Adsorbent for malignant tumors as well as preparation method and application thereof
By developing an adsorbent loaded with polyphenol compounds or biphenyl derivatives, using its selective adsorption and removal of TGF-β and PD-L1, the problem of tumor immune escape was solved, the success rate of tumor treatment was improved, and the adverse effects were reduced.
Patent Information
- Application Number
- CN202411879763.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-28
AI Technical Summary
Existing tumor treatment methods are difficult to effectively deal with immunosuppressive substances such as TGF-β and PD-L1 released by tumor cells, resulting in tumor immune escape, thereby reducing the success rate of treatment.
An adsorbent is developed to use an ultra-high crosslinked macroporous adsorption resin containing chloromethyl as a carrier, solid-carrying polyphenol compounds or biphenyl derivatives, and selective adsorption and removal of TGF-β and PD-L1 through the synergistic effect of the carrier and the solid-carrying adsorption ligand on the carrier.
This adsorbent can effectively reduce the immune escape of tumors and improve the success rate of tumor treatment. At the same time, polyphenol compounds or biphenyl derivatives are connected to the carrier through chemical bonds, which avoids entering the body and reduces adverse effects.
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Figure CN119700757A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of blood purification, and in particular to an adsorbent for malignant tumors, a preparation method thereof and an application thereof. Background Art
[0002] In recent years, many research reports have pointed out that the progression of malignant tumors is related to the immunosuppression and immune escape of tumors. Tumor immune escape refers to the phenomenon that tumor cells evade the recognition and attack of the body's immune system through various mechanisms, thereby being able to survive and proliferate in the body.
[0003] Transforming growth factor-β (TGF-β) is a representative substance related to tumor immunosuppression. TGF-β is a multifunctional protein that can affect the growth, differentiation, apoptosis and immune regulation of various cells. It has 5 subtypes (TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5). Among them, transforming growth factor β1 (TGF-β1) is a cell growth and differentiation regulator. Studies have shown that it is abnormally expressed in various tumors such as liver cancer, breast cancer, gastric cancer, colorectal cancer, and prostate cancer, and plays an important role in the metastasis of cancer cells. If TGF-β is removed or reduced, the TGF-β signaling channel is blocked or eliminated, the signal that cancer cells are protected from the attack of the immune system is released, and the patient's immunity is improved, it is expected to degenerate the tumor or inhibit the progression of cancer. In addition, the overexpression of programmed death ligand 1 (PD-L1) plays a role in escaping the immune response in tumors. The full length of PD-L1 is encoded by 7 exons and is a type 1 transmembrane protein. After PD-L1 on the surface of tumor cells binds to the programmed death molecule 1 (PD-1) receptor on the surface of T cells, it can inhibit the activity of T cells, inhibit T cell proliferation, and induce T cell apoptosis, thereby causing tumor immune escape. More and more studies have found that high concentrations and soluble PD-L1 exist in the plasma of patients with various tumors, and can play the same immunosuppressive function as PD-L1 on the cell membrane surface, which may be one of the reasons for the low response rate of PD-1 / PD-L1 therapy. However, existing tumor treatment methods do not have a good treatment plan for immunosuppressive substances released by tumor cells and substances that help tumor immune escape. Summary of the invention
[0004] The present invention aims to solve the above-mentioned defects of the prior art and provide an adsorbent for malignant tumors and a preparation method thereof. The adsorbent for malignant tumors can remove transforming growth factor-β (TGF-β) and programmed death ligand 1 (PD-L1) that help tumor metastasis and escape, and can reduce the immune escape of malignant tumors, thereby helping to improve the success rate of malignant tumor treatment.
[0005] To solve the above problems, the first aspect of the present invention provides an adsorbent for malignant tumors, wherein the adsorbent uses an ultra-high cross-linked macroporous adsorption resin containing chloromethyl as a carrier, and an adsorption ligand is immobilized on the carrier, and the adsorption ligand is a polyphenol compound or a biphenyl derivative, and the polyphenol compound or the biphenyl derivative reacts with the chloromethyl group and is immobilized on the carrier.
[0006] The second aspect of the present invention provides a method for preparing an adsorbent for malignant tumors, which is used to prepare the adsorbent as described in the first aspect, and the preparation method comprises the following steps:
[0007] Preparation of ultra-high cross-linked macroporous adsorption resin containing chloromethyl;
[0008] The ultra-high cross-linked macroporous adsorption resin containing chloromethyl groups is reacted with polyphenol compounds or biphenyl derivatives, and the polyphenol compounds or biphenyl derivatives are connected to the ultra-high cross-linked macroporous adsorption resin through chloromethyl groups to prepare an adsorbent for malignant tumors.
[0009] The third aspect of the present invention provides an adsorbent for malignant tumors as described in the first aspect, or use of the adsorbent prepared by the preparation method as described in the second aspect in blood purification.
[0010] The adsorbent for malignant tumors described in the present invention uses an ultra-high cross-linked macroporous adsorption resin containing chloromethyl as a carrier, and the chloromethyl on the carrier reacts with polyphenol compounds or biphenyl derivatives to immobilize the polyphenol compounds or biphenyl derivatives on the carrier. The polyphenol compounds or biphenyl derivatives in the adsorbent have a strong binding force to PD-L1, and the carrier has a steric hindrance effect. Through the synergistic effect of the two, selective adsorption and removal of PD-L1 can be achieved. The carrier itself has a rich pore structure and is a hydrophobic skeleton. Through the combination of the sieving effect of the carrier pores and the hydrophobic effect, TGF-β can be adsorbed. Through the carrier and the adsorption ligand immobilized on the carrier, the adsorbent can achieve simultaneous adsorption of TGF-β and PD-L1. In addition, the polyphenol compounds or biphenyl derivatives and the carrier are connected by chemical bonds, so that the polyphenol compounds or biphenyl derivatives can be firmly combined with the carrier, thereby improving the structural strength of the adsorbent and thus improving the safety of the adsorbent. At the same time, it can also prevent the polyphenol compounds or biphenyl derivatives from entering the body, thereby reducing the adverse effects caused by the direct use of polyphenol compounds or biphenyl derivatives.
[0011] The preparation method of the adsorbent for malignant tumors of the present invention has a simple preparation process, mild reaction conditions, high safety during the reaction process, low production cost, and is conducive to industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1A process flow chart for preparing an adsorbent for malignant tumors provided in an embodiment of the present invention. DETAILED DESCRIPTION
[0013] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.
[0014] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.
[0015] In addition, the terms "comprising", "including", "containing", and "having" are not restrictive, that is, other steps and other components that do not affect the results can be added. Unless otherwise specified, materials, equipment, and reagents are commercially available.
[0016] In addition, although the present invention describes each step in the preparation process in the form of S110 and S120, this description is only for ease of understanding, and the form of S110 and S120 does not limit the sequence of the steps.
[0017] The first aspect of the embodiments of the present application provides an adsorbent for malignant tumors, wherein the adsorbent uses an ultra-high cross-linked macroporous adsorption resin containing chloromethyl as a carrier, and an adsorption ligand is immobilized on the carrier. The adsorption ligand is a polyphenol compound or a biphenyl derivative. The polyphenol compound or the biphenyl derivative reacts with the chloromethyl group and is immobilized on the carrier.
[0018] The adsorbent for malignant tumors provided in the embodiments of the present application uses an ultra-high cross-linked macroporous adsorption resin containing chloromethyl as a carrier, and the chloromethyl on the carrier reacts with a polyphenol compound or a biphenyl derivative to immobilize the polyphenol compound or the biphenyl derivative on the carrier. The polyphenol compound or the biphenyl derivative in the adsorbent has a strong binding force to PD-L1, and the carrier has a steric hindrance effect. Through the synergistic effect of the two, selective adsorption and removal of PD-L1 can be achieved. The carrier itself has a rich pore structure and is a hydrophobic skeleton. Through the combination of the sieving effect of the carrier pores and the hydrophobic effect, TGF-β can be adsorbed. Through the carrier and the adsorption ligand immobilized on the carrier, the adsorbent can achieve simultaneous adsorption of TGF-β and PD-L1. In addition, the polyphenol compounds or biphenyl derivatives and the carrier are connected by chemical bonds, so that the polyphenol compounds or biphenyl derivatives can be firmly combined with the carrier, thereby improving the structural strength of the adsorbent and thus improving the safety of the adsorbent. At the same time, it can also prevent the polyphenol compounds or biphenyl derivatives from entering the body, thereby reducing the adverse effects caused by the direct use of polyphenol compounds or biphenyl derivatives.
[0019] Based on the above embodiment, as an optional implementation, the polyphenol compound is 5,6,7-trihydroxyflavone, the biphenyl derivative is one or a combination of IMMH-010 (Amedifen), INCB086550, BMS-8 and Incyte-001, and several substances in the biphenyl derivatives can be used as PD-L1 inhibitors, all of which are purchased from the market. Therefore, the above polyphenol compounds and biphenyl derivatives can be grafted onto the chloromethyl group of the carrier through an alkylation reaction, and can have a strong binding ability with PD-L1, thereby having a good chemical inhibition effect on PD-L1.
[0020] Based on the above embodiment, as an optional embodiment, the specific surface area of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is in the range of 600m 2 / g to 1500m 2 / g, pore volume range is 1.2cm 3 / g to 2.0cm 3 / g, the average pore size range is 2nm to 15nm, and the particle size range of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 0.4mm to 2mm. Preferably, the specific surface area range of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 700m 2 / g to 1200m 2 / g, pore volume range is 1.4cm 3 / g to 1.9cm 3 / g, the average pore size ranges from 4nm to 10nm, and the particle size range of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 0.6mm to 1.2mm. The ultra-high cross-linked macroporous adsorption resin containing chloromethyl has abundant mesopores and micropores, wherein the surface area occupied by the micropores is 300m 2 / g to 800m 2 / g, the surface area occupied by medium and large pores is 200m 2 / g to 500m 2 / g, the pore volume occupied by micropores is 0.3cm 3 / g to 0.6cm 3 / g, the pore volume occupied by medium and large pores ranges from 0.8cm 3 / g to 1.5cm 3 Therefore, the specific surface area and pore volume of the mesopores and micropores are within the above ranges, which can improve the adsorption capacity of TGF-β.
[0021] Based on the above embodiment, as an optional implementation, the content of chloromethyl in the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 1.5% to 5.5%. Therefore, the content of chloromethyl in the ultra-high cross-linked macroporous resin is within the above range, which can ensure that there are enough chloromethyl groups to carry out grafting reaction with polyphenol compounds and biphenyl derivatives, so that the grafting amount of polyphenol compounds or biphenyl derivatives on the carrier is maintained within a suitable range.
[0022] Figure 1 The process flow chart of preparing an adsorbent for malignant tumors provided in the examples of the present application is shown in FIG. Figure 1 As shown, the second aspect of the embodiment of the present application provides a method for preparing an adsorbent for malignant tumors, which is used to prepare the adsorbent as described in the first aspect, and the preparation method comprises the following steps:
[0023] Step S110, preparing an ultra-high cross-linked macroporous adsorption resin containing chloromethyl groups.
[0024] Specifically, a polystyrene-based macroporous resin is chloromethylated to obtain a chloromethylated macroporous adsorption resin; the chloromethylated macroporous adsorption resin is post-crosslinked to obtain an ultra-high crosslinked macroporous adsorption resin containing chloromethyl groups.
[0025] Based on the above embodiment, as an optional implementation, a polystyrene-based macroporous resin is chloromethylated to obtain a chloromethylated macroporous adsorption resin, comprising the following steps:
[0026] The polystyrene-based macroporous resin and chloromethyl ether are mixed and allowed to stand at room temperature for 4 to 5 hours, and then anhydrous zinc chloride is added under stirring, and the mixture is reacted at 50° C. to 52° C. for 8 to 24 hours to obtain a chloromethylated macroporous adsorption resin. The mass of the chloromethyl ether is 4 to 6 times the mass of the polystyrene-based macroporous resin, and the mass of the anhydrous zinc chloride is 0.5 to 1.5 times the mass of the polystyrene-based macroporous resin. Thus, it is possible to ensure that a sufficient amount of chloromethyl groups are introduced into the polystyrene-based macroporous resin. The chlorine content in the chloromethylated macroporous adsorption resin in this embodiment is 5% to 25%.
[0027] The polystyrene-based macroporous resin is prepared by suspension polymerization of styrene monomers and polyvinyl crosslinking agents. Based on the above embodiment, as an optional implementation, the polystyrene-based macroporous resin is prepared by the following method:
[0028] Styrene monomers, polyvinyl crosslinkers, porogens and initiators are mixed to form an oil phase, and the oil phase is added to a dispersion medium. A suspension polymerization reaction is carried out at 50°C to 100°C under stirring. After the reaction is carried out for 12h to 20h, the suspension polymerization product is cleaned and dried to obtain a polystyrene-based macroporous resin. Preferably, the temperature of the suspension polymerization reaction is 70°C to 95°C, and the reaction time is 14h to 18h. In the specific operation, the temperature can be gradually increased in stages for reaction. After the oil phase forms uniform droplets of a certain size in the dispersion medium, the temperature is increased to 75°C for polymerization reaction for 3h, then increased to 80°C for reaction for 7h, and then increased to 85°C. The reaction is continued for 6h before stopping the reaction. As a result, the polystyrene-based macroporous resin prepared by this method has two pore structures of rich mesopores and micropores, and the polystyrene-based macroporous resin has an ultra-high specific surface area, so that it has good adsorption capacity for TGF-β.
[0029] The styrene monomer is selected from at least one of styrene, methyl styrene and ethyl styrene, preferably, the styrene monomer is styrene; the polyvinyl crosslinking agent is selected from at least one of divinylbenzene, divinyltoluene, divinylxylene and divinylethylbenzene, preferably, the polyvinyl crosslinking agent is divinylbenzene. The styrene monomer accounts for 20% to 92% of the total mass of the styrene monomer and the polyvinyl crosslinking agent, and the polyvinyl crosslinking agent accounts for 8% to 80% of the total mass of the styrene monomer and the polyvinyl crosslinking agent. Therefore, by using monovinyl styrene monomer and polyvinyl crosslinking agent as reaction monomers, the polystyrene-based macroporous resin is partially crosslinked, which is beneficial to improve the mechanical strength of the polystyrene-based macroporous resin and the structural stability in organic solvents.
[0030] The porogen is one or a combination of aromatic hydrocarbons, alkanes, higher alcohols, higher ketones, and esters; wherein the aromatic hydrocarbons are selected from toluene and xylene; the alkanes are selected from n-heptane, 200# gasoline, and solid paraffin; the higher alcohols are selected from butanol, hexanol, cyclohexanol, isooctyl alcohol, n-octanol, and methyl isobutyl carbinol; the higher ketones are selected from methyl isobutyl ketone, 2-hexanone, diisobutyl ketone, and methyl tert-butyl ketone; and the esters are selected from butyl acetate, ethyl acetate, and butyl butyrate. As an optional embodiment, the mass of the porogen can be 70% to 230% of the total mass of the styrene monomer and the polyvinyl crosslinking agent.
[0031] The initiator is at least one of benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, and tert-amyl peroxy-2-ethylhexanoate. Preferably, the initiator is benzoyl peroxide. As an optional embodiment, the mass of the initiator is 0.5% to 1.5% of the total mass of the styrene monomer and the polyvinyl crosslinking agent. Therefore, the use of the above initiator can effectively initiate polymerization, and the price of the initiator is relatively low.
[0032] The dispersion medium is water, and the volume ratio of the dispersion medium to the oil phase is 1:1 to 3:1. The dispersion medium contains a dispersant, which is selected from at least one of gelatin, polyvinyl alcohol, and carboxymethyl cellulose. Preferably, the dispersion medium is gelatin. As an optional embodiment, the mass of the dispersant is 0.5% to 2% of the mass of the dispersion medium. Therefore, the use of the above dispersion medium is safe and environmentally friendly, and can achieve suspension polymerization of polystyrene-based macroporous resin.
[0033] Based on the above embodiment, as an optional implementation method, the chloromethylated macroporous adsorption resin is subjected to a post-crosslinking reaction to obtain an ultra-high cross-linked macroporous adsorption resin containing chloromethyl, comprising the following steps:
[0034] Add dichloroethane or nitrobenzene to the chloromethylated macroporous adsorption resin, let it stand for 4 hours to 5 hours at 38°C to 42°C to swell, then add ferric chloride or zinc chloride under stirring, and dichloroethane or nitrobenzene and the chloromethyl groups on the chloromethylated macroporous adsorption resin undergo post-crosslinking reaction to prepare an ultra-high crosslinked macroporous adsorption resin containing chloromethyl groups. Thus, by subjecting the chloromethylated macroporous adsorption resin to secondary crosslinking treatment, an ultra-high crosslinked network structure can be formed, which greatly enriches the pore structure of the macroporous adsorption resin and increases its specific surface area, which is beneficial to improving the adsorption capacity of TGF-β, and the macroporous adsorption resin after the secondary crosslinking treatment has better stability, high crosslinking density, and high strength, which is beneficial to improving the structural stability of the carrier. In addition, there are more chloromethyl residues on the ultra-high crosslinked macroporous adsorption resin after the post-crosslinking treatment, which can subsequently react with polyphenol compounds or biphenyl derivatives.
[0035] Based on the above embodiment, as an optional implementation, if ethylene dichloride is added to the chloromethylated macroporous adsorption resin for post-crosslinking reaction, the temperature of the post-crosslinking reaction is 78°C to 80°C, and the reaction time is 6h to 8h. If nitrobenzene is added to the chloromethylated macroporous adsorption resin for post-crosslinking reaction, the temperature of the post-crosslinking reaction is 115°C to 125°C, and the reaction time is 8h to 12h.
[0036] On the basis of the above embodiment, as an optional implementation manner, the mass of ethylene dichloride is 5 to 7 times the mass of the chloromethylated macroporous adsorption resin, the mass of nitrobenzene is 5 to 7 times the mass of the chloromethylated macroporous adsorption resin; the mass of ferric chloride is 0.1 to 0.5 times the mass of the chloromethylated macroporous adsorption resin, and the mass of zinc chloride is 0.1 to 0.5 times the mass of the chloromethylated macroporous adsorption resin.
[0037] The specific surface area of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl prepared by the method of this embodiment is in the range of 600m 2 / g to 1500m2 / g, pore volume range is 1.2cm 3 / g to 2.0cm 3 / g, the average pore size range is 2nm to 15nm, and the particle size range of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 0.4mm to 2mm. Preferably, the specific surface area range of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 700m 2 / g to 1200m 2 / g, pore volume range is 1.4cm 3 / g to 1.9cm 3 / g, the average pore size ranges from 4nm to 10nm, and the particle size range of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 0.6mm to 1.2mm. The ultra-high cross-linked macroporous adsorption resin containing chloromethyl has abundant mesopores and micropores, wherein the surface area occupied by the micropores is 300m 2 / g to 800m 2 / g, the surface area occupied by medium and large pores is 200m 2 / g to 500m 2 / g, the pore volume occupied by micropores is 0.3cm 3 / g to 0.6cm 3 / g, the pore volume occupied by medium and large pores ranges from 0.8cm 3 / g to 1.5cm 3 The content of chloromethyl in the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 1.5% to 5.5%.
[0038] Step S120, reacting the ultra-high cross-linked macroporous adsorption resin containing chloromethyl groups with polyphenol compounds or biphenyl derivatives, wherein the polyphenol compounds or biphenyl derivatives are connected to the ultra-high cross-linked macroporous adsorption resin via the chloromethyl groups, to prepare an adsorbent for malignant tumors.
[0039] Specifically, after mixing an ultra-high cross-linked macroporous adsorption resin containing chloromethyl, nitrobenzene and polyphenol compounds or biphenyl derivatives, the mixture is stirred at 38°C to 42°C for 1 to 2 hours, and then ferric chloride is added and reacted at 110°C to 120°C for 3 to 4 hours to allow the polyphenol compounds or biphenyl derivatives to react with the chloromethyl groups and connect the polyphenol compounds or biphenyl derivatives to the ultra-high cross-linked macroporous adsorption resin.
[0040] In this embodiment, the chloromethyl groups on the ultra-high cross-linked macroporous adsorption resin undergo an alkylation reaction with the polyphenol compounds or biphenyl derivatives, and the polyphenol compounds or biphenyl derivatives are immobilized on the ultra-high cross-linked macroporous adsorption resin, so that the polyphenol compounds or biphenyl derivatives and the ultra-high cross-linked macroporous adsorption resin are connected by chemical bonds, which improves the stability of the connection between the two, is beneficial to improving the structural stability of the adsorbent, reduces the risk of the polyphenol compounds or biphenyl derivatives falling off, and improves the safety of the adsorbent.
[0041] Based on the above embodiment, as an optional implementation, the amount of the polyphenol compound or biphenyl derivative is 0.01 to 0.1 times the mass of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl. Therefore, the amount of the polyphenol compound or biphenyl derivative within the above range can increase the grafting rate of the polyphenol compound or biphenyl derivative on the ultra-high cross-linked macroporous adsorption resin, which is beneficial to improve the PD-L1 clearance ability.
[0042] Based on the above embodiment, as an optional implementation, the amount of nitrobenzene is 3 to 5 times the mass of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl; the amount of ferric chloride is 0.05 to 0.3 times the mass of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl. Therefore, the amount of nitrobenzene and ferric chloride used within the above range is conducive to improving the activity and reaction efficiency of the alkylation reaction.
[0043] After the alkylation reaction is completed and before the adsorbent for malignant tumors is obtained, the method further includes: washing the alkylation reaction product with a 2% by mass hydrochloric acid solution for three times, extracting with acetone to remove the solvent and the reacted substances, and drying to obtain the adsorbent for malignant tumors. This is conducive to improving the safety of the adsorbent.
[0044] The preparation method of the adsorbent for malignant tumors provided in the embodiment of the present application first prepares an ultra-high cross-linked macroporous adsorption resin containing chloromethyl, the ultra-high cross-linked macroporous adsorption resin containing chloromethyl has a rich pore structure, and is a hydrophobic skeleton, which can adsorb TGF-β by combining the sieving effect of the pores and the hydrophobic effect, and then uses chloromethyl to immobilize the polyphenolic compound or biphenyl derivative on the ultra-high cross-linked macroporous adsorption resin, and the polyphenolic compound or biphenyl derivative has a strong binding force on PD-L1 and the steric hindrance effect of the carrier. The two synergistic effects can achieve selective adsorption and removal of PD-L1, so that the adsorbent can achieve simultaneous adsorption of TGF-β and PD-L1. In addition, the polyphenolic compound or biphenyl derivative and the carrier are connected by chemical bonds, so that the polyphenolic compound or biphenyl derivative and the carrier can be firmly combined, which improves the structural strength of the adsorbent, thereby improving the safety of the use of the adsorbent, and at the same time, it can prevent the polyphenolic compound or biphenyl derivative from entering the body, reducing the adverse effects of direct use of the polyphenolic compound or biphenyl derivative. The preparation method provided by the present invention has the advantages of simple preparation process, mild reaction conditions, high safety during the reaction process, low production cost, and is conducive to industrialized mass production.
[0045] The third aspect of the present application provides an application of an adsorbent for malignant tumors in blood purification, wherein the adsorbent for malignant tumors is the adsorbent described in the first aspect, or is the adsorbent prepared by the preparation method described in the second aspect. The adsorbent for malignant tumors is used for extracorporeal circulation of blood to remove TGF-β and PD-L1, effectively reducing TGF-β and PD-L1 in the blood. The adsorbent is used in vitro and will not enter the body, and has low harmful effects on the human body. It can avoid polyphenol compounds or biphenyl derivatives directly entering the human body through intravenous injection, resulting in various immune-related adverse reactions and liver toxicity.
[0046] In order to further explain the present invention in detail, the present invention will be further explained in conjunction with specific examples. The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified; the materials, reagents, etc. used in the examples of the present invention are purchased from the market unless otherwise specified.
[0047] Example 1
[0048] This embodiment provides a method for preparing an adsorbent for malignant tumors, comprising the following steps:
[0049] (1) Preparation of chloromethyl modified macroporous adsorption resin: 600 mL of 1.5 wt% gelatin aqueous solution was added to a 1000 mL three-necked flask, 12 g of styrene, 38 g of divinylbenzene (DVB) with a purity of 63 wt%, 40 g of toluene, 45 g of 200# gasoline, and 0.5 g of benzoyl peroxide were mixed to form an oil phase, and the oil phase was slowly added to the gelatin aqueous solution. Under mechanical stirring, the temperature was raised to 75°C for reaction for 5 h, and then the temperature was raised to 80°C for reaction overnight. After the reaction was completed, the mixture was cooled, extracted with acetone for 12 h, washed with water until there was no acetone smell, filtered, dried, sieved, and the resin with a particle size of 0.6 mm to 1.2 mm was selected to obtain a polystyrene-based macroporous resin. In a 500mL three-necked flask, 20g of polystyrene-based macroporous resin and 100g of chloromethyl ether were added, and the mixture was allowed to stand at room temperature for 4h. The stirrer was started, 20g of anhydrous zinc chloride was added, and the temperature was raised to 50°C for reaction for 24h. After the reaction was completed, the mixture was cooled to room temperature, the mother liquor was filtered out, and methanol was extracted for 12h. The mixture was washed with water until there was no methanol smell, filtered, and dried to obtain a light yellow chloromethylated macroporous adsorption resin. The chlorine content in the chloromethylated macroporous adsorption resin was 12.1% as tested by the Volhard method.
[0050] (2) Preparation of ultra-high cross-linked macroporous adsorption resin containing chloromethyl: Take 20g of the chloromethylated macroporous adsorption resin obtained in step (1), add 140g of nitrobenzene, let it stand at 40°C for 4h to swell, then add 10g of zinc chloride under mechanical stirring, heat and react at 125°C for 10h, the chloromethyl undergoes Friedel-Crafts alkylation reaction to form an ultra-high cross-linked network, and obtain an ultra-high cross-linked macroporous adsorption resin, and a certain amount of chloromethyl remains on the surface and in the skeleton of the ultra-high cross-linked macroporous adsorption resin. It has been determined that the specific surface area of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is in the range of 980m 2 / g, pore volume 1.75cm 3 / g, the average pore size is 7.1nm. The residual chlorine content tested by Volhard method is 3.5%.
[0051] (3) Grafting 5,6,7-trihydroxyflavone: Take 50 g of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl obtained in step (2), add 200 g of nitrobenzene, add 2.5 g of 5,6,7-trihydroxyflavone, stir at 40°C for 1 hour, then add 10 g of ferric chloride, slowly raise the temperature to 110°C, stir and react for 4 hours, and graft 5,6,7-trihydroxyflavone onto the ultra-high cross-linked macroporous adsorption resin. After the reaction is completed, filter out the mother liquor, remove the reaction mother liquor, add 2% hydrochloric acid aqueous solution to wash 3 times, and then use acetone to extract to remove the solvent and unreacted chemicals to obtain an adsorbent for malignant tumors.
[0052] Example 2
[0053] This embodiment provides a method for preparing an adsorbent for malignant tumors, comprising the following steps:
[0054] (1) Preparation of chloromethyl modified macroporous adsorption resin: 600 mL of 1.5 wt% gelatin aqueous solution was added to a 1000 mL three-necked flask, 34 g of styrene, 16 g of 63 wt% pure divinylbenzene (DVB), 20 g of toluene, 20 g of isooctyl alcohol, 40 g of methyl isobutyl ketone, and 0.5 g of tert-butyl peroxy-2-ethylhexanoate were mixed to form an oil phase, the oil phase was slowly added to the gelatin aqueous solution, the temperature was raised to 75°C for reaction for 5 h under mechanical stirring, and then the temperature was raised to 80°C for reaction overnight. After the reaction was completed, the mixture was cooled, extracted with acetone for 12 h, washed with water until there was no acetone smell, filtered, dried, sieved, and the resin with a particle size of 0.6 mm to 1.2 mm was selected to obtain a polystyrene-based macroporous resin. In a 500mL three-necked flask, 20g of polystyrene-based macroporous resin and 100g of chloromethyl ether were added, and the mixture was allowed to stand at room temperature for 4h. The stirrer was started, 20g of anhydrous zinc chloride was added, and the temperature was raised to 50°C for reaction for 24h. After the reaction was completed, the mixture was cooled to room temperature, the mother liquor was filtered out, and methanol was extracted for 12h. The mixture was washed with water until there was no methanol smell, filtered, and dried to obtain a light yellow chloromethylated macroporous adsorption resin. The chlorine content in the chloromethylated macroporous adsorption resin was 14.3% as tested by the Volhard method.
[0055] (2) Preparation of ultra-high cross-linked macroporous adsorption resin containing chloromethyl: Take 20g of the chloromethylated macroporous adsorption resin obtained in step (1), add 140g of nitrobenzene, and let it swell at 40°C for 4h. Then, under mechanical stirring, add 8g of zinc chloride, and heat to react at 120°C for 8h. The chloromethyl groups undergo Friedel-Crafts alkylation reaction to form an ultra-high cross-linked network, thereby obtaining an ultra-high cross-linked macroporous adsorption resin, and a certain amount of chloromethyl groups remain on the surface and in the skeleton of the ultra-high cross-linked macroporous adsorption resin. It has been determined that the specific surface area of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl groups is in the range of 1090m 2 / g, pore volume 1.97cm 3 / g, the average pore size is 7.2nm. The residual chlorine content tested by Volhard method is 4.1%.
[0056] (3) Grafting IMMH-010: Take 50 g of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl obtained in step (2), add 200 g of nitrobenzene, add 1 g of IMMH-010, stir at 40°C for 1 h, then add 10 g of ferric chloride, slowly heat to 110°C, stir and react for 4 h, and graft IMMH-010 onto the ultra-high cross-linked macroporous adsorption resin. After the reaction is completed, filter out the mother liquor, remove the reaction mother liquor, add 2% hydrochloric acid aqueous solution to wash 3 times, and then use acetone to extract to remove the solvent and unreacted chemicals to obtain an adsorbent for malignant tumors.
[0057] Example 3
[0058] This embodiment provides a method for preparing an adsorbent for malignant tumors, comprising the following steps:
[0059] (1) Preparation of chloromethyl modified macroporous adsorption resin: 600 mL of 1.5 wt% gelatin aqueous solution was added to a 1000 mL three-necked flask, 50 g of 63 wt% pure divinylbenzene (DVB), 50 g of toluene, 20 g of solid paraffin, 35 g of butyl butyrate, and 0.5 g of tert-butyl peroxy-2-ethylhexanoate were mixed to form an oil phase, the oil phase was slowly added to the gelatin aqueous solution, the temperature was raised to 75°C under mechanical stirring for reaction for 5 h, and then the temperature was raised to 80°C for reaction overnight. After the reaction was completed, the mixture was cooled, extracted with acetone for 12 h, washed with water until there was no acetone smell, filtered, dried, sieved, and the resin with a particle size of 0.6 mm to 1.2 mm was selected to obtain a polystyrene-based macroporous resin. In a 500mL three-necked flask, 20g of polystyrene-based macroporous resin and 100g of chloromethyl ether were added, and the mixture was allowed to stand at room temperature for 4h. The stirrer was started, 20g of anhydrous zinc chloride was added, and the temperature was raised to 50°C for reaction for 24h. After the reaction was completed, the mixture was cooled to room temperature, the mother liquor was filtered out, and methanol was extracted for 12h. The mixture was washed with water until there was no methanol smell, filtered, and dried to obtain a light yellow chloromethylated macroporous adsorption resin. The chlorine content in the chloromethylated macroporous adsorption resin was 12.1% as tested by the Volhard method.
[0060] (2) Preparation of ultra-high cross-linked macroporous adsorption resin containing chloromethyl: Take 20g of the chloromethylated macroporous adsorption resin obtained in step (1), add 140g of nitrobenzene, let it stand at 40°C for 4h to swell, then add 8g of zinc chloride under mechanical stirring, heat and react at 120°C for 8h, the chloromethyl undergoes Friedel-Crafts alkylation reaction to form an ultra-high cross-linked network, and obtain an ultra-high cross-linked macroporous adsorption resin, and a certain amount of chloromethyl remains on the surface and in the skeleton of the ultra-high cross-linked macroporous adsorption resin. It has been determined that the specific surface area of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is in the range of 1110m 2 / g, pore volume 2.08cm 3 / g, the average pore size is 7.5nm. The residual chlorine content tested by Volhard method is 2.5%.
[0061] (3) Grafting INCB086550: Take 50 g of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl obtained in step (2), add 200 g of nitrobenzene, add 1 g of INCB086550, stir at 40° C. for 1 h, then add 10 g of ferric chloride, slowly raise the temperature to 110° C., stir and react for 4 h, and graft INCB086550 onto the ultra-high cross-linked macroporous adsorption resin. After the reaction is completed, filter out the mother liquor, remove the reaction mother liquor, add 2% hydrochloric acid aqueous solution to wash three times, and then use acetone to extract to remove the solvent and unreacted chemicals to obtain an adsorbent for malignant tumors.
[0062] Example 4
[0063] This embodiment provides a method for preparing an adsorbent for malignant tumors, comprising the following steps:
[0064] (1) Preparation of chloromethyl modified macroporous adsorption resin: 600 mL of 1.5 wt% gelatin aqueous solution was added to a 1000 mL three-necked flask, 50 g of 80 wt% pure divinylbenzene (DVB), 60 g of toluene, 20 g of solid paraffin, 20 g of methyl isobutyl carbinol, and 0.5 g of tert-butyl peroxy-2-ethylhexanoate were mixed to form an oil phase, the oil phase was slowly added to the gelatin aqueous solution, the temperature was raised to 75°C for reaction for 5 h under mechanical stirring, and then the temperature was raised to 80°C for reaction overnight. After the reaction was completed, the mixture was cooled, extracted with acetone for 12 h, washed with water until there was no acetone smell, filtered, dried, sieved, and the resin with a particle size of 0.6 mm to 1.2 mm was selected to obtain a polystyrene-based macroporous resin. In a 500mL three-necked flask, 20g of polystyrene-based macroporous resin and 100g of chloromethyl ether were added, and the mixture was allowed to stand at room temperature for 4h. The stirrer was started, 20g of anhydrous zinc chloride was added, and the temperature was raised to 50°C for reaction for 24h. After the reaction was completed, the mixture was cooled to room temperature, the mother liquor was filtered out, and methanol was extracted for 12h. The mixture was washed with water until there was no methanol smell, filtered, and dried to obtain a light yellow chloromethylated macroporous adsorption resin. The chlorine content in the chloromethylated macroporous adsorption resin was 10.3% as tested by the Volhard method.
[0065] (2) Preparation of ultra-high cross-linked macroporous adsorption resin containing chloromethyl: Take 20g of the chloromethylated macroporous adsorption resin obtained in step (1), add 140g of nitrobenzene, let it stand at 40°C for 4h to swell, then add 10g of zinc chloride under mechanical stirring, heat and react at 120°C for 12h, the chloromethyl undergoes Friedel-Crafts alkylation reaction to form an ultra-high cross-linked network, and obtain an ultra-high cross-linked macroporous adsorption resin, and a certain amount of chloromethyl remains on the surface and in the skeleton of the ultra-high cross-linked macroporous adsorption resin. It has been determined that the specific surface area of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is in the range of 990m 2 / g, pore volume 1.79cm 3 / g, the average pore size is 8.1nm. The residual chlorine content tested by Volhard method is 2.8%.
[0066] (3) Grafting BMS-8: Take 50 g of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl obtained in step (2), add 200 g of nitrobenzene, add 1 g of BMS-8, stir at 40°C for 1 h, then add 10 g of ferric chloride, slowly raise the temperature to 110°C, stir and react for 4 h, and graft BMS-8 onto the ultra-high cross-linked macroporous adsorption resin. After the reaction is completed, filter out the mother liquor, remove the reaction mother liquor, add 2% hydrochloric acid aqueous solution to wash 3 times, and then use acetone to extract to remove the solvent and unreacted chemicals to obtain an adsorbent for malignant tumors.
[0067] Test example
[0068] 2 mL of the adsorbent for malignant tumors prepared in Examples 1 to 4, 2 mL of the commercially available Jianfan HA resin and 2 mL of the commercially available Boxin MG resin were accurately measured respectively, and each group of adsorbents and commercially available resins were added to the pre-prepared bovine plasma, which contained TGF-β and PD-L1. After shaking at 37°C for 2 hours, the adsorption was completed, and the bovine plasma was taken for detection. The concentrations of TGFβ and PD-L1 after adsorption were detected respectively, and the adsorption rate of each group of adsorbents and commercially available resins was calculated. The calculation formula of the adsorption rate is: adsorption rate = [(m1-m2) / m1] × 100%, wherein m1 is the concentration of the adsorbed substance in the dialysate before adsorption, and m2 is the concentration of the adsorbed substance after the dialysate is adsorbed by the adsorbent. The adsorption rate of the adsorbents in each embodiment to the adsorbed substance is shown in Table 1.
[0069] Table 1
[0070]
[0071]
[0072] It can be seen from Table 1 that compared with commercially available adsorbents, the adsorbents for malignant tumors in Examples 1 to 4 have good adsorption and clearance capabilities for TGF-β and PD-L1, indicating that the adsorbents for malignant tumors provided in the present invention can clear transforming growth factor-β (TGF-β) and programmed death ligand 1 (PD-L1) that help tumor metastasis and escape, can reduce the immune escape of malignant tumors, and can assist in the treatment of malignant tumors to help improve the success rate of malignant tumor treatment.
[0073] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.
Claims
1. An adsorbent for malignant tumors, characterized in that: The adsorbent uses a super-high cross-linked macroporous adsorption resin containing chloromethyl as a carrier, and an adsorption ligand is immobilized on the carrier. The adsorption ligand is a polyphenol compound or a biphenyl derivative. The polyphenol compound or the biphenyl derivative reacts with the chloromethyl group and is immobilized on the carrier.
2. The adsorbent for malignant tumors according to claim 1, characterized in that: The polyphenol compound is 5,6,7-trihydroxyflavone, and the biphenyl derivative is one or a combination of several of IMMH-010, INCB086550, BMS-8 and Incyte-001.
3. The adsorbent for malignant tumors according to claim 1, characterized in that: The ultra-high cross-linked macroporous adsorption resin containing chloromethyl has mesopores and micropores, and the surface area occupied by the micropores is 300m 2 / g to 800m 2 / g, the surface area occupied by the mesopores is 200m 2 / g to 500m 2 / g, the pore volume range of the micropores is 0.3cm 3 / g to 0.6cm 3 / g, the pore volume range of the medium and large pores is 0.8cm 3 / g to 1.5cm 3 / g.
4. The adsorbent for malignant tumors according to claim 1, characterized in that: The content of chloromethyl in the ultra-high cross-linked macroporous adsorption resin containing chloromethyl is 1.5% to 5.5%.
5. A method for preparing an adsorbent for malignant tumors, characterized in that: For preparing the adsorbent according to any one of claims 1 to 4, the preparation method comprises the following steps: Preparation of ultra-high cross-linked macroporous adsorption resin containing chloromethyl; The ultra-high cross-linked macroporous adsorption resin containing chloromethyl groups is reacted with polyphenol compounds or biphenyl derivatives, and the polyphenol compounds or biphenyl derivatives are connected to the ultra-high cross-linked macroporous adsorption resin through chloromethyl groups to prepare an adsorbent for malignant tumors.
6. The preparation method according to claim 5, characterized in that: The ultra-high cross-linked macroporous adsorption resin containing chloromethyl groups is reacted with a polyphenol compound or a biphenyl derivative, wherein the polyphenol compound or the biphenyl derivative is connected to the ultra-high cross-linked macroporous adsorption resin via the chloromethyl groups, comprising: The ultra-high cross-linked macroporous adsorption resin containing chloromethyl, nitrobenzene and polyphenol compounds or biphenyl derivatives are mixed, stirred at 38° C. to 42° C. for 1 h to 2 h, and then ferric chloride is added and reacted at 110° C. to 120° C. for 3 h to 4 h to allow the polyphenol compounds or biphenyl derivatives to react with the chloromethyl and connect the polyphenol compounds or biphenyl derivatives to the ultra-high cross-linked macroporous adsorption resin.
7. The preparation method according to claim 6, characterized in that: The amount of the polyphenol compound or biphenyl derivative is 0.01 to 0.1 times the mass of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl.
8. The preparation method according to claim 6, characterized in that: The amount of nitrobenzene used is 3 to 5 times the mass of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl; The amount of ferric chloride used is 0.05 to 0.3 times the mass of the ultra-high cross-linked macroporous adsorption resin containing chloromethyl.
9. The preparation method according to claim 5, characterized in that: The method for preparing the ultra-high cross-linked macroporous adsorption resin containing chloromethyl comprises: Chloromethylation of polystyrene-based macroporous resin to obtain chloromethylation-modified macroporous adsorption resin; The chloromethyl-modified macroporous adsorption resin is subjected to a post-crosslinking reaction to obtain an ultra-high crosslinked macroporous adsorption resin containing chloromethyl groups.
10. Use of an adsorbent for malignant tumors as claimed in any one of claims 1 to 4, or an adsorbent prepared by the preparation method as claimed in any one of claims 5 to 9 in blood purification.