Application of trans-cyclooctene group adsorbent based on biological orthogonal reaction in removing circulating tumor cells

Abstract

The invention relates to the technical field of medical adsorbents for blood perfusion, and discloses an application of a trans-cyclooctene group adsorbent based on a biological orthogonal reaction in preparation of a circulating tumor cell clearing preparation. The adsorbent adopts sephadex microspheres as a carrier, the surface of the carrier is modified with a trans-cyclooctene (TCO) group, a tetrazine (Tz) group is taken in by CTCs through modified mannose, the Tz group is expressed on the surface of a CTCs cell membrane in the form of glycoprotein through a Roseman-Warden pathway in cells, TCO and Tz are subjected to a biological orthogonal reaction specifically, and the adsorption capacity of the adsorbent is improved. The sephadex microsphere adsorbent can effectively enrich and remove CTCs in blood of the whole body of a patient through adsorption, and compared with a conventional single-antibody or multi-antibody capture technology, the sephadex microsphere adsorbent does not depend on antibody-antigen binding, and the capture efficiency of the CTCs can be remarkably improved through a biological orthogonal reaction.

Description

Technical Field

[0001] This invention relates to the field of medical adsorbent technology for hemoperfusion, specifically to the application of a trans-cyclooctene group adsorbent based on bioorthogonal reaction in clearing circulating tumor cells. Background Technology

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

[0003] Currently, methods for capturing CTCs are mainly divided into physical and biological methods. Physical methods separate CTCs based on differences in size, optical and acoustic properties, but their long capture time and high cost limit their in vivo application. Biological methods rely on antibody technology, using antibody-conjugated vectors to target specific biomarkers for more precise CTC capture. Some studies have combined physical and biological methods to develop systems using microfluidic microarrays and antibody-functionalized magnetic beads, but these technologies are mainly used for in vitro detection and still require integration with traditional therapies.

[0004] Hemoperfusion, as a blood purification technique, effectively removes exogenous and endogenous toxins and metabolic waste from the whole blood through the adsorption of adsorbents within the hemoperfusion device. It has been widely used in the treatment of renal failure, liver failure, drug poisoning, and autoimmune diseases. However, current hemoperfusion techniques can only effectively remove medium- and large-molecule toxic substances from the whole blood and cannot effectively remove central toxic cells (CTCs).

[0005] In previous tumor metastasis suppression, the clearance efficiency of CTCs in the blood circulation was often low by using a single target. Different CTCs carry different characteristic receptors, and there is no widely applicable receptor. Furthermore, the effect of inhibiting tumor metastasis is limited due to factors such as the complex blood environment and the lack of CTC receptors caused by epithelial-mesenchymal transition (EMT).

[0006] In order to comprehensively and effectively remove circulating tumor cells (CTCs) from the patient's systemic blood circulation, prevent distant metastasis of tumors, and thus achieve 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

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and propose an application of a trans-cyclooctene group adsorbent based on bioorthogonal reaction in the clearance of circulating tumor cells;

[0008] The designed adsorbent uses dextran gel microspheres and their analogues as a carrier. The carrier surface is modified with trans-cyclooctene (TCO) groups, while the tetrazine (Tz) groups are taken up by cytotoxic tumor cells (CTCs) via modified mannose (Ac4ManNTz). Intracellularly, through the Roseman-Warren pathway, the Tz groups are expressed on the CTC cell membrane surface as glycoproteins. TCO and Tz can specifically undergo a bioorthogonal reaction. When peripheral blood flows through the adsorbent, CTCs are selectively adsorbed. This adsorbent not only removes CTCs from the systemic bloodstream but also releases enriched CTCs, supporting subsequent molecular analysis and metastatic potential assessment, thus playing an effective role in preventing tumor metastasis and recurrence and improving cancer treatment outcomes.

[0009] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a compound for clearing circulating tumor cells, the chemical name of which is (3S,5S,6R)-3-(2-((1,2,4,5-tetraazine-3-yl)thio)acetamido)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triacyltriacetate.

[0011] The chemical formula is shown in formula (1):

[0012]

[0013] Furthermore, a method for preparing a compound for clearing circulating tumor cells is provided, the synthetic route of which is shown below:

[0014]

[0015] The method includes the following steps:

[0016] Step S1: 3,6-Di(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetraazine was dissolved in acetonitrile, and mercaptoacetic acid was added dropwise at room temperature; the reaction mixture was stirred at room temperature for 1 h, the precipitate was filtered off, and the precipitate was washed with acetonitrile to obtain intermediate compound 2; wherein, the molar ratio of 3,6-di(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetraazine to mercaptoacetic acid was 1:2.8;

[0017] Step S2: A methanol solution of hydrazine hydrate is added dropwise to a methanol solution of intermediate compound 2 at room temperature. The mixture is stirred at room temperature for 2 hours. After the reaction is complete, the precipitate is collected by filtration to obtain intermediate compound 3. The molar ratio of hydrazine hydrate to intermediate compound 2 is 2.84:1.

[0018] Step S3: Add copper sulfate solution dropwise to the PBS buffer solution of intermediate compound 3 at room temperature, stir for 2 hours, and then extract the precipitate with diethyl ether continuously or multiple times to obtain intermediate compound 4; wherein the molar ratio of copper sulfate to intermediate compound 3 is 2.9:1.

[0019] Step S4: In the reaction flask, mannosamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added. The flask was evacuated with nitrogen, methanol was added to dissolve the compounds, triethylamine was added, and a methanol solution of intermediate compound 4 was added dropwise at 0°C. The reaction was then allowed to proceed to room temperature for 4 hours. After the reaction was complete, the methanol was removed by vortexing, and the crude product was separated by column chromatography to obtain intermediate compound 5. The molar ratio of mannosamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, triethylamine, and the intermediate compound was 1.2:2.0:2.5:1.5:1.

[0020] Step S5: Bake the flask, add DMAP to the reaction flask, replace the nitrogen gas, add a pyridine solution of intermediate compound 5, add acetic anhydride dropwise at room temperature, stir the reaction for 4 hours, after the reaction is completed, extract, and separate by column chromatography to obtain Ac4ManNTz, wherein the molar ratio of DMAP, intermediate compound 5 and acetic anhydride is 0.1:1:10.

[0021] Preferably, the solvents used for column separation in step S4 are dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 5:1.

[0022] Furthermore, in step S5, the solvents for gradient elution are petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 2:1 to 1:1.

[0023] Secondly, the present invention provides an adsorbent for clearing circulating tumor cells, the adsorbent comprising a dextran microsphere carrier, the surface of which is modified with trans-cyclooctene groups.

[0024] A method for preparing an adsorbent for clearing circulating tumor cells is provided, the method comprising the following steps:

[0025] (1) Preparation of epoxy group modified dextran gel microspheres: After swelling the dextran microspheres, they were washed with ultrapure water and then dried. 900 mg of the dried dextran microspheres (Dex), 1.5 mL of epichlorohydrin, 3 mL of 0.4 M NaOH, and 10.5 mL of DMSO were added to a 50 mL centrifuge tube and reacted at 100 rpm at room temperature for 5-7 h. After washing, Dex-Epo microspheres were obtained.

[0026] (2) Preparation of amino-modified dextran gel microspheres: Dex-Epo microspheres were added to 15 mL of ammonia water, reacted at 40 °C for 1 h, and then washed to obtain Dex-NH2 microspheres.

[0027] (3) Grafting of TCO: Dex-NH2 microspheres, TCO-PEG-NHS, and 15 mL of 2-morpholine ethanesulfonic acid solution were added to an eggplant-shaped flask and reacted at room temperature for 5 h. After washing, Dex-TCO adsorbent was obtained. The molar ratio of Dex-NH2 microspheres to TCO-PEG-NHS was 1:1.5-2.

[0028] Thirdly, the present invention provides a composition for clearing circulating tumor cells, the composition comprising the compound of claim 1 and the adsorbent of claim 5.

[0029] Fourthly, this invention provides the application of a trans-cyclooctene group adsorbent based on bioorthogonal reaction in the preparation of a formulation for clearing circulating tumor cells (CTCs). The adsorbent is characterized by expressing the Tz group in the compound onto the CTC cell membrane surface via the Roseman-Warren pathway, utilizing the specific bioorthogonal reaction between the TCO and Tz groups in the adsorbent to clear CTCs from the bloodstream.

[0030] The compound Ac4ManNTz can be administered via intratumoral injection or as a formulation, and the adsorbent can be administered via perfusion.

[0031] This invention offers the following advantages: The dextran gel microsphere adsorbent provided by this invention can effectively enrich and remove central cytokines (CTCs) from the patient's systemic blood through adsorption. Compared with previous single-antibody or multi-antibody capture technologies, this invention does not rely on antibody-antigen binding and significantly improves the capture efficiency of CTCs through bioorthogonal reactions, exhibiting excellent and wide applicability. Using this adsorbent for hemoperfusion therapy can serve as an alternative or supplement to existing clinical surgical resection, chemotherapy, or radiotherapy, effectively reducing the risk of distant tumor metastasis, minimizing patient harm, and improving cure rates. Simultaneously, CTCs can be collected from the adsorbent, providing valuable guidance for patient diagnosis and treatment. Attached Figure Description

[0032] Figure 1 Synthetic pathway diagram of adsorbents used to remove circulating tumor cells;

[0033] Figure 2 A trend graph showing the density of epoxy groups in dextran microspheres at different times;

[0034] Figure 3A trend graph showing the epoxidation density of dextran microspheres under different NaOH concentrations;

[0035] Figure 4 A trend graph showing the epoxidation density of dextran microspheres under different epichlorohydrin concentrations;

[0036] Figure 5 A trend graph showing the amino density of dextran microspheres at different times and temperatures;

[0037] Figure 6 A trend graph showing the TCO group density of dextran microspheres at different times;

[0038] Figure 7 This is a static capture image (bright field) of the adsorbent microspheres;

[0039] Figure 8 The 1H NMR spectrum is for Ac4ManNTz.

[0040] Figure 9 The carbon NMR spectrum of Ac4ManNTz;

[0041] Figure 10 Expression of Tz groups on the cell surface at different concentrations and time points;

[0042] Figure 11 Static capture efficiency of adsorbent microspheres at different time points (ANOVA, *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.);

[0043] Figure 12 Static capture efficiency of adsorbent microspheres at different packing amounts (ANOVA, *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.);

[0044] Figure 13 Static capture efficiency of adsorbent microspheres and Dex microspheres under different cell conditions;

[0045] Figure 14 Static capture efficiency of adsorbent microspheres under different media (ANOVA, *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.);

[0046] Figure 15 This is a graph evaluating the blood compatibility of the adsorbent microspheres.

[0047] Figure 16Figure 1. Efficacy of multivalent ligand-modified microspheres in removing CTCs in a rat model and its in vivo biosafety assessment.

[0048] Figure 17 A graph showing the ability of adsorbent microspheres to release CTCs;

[0049] Figure 18 To release the number of subsequent generations;

[0050] Figure 19 A graph showing the proliferation activity of CTCs after release;

[0051] Figure 20 Calcein / PI double staining plot of CTCs after release;

[0052] Figure 21 This is a diagram showing the expression of surface Tz groups in CTCs before and after release.

[0053] Figure 22 This is a schematic diagram of the expression of tetrazine groups on the cell surface. Detailed Implementation

[0054] 1. Adsorbents used to remove circulating tumor cells

[0055] An adsorbent for scavenging circulating tumor cells, the adsorbent comprising a dextran microsphere carrier, the surface of which is modified with trans-cyclooctene groups; its preparation method is as follows:

[0056] (1) Preparation of epoxy group modified dextran gel microspheres: After swelling the dextran microspheres with ultrapure water, they were washed three times with ultrapure water. After each washing, they were dried by vacuum filtration funnel. 900 mg of dextran microspheres Dex, 1.5 mL of epichlorohydrin (ECH), 3 mL of 0.4 M NaOH and 10.5 mL of DMSO were added to a 50 mL centrifuge tube and reacted at 100 rpm for 6 h at room temperature. Then, the mixture was transferred to a glass filter funnel and washed with 400 mL of ultrapure water to obtain Dex-Epo microspheres.

[0057] (2) Preparation of amino-modified dextran gel microspheres: Add all the Dex-Epo microspheres obtained in step (1) and 15 mL of ammonia water to a 25 mL flask, react at 40 °C for 1 h, then transfer them to a glass filter funnel and wash them with 400 mL of ultrapure water to obtain Dex-NH2 microspheres.

[0058] (3) Grafting of TCO: All Dex-NH2 microspheres, TCO-PEG-NHS and 15 mL of 2-morpholinoethanesulfonic acid (MES) solution obtained in step (2) were added to an eggplant-shaped flask and reacted at room temperature for 5 h. After washing, Dex-TCO adsorbent was obtained. The molar ratio of Dex-NH2 microspheres to TCO-PEG-NHS was 1:2.

[0059] The synthetic route of the adsorbent is as follows Figure 1 As shown. Figures 2-4 The density of epoxy groups on the surface of dextran under different conditions, such as Figure 2 As shown, the density of epoxy groups on the microsphere surface gradually increased from 1 to 6 hours, reaching a maximum density of 122.35 ± 1.12 μmol / g at 6 hours. Figure 3 As shown, the density of epoxy groups on the microsphere surface gradually increased in the range of 0–0.4 mol / L NaOH, reaching a maximum density of 122.59 ± 0.15 μmol / g at 0.4 mol / L, and then gradually decreased. Figure 4 As shown, the density of epoxy groups on the microsphere surface gradually increased from 0-10% ECH (0-10% ECH is the ratio of ECH to the total liquid volume), reaching a maximum density of 122.05 ± 1.68 μmol / g at 10%, and then gradually decreased. Figure 5 As shown, the maximum density of the amino group was reached at 40℃ for 1 hour, which was 93.34 ± 2.99 μmol / g. Figure 6 As shown, the maximum TCO density was reached after 5 hours of reaction, which was 85.66 ± 1.29 μmol / g.

[0060] 2. Compounds used to eliminate circulating tumor cells

[0061] The synthesis route is shown below:

[0062]

[0063] The method includes the following steps:

[0064] Step S1: 3,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetraazine (92 mmol) was dissolved in acetonitrile, and mercaptoacetic acid (2.8 equiv) was added dropwise at room temperature; the reaction mixture was stirred at room temperature for 1 h, the precipitate was filtered off, and the precipitate was washed with acetonitrile to obtain intermediate compound 2;

[0065] In step S2, a methanol solution (3.6 mol / L) of hydrazine hydrate (2.84 equiv) was added dropwise to a methanol solution (0.2 mol / L) of intermediate compound 2 at room temperature. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the precipitate was collected by filtration to obtain intermediate compound 3.

[0066] In step S3, copper sulfate solution (0.35 mol / L, 2.9 equiv) was added dropwise to the PBS buffer solution (0.33 mol / L) of intermediate compound 3 at room temperature. During this process, a black precipitate was immediately formed and nitrogen gas was generated. After stirring for 2 hours, the mixture was extracted with ether continuously or multiple times to obtain intermediate compound 4.

[0067] Step S4: In the reaction flask, mannosamine (1.2 equiv), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.0 equiv), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.5 equiv) were added. The flask was purged with nitrogen, methanol was added to dissolve the compounds, and triethylamine (1.5 equiv) was added. A methanol solution of intermediate compound 4 was added dropwise at 0°C, and the reaction was allowed to proceed to room temperature for 4 hours. After the reaction was complete, the methanol was removed by vortexing, and the crude product was separated by column chromatography using dichloromethane:methanol = 5:1 (volume ratio) to obtain intermediate compound 5.

[0068] Step S5: Bake the flask, add DMAP (0.1 equiv) to the reaction flask, replace with nitrogen, add pyridine solution of intermediate compound 5, add acetic anhydride (10 equiv) dropwise at room temperature, stir the reaction for 4 h, after the reaction is completed, extract with 1 mol / L hydrochloric acid and ethyl acetate in sequence, and separate by column chromatography with petroleum ether:ethyl acetate = 2:1 (volume ratio) to obtain Ac4ManNTz.

[0069] Figure 8 and Figure 9 The proton and carbon NMR spectra, respectively, characterize the successful synthesis of Ac4ManNTz.

[0070] Tz group expression on cell surface: 50,000 MDA-MB-231 cells were seeded in a confocal dish and cultured in DMEM medium (100 μM) containing Ac4ManNTz for 48 h. After removing the medium, the cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and blocked with 5% donkey serum for 30 min at room temperature. The cells were then incubated with TCO-FITC at room temperature for 1 h. After washing three times with PBS, the cells were stained with DAPI in the dark for 5 min at room temperature. The cells were washed three times with PBS and observed using a laser confocal microscope. Figure 22 As shown.

[0071] like Figure 10 As shown, the incubation time was first controlled at 48 h to investigate the density of Tz groups at different concentrations. Then, the concentration was controlled at 100 μM to investigate the density of Tz groups at different times. The results showed that the density of Tz groups on the cell surface was the highest at a concentration of 100 μM and a culture time of 48 h.

[0072] 3. Static capture experiment of CTCs

[0073] Place 50 mg of Dex-TCO into a 2 mL EP tube, then add 2 × 10⁻⁶ mg of the EP tube. 5 MDA-MB-231 cells (expressing Tz groups on the cell surface) were collected and diluted with PBS to 1 mL. The cells and Dex-TCO were gently mixed and then added to a 24-well plate. The plate was incubated at 37°C for 20 min. After 20 min, the microspheres were filtered through a 200-mesh cell filter, and the capture efficiency was calculated by visual counting.

[0074] Figure 11-14 To evaluate the capture efficiency of adsorbent microspheres under static conditions, such as... Figure 11 As shown, the adsorbent microspheres reached their maximum capture efficiency of 97.92 ± 1.91% after 20 minutes, and the capture efficiency did not change significantly with increasing capture time. Figure 12 As shown, the capture efficiency peaked at a loading volume of 50 mg, at 98.75 ± 1.25%, and remained relatively stable with increasing loading volume. Figure 13 As shown, the adsorbent microspheres exhibit capture efficiencies exceeding 90% for a variety of different tumor cells (with Tz group expression on the cell surface), while Dex shows a capture efficiency of approximately 15%. This demonstrates the excellent broad applicability of the adsorbent microspheres of the present invention. Figure 14 As shown, the capture efficiency did not change much in PBS, DMEM, and 20% serum media, but it decreased further in 50% serum and whole blood media. However, the capture efficiency in whole blood still exceeded 75%, which may be because non-specific protein adsorption caused some binding sites to be masked.

[0075] 4. Blood compatibility evaluation experiment of adsorbent microspheres

[0076] Protein adsorption: After soaking 50 mg of Biosky and Dex-TCO in saline solution, 1 mL of 1 mg / mL FITC-HSA solution (diluted with PBS) was added, and the mixture was incubated with the microspheres at 37°C for 1 h. The protein concentration in the supernatant was determined using a BCA kit. Alternatively, after soaking 50 mg of Biosky and Dex-TCO in saline solution, 1 mL of 1 mg / mL FITC-HSA solution (diluted with PBS) was added, and the mixture was incubated with the microspheres at 37°C for 1 h. The supernatant was discarded, and the microspheres were washed 1-2 times. The microspheres were then photographed using an inverted fluorescence microscope.

[0077] Platelet activation and complement activation: Take 50mg Biosky and Dex-TCO, soak them in saline solution, collect blood in EDTA anticoagulant tubes, aliquot the blood, incubate with microspheres at 37℃ for 1 hour, centrifuge at 2500g, 4℃ for 10 minutes, collect the supernatant plasma, and perform ELISA.

[0078] Hemolysis rate: After soaking 50 mg of Biosky and Dex-TCO in the solution and removing the saline solution, blood was collected using EDTA anticoagulant tubes. The lower red blood cells were collected by centrifugation at 2000 rpm for 10 min. The red blood cells were resuspended in an appropriate amount of saline solution (positive control was diluted with pure water, and negative control was diluted with saline solution). 1 mL of the negative control was added to Biosky and Dex-TCO solution and incubated at 37℃ for 3 h. After centrifugation at 3000 rpm for 5 min, the absorbance of each group was measured at 540 nm and photographed.

[0079] Complete blood count: Take 50mg Biosky and Dex-TCO, soak them in saline solution, collect blood using EDTA anticoagulant tubes, aliquot the blood, incubate with the adsorbent microspheres at 37℃ for 1 hour, collect the remaining whole blood, and perform complete blood count analysis using a blood cell analyzer.

[0080] Coagulation panel: Take 50mg Biosky and Dex-TCO, soak in the solution and remove the saline. Collect blood using sodium citrate anticoagulant tubes, centrifuge at 4000rpm for 15min, take the supernatant PPP and microspheres and incubate at 37℃ for 1h before performing coagulation panel test.

[0081] Cytotoxicity assay: MDA-MB-231 cells (expressing Tz groups on the cell surface) were grown in DMEM supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin) in a humidified incubator at 37°C and 5% CO2. The cytotoxicity of Dex-TCO was assessed using a CCK-8 cell viability assay kit. Before measurement, 2 g of Dex-TCO was pre-soaked in DMEM (10 mL) and incubated at room temperature for 24 h. The supernatant was then filtered, and bacteria were removed using a 0.22 μm microporous membrane to obtain the supernatant. Cells were then cultured at the desired density (1 × 10⁶ cells per well). 4 Cells were seeded in 96-well plates for 24 h. After washing with PBS, 100 μL of supernatant was added to the culture medium, and the plates were incubated at 37°C for 24 h under the above conditions. The control group was incubated with 100 μL of DMEM medium without Dex-TCO, while the blank group contained only DMEM medium and no cells. Then, 10 μL of CCK-8 solution was added and the plates were incubated for 4 h. OD values ​​were obtained at 450 nm using a microplate reader. Each sample was tested in six replicates. Cell viability was calculated.

[0082] Figure 15 For the evaluation of the blood compatibility of the adsorbent microspheres. For example... Figure 15 As shown in Figure A, the hemolysis rate of Dex-TCO is lower than that of Biosky (a commercially available blood perfusion adsorbent), indicating that Dex-TCO not only meets the requirements of the Applied Materials Hemolysis Test (ASTM, F756-2008), but also has better erythrocyte compatibility. Figure 15The results showed that Dex-TCO had a lower protein adsorption capacity compared to Biosky. Figure 15 C-fluorescence imaging further confirmed the low protein adsorption capacity of Dex-TCO. Figure 15 Results showed that Dex-TCO did not cause significant changes in blood cell count or size. Figure 15 E and Figure 15 F investigated the coagulation properties of Dex-TCO, including activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB). The results showed that Dex-TCO hardly caused whole blood coagulation. Figure 15 G investigated the effect of Dex-TCO on platelet activation. The results showed that Dex-TCO did not induce platelet activation, because platelet activation releases PF4 when the biological material is exposed to blood. Figure 15 H and Figure 15 The study found that complement activation was poor, with no significant increase in complement fragments 3a and 5a, indicating that Dex-TCO does not induce complement activation. Figure 15 J investigated the cytocompatibility of Dex-TCO. The results showed that these cells maintained over 90% of their proliferative activity. These results indicate that the functionalized microspheres possess excellent blood compatibility.

[0083] 5. In vivo circulation experiment of rat model

[0084] Nine-week-old male SD rats (280-300g) were housed for 7 days under pathogen-free conditions. The experimental procedure was performed under general anesthesia induced by intramuscular injection of 5% pentobarbital (10mg / kg). The procedure was as follows: First, a hemoperfusion device was installed, and the catheter was initially filled with heparin sodium solution. Air bubbles were removed from the catheter, and an indwelling needle was attached to the end of the catheter. The indwelling needle was then inserted into the abdominal aorta and inferior vena cava, and the needle and blood vessels were secured with sutures. At the end of the procedure, the peristaltic pump was turned on (10mL / min), and then 2×10⁻⁶ mmol / L was injected through the T-port of the catheter. 5 Hoechst 33342-stained MDA-MB-231 cells (expressing Tz groups on the cell surface) were perfused with an adsorbent-filled perfusion column. The circulation time was 20 min, with 1 mL blood samples collected at 1 min and 20 min through the T-port of the catheter to calculate the capture efficiency. During this period, heparin sodium solution was injected every 5 min through the T-port of the catheter. Blood sample processing was as follows: First, the collected blood samples were centrifuged at 2000 rpm for 3 min. Next, erythrocytes were lysed using erythrocyte lysis buffer, and the remaining cells were resuspended in 200 μL PBS to calculate the capture efficiency of cancerous CTCs. Finally, fluorescence intensity was measured using a multi-well microplate detector to calculate the capture efficiency.

[0085] To investigate the in vivo toxicity of the adsorbent, blood samples were collected from the tail vein on days 1, 7, and 14. Routine blood tests and biochemical analyses were performed using an automated blood cell analyzer and an automated biochemical analyzer. Rats' body weight was monitored daily post-surgery. Rats were sacrificed on day 14, and major organs, including the heart, liver, spleen, lungs, and kidneys, were collected and stained with H&E to observe changes in these organs.

[0086] Figure 16 The efficacy of multivalent ligand-modified microspheres in removing CTCs in a rat model and their in vivo biosafety assessment. Figure 16 A is a diagram of the rat in vivo circulation model. Figure 16 B is a photograph of a rat circulating in vivo. Figure 16 The results showed that the adsorbent microspheres had an efficiency of 70.4% in removing CTCs from the whole bloodstream, which was much greater than the efficiency of Dex in removing CTCs. Figure 16 D recorded the results of routine blood tests on days 1, 7, and 14 after the circulatory surgery, and the results showed that the immunoadsorbent did not induce a significant immune response. Figure 16 E shows that on days 1, 7, and 14 post-surgery, the rats' blood biochemical parameters, including liver function, total protein, blood lipids, blood glucose, and kidney function, did not show significant changes. Figure 16 F monitored the changes in rat body weight 14 days after surgery. The results showed that the weight decreased slightly after surgery, which may be related to the slight blood loss during surgery, but the weight recovered quickly. Figure 16 H&E staining of G showed no significant damage to any of the five major organs. Overall, the results indicate that the immunoadsorbent has no significant toxic side effects in vivo and exhibits good biocompatibility.

[0087] 6. Experiment on the ability of adsorbent microspheres to release CTCs

[0088] CTCs were released from Dex-TCO: After cycling, the Dex-TCO was removed from the capture column, incubated with 0.25% trypsin for 60 s at room temperature, and then DMEM containing 10% serum was added to terminate digestion. The mixture was filtered through a 200-mesh cell filter, and the cell pellet was collected from the filtrate, centrifuged at 2000 rpm for 3 min, and then used for reculture.

[0089] Calcein AM / PI double staining method: The released cells were co-stained with calcein AM and PI for 30 min, washed three times with PBS, placed on a glass slide, covered with a coverslip, and observed under an inverted fluorescence microscope.

[0090] Cell viability: Released cells were seeded into 96-well plates with 200 μL of DMEM (containing 10% FBS) per well and cultured for 4 consecutive days. Cell viability was measured daily using a CCK-8 assay kit, and the OD value at 450 nm was recorded using a microplate reader.

[0091] 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% donkey serum for 30 min at room temperature. Cells were then incubated with TCO-FITC at room temperature for 1 h. After washing three times with PBS, cells were stained with DAPI in the dark for 5 min at room temperature. Cells were washed three times with PBS and observed using a laser confocal microscope.

[0092] Figure 17 The ability of the adsorbent microspheres to release CTCs was measured, with a release rate of 90.71 ± 3.22%. Figure 18 The number of cell passages within 12 days after release was measured. There was no significant difference between the release group and the normal group, indicating good proliferation activity. Figure 19 The results showed that the released cells also had a higher proliferative capacity compared with tumor cells that were not captured / released. Figure 20 Co-staining with calcein (green live cells) and propidium iodide (PI) (red dead cells) showed that the released cells maintained good cell stock. Figure 21 The results showed that, compared with the control group, the released cells still expressed Tz groups on their surface. These results indicate that it is feasible to use adsorbents to release captured CTCs to provide tumor samples for tumor diagnosis and detection.

[0093] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A compound for eliminating circulating tumor cells, characterized in that, The chemical name of the compound is 3S,5S,6R)-3-(2-((1,2,4,5-tetraazine-3-yl)thio)acetamido)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triacyltriacetate. The chemical formula is shown in formula (1):

2. The method for preparing the compound according to claim 1, characterized in that, The synthesis route is shown below: The method includes the following steps: Step S1: 3,6-Di(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetraazine was dissolved in acetonitrile, and mercaptoacetic acid was added dropwise at room temperature; the reaction mixture was stirred at room temperature for 1 h, the precipitate was filtered off, and the precipitate was washed with acetonitrile to obtain intermediate compound 2; wherein, the molar ratio of 3,6-di(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetraazine to mercaptoacetic acid was 1:2.8; Step S2: A methanol solution of hydrazine hydrate is added dropwise to a methanol solution of intermediate compound 2 at room temperature. The mixture is stirred at room temperature for 2 hours. After the reaction is complete, the precipitate is collected by filtration to obtain intermediate compound 3. The molar ratio of hydrazine hydrate to intermediate compound 2 is 2.84:

1. Step S3: Add copper sulfate solution dropwise to the PBS buffer solution of intermediate compound 3 at room temperature, stir for 2 hours, and then extract the precipitate with diethyl ether continuously or multiple times to obtain intermediate compound 4; wherein the molar ratio of copper sulfate to intermediate compound 3 is 2.9:

1. Step S4: In the reaction flask, mannosamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added. The flask was evacuated with nitrogen, methanol was added to dissolve the compounds, triethylamine was added, and a methanol solution of intermediate compound 4 was added dropwise at 0°C. The reaction was then allowed to proceed to room temperature for 4 hours. After the reaction was complete, the methanol was removed by vortexing, and the crude product was separated by column chromatography to obtain intermediate compound 5. The molar ratio of mannosamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, triethylamine, and the intermediate compound was 1.2:2.0:2.5:1.5:

1. Step S5: Bake the flask, add DMAP to the reaction flask, replace the nitrogen gas, add a pyridine solution of intermediate compound 5, add acetic anhydride dropwise at room temperature, stir the reaction for 4 hours, after the reaction is completed, extract, and separate by column chromatography to obtain Ac4ManNTz, wherein the molar ratio of DMAP, intermediate compound 5 and acetic anhydride is 0.1:1:

10.

3. The method for preparing the compound according to claim 2, characterized in that, In step S4, the solvents used for column separation are dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 5:

1.

4. The method for preparing the compound according to claim 2, characterized in that, In step S5, the solvents for gradient elution are petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 2:1 to 1:

1.

5. An adsorbent for clearing circulating tumor cells, characterized in that, The adsorbent includes a dextran microsphere carrier, the surface of which is modified with trans-cyclooctene groups.

6. The method for preparing the adsorbent for clearing circulating tumor cells according to claim 5, characterized in that, The method includes the following steps: (1) Preparation of epoxy group modified dextran gel microspheres: After swelling the dextran microspheres, they were washed with ultrapure water and then dried. 900 mg of the dried dextran microspheres (Dex), 1.5 mL of epichlorohydrin, 3 mL of 0.4 M NaOH, and 10.5 mL of DMSO were added to a 50 mL centrifuge tube and reacted at 100 rpm at room temperature for 5-7 h. After washing, Dex-Epo microspheres were obtained. (2) Preparation of amino-modified dextran gel microspheres: Dex-Epo microspheres were added to 15 mL of ammonia water, reacted at 40 °C for 1 h, and then washed to obtain Dex-NH2 microspheres. (3) Grafting of TCO: Dex-NH2 microspheres, TCO-PEG-NHS, and 15 mL of 2-morpholine ethanesulfonic acid solution were added to an eggplant-shaped flask and reacted at room temperature for 5 h. After washing, Dex-TCO adsorbent was obtained. The molar ratio of Dex-NH2 microspheres to TCO-PEG-NHS was 1:1.5-2.

7. A composition for eliminating circulating tumor cells, characterized in that, The composition comprises the compound of claim 1 and the adsorbent of claim 5.

8. The application of a trans-cyclooctene group adsorbent based on bioorthogonal reaction in the preparation of a formulation for clearing circulating tumor cells, characterized in that, In CTCs, the Tz group in the compound of claim 1 is expressed on the cell membrane surface of CTCs in the form of a glycoprotein via the Roseman-Warren pathway. The TCO group and Tz group in the adsorbent of claim 5 undergo a specific bioorthogonal reaction, thereby clearing CTCs from the bloodstream.

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