A double network hydrogel surface and its preparation method and application

Abstract

The present invention relates to the technical field of biopolymer materials, and in particular to a double network hydrogel surface and its preparation method and application. The structure of the double network hydrogel is a micron-sized honeycomb structure with a pore size of 21-32 μm; the double network hydrogel is composed of nanoparticles, and the size of the nanoparticles is 400nm-760nm; the double network hydrogel has a synergistic effect of cell imprinting and aptamer affinity, can accurately match the structure of target cells, and realize efficient capture and cultivation of live target cells; high stability, low degradation rate, and good adhesion, growth and proliferation ability. The preparation method has universality and strong operability, and can realize the controllable preparation of hydrogel; the capture efficiency of the double network hydrogel for MCF-7 and Hela cells is as high as 93±3% and 90±2%, respectively, and the captured cancer cells still have a high viability on the hydrogel surface, which is expected to be applied to CTC culture and personalized drug sensitivity testing, and has good development and application value.

Description

Technical Field

[0001] The invention relates to the technical field of biopolymer materials, and in particular to a double network hydrogel surface and a preparation method and application thereof. Background Art

[0002] Cancer, or malignant tumor, is a disease caused by the unlimited proliferation of cancer cells due to abnormal control of cell division and proliferation mechanisms. Cancer cells can infiltrate normal tissues around the tumor, or they can be transferred to other tissues in the body through the circulatory system and lymphatic system, causing the disease to get out of control. Circulating tumor cells (CTCs) released from the tumor and passing through the peripheral blood and lymphatic system are the cause of cancer metastasis. Accurately measuring the level of CTCs in peripheral blood can provide valuable tumor information, reduce false positive results and reduce invasiveness. Therefore, it is very important to capture cancer cells in a targeted manner.

[0003] In the current existing technology, various fine material surfaces have been created to effectively capture rare CTCs by utilizing the unique biophysical and biochemical properties of CTCs and the progress of nanotechnology. However, due to the use of antibody-based strategies or lack of selective physical methods, the currently disclosed biomaterials have problems such as low CTC capture ability, easy release after capture, easy degradation of biomaterials, and unstable structure. Therefore, it is of great development and application value to prepare a stable biomaterial with low degradation rate to achieve selective and efficient capture and cultivation of cancer cells. Summary of the invention

[0004] In order to address the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a double network hydrogel surface that can accurately match the structure of target cells, thereby achieving efficient capture and cultivation of living cells.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned double-network hydrogel surface, which is stable, efficient, universal and highly repeatable.

[0006] The third object of the present invention is to provide an application of the above-mentioned double-network hydrogel surface, which can efficiently capture cancer cells, is widely used in biomedical engineering, and has good development and application value.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] A double network hydrogel surface, wherein the structure of the double network hydrogel surface is a micrometer-level honeycomb structure with a pore size of 21-32 μm; the double network hydrogel surface is composed of nanoparticles with a size of 400nm-760nm; the double network hydrogel surface has a synergistic effect of cell imprinting and aptamer affinity.

[0009] Furthermore, the aptamer is DNA, RNA, siRNA (short interfering RNA), Anti-PD-L1, shRNA (short hairpin RNA), lectin or epidermal growth factor.

[0010] A method for preparing the above-mentioned double network hydrogel surface, the preparation method adopts the following steps:

[0011] (1) Preparation of cell templates: culturing cancer cells, washing, and fixing the cells with a cell fixative to obtain a cell template;

[0012] (2) Add deionized water to the mixture of compound monomers, polymers and initiators to dissolve, and add methacrylamide (MBAA ) Mixing, polymerizing with the cell template to obtain a composite hydrogel; placing the composite hydrogel in an inorganic cationic solution to perform a cross-linking reaction, washing, incubating, and preparing a hydrogel template;

[0013] (3) soaking the hydrogel template prepared in step (2) in a solution modified with an amino or carboxyl functional group polymer, and then soaking it in a sulfo-NHS / EDC mixture, washing it, covering the cell imprinting surface with an aptamer solution, and washing it to obtain a double network hydrogel surface.

[0014] Furthermore, in step (1), the density of the cultured cancer cells is 70-90%.

[0015] Furthermore, in step (2), the compound monomer is acrylamide (AM), acrylic acid (AA), cellulose, ethylene oxide (EO), methyl methacrylate (MMA), ethylene glycol dimethacrylate (EGDMA), propane or antimony trioxide; the polymer is polypropylene (PP), chitosan (CS), polycarbonate (PC), polyamide (PA), polyvinyl alcohol (PVA) or polyethylene glycol (PEG); the initiator is one or more of ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED) and hydrogen peroxide (H2O2); the mass ratio of the compound monomer, polymer and initiator is 1.8:(0.5-0.75):0.0312.

[0016] Furthermore, in step (2), the inorganic cation solution is sodium ion, iron ion, aluminum ion or calcium ion; the concentration of the inorganic cation is 0.5-1 mol / L, and the cross-linking reaction time is 0.5-1h.

[0017] Furthermore, in step (3), the polymer containing amino or carboxyl functional groups is polyaspartic acid (PASP), collagen protein, phosphoprotein, plasma protein, poly-D-lysine (Poly-D-lysine) or casein; and the polymerization reaction is carried out in a deoxygenated or vacuum environment at a temperature of 65-75°C and a time of 0.5-2h.

[0018] Furthermore, in step (3), the concentration of the aptamer solution is 5-10 μg / mL, and the time for the aptamer solution to cover the cell imprint surface is 0.5-1 h; the concentration ratio of sulfo-NHS to EDC in the sulfo-NHS / EDC mixture is 1:(2-3), wherein the concentration of sulfo-NHS is 1 mg / mL; the time for immersion in the sulfo-NHS / EDC mixture is 1-2 h; the washing in steps (1), (2), and (3) are all performed using PBS; the incubation in step (2) is performed using 0.25% trypsin at 37° C.; the fixative in step (1) is 4% paraformaldehyde (PFA), 70% ethanol, or 10% formalin.

[0019] Furthermore, the cancer cells in step (1) are breast cancer cells (MCF-7), cervical cancer cells (Hela), esophageal cancer cells (ECA-109), gastric cancer cells, colorectal cancer cells, lung cancer cells or liver cancer cells.

[0020] An application of the double-network hydrogel surface as described above, wherein the hydrogel surface can be applied to CTC culture.

[0021] Furthermore, the hydrogel surface can be applied to compare gene expression profiles of primary tumors, CTCs and metastatic lesions, or to study the expression of different surface markers of CTCs, or to test drug sensitivity of CTC culture.

[0022] Beneficial Effects

[0023] (1) The present invention provides a double network hydrogel surface with synergistic effects of cell imprinting and aptamer affinity. By introducing cell imprinting into the hydrogel, the structure of cancer cells can be accurately matched; by introducing aptamers into the hydrogel, cancer cells can be targeted; the double network (DN) hydrogel contains a typical micron-scale honeycomb structure with a pore size of 21-32 μm, which makes it more stable and has a lower degradation rate; and the double network hydrogel is composed of nanoparticles with a size of 400nm-760nm. The unique structural composition enables it to accurately match target cells, thereby achieving efficient capture and cultivation of cells.

[0024] (2) The preparation method of the double network hydrogel surface provided by the present invention is stable, efficient and highly repeatable; in addition, the preparation method provided by the present invention has the characteristics of universality and strong operability. By rationally regulating the composition of the compounds, the double network hydrogel can be modified to have cell imprinting and aptamer affinity, thereby realizing the controllable preparation of the method.

[0025] (3) The double-network hydrogel surface provided by the present invention has good adhesion, growth and proliferation capabilities, and can efficiently capture cancer cells. The capture efficiency of MCF-7 and Hela cells is as high as 93±3% and 90±2%, respectively. In addition, the captured cancer cells still have a high survival ability on the hydrogel surface. It is widely used in biomedical engineering and has good development and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the process for preparing double-network hydrogel surfaces with cell imprinting and aptamers.

[0027] Figure 2 It is the SEM picture of comparative example 1-3 and embodiment 1-2; Figure 2 a and Figure 2 a1 is the SEM image of comparative example 2, Figure 2 b and Figure 2 b1 is the SEM image of comparative example 1, Figure 2 c and Figure 2 c1 is the SEM image of Example 1, Figure 2 d and Figure 2 d1 is the SEM image of Example 2, Figure 2 e and Figure 2 e1 is the SEM image of Comparative Example 3;

[0028] Figure 3 is the UV spectrum of the double network hydrogel surface modified with different contents of aptamers prepared in Example 1;

[0029] Figure 4 is the Hoechst staining image of cells in Example 1; Figure 4 a is the Hoechst staining image before cell removal. Figure 4 b is the composite image before cell removal, Figure 4 c is the Hoechst staining image before cell removal. Figure 4 d is the composite image after cell removal;

[0030] Figure 5 It is a statistical diagram of the swelling rate and degradation rate of Comparative Example 1 and Examples 1-2; Figure 5 a is a statistical diagram of the swelling ratios of Example 1 and Examples 1-2, Figure 5 b is a statistical graph of degradation rates of Comparative Example 1 and Examples 1-2;

[0031] Figure 6 is a statistical diagram of the capture efficiency of the double network hydrogel surface prepared in Example 2 for cancer cells at different incubation times;

[0032] Figure 7 It is a statistical graph of cancer cell capture efficiency and cancer cell proliferation quantity; Figure 7 a is a statistical diagram of cancer cell capture efficiency of comparative example 1-2 and embodiment 1-2, Figure 7 b is a statistical diagram of the number of cancer cell proliferations in Comparative Example 2 and Example 2;

[0033] Figure 8 These are the microscope pictures and SEM pictures of cancer cell adhesion in Comparative Example 2 and Example 2; Figure 8 a1. Figure 8 a2 and Figure 8 a3 are the microscopic images of ECA-109, Hela and MCF-7 growing on ordinary culture dishes. Figure 8 b1. Figure 8 b2 and Figure 8 b3 are the microscopic images of the growth states of ECA-109, Hela and MCF-7 on the surface of Comparative Example 2, Figure 8 c1. Figure 8 c2 and Figure 8 c3 are the microscopic images of the growth states of ECA-109, Hela and MCF-7 on the surface of Example 2, Figure 8 d1, Figure 8 d2 and Figure 8 d3 are SEM images of the growth states of ECA-109, Hela and MCF-7 on the surface of Example 2. DETAILED DESCRIPTION

[0034] In order to further describe the present invention in detail, the following specific implementation examples are given, but they are only used to illustrate the present invention and make the steps clearer, rather than to limit the scope of application of the present invention.

[0035] Comparative Example 1

[0036] Preparation of cell templates: Before making cell imprints, cells need to be cultured to a density of 70%-90%. We cultured MCF-7 cells in a 100mm×20mm cell culture dish until the cell density reached 70%-90%. Before collecting the cells, wash the cells 3 times with PBS to remove impurities and residual serum components in the culture medium. Then, fix the cells with 4% paraformaldehyde (PFA) at room temperature for 10 minutes to fix the cells on the culture dish. Subsequently, wash the cells with PBS to remove PFA. This step is to remove excess PFA and place the cells in PBS for continued imprinting.

[0037] Preparation of cell-imprinted hydrogel surfaces: 1.8 g AM, 0.03 g APS, and 120 μL 10 mg ml -1 TEMED was added to 10 mL of pure water. The mixture was heated to 60 °C and vibrated on an oscillator for 20 min to form a transparent solution. Subsequently, 120 μL of MBAA was added, mixed well, and the mixture was quickly transferred to a culture dish with a cell template and heated at 70 °C for 2 h to obtain a PAM hydrogel. The PAM hydrogel was peeled off and soaked in PBS until it swelled to its maximum level. In order to remove unreacted monomers and deplete the cell template, the hydrogel was washed with PBS and incubated with 0.25% trypsin solution at 37 °C. After the cells were completely removed, the trypsin solution was replaced with PBS and cell culture medium for washing and cut into 24 mm 2 circular shape.

[0038] Preparation of PAM hydrogel (CMD-CIDH1) surface with cell imprints and aptamers: First, the cell imprints cut into circular shapes were immersed in a 0.1 mg / mL poly-D-lysine solution for 12 h. Then, the poly-D-lysine-modified cell imprints were immersed in a solution containing a mixture of 1 mg / mL sulfo-NHS and 2 mg / mL EDC for 2 h and washed twice with a PBS solution. Next, the cell imprint surface was covered with a 10 μg / mL polycyclic DNA aptamer (CMD) solution, left for 1 h, and washed twice with a PBS solution to obtain a CMD-CIDH1 surface. The molar ratio of poly-D-lysine to CMD was 0.6.

[0039] Figure 2 (b and b1) are SEM images of CMD-CIDH1. It can be seen from the figures that CMD-CIDH1 has a typical honeycomb structure and nanoparticles (NPs), where the pore size of CMD-CIDH1 is 40 μm and the size of the nanoparticles is 1300 nm.

[0040] Comparative Example 2

[0041] Preparation of cell-free DN hydrogel surface: 1.8 g AM, 0.75 g short-chain CS (90% deacetylation), 0.03 g APS and 120 μL 10 mg mL -1TEMED was added to 10 mL of pure water. The mixture was heated to 60 °C and vibrated on an oscillator for about 20 min to allow the mixture to form a transparent solvent. After the mixture was deoxygenated, 120 μL of MBAA was added, mixed well, and the mixture was quickly transferred to a culture dish and heated at 70 °C for 2 h to obtain a PAM-CS composite hydrogel. The composite hydrogel was soaked in a 1 M CaCl2 solution for 20 min. After standing for 5 h, a uniform PAM-CS DN hydrogel without a cell template was obtained. The DN hydrogel was peeled off and soaked in PBS until it swelled to the maximum level. In order to remove unreacted monomers and deplete cell templates, the hydrogel was washed with PBS and incubated with 0.25% trypsin solution at 37 °C. After the cells were completely removed, the trypsin solution was replaced with PBS and cell culture medium for washing and cut into 24 mm 2 circular shape.

[0042] Preparation of DN hydrogel (CMD-NCIDH) surface with aptamer: The preparation process is the same as that of Comparative Example 1.

[0043] Figure 2 (a and a1) are SEM images of CMD-NCIDH. Figure 2 a and Figure 2 As can be seen from a1, CMD-NCIDH has a mesh structure with a smooth surface and no nanoparticles.

[0044] Comparative Example 3

[0045] Preparation of cell template: The preparation process is the same as that of Comparative Example 1.

[0046] Preparation of cell-imprinted DN hydrogel surface: 1.8 g AM, 1.5 g short-chain CS (90% deacetylation), 0.03 g APS and 120 μL 10 mg mL -1 TEMED was added to 10 ml of pure water. The mixture was heated to 60 °C and vibrated on an oscillator for about 20 minutes to allow the mixture to form a transparent solvent. After the mixture was deoxygenated, 120 μL of MBAA was added, mixed well, and the mixture was quickly transferred to the above-mentioned culture dish with the cell template and heated at 70 °C for 2 hours to obtain the PAM-CS composite hydrogel. The composite hydrogel was soaked in a 1M CaCl2 solution for 20 minutes, and after standing for 5 hours, a uniform PAM-CSDN hydrogel with a cell template was obtained. The DN hydrogel was peeled off and soaked in PBS until it swelled to the maximum level. In order to remove unreacted monomers and deplete the cell template, the hydrogel was washed with PBS and incubated with 0.25% trypsin solution at 37 °C. After the cells were completely removed, they were washed with PBS and cell culture medium instead of the trypsin solution and cut into 24 mm 2 circular shape.

[0047] Preparation of DN hydrogel (CMD-CIDH4) surface with cell imprinting and aptamer: The preparation process is the same as that of Comparative Example 1.

[0048] Figure 2 (e and e1) are SEM images of CMD-CIDH4. It can be seen from the figures that the typical honeycomb structure of the hydrogel has been replaced by an irregular structure. In addition, the NPs disappeared and were replaced by a denser structure.

[0049] Example 1

[0050] Preparation of cell template: The preparation process is the same as that of Comparative Example 1.

[0051] Preparation of cell-imprinted DN hydrogel surface: 1.8 g AM, 0.5 g short-chain CS (90% deacetylation), 0.03 g APS and 120 μL 10 mg mL -1 TEMED was added to 10 mL of pure water. The mixture was heated to 60 °C and vibrated on an oscillator for about 20 minutes to allow the mixture to form a transparent solvent. After the mixture was deoxygenated, 120 μL of MBAA was added, mixed well, and the mixture was quickly transferred to the above-mentioned culture dish with the cell template and heated at 70 °C for 2 hours to obtain the PAM-CS composite hydrogel. The composite hydrogel was soaked in a 1M CaCl2 solution for 20 minutes, and after standing for 5 hours, a uniform PAM-CSDN hydrogel with a cell template was obtained. The DN hydrogel was peeled off and soaked in PBS until it swelled to the maximum level. In order to remove unreacted monomers and deplete the cell template, the hydrogel was washed with PBS and incubated with 0.25% trypsin solution at 37 °C. After the cells were completely removed, they were washed with PBS and cell culture medium instead of the trypsin solution and cut into 24 mm 2 circular shape.

[0052] Preparation of DN hydrogel surface with cell imprinting and aptamers (CMD-CIDH2): The preparation process was the same as that of Comparative Example 1; the molar ratios of poly-D-lysine to CMD were 0.6, 0.8 and 1.0 respectively. Figure 3 In addition, the data characterizations related to the rest of Example 1 are all the results when the molar ratio of poly-D-lysine to CMD is 0.6.

[0053] Figure 2 (c and c1) are SEM images of CMD-CIDH2. It can be seen from the images that CMD-CIDH2 has a typical honeycomb structure and nanoparticles, where the pore size of CMD-CIDH2 is 32 μm and the size of the nanoparticles is 760 nm.

[0054] Figure 3UV spectra of DN hydrogel surfaces modified with different amounts of aptamers. The peak at 260 nm indicates that CMD has been successfully modified on the DN hydrogel surface. The different amounts of aptamer modification lead to changes in peak shape and peak value.

[0055] Figure 4 The cell removal rates before and after the cell template preparation are shown, and the Hoechst staining image shows that the cell removal efficiency is 100%.

[0056] Example 2

[0057] Preparation of cell template: The preparation process is the same as that of Comparative Example 1.

[0058] Preparation of cell-imprinted DN hydrogel surface: 1.8 g AM, 0.75 g short-chain CS (90% deacetylation), 0.03 g APS and 120 μL 10 mg mL -1 TEMED was added to 10 ml of pure water. The mixture was heated to 60 °C and vibrated on an oscillator for about 20 minutes to allow the mixture to form a transparent solvent. After the mixture was deoxygenated, 120 μL of MBAA was added, mixed well, and the mixture was quickly transferred to the above-mentioned culture dish with the cell template and heated at 70 °C for 2 hours to obtain the PAM-CS composite hydrogel. The composite hydrogel was soaked in a 1M CaCl2 solution for 20 minutes, and after standing for 5 hours, a uniform PAM-CSDN hydrogel with a cell template was obtained. The DN hydrogel was peeled off and soaked in PBS until it swelled to the maximum level. In order to remove unreacted monomers and deplete the cell template, the hydrogel was washed with PBS and incubated with 0.25% trypsin solution at 37 °C. After the cells were completely removed, they were washed with PBS and cell culture medium instead of the trypsin solution and cut into 24 mm 2 circular shape.

[0059] Preparation of DN hydrogel (CMD-CIDH3) surface with cell imprinting and aptamer: The preparation process is the same as that of Comparative Example 1.

[0060] Figure 2 (d and d1) are SEM images of CMD-CIDH3. It can be seen from the images that CMD-CIDH3 has a typical honeycomb structure and nanoparticles, where the pore size of CMD-CIDH3 is 21 μm and the size of the nanoparticles is 400 nm.

[0061] Performance Testing

[0062] 1. Stability test

[0063] The swelling rate and degradation rate (DR) of the hydrogel were detected. CMD-CIDH1-3 was placed in PBS solution at 37°C for 10 h, and CMD-CIDH1-3 still showed good swelling properties. This is because CS interacted with the polymer, increasing the cross-linking density of the hydrogel, thereby increasing the porosity of the hydrogel, and further improving the swelling properties of the hydrogel to a certain extent.

[0064] CMD-CIDH1-3 was freeze-dried, and the initial mass W0 of CMD-CIDH1-3 was recorded. Then, CMD-CIDH1-3 was placed in a PBS solution at 37°C, and its swelling changes were observed. At 2h, 6h, 12h, 24h, 48h, and 72h, filter paper was used to absorb the moisture on the surface of each example, and samples were taken and their weights W were recorded. i When the weight no longer changes, the expansion equilibrium is reached. Three samples were tested in each group.

[0065] The calculation formula of hydrogel swelling degree (W) is as follows:

[0066] W=(W i -W0) / W0×100%

[0067] The results are as follows Figure 5 As shown by Figure 5 It can be seen from a that the swelling rates of CMD-CIDH1-3 are 92%, 86% and 81% respectively. This is because the pore size of CMD-CIDH1 is 40 μm, and the size of the nanoparticles is 1300 nm. The larger pore size and nanoparticle size make its swelling rate higher.

[0068] After freeze-drying CMD-CIDH1-3, its original mass M0 was recorded. Then, CMD-CIDH1-3 was placed in a 37°C PBS degradation solution and incubated under an ultraviolet lamp. The degradation solution was changed every day and the freeze-dried CMD-CIDH1-3 was weighed and its mass M0 was recorded. i Three samples were tested in each group.

[0069] The hydrogel degradation rate (DR) can be calculated by the following formula:

[0070] DR=(M0-M i ) / M0×100%

[0071] Depend on Figure 5 As can be seen in b, CMD-CIDH1 was completely degraded on the 12th day, and CMD-CIDH2-3 was completely degraded on the 14th day.

[0072] 2. Cell capture efficiency and proliferation test

[0073] PD-L1-positive MCF-7 and Hela cells, as well as PD-L1-negative human esophageal cancer cell ECA-109, were selected to evaluate the sensitivity of CMD-CIDH3 surface as a target cell recognition. First, the capture efficiency of CMD-CIDH3 surface on the above cells was determined after different incubation times. Figure 6 The results showed that after 90 minutes of incubation, the capture efficiency of MCF-7 and Hela cells on the CMD-CIDH3 surface was significantly improved, reaching 93±3% and 90±2%, respectively, while the capture efficiency of PD-L1-negative ECA-109 cells was less than 7%. The results showed that the CMD-CIDH3 surface has high sensitivity and specificity in recognizing specific cell lines.

[0074] PD-L1 positive MCF-7 and Hela cells were selected to detect the capture efficiency of target cells by CMD-NCIDH and CMD-CIDH1-3 surfaces. The results are as follows Figure 7 As shown in Figure 2, the capture efficiency of MCF-7 and Hela cells on the CMD-CIDH2-3 surface is much higher than that on the CMD-NCIDH and CMD-CIDH1 surfaces ( Figure 7 a); Then the activity of the target cells captured on the surface of CMD-CIDH3 was detected, and further cultured and propagated. Figure 7 b shows that the proliferation number of MCF-7 cells captured on the CMD-CIDH3 surface was similar to that of untreated cells, increasing exponentially over time, and the slopes of the linear plots of their proliferation logarithms were similar, 0.043 (R 2 =0.994) and 0.045 (R 2 =0.996), indicating that these cells still have a high viability.

[0075] 3. Cell Growth Status Test

[0076] PD-L1-positive MCF-7 and Hela cells, as well as PD-L1-negative human esophageal cancer cell ECA-109, were selected to detect the growth status of different types of cancer cells captured by CMD-CIDH3. The results are as follows Figure 8 As shown, Figure 8 a shows the above ECA-109 ( Figure 8 a1)、Hela( Figure 8 a2) and MCF-7( Figure 8 a3) Growth status on ordinary culture dishes, Figure 8 b shows the above ECA-109 ( Figure 8 b1)、Hela( Figure 8 b2) and MCF-7( Figure 8 b3) Growth states on the CMD-NCIDH surface, respectively, Figure 8c shows the above ECA-109 ( Figure 8 c1)、Hela( Figure 8 c2) and MCF-7( Figure 8 c3) Growth states on the CMD-CIDH3 surface, Figure 8 d is the above ECA-109 ( Figure 8 d1)、Hela( Figure 8 d2) and MCF-7( Figure 8 d3) SEM images of the growth states on the CMD-CIDH3 surface. The results show that the three cells are round on the CMD-NCIDH surface and are not fully stretched. On the CMD-CIDH3 surface, the three cells have tail feet and are spread out, which is the same as the spread state of ordinary culture dishes. At the same time, the SEM images also verify that the three cells have good adhesion, growth and proliferation on the CMD-CIDH3 surface.

[0077] The above embodiments can partially demonstrate the content of the present invention, but the present invention is not limited to the monomers described in the embodiments, that is, the description of the above embodiments is not a limitation of the present invention. Those skilled in the art should understand that improvements to the present invention, equivalent replacement of raw materials of the products of the present invention, addition of auxiliary components, or direct or indirect application in other related technical fields should all be included in the scope of patent protection of the present invention.

Claims

1. A method for preparing a double network hydrogel surface, characterized in that: The structure of the double network hydrogel surface is a micron-scale honeycomb structure with a pore size of 21-32 μm; the double network hydrogel surface is composed of nanoparticles with a size of 400nm-760nm; the double network hydrogel surface has a synergistic effect of cell imprinting and aptamer affinity; the preparation method adopts the following steps: (1) Preparation of cell template: Cultivate cancer cells until the cell density reaches 70-90%, wash, and fix the cells on the culture dish using cell fixative to obtain a cell template; (2) Add 1.8 g acrylamide, 0.5 g or 0.75 g of short-chain chitosan with a deacetylation degree of 90% and an initiator mixture to 10 mL of pure water to obtain a mixture, heat it to 60°C, and vibrate it on an oscillator for 20 min to form a transparent solvent; after the mixture is deoxygenated, add 120 μL of MBAA and mix well, then quickly transfer it to the culture dish of the cell template in step (1) and heat it at 70°C for 2 h to obtain a PAM-CS composite hydrogel; soak the PAM-CS composite hydrogel in 1 M CaCl2 solution for 20 min, and after leaving it for 5 h, obtain a PAM-CS DN hydrogel with a cell template; peel off the PAM-CS DN hydrogel and soak it in PBS until it swells to the maximum level; the initiator mixture consists of 0.03 g APS and 120 μL 10 mg / mL TEMED; (3) The PAM-CS DN hydrogel prepared in step (2) is immersed in a solution containing poly-D-lysine, and then immersed in a sulfo-NHS / EDC mixture, washed, and the cell imprinting surface is covered with an aptamer solution, washed, and a double network hydrogel surface is obtained.

2. The preparation method according to claim 1, characterized in that: The cancer cells described in step (1) are breast cancer cells, cervical cancer cells, esophageal cancer cells, gastric cancer cells, colorectal cancer cells, lung cancer cells or liver cancer cells.

3. The preparation method according to claim 1, characterized in that: In step (2), the PAM-CS DN hydrogel is peeled off and immersed in PBS until it swells to the maximum level, and then the following steps are included: in order to remove unreacted monomers and exhaust the cell template, the PAM-CS DN hydrogel is washed with PBS and incubated with 0.25% trypsin solution at 37°C; after the cells are completely removed, the trypsin solution is replaced by PBS and cell culture medium for washing.

4. The preparation method according to claim 1, characterized in that: In step (3), the concentration of the aptamer solution is 5-10 μg / mL, and the time for the aptamer solution to cover the cell imprint surface is 0.5-1h; the concentration ratio of sulfo-NHS to EDC in the sulfo-NHS / EDC mixture is 1:(2-3), wherein the concentration of sulfo-NHS is 1 mg / mL; the immersion time in the sulfo-NHS / EDC mixture is 1-2h; and the washing in steps (1) and (3) is performed using PBS.

5. Application of the double network hydrogel surface prepared by the method according to any one of claims 1 to 4, characterized in that: The hydrogel surface is applied to CTC culture.

6. The use according to claim 5, characterized in that The application is drug sensitivity testing of CTC culture.

Images