Microsphere adsorbent for removing circulating tumor cells through blood perfusion as well as preparation method and application of microsphere adsorbent

Abstract

The invention discloses a microsphere adsorbent for removing circulating tumor cells through blood perfusion and a preparation method and application of the microsphere adsorbent, and relates to the technical field of medical adsorbents for blood perfusion, the microsphere adsorbent comprises a carrier and ligands grafted to the surface of the carrier, the carrier is composed of agarose gel microspheres 2B and analogues thereof, the ligands comprise HA-N3 and CE, hA grafted on the microspheres by using an azide-alkyne click chemical reaction can be specifically combined with a transmembrane adhesion glycoprotein CD44 receptor, and CE is grafted on the HA through an amidation reaction and is specifically combined with a glucose transporter 1GLUT1 receptor on the surface of CTCs. When peripheral blood flows through the device filled with the adsorbent, the CTCs are selectively adsorbed. According to the adsorbent, the capturing efficiency of CTCs is remarkably improved, the adsorbent is used for blood perfusion treatment, the adsorbent can be used as a substitute or supplement for existing clinical surgical resection, chemotherapy or radiotherapy, the risk of tumor distal metastasis is effectively reduced, patient injury is reduced, and the cure rate is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of medical adsorbents for blood perfusion, and in particular to a microsphere adsorbent for removing circulating tumor cells by blood perfusion, and a preparation method and application thereof. Background Art

[0002] Circulating tumor cells (CTCs) play a vital role in the process of tumor metastasis. Although conventional treatment can prolong the survival of cancer patients, the tumor-related mortality rate remains high due to the limitations of surgical treatment and tumor resistance. Therefore, preventing CTCs from spreading to distant organs through blood circulation is the key to reducing the risk of tumor metastasis and recurrence.

[0003] Currently, the methods for capturing CTCs are mainly divided into physical methods and biological methods. Physical methods separate CTCs based on differences in size, density, etc., but the long capture cycle and high cost limit their in vivo applications. Biological methods rely on antibody technology, using antibody-coupled devices or magnetic beads to target specific biomarkers to more accurately capture CTCs. Some studies have combined physical and biological methods to develop a system of microfluidic microarrays and antibody-functionalized magnetic beads, but these technologies are mainly used for in vitro detection and still need to be combined with traditional treatments.

[0004] As a method of blood purification technology, hemoperfusion can effectively remove exogenous and endogenous toxins and metabolic wastes in the blood through the adsorption of adsorbents in the hemoperfusion device. It has been widely used in the treatment of renal failure, liver failure, drug poisoning and autoimmune diseases. However, the existing hemoperfusion technology can only effectively remove medium-molecule and large-molecule toxic substances in the blood, and cannot effectively remove CTCs. In the past tumor metastasis inhibition, the removal efficiency of CTCs in the blood circulation by using a single targeted ligand is often low, and the effect of inhibiting tumor metastasis is limited due to factors such as the complex blood environment and the loss of CTCs receptors caused by epithelial-mesenchymal transition (EMT).

[0005] In order to comprehensively and effectively remove CTCs in the patient's systemic blood circulation, prevent distant metastasis of the tumor, and thus achieve a radical cure of cancer, it is necessary to develop a blood perfusion adsorbent that can capture and remove circulating tumor cells. Summary of the invention

[0006] The purpose of the present invention is to solve the deficiencies in the prior art and to provide a microsphere adsorbent for removing circulating tumor cells by blood perfusion, and a preparation method and application thereof.

[0007] In order to solve the above technical problems, the present invention adopts the following technical scheme: a microsphere adsorbent for removing circulating tumor cells by blood perfusion, comprising a carrier and a ligand grafted to the surface of the carrier, wherein the carrier is composed of agarose gel microspheres 2B and its analogs, and the ligand comprises azid hyaluronic acid (HA-N 3 ) and 4-aminophenyl-β-D-cellobiose (CE), the HA-N 3 The CE is grafted onto the carrier through any one or more of click chemistry reaction, epoxidation reaction, epoxidation ring-opening reaction, amide reaction, and carboxyl activation reaction.

[0008] Furthermore, the carrier is dibenzocyclooctyne-amino (DBCO-NH 2 ) modified agarose gel microspheres, the diameter of the microspheres is 60μm to 180μm.

[0009] Furthermore, the azid hyaluronic acid (HA- 3 ) The grafting ratio of the azidation group is 1% to 10%, and the average protein molecular weight is 7 to 600 KDa.

[0010] Further, the click chemistry reaction is one of copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), oxazolidinone-azide cycloaddition (Oxyamine-Azide Cycloaddition), Diels-Alder reaction, thiol-ene addition (Thiol-Ene Reaction), and thiol-yne addition (Thiol-Yne Reaction).

[0011] Furthermore, the HA-N 3 The CE was grafted onto the microspheres via an azide-alkyne cycloaddition reaction, and the CE was grafted onto the HA via an amidation reaction.

[0012] Furthermore, the preparation method of the microsphere adsorbent for removing circulating tumor cells by blood perfusion comprises the following steps:

[0013] (1) Dibenzocyclooctyne-amino (DBCO-NH 2 ) Preparation of modified agarose gel microspheres: Epichlorohydrin activated microspheres, activated microspheres, DBCO-NH 2Add to a solution of NaOH and DMSO in a volume ratio of 1:2, adjust the pH, stir and react at room temperature, filter after the reaction is completed, and wash with ultrapure water to obtain DBCO-NH 2 Modified microspheres;

[0014] (2) Hyaluronic acid azidate (HA-N 3 ) grafting: DBCO-NH 2 Modified microspheres, HA-N 3 and ultrapure water were added to the flask and reacted at room temperature. After the reaction was completed, filtration was performed and ultrapure water was used to wash to obtain HA-N 3 Grafted microspheres;

[0015] (3) Grafting of 4-aminophenyl-β-D-cellobiose (CE): HA-N 3 The grafted microspheres, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were added to the 2-morpholineethanesulfonic acid (MES) solution and stirred at room temperature to activate the carboxyl groups on the microspheres. Then, 4-aminophenyl-β-D-cellobioside was added to the flask and reacted at room temperature. After the reaction was completed, the solution was filtered and washed with ultrapure water to obtain CE-grafted microspheres.

[0016] Furthermore, in step (1), the microspheres and DBCO-NH 2 The mass ratio of the feed amount is 60:1-18:1; in the step (2), DBCO-NH 2 Modified microspheres and HA-N 3 The mass ratio of the feed amount is 120:1-60:1; in the step (3), HA-N 3 The mass ratio of the grafted microspheres to the 4-aminophenyl-β-D-cellobioside is 180:1-900:11.

[0017] Furthermore, the microsphere adsorbent used for blood perfusion to remove circulating tumor cells is used in the preparation of a preparation for capturing CTCs in tumors.

[0018] Beneficial effects:

[0019] The agarose gel microsphere adsorbent provided by the present invention can effectively enrich and remove CTCs in the patient's blood through adsorption. Compared with the previous single ligand or multi-ligand capture technology, the present invention modifies the multivalent ligand on the microsphere through the azide-alkyne reaction, which significantly improves the capture efficiency of CTCs. The use of this adsorbent for hemoperfusion therapy can be used as a substitute or supplement for existing clinical surgical resection, chemotherapy or radiotherapy, effectively reducing the risk of distant tumor metastasis, reducing patient harm, and improving the cure rate. At the same time, CTCs can also be collected from the adsorbent to provide valuable guidance for the diagnosis and treatment of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an enlarged image of the multivalent ligand-modified microspheres.

[0021] Figure 2 The synthetic route of multivalent ligand-modified microspheres.

[0022] Figure 3 Characterization of multivalent ligand-modified microspheres.

[0023] Figure 4 Characterization of CTCs captured by multivalent ligand-modified microspheres.

[0024] Figure 5 To evaluate the capture efficiency of multivalent ligand-modified microspheres under static conditions and compare them with microspheres synthesized by other methods.

[0025] Figure 6 Evaluation of blood compatibility and biocompatibility of multivalent ligand modified microspheres.

[0026] Figure 7 To evaluate the effect of multivalent ligand-modified microspheres on the removal of CTCs in a rat model and its in vivo biosafety.

[0027] Figure 8 The ability of multivalent ligand-modified microspheres to release CTCs. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood. The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0029] Example 1

[0030] First, agarose microspheres were activated with epichlorohydrin: 900 mg of washed and suction-dried agarose microspheres 2B were suspended in 10.5 mL of dimethyl sulfoxide (DMSO), mixed with 3 mL of 0.4 M sodium hydroxide solution (0.4 M NaOH), and then 1.5 mL of epichlorohydrin was added. The suspension was shaken and cultivated at 100 rpm for 6 hours at room temperature (RT). It was then transferred to a glass filter funnel and washed with 200 mL of ultrapure water to obtain epoxidized microspheres (AG-Epo).

[0031] 900 mg AG-Epo, 33.4 mg DBCO-NH, 2.5 mL 0.4 M NaOH and 10 mL DMSO were added to a 50 mL flask and reacted at room temperature for 5 hours. During this process, the pH value of the solution was adjusted to 10-11 using 0.4 M NaOH. It was then transferred to a glass filter funnel and washed with 200 mL ultrapure water to obtain DBCO-NH 2 Modified microspheres (AG-DBCO).

[0032] Combine AG-DBCO, 10mg HA-N 3 and 10 mL of ultrapure water were added to a 25 mL flask and reacted at room temperature for 1 hour. Then it was transferred to a glass filter funnel and washed with 200 mL of ultrapure water to obtain HA-N 3 Grafted microspheres (AG-HA).

[0033] AG-HA, 5.75 mg NHS, 9.59 mg EDC and MES were added to a 25 mL flask to activate the carboxyl groups on AG-HA for 1 hour. Then 6.9 mg 4-aminophenyl-β-D-cellobioside was added to the flask and reacted at room temperature for 24 hours. After the reaction was completed. Then it was transferred to a glass filter funnel and washed with 200 mL ultrapure water to obtain CE-grafted microspheres (AG-HA-CE).

[0034] The above HA-N 3 The click chemistry reaction used is the copper-free catalyzed azide-alkyne cycloaddition (SPAAC, Strain-Promoted Azide-Alkyne Cycloaddition) reaction. The same effect can also be achieved by using copper-catalyzed azide-alkyne cycloaddition (CuAAC, Copper-Catalyzed Azide-Alkyne Cycloaddition), oxazolidinone-azide cycloaddition (Oxyamine-Azide Cycloaddition), Diels-Alder reaction, thiol-ene addition (Thiol-Ene Reaction), thiol-yne addition (Thiol-Yne Reaction) and other reaction methods.

[0035] Figure 3 The characterization results of multivalent ligand-modified microspheres. Figure 3 As shown in A, when the molecular weight of HA is 60KDa, AG-HA has the highest capture efficiency for cancer cells. Figure 2 The multivalent ligand-modified microspheres AG-HA-CE were synthesized by a synthetic route. 2Grafting was then performed with HA-N by click chemistry reaction. 3 Grafting to obtain AG-HA. Grafting AG-HA with CE through amidation reaction to obtain AG-HA-CE. 2 Feeding amount( Figure 3 B) and reaction time ( Figure 3 C) DBCO-NH 2 The effect of grafting rate on the DBCO-NH 2 The grafting rate of HA-N was the highest, at 40.1%. 3 dose( Figure 3 D) and reaction time ( Figure 3 E) To HA-N 3 The best dosage and reaction time were determined by the effect of the grafting rate. When the dosage was 10 mg and the reaction time was 1 h, the grafting rate was the highest at 55.6%. Figure 3 F investigated the effect of CE dosage on the reaction grafting rate and found that the grafting rate was the highest when 6.9 mg CE was added, which was 74.4%.

[0036] Example 2

[0037] Static capture of CTCs: 50 mg of AG-HA-CE was placed in a 2 ml EP tube, and then 2 × 10 5 4T1 cells and supplemented with DMEM to 1 ml. Gently pipette the cells and microspheres, then add them to a 24-well plate and incubate in a 37°C incubator for 30 minutes. After 30 minutes, filter the microspheres through a cell strainer and calculate the capture efficiency using visual counting or fluorescence counting.

[0038] Figure 5 To evaluate the capture efficiency of multivalent ligand-modified microspheres under static conditions, and to compare them with microspheres synthesized by other methods. Figure 5 As shown in A, the capture ability of microspheres (AG-BSA, AG-HA, AG-HA-CE) modified by azide-alkyne (click chemistry) reaction at different time points was investigated. The results showed that AG-HA-CE had the highest capture efficiency, reaching a peak of 97.5% at 30 minutes, which was significantly better than AG-BSA and AG-HA. Figure 5 B shows the microspheres modified by esterification reaction (AG-Rg 3 AG-HA, AG-HA-Rg 3 ) capture capacity, among which AG-HA-Rg 3 A peak capture efficiency of 90% was achieved at 120 min. Figure 5C shows the capture efficiency of different antibody-modified microspheres (SMs-EGFR, SMs-EpCAM, SMs-EE (EGFR & EpCAM)), among which SMs-EE reached a peak capture efficiency of 73.3% at 60 minutes. 3 In comparison, the azide-alkyne (click chemistry) modified multivalent ligand microspheres showed higher capture efficiency and shorter capture time (the amount of microspheres in Figures A, B, and C was the same). Figure 5 As shown in D, AG-HA-CE can effectively capture a variety of tumor cells, with a capture efficiency of 95.1±1.25%. The multivalent ligand microspheres modified by click chemistry significantly enhanced the capture efficiency of CTCs, making the enrichment process faster and more efficient.

[0039] Example 3

[0040] The erythrocyte compatibility of AG-HA-CE was evaluated by hemolysis test. Blood from rats was collected using a vacuum tube containing sodium citrate. Whole blood was centrifuged at 2000rpm for 10 minutes to separate red blood cells (RBC) from plasma, and the centrifugation process was repeated until the supernatant was clear and colorless. The separated erythrocyte suspension was prepared into a 2% (V / V) erythrocyte suspension with normal saline. The diluted erythrocyte suspension was added to 50 mg AG-HA-CE pre-soaked in PBS overnight and incubated at 37°C for 3 hours. Normal saline was selected as a negative control and deionized water as a positive control. The suspension was then centrifuged at 3000rpm for 5 minutes, and the absorbance of hemoglobin released in the suspension was measured at 540nm using a microplate reader, and the hemolysis rate was then calculated.

[0041] Protein adsorption experiments were performed with HSA solution under static conditions. First, 50 mg of AG-HA-CE was pre-soaked in PBS overnight and then incubated at 37°C for 1 hour. The microspheres were then immersed in a PBS solution containing HSA at a concentration of 1 mg / mL and incubated at 37°C for one hour. The supernatant protein concentration was determined using a BCA kit. The protein adsorption amount (μg / mg) of AG-HA-CE was calculated.

[0042] Platelet activation was assessed by ELISA using a rat platelet factor 4 (PF4) kit. AG-HA-CE (10 mg) was pre-soaked in PBS and placed in a 1.5 mL EP tube overnight. Then, PBS was removed and 150 μL of rat whole blood was added. After incubation at 37°C for 1 hour, the whole blood was centrifuged at 2500 g for 10 minutes (2-8°C) to obtain plasma. Next, an ELISA was performed according to the instructions of the rat platelet factor 4 (PF4) kit.

[0043] Routine blood test evaluation: First, 50 mg of AG-HA-CE was pre-soaked in a 1.5 mL PBS EP tube (pH = 7.4) overnight and equilibrated at 37 ° C for 1 hour. Secondly, fresh whole blood of rats was collected using EDTA-K2 anticoagulation blood collection tubes. After removing PBS, 500 μL of fresh rat whole blood was introduced into each test tube. Then, AG-HA-CE was incubated with whole blood at 37 ° C for 60 minutes, and the remaining blood was collected. Routine blood test evaluation was performed using an automated hematology cell analyzer according to the instructions provided.

[0044] PPP coagulation analysis: 10 mL of rat blood was added to a sodium citrate anticoagulation collection tube and then centrifuged at 4000 rpm for 15 minutes to obtain PPP. Then 0.5 mL of PPP was added to 50 mg of AG-HA-CE in a 1.5 mL EP tube and gently shaken at 37°C for 1 hour. PPP without AG-HA-CE was treated as a control. The test was performed by an automated coagulation analyzer RAC-1830.

[0045] Cytotoxicity assay: 4T1 and L02 cells were grown in DMEM supplemented with 10% FBS and 1% antibiotics (penicillin-streptomycin) at 37°C in a humidified incubator with 5% CO2. The cytotoxicity of AG-HA-CE was evaluated by the CCK-8 cell viability test kit. Before measurement, 2 g of AG-HA-CE was pre-soaked in DMEM (10 mL) and incubated at 37°C for 24 hours. The supernatant was then filtered and sterilized with a 0.22 μm microporous membrane to obtain the supernatant. The desired density of cells (1×104 cells per well) was inoculated in a 96-well plate for 24 hours. After washing with PBS, 100 μL of the supernatant was added to the culture medium and incubated at 37°C for 24 hours under the above conditions. The control group was incubated with 100 μL of DMEM culture medium without AG-HA-CE, and the blank group was incubated with DMEM culture medium and CCK-8 without cells. Then, CCK-8 solution (10 μL) was added and incubated for 4 hours. OD values ​​were obtained at 450 nm using a microplate reader. Each sample was tested six times in parallel. Cell viability was calculated.

[0046] Figure 6 To evaluate the blood compatibility and biocompatibility of multivalent ligand-modified microspheres. Figure 6 As shown in Figure A, the hemolysis rate of AG-HA-CE (0.26±1.1%) is lower than that of Biosky (2.04±0.47%) (a commercially available hemoperfusion adsorbent). This indicates that AG-HA-CE not only meets the requirements of the applied material hemolysis test (ASTM, F756-2008), but also has better red blood cell compatibility. Figure 6B The results showed that the plasma protein adsorption of AG-HA-CE was lower, only 3.9 μg / mg, compared with Biosky (7.7 μg / mg). Figure 6 C fluorescence imaging further demonstrated the lower HSA adsorption capacity of AG-HA-CE. Figure 6 D The results showed that AG-HA-CE did not cause significant changes in blood cell counts or their sizes. Figure 6 E The coagulation properties of AG-HA-CE were investigated, including activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and fibrinogen (FIB), indicating that AG-HA-CE hardly induced whole blood coagulation. Figure 6 F The effect of AG-HA-CE on platelet activation was investigated. The results showed that AG-HA-CE did not induce platelet activation because platelet activation would release PF4 when the biomaterial was exposed to blood. Figure 6 G The cell compatibility of AG and AG-HA-CE was investigated. The results showed that these cells maintained more than 90% of their proliferation activity. These results indicate that AG-HA-CE has excellent blood compatibility and biocompatibility.

[0047] Example 4

[0048] In vivo circulation experiment in rat model: SD rats (male, 280-300 g) were housed under pathogen-free conditions for 7 days. The experimental procedures were performed under general anesthesia. The surgical procedure was as follows: First, the blood perfusion device was installed and the sodium heparin solution was filled in the catheter. The air bubbles were removed from the catheter and an indwelling needle was connected to the end of the catheter; the indwelling needle was then inserted into the abdominal aorta and inferior vena cava, and the indwelling needle and blood vessels were fixed with surgical sutures. At the end of the operation, the peristaltic pump was turned on (10 mL / min), and then 2×10 5 4T1 cells stained with Hoechst33342. The circulation time was 30 minutes, and 1 ml of blood samples were collected through the T-port of the catheter at the 1st and 30th minutes to calculate the capture efficiency. During this period, heparin sodium solution was injected through the T-port of the catheter every 5 minutes. The blood samples were processed as follows: First, the collected blood samples were centrifuged at 2000rpm for 3min. Next, for cell precipitation, red blood cells were lysed using red blood cell lysis buffer, and then the remaining cells were resuspended with 200μL PBS to calculate the capture efficiency of cancer cells. Finally, the fluorescence intensity was measured using a multifunctional enzyme analyzer to calculate the capture efficiency.

[0049] To investigate the in vivo toxicity of the adsorbent, blood samples were collected from the tail vein on days 1, 7, and 14, and routine blood tests and blood biochemical analysis were performed by an automatic blood cell analyzer and an automatic biochemical analyzer (Chemray 240, Mindray Biomedical Electronics Co., Ltd., Shenzhen, China). The body weight of the rats was monitored once a day after surgery. The rats were sacrificed on day 14, and then the major organs, including the heart, liver, spleen, lung, and kidney, were collected and stained with hematoxylin and eosin (H&E).

[0050] Figure 7 To evaluate the effect of multivalent ligand-modified microspheres on the removal of CTCs in a rat model and its in vivo biosafety. Figure 7 A shows that the efficiency of AG-HA-CE microspheres in removing CTCs from systemic blood was 64.2%. Figure 7 B records the results of routine blood tests on days 1, 7, and 14 after the circulation surgery, which showed that the immunoadsorbent did not induce a significant immune response. Figure 7 C shows that the blood biochemical parameters of rats on days 1, 7, and 14 after surgery, including liver function, total protein, blood lipids, blood sugar, and renal function, did not show significant changes. Figure 7 The H&E staining results of D showed that there was no obvious damage to the five major organs. The overall results showed that the immunoadsorbent had no obvious toxic side effects in vivo and had good biosafety.

[0051] Example 5

[0052] Release of CTCs from AG-HA-CE: After cycling, AG-HA-CE was removed from the capture column, incubated with 0.25% trypsin at room temperature for 90 s, then diluted with DMEM, filtered through a 300-mesh sieve, and then the cell pellet was collected from the filtrate, centrifuged at 2000 rpm for 3 min, and then used for reculture.

[0053] Calcein AM / PI double staining method: The released cells were co-stained with Calcein AM and PI for 15 min, washed three times with PBS, added to a slide, covered with a cover glass, and photographed under an inverted fluorescence microscope.

[0054] Cell viability: The released cells were seeded into 96-well plates with 100 μL DMEM (containing 10% FBS) per well and cultured for 4 days. The cell viability was measured every day using a CCK-8 kit and the OD value at 450 nm was recorded using a microplate reader (Thermo, USA).

[0055] Cell surface protein expression after release: cells were fixed with 4% paraformaldehyde for 20 min, then washed three times with PBS and blocked with 5% BSA for 30 min at 37°C. Cells were then incubated with CD44 monoclonal antibody or GLUT1 monoclonal antibody overnight at 4°C. After washing three times with PBS, cells were incubated with Alexa Fluor 647-labeled goat anti-mouse IgG or Alexa Fluor The cells were incubated with goat anti-rabbit IgG labeled with 647 for 1 hour. After washing with PBS three times, the cells were stained with DAPI for 5 minutes in the dark at room temperature. The cells were washed three times with PBS and observed by CLSM.

[0056] Figure 8 The ability of multivalent ligand-modified microspheres to release CTCs. Figure 8 A The results show that the released cells have a higher proliferation capacity compared with tumor cells without capture / release treatment. Figure 8 B Co-staining with calcein (green live cells) and propidium iodide (PI) (red dead cells) showed that the released cells maintained good cell viability. Figure 8 C results show that compared with negative cells, the cell surface after release still highly expressed CD44 and GLUT1 proteins, indicating that the adsorbent has little effect on cell surface protein expression. The above results show that it is feasible to use adsorbents to release captured CTCs to provide tumor samples for tumor diagnosis and detection.

[0057] The designed adsorbent carrier must be sterilized by high-pressure steam in advance. All activation and ligand grafting processes are carried out in a sterile room or clean bench. During the operation, it is isolated from the outside world to avoid the invasion of microorganisms and ensure that the sterility level of the adsorbent is within 10 -6 .

[0058] All adsorbents produced must be packaged under aseptic conditions into sterile containers that have been sterilized or aseptically treated in advance, and stored in a refrigerated environment after sealing.

[0059] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A microsphere adsorbent for removing circulating tumor cells by blood perfusion, comprising a carrier and a ligand grafted to the surface of the carrier, It is characterized in that The carrier is composed of agarose gel microspheres 2B or its analogs, and the ligand includes azid hyaluronic acid HA-N 3 and 4-aminophenyl-β-D-cellobiose CE, the HA-N 3 The CE is grafted onto the carrier through any one or more of click chemistry reaction, epoxidation reaction, epoxidation ring-opening reaction, amide reaction, and carboxyl activation reaction.

2. The microsphere adsorbent for removing circulating tumor cells by hemoperfusion according to claim 1, Features: The carrier is dibenzocyclooctyne-amino DBCO-NH 2 Modified agarose gel microspheres, the diameter of the microspheres is 60μm to 180μm.

3. The microsphere adsorbent for removing circulating tumor cells by hemoperfusion according to claim 1, Features: The azid hyaluronic acid HA-N 3 The grafting ratio of the azide group is 1% to 10%, and the average protein molecular weight is 7 to 600 KDa.

4. The microsphere adsorbent for removing circulating tumor cells by hemoperfusion according to claim 1, Features: The click chemistry reaction is one of copper-catalyzed azide-alkyne cycloaddition, copper-free catalyzed azide-alkyne cycloaddition, oxazolidinone-azide cycloaddition, Diels-Alder reaction, thiol-ene addition, and thiol-alkyne addition.

5. The microsphere adsorbent for removing circulating tumor cells by blood perfusion and the preparation method thereof according to claim 4, Features: The HA-N 3 The CE was grafted onto the microspheres via an azide-alkyne addition reaction, and the CE was grafted onto the HA via an amidation reaction.

6. The method for preparing the microsphere adsorbent for removing circulating tumor cells by hemoperfusion according to claim 1, Features: The preparation method comprises the following steps: (1) Dibenzocyclooctyne-amino DBCO-NH 2 Preparation of modified agarose gel microspheres: Epichlorohydrin activated microspheres, activated microspheres, DBCO-NH 2 Add to a solution of NaOH and DMSO in a volume ratio of 1:2, adjust the pH, stir and react at room temperature, filter after the reaction is completed, and wash with ultrapure water to obtain DBCO-NH 2 Modified microspheres; (2) Azide hyaluronic acid HA-N 3 Grafting: DBCO-NH 2 Modified microspheres, HA-N 3 and ultrapure water were added to the flask and reacted at room temperature. After the reaction was completed, filtration was performed and ultrapure water was used to wash to obtain HA-N 3 Grafted microspheres; (3) Grafting of 4-aminophenyl-β-D-cellobiose CE: HA-N 3 The grafted microspheres, N-hydroxysuccinimide NHS, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC were added to the 2-morpholineethanesulfonic acid MES solution and stirred at room temperature to activate the carboxyl groups on the microspheres. Then, 4-aminophenyl-β-D-cellobioside was added to the flask and reacted at room temperature. After the reaction was completed, the microspheres were filtered and washed with ultrapure water to obtain CE-grafted microspheres.

7. The method for preparing the microsphere adsorbent for removing circulating tumor cells by hemoperfusion according to claim 6, Features: In step (1), the microspheres and DBCO-NH 2 The mass ratio of the feed amount is 60:1-18:1; in the step (2), DBCO-NH 2 Modified microspheres and HA-N 3 The mass ratio of the feed amount is 120:1-60:1; in the step (3), HA-N 3 The mass ratio of the grafted microspheres to the 4-aminophenyl-β-D-cellobioside is 180:1-900:

11.

8. Use of the microsphere adsorbent for removing circulating tumor cells by hemoperfusion according to any one of claims 1 to 5 in preparing a preparation for capturing CTCs in tumors.

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