taking out 5mL of solution after shaking and mixing uniformly for 1 hour, adding the solution into a cell culture dish fixed with cells, and shaking at 300rpm on a shaking table for 5 hours to enable functional monomers in the solution to search target molecules on the surfaces of the cells. Subsequently, the dishes were transferred to an oven, initially held at 30 ℃ for 12 hours, then started to warm to 80 ℃, held for 6 hours and then cooled to 30 ℃ for 2 days (including the preceding 18 hours), with both warm and cool rates of 5 ℃/hour. Then obtaining the hydrogel P4-cell with cell blotting and functional groups. The gel P4-cell is washed by ultrapure water, ultrapure water is slowly added from the edge of the inner wall of the cell culture dish, so that the P4-cell hydrogel is separated from the culture dish, the P4-cell hydrogel is taken out, soaked in 10ml of ultrapure water, dialyzed and purified for 7 days, and the soaking solution is changed every half day. Thereafter, the gel was taken out and immersed in 10ml of pancreatin solution (Trypsin EDTA solution:0.25% Trypsin) &0.02% EDTA; pH-8.0 (Viva Cell)), after 4 hoursThe gel was rinsed 3 times with 5 ml of phosphate buffered solution (pbs=10 mm, ph=7.4) and stored in 10 ml of PBS solution (pbs=10 mm, ph=7.4). The process is to remove the cell template, unreacted monomers and AIBN residues, so as to prepare the intelligent response molecularly imprinted hydrogel carrying functional monomers and cell micro morphology. The microscopic morphology of the cell blot indicated by the white arrows shown in fig. 7 can be seen by an optical microscope.

Application instance

Example 10 anti-protein adsorption of the gel matrix of Intelligent responsive hydrogels

Firstly, 3 milliliters of blood samples (healthy physical examination crowd) are taken from a second people hospital affiliated to university of Dalian medical science, according to the density difference of cells (the densities of red blood cells and granulocytes are 1.090g/mL, the densities of lymphocytes and mononuclear cells are 1.075-1.090 g/mL, and the densities of platelets are 1.030-1.035 g/mL), the cells are distributed according to the corresponding density gradient through centrifugation, so that the cells are separated from blood, the residual solution is collected, and the protein content in the solution is measured through a BCA kit. From this, 4 mg of protein was taken and diluted in 13 mL of ultrapure water (initial concentration of protein 0.3 mg/mL), and dispensed into 12 2 mL glass vials (1 mL of solution per vial). P1, P2 and P3 were cut into cubes of 1 cm. Times.1 cm (3 parts each, 70 mg in weight) and added to the different glass vials described above, respectively, while 3 blank groups were set (without any material added). After 4 hours, the solution in the glass vial was aspirated, the absorbance (OD) was determined by a microphotometer (nanodrop) and the protein content was calculated as A 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) the P1, P2 and P3 protein adsorption contents were calculated. The specific calculation process is as follows: first, a standard curve was prepared, and 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL solutions of bovine serum albumin (Bovine serum albumin, BSA) were prepared and OD values were measured at 0.0195, 0.0425, 0.0835, 0.158, 0.2445, 0.3085, and 0.39. Drawing a standard curve in Excel according to the OD value and the concentration to obtain an equation of y=0.7741x+0.0039; r is R ² =0.999. Then soak 4After an hour, 100. Mu.l of the solution was removed from 12 glass vials and 2. Mu.l of the OD was measured. OD values of control groups were 0.222, 0.209, 0.213; the OD value of P1 is 0.211, 0.1995 and 0.202; the OD value of P2 is 0.219, 0.206 and 0.209; the OD value of P3 was 0.219, 0.208, 0.211. The corresponding protein concentrations were control: 0.2817, 0.2649, 0.2701mg/mL; group P1: 0.2675, 0.2526, 0.2559mg/mL; group P2: 0.2778, 0.2610, 0.2649mg/mL; group P3: 0.27787, 0.2636, 0.2675mg/mL. Subtracting the protein concentration of the sample group from the protein concentration of the control group (respectively corresponding to the protein concentration of the sample group one by one in sequence) to obtain the protein concentrations adsorbed by the P1, P2 and P3 gel, wherein the volumes of the protein concentrations are respectively 1 milliliter, so that the adsorbed protein amounts are P1-14.21, 12.27 and 14.21 mug; p2-3.87, 5.16 μg; p3-0.38, 0.13 and 0.26 mug, the corresponding mass adsorption ratio is P1-0.203, 0.175 and 0.203 mug/mg; p2-0.055, 0.073 μg/mg; p3-0.005, 0.002, 0.004. Mu.g/mg. As can be seen from fig. 8, the P3 hydrogel scaffold has excellent protein adsorption resistance.

EXAMPLE 11 processability of Intelligent responsive hydrogel P4

P4 gel (diameter is 10cm, height is 10 cm) is fixed on a probe of a texture analyzer (Shanghai Paul, TA. XTC-20), a certain force is axially applied on a cylinder through the texture analyzer, the probe moves downwards at a constant speed to apply pressure on the cylinder, the probe moves upwards after the pressure reaches a set value, and the change of the height of the cylinder before and after the pressure is observed and measured under a microscope, so that the mechanical property of the hydrogel is reflected. The damage to the smart responsive hydrogel from compression and rebound in 50 cycles was tested at a rate of 1mm/s under 15% strain (quantitatively describing the degree of hydrogel expansion, compression being 15% of the gel height, i.e. the gel height compressed from 10cm to 8.5 cm), after compression of the gel to a fixed position for a further 2 s. From the experimental results, no damage occurred to P4 after 50 cycles, only a 7% decrease in mechanical properties (initial gel height versus gel height change after 50 cycles) was measured, indicating that P4 has excellent plasticity (see fig. 9) and is suitable for processing to prepare biomedical materials.

Example 12 availability of Intelligent responsive hydrogels P4+ for CTCs Capture re-release cycle

Will 10 ⁵ 1 ml blood sample (healthy physical examination patient) with a single SMMC-7721 cell doping density was added dropwise to a P4+ hydrogel (area: 14 cm) ² Height: 0.7 cm) surface, cell incubator incubated for 30 min, followed by washing of unbound cells with PBS solution (pbs=10 mm, ph=7.4, the same applies below), collection and calculation of capture rate by observing the number of CTCs in PBS. Then, 0.2 ml of pancreatin solution [ Trypsin EDTA solution:0.1-0.5% Trypsin, preheated at 37 ℃ in advance, was added&0.005% -0.05% EDTA; the pH value is 8.0 (Viva Cell), the mixture is digested for 1 minute, 1 milliliter of Cell culture medium preheated at 37 ℃ in advance is immediately added, and a pipetting gun is used for sucking the Cell culture medium to blow off cells adhered to the surface of the hydrogel; the solution was collected and centrifuged at 1000rpm for 3 minutes, the supernatant was decanted to leave a cell pellet, the cells were resuspended in 100. Mu.L of cell culture medium, and the cell count was counted by a hemocytometer to calculate the release rate. This procedure was repeated 10 times, 3 groups were run in single replicates, and the parallel runs were repeated 3 times. Specific experimental data for 1 cycle are described below: first, the capture rate was calculated by centrifuging the obtained PBS solution at 1000rpm for 3 minutes, adding 100. Mu.l of cell culture medium [ Dulbecco's modified eagle medium, DMEM (Gibco), 10% Fetal bovine serum, FBS (ExCell Bio) and 1% penicillin-streptomycin, 100X (Biyun days), the following cell culture medium was composed here ], resuspension of cells, taking 10. Mu.l, and counting the number of cells (N1=24, 26, 27) with a blood cell counting plate, the number of cells in 100. Mu.l of cell culture medium was 6X 10 ³ 、6.5×10 ³ 、6.75×10 ³ Dividing the total number of seeded cells by the respective cell number gives a percentage of cells not captured of 6%, 6.5%, 6.75%, so the P4+ hydrogel pair 10 ⁵ The capture rate of the SMMC-7721 cells is 94.0%, 93.5% and 93.3%. Then, the release rate was calculated, and the number of cells trapped on the surface of the p4+ hydrogel was calculated as 94000, 93500 and 93250 from the trapping rate. Then, the number of cells released from the gel surface was counted by a hemocytometer (n2=353, 360, 347), and the number of released cells was 88250, 90000, 86750, so that the release rate was 9388%, 96.26% and 93.03%. From fig. 10, it can be seen that the intelligent responsive hydrogel has good capturing efficiency and reusability, can reduce the detection cost, is hopeful to be developed for selective enrichment of CTCs, and promotes the development of cancer detection to the popularization.

Claims

A ctcs captured polymeric material characterized by: the polymer consists of a network skeleton (derived from PEGDMA, a bracket part corresponding to a subscript y, y=0.01-0.5 (preferably y=0.1-0.4), n=1-15 integers (preferably n=3-13)) and a CTCs binding unit (one or more than two of AHH, AH and APBA); their number average molecular weight ranges are: p4=5000 to 20000Da (preferably 8000 to 18700), p5=5000 to 20000Da (preferably 7000 to 16400), p6=5000 to 20000Da (preferably 7000 to 17000), and the molecular structure thereof is represented by one or more of the following (the polymerization process PEGDMA and CTCs binding units are randomly copolymerized):
2. A method of preparing a CTCs-captured polymeric material of claim 1, wherein: the specific process is as follows:

(1) Synthesis of Poly (polyethylene glycol dimethacrylate-co-methylpropenyl bis-histidine) [ Poly (PP-co-acrylic acid bishistididine), PP-co-AHH, P4]: PEGDMA (5-50 mL) and AHH (5-100 g) are added into a 10-200 mL beaker in sequence, and the material mol ratio is 0.5:1 to 3:1, simultaneously adding 10-200 mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding initiator AIBN (100-1000 mg), shaking and mixing uniformly for 30-2880 minutes, pouring into a 10-100 mL glass sample bottle, sealing a bottle mouth, placing in an oven, initially keeping the temperature at 10-50 ℃ for 3-20 hours, then starting to rise to 60-150 ℃, keeping the temperature for 2-10 hours and then cooling to 10-50 ℃ for 2-9 days (including the previous 5-30 hours), wherein the heating and cooling rates are 1-10 ℃/hour; carefully breaking the glass bottle filled with P4, taking out the P4, soaking in 5-50 ml of ultrapure water, dialyzing and purifying for 1-14 days to remove unreacted monomers, and changing the soaking solution every half day to one day during the period to obtain gel P4;

or, (2) synthesizing Poly (polyethylene glycol dimethacrylate-co-methylpropenyl histidine) [ Poly (PP-co-acrylic amide) PP-co-AH, P5]: PEGDMA (5-50 mL) and AH (1-500 g) are added into a 10-200 mL beaker in sequence, and the material mol ratio is 0.5:1 to 3:1, simultaneously adding 10-200 mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding initiator AIBN (100-1000 mg), shaking and mixing uniformly for 30-2880 minutes, pouring into a 10-100 mL glass sample bottle, sealing a bottle mouth, placing in an oven, initially keeping the temperature at 10-50 ℃ for 3-20 hours, then starting to rise to 60-150 ℃, keeping the temperature for 2-10 hours and then cooling to 10-50 ℃ for 2-9 days (including the previous 5-30 hours), wherein the heating and cooling rates are 1-10 ℃/hour; carefully breaking the glass bottle filled with P5, taking out the P5, soaking in 5-50 ml of ultrapure water, dialyzing and purifying for 1-14 days to remove unreacted monomers, and changing the soaking solution every half day to one day during the period to obtain gel P5;

Or, (3) synthesizing Poly (polyethylene glycol dimethacrylate-co-acrylated 3-aminophenylboric acid) [ Poly (PP-co-3- (acrylic amide) phenylboronic acid, PP-co-APBA, P6 ]. The PEGDMA (5-50 mL) and APBA (5-100 g) are sequentially added into a 10-200 mL beaker, the molar ratio of substances is 0.5:1-3:1, meanwhile, 10-200 mL of ultrapure water is added as a solvent, the mixture is ultrasonically dissolved and uniformly mixed, then initiator AIBN (100-1000 mg) is added, after shaking and uniformly mixing for 30-2880 minutes, the mixture is poured into a 10-100 mL glass sample bottle, the bottle mouth is sealed, the bottle mouth is placed into an oven, the mixture is kept at 10-50 ℃ for 3-20 hours initially, then the temperature is raised to 60-150 ℃ for 2-10 hours, the temperature is lowered to 10-50 ℃ for 2-9 days (including the previous 5-30 hours), the mixture is carefully soaked in the glass bottle with P6, the mixture is not broken and is removed in the dialysis bottle with the shaking and the solution after the shaking and the mixture is removed for 6-14 days, and the gel is not broken and the mixture is removed every one day.
3. A method of preparing a CTCs-captured polymeric material of claim 1, wherein: the specific process of material surface cell blotting is as follows:

(1) Preparation of an intelligent responsive molecularly imprinted hydrogel p4+: first 10 will be ³ ～10 ⁶ Placing individual tumor cells (liver cancer or lung cancer or breast cancer or cervical cancer or prostate cancer or colon cancer or ovarian cancer) into a cell culture dish with the diameter of 30-150 mm and the height of 30-150 mm, adding 1-30 mL of cell culture medium, and culturing for 2-48 hours; removing the culture medium, washing for 1-10 times by using PBS (phosphate buffer solution), adding 0.1-5 mL of paraformaldehyde solution (with the mass concentration of 0.5-10%) which is pre-prepared and coated with a film (0.1-0.8 μm), fixing for 1-15 minutes at the temperature of 4-37 ℃, removing the fixing solution, and washing for 1-10 times by using PBS; then, PEGDMA (5-50 mL) and AHH (5-100 g) were added sequentially to a 10-200 mL beaker at a mass molar ratio of 0.5:1 to 3:1, simultaneously adding 10-200 mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding an initiator AIBN (100-1000 mg), shaking and mixing uniformly for 30-2880 minutes, taking out 1-200 mL of solution, adding the solution into a cell culture dish with fixed cells, and shaking at 300rpm on a shaking table for 0.5-10 hours to enable functional monomers in the solution to search target molecules on the surfaces of the cells; transferring the culture dish into a baking oven, keeping the temperature at 10-50 ℃ for 3-20 hours initially, then starting to heat to 60-150 ℃ for 2-10 hours, and then cooling to 10-50 ℃ for 2-9 days (including the previous 5-30 hours), wherein the heating and cooling rates are 1-10 ℃/hour; then obtaining hydrogel P4-cell with cell imprinting and functional groups; flushing the gel P4-cell with ultrapure water, slowly adding ultrapure water from the edge to enable the P4-cell hydrogel to fall off from the culture dish, taking out the P4-cell hydrogel, soaking the gel in 1-50 ml of ultrapure water, dialyzing and purifying for 1-14 days, and changing the soaking solution every half day to one day during the period; then taking out the gel, soaking into 1-50 ml pancreatin solution [ Trypsin EDTA solution:0.1-0.5%Trypsin &0.005% -0.05% EDTA; pH is 8.0 (Viva Cell), after 0.1-10 hours, the gel is washed 1-10 times by 0.5-10 ml of phosphate buffer solution (PBS=10 mM, pH=7.4) and stored1-100 ml PBS solution; the process is to remove the cell template, unreacted monomers and AIBN residues, so as to prepare the intelligent response molecularly imprinted hydrogel P4+ carrying functional monomers and cell micro morphology;

or, (2) preparing the intelligent response molecularly imprinted hydrogel P5+: first 10 will be ³ ～10 ⁶ Placing individual tumor cells (liver cancer or lung cancer or breast cancer or cervical cancer or prostate cancer or colon cancer or ovarian cancer) into a cell culture dish with the diameter of 30-150 mm and the height of 30-150 mm, adding 1-30 mL of cell culture medium, and culturing for 2-48 hours; removing the culture medium, washing for 1-10 times by using PBS (phosphate buffer solution), adding 0.1-5 mL of paraformaldehyde solution (with the mass concentration of 0.5-10%) which is pre-prepared and coated with a film (0.1-0.8 μm), fixing for 1-15 minutes at the temperature of 4-37 ℃, removing the fixing solution, and washing for 1-10 times by using PBS; then, PEGDMA (5-50 mL) and AH (1-500 g) were added sequentially to a 10-200 mL beaker at a molar ratio of 0.5:1 to 3:1, simultaneously adding 10-200 mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding an initiator AIBN (100-1000 mg), shaking and mixing uniformly for 30-2880 minutes, taking out 1-200 mL of solution, adding the solution into a cell culture dish with fixed cells, and shaking at 300rpm on a shaking table for 0.5-10 hours to enable functional monomers in the solution to search target molecules on the surfaces of the cells; transferring the culture dish into a baking oven, keeping the temperature at 10-50 ℃ for 3-20 hours initially, then starting to heat to 60-150 ℃ for 2-10 hours, and then cooling to 10-50 ℃ for 2-9 days (including the previous 5-30 hours), wherein the heating and cooling rates are 1-10 ℃/hour; then obtaining hydrogel P5-cell with cell imprinting and functional groups; flushing the gel P5-cell with ultrapure water, slowly adding ultrapure water from the edge to enable the P5-cell hydrogel to fall off from the culture dish, taking out the P5-cell hydrogel, soaking the gel in 1-50 ml of ultrapure water, dialyzing and purifying for 1-14 days, and changing the soaking solution every half day to one day during the period; then taking out the gel, soaking the gel into 1-50 ml of pancreatin solution, washing the gel with 0.5-10 ml of phosphate buffer solution (PBS-10 mM, pH-7.4) for 1-10 times after 0.1-10 hours, and storing the gel in 1-100 ml of PBS solution; the above process is to remove cell templates, unreacted monomers and AIBN Residues, so as to prepare intelligent response molecularly imprinted hydrogel P5+ carrying functional monomers and cell micro morphology;

or, (3) preparing intelligent response molecularly imprinted hydrogel P6+: first 10 will be ³ ～10 ⁶ Placing individual tumor cells (liver cancer or lung cancer or breast cancer or cervical cancer or prostate cancer or colon cancer or ovarian cancer) into a cell culture dish with the diameter of 30-150 mm and the height of 30-150 mm, adding 1-30 mL of cell culture medium, and culturing for 2-48 hours; removing the culture medium, washing for 1-10 times by using PBS (phosphate buffer solution), adding 0.1-5 mL of paraformaldehyde solution (with the mass concentration of 0.5-10%) which is pre-prepared and coated with a film (0.1-0.8 μm), fixing for 1-15 minutes at the temperature of 4-37 ℃, removing the fixing solution, and washing for 1-10 times by using PBS; then, PEGDMA (5-50 mL) and APBA (5-100 g) were added in sequence to a 10-200 mL beaker at a mass molar ratio of 0.5:1 to 3:1, simultaneously adding 10-200 mL of ultrapure water as a solvent, carrying out ultrasonic dissolution and shaking and mixing uniformly, then adding an initiator AIBN (100-1000 mg), shaking and mixing uniformly for 30-2880 minutes, taking out 1-200 mL of solution, adding the solution into a cell culture dish with fixed cells, and shaking at 300rpm on a shaking table for 0.5-10 hours to enable functional monomers in the solution to search target molecules on the surfaces of the cells; transferring the culture dish into a baking oven, keeping the temperature at 10-50 ℃ for 3-20 hours initially, then starting to heat to 60-150 ℃ for 2-10 hours, and then cooling to 10-50 ℃ for 2-9 days (including the previous 5-30 hours), wherein the heating and cooling rates are 1-10 ℃/hour; then obtaining hydrogel P6-cell with cell imprinting and functional groups; washing the gel P6-cell with ultrapure water, slowly adding ultrapure water from the edge to enable the P6-cell hydrogel to fall off from the culture dish, taking out the P6-cell hydrogel, soaking the gel in 1-50 ml of ultrapure water, dialyzing and purifying for 1-14 days, and changing the soaking solution every half day to one day during the period; then taking out the gel, soaking the gel into 1-50 ml of pancreatin solution, washing the gel with 0.5-10 ml of phosphate buffer solution (PBS-10 mM, pH-7.4) for 1-10 times after 0.1-10 hours, and storing the gel in 1-100 ml of PBS solution; the process is to remove the cell template, unreacted monomer and AIBN residue, thereby preparing the intelligent response molecular imprinting carrying the functional monomer and the cell micro morphology Hydrogels p6+.
4. A method for preparing CTCs binding units in CTCs capturing intelligent responsive polymer biomaterials according to claim 1, the synthetic route of which is schematically as follows:

the preparation process comprises the following steps:

(1) Synthesis of methacryloylated Di-histidine:

sodium hydroxide (1-10 g) is added into 1-100 mL of ultra-pure water solution containing bis-histidine (10-500 mg), the mixture is stirred for 1-24 hours at 15-60 ℃, then methacryloyl chloride (0.01-1 mL) is added into the mixture, and stirring is continued for 1-3 days at 15-60 ℃; after evaporating the solvent, adding 10-100 ml of methanol, dissolving by ultrasonic for 10-60 minutes, centrifuging at 2000-8000 rpm for 5-30 minutes, sucking the supernatant, and discarding the precipitate. Then, freeze-drying the supernatant to obtain a white powdery product;

(2) Synthesis of methacryloylated histidine:

sodium hydroxide (1-20 g) is added into 1-100 mL of ultrapure water solution containing histidine (1-50 g), the mixture is stirred for 1-24 hours at 15-60 ℃, then methacryloyl chloride (1-10 mL) is added into the mixture, and stirring is continued for 1-3 days at 15-60 ℃; after evaporating the solvent, adding 10-100 ml of methanol, dissolving by ultrasonic for 10-60 minutes, centrifuging at 2000-8000 rpm for 5-30 minutes, sucking the supernatant, and discarding the precipitate. Then, freeze-drying the supernatant to obtain a white powdery product;

(3) Synthesis of acrylated 3-aminophenylboronic acid:

sodium bicarbonate (1-30 g) is added into 1-100 mL of ultrapure water containing 3-aminophenylboric acid (1-50 g) and tetrahydrofuran (volume ratio of 0.05:1-10:1), the mixture is stirred for 1-24 hours at 15-60 ℃, then acryloyl chloride (0.01-1 mL) is added into the mixture, and stirring is continued for 1-3 days at 15-60 ℃; after evaporating the solvent, adding 10-100 ml of methanol, dissolving by ultrasonic for 10-60 minutes, centrifuging at 2000-8000 rpm for 5-30 minutes, sucking the supernatant, and discarding the precipitate. Thereafter, the supernatant was freeze-dried to obtain a white powdery product.
5. Use of the polymeric material of claim 1 for the enrichment, capture or isolation of CTCs or leukocytes in blood or plasma.
6. The use according to claim 5, characterized in that: the polymeric material may target CTCs or leukocytes;

the CTCs tumor cells are one or more than two of liver cancer, lung cancer, breast cancer, cervical cancer, prostatic cancer, colon cancer or ovarian cancer cells.
7. Use of the polymeric material of claim 1 for the accurate capture and release of CTCs in blood, characterized in that: the specific process is as follows:

The capturing process comprises the following steps:

0.1-5 ml of whole blood sample containing tumor cells or leucocytes is doped and inoculated on the surface of hydrogel (area: 1-50 cm) ² Height: 0.01-1 cm), and culturing the cells in a cell culture box for 0.5-24 hours; then, 0.5-10 ml of PBS solution is used for flushing the surface of the hydrogel to remove red blood cells, unbound tumor cells and WBCs;

then (release process), adding 0.1-5 ml pancreatin solution [ Trypsin EDTA solution:0.1-0.5%Trypsin &0.005% -0.05%EDTA preheated at 20-40 ℃; the pH value is 8.0 (Viva Cell), the mixture is digested for 0.1 to 5 minutes, 1 to 20 milliliters of Cell culture medium preheated at 20 to 40 ℃ in advance is immediately added, and a pipetting gun is used for sucking the Cell culture medium and blowing off cells adhered to the surface of the hydrogel; collecting the solution and centrifuging at 1000rpm for 1-3 minutes, pouring off the supernatant, and leaving a cell precipitate; the cells were resuspended in 100-5000 microliters of cell culture medium and the cell count plate was used to calculate the cell number and thus the release rate.