Functional polymers and endotoxin separation materials targeting endotoxins, their preparation and application

Abstract

This invention provides a functional polymer targeting endotoxins and its applications, belonging to the fields of materials science, polymer science, and biomedical engineering. The invention designs and synthesizes a functional polymer targeting endotoxins through free radical polymerization. The functional polymer consists of an antifouling unit, an endotoxin-binding unit, and a coupling unit connecting the polymer and the endotoxin-binding unit, wherein the endotoxin-binding unit is an affinity polypeptide targeting endotoxins. In vitro and in vivo endotoxin clearance experiments show that the functional polymer exhibits high specificity and affinity for endotoxins, while also possessing good antifouling, anti-interference, and blood compatibility properties. It can achieve precise clearance of endotoxins from the blood and can be widely applied in various fields of biomedicine, such as bioseparation, biodetection, blood purification, and disease diagnosis and treatment.

Description

Technical Field

[0001] This invention provides a functional polymer targeting endotoxins and its applications, belonging to the fields of materials science, polymer science, and biomedical engineering. This invention designs and synthesizes a functional polymer targeting bacterial endotoxins through free radical polymerization. The functional polymer exhibits high specificity and affinity for endotoxins, while also possessing good anti-fouling properties, anti-interference properties, and blood compatibility, enabling in-situ and precise removal of endotoxins from the bloodstream. The endotoxin-targeting functional polymer of this invention can be widely applied in various fields of biomedicine, such as bioseparation, biodetection, blood purification, and disease diagnosis and treatment. Background Technology

[0002] Sepsis is a critical illness with a high incidence and mortality rate. Nearly 50 million new cases of sepsis are diagnosed worldwide each year, resulting in 10 million deaths, accounting for one-fifth of all deaths globally. It is the leading cause of death for ICU patients worldwide and seriously threatens human life and health.

[0003] Endotoxins, species-specific lipopolysaccharides (LPS) produced by Gram-negative bacteria, are the most important pathogen-associated molecular pattern (PAMP) in the pathogenesis and progression of sepsis. Endotoxins are highly toxic inflammatory and pyrogenic substances, first discovered and proposed by French scientist Pfeiffer in his research on Vibrio cholerae. They are mainly released in large quantities after bacterial death due to cell wall lysis, inducing a host's innate immune response and causing the expression and systemic release of various cytokines such as tumor necrosis factor and interleukins, as well as other inflammatory mediators. Especially when the body is under stress, such as from severe trauma (burns), infection, or major surgery, the body's barrier function is compromised, allowing large amounts of endotoxins to enter the bloodstream from the intestines or site of infection, triggering a cascade of inflammatory reactions and leading to an excessive immune response, which can easily induce sepsis. Further progression of the disease can cause a series of critical illnesses, including septic shock, disseminated intravascular coagulation, acute respiratory distress syndrome, systemic inflammatory response syndrome, and multiple organ failure, ultimately leading to death.

[0004] Timely removal of endotoxins from the blood is crucial for the treatment of sepsis. However, the structural variability of endotoxins and their extremely low threshold for inducing sepsis pose significant challenges to the specific removal of endotoxins from the blood. Effective removal of endotoxins remains a hot topic and a difficult area of ​​research in clinical diagnosis, biomedicine, and other fields. Currently, some natural and artificial ligands have been reported for endotoxin binding. Although most ligands perform well in in vitro experiments, their lack of specificity leads to significant loss of supportive therapies and other immunologically active substances during in vivo application for endotoxin removal from the blood, often making blood purification less effective in sepsis treatment. Therefore, there is an urgent need to develop separation materials with high specificity, strong binding force, and good blood compatibility to achieve in-situ, rapid, and precise removal of endotoxins from the blood.

[0005] Phage display technology is a rapid, efficient, and high-throughput ligand screening method. This technology involves inserting the DNA sequence of a foreign protein or peptide into an appropriate position in the structural gene of the phage coat protein, allowing the foreign gene to be expressed along with the coat protein. Simultaneously, the foreign protein is displayed on the phage surface as the phage reassembles. The displayed peptide or protein maintains a relatively independent spatial structure and biological activity, facilitating the recognition and binding of target molecules. After incubation of the peptide library with the target molecule on a solid phase for a certain period, unbound free phages are washed away. Then, phages bound to the target molecule are eluted using competitive receptors or acid. The eluted phages infect host cells, multiply, and undergo another round of elution. After 3-5 rounds of "adsorption-elution-amplification," phages specifically bound to the target molecule are highly enriched. Finally, DNA sequencing is used to obtain the amino acid sequence of the foreign protein or peptide, thereby screening for the specific binding ligands of the target molecule.

[0006] By combining the design of functional polymers and integrating the screened specific binding ligands with functional monomers, different functions can be integrated on demand, thereby obtaining functional polymers that possess target molecule specific binding properties, anti-fouling properties, blood compatibility, or other properties. This enables the specific removal of endotoxins or other target toxins from the blood, which has important positive significance for promoting the development of the field of precision blood purification. Summary of the Invention

[0007] The purpose of this invention is to fill the technological gap in the field of precise endotoxin separation by providing a functional polymer capable of specifically binding to endotoxins. This functional polymer exhibits high specificity and affinity for endotoxins, along with good anti-fouling, anti-interference, and blood compatibility properties. This provides guidance for the development of fields involving precise endotoxin separation, such as blood purification and biomedicine. The technical solution of this invention is as follows:

[0008] Firstly, the object of this invention is to provide a functional polymer capable of targeting endotoxins.

[0009] Specifically, the functional polymer targeting endotoxin consists of an antifouling unit (PEGMEA, x in parentheses, x = 1 to 10000), an endotoxin-binding unit (PEP), and a coupling unit (GMA, y in parentheses excluding PEP, y = 1 to 10000) for connecting the polymer and the endotoxin-binding unit. Its molecular structure is shown below (PEGMEA and GMA undergo random copolymerization during the polymerization process, and the xy parts are randomly linked):

[0010]

[0011] More specifically, the antifouling unit in the functional polymer is polyethylene glycol monomethyl ether acrylate (PEGMEA); the endotoxin binding unit is an endotoxin affinity polypeptide with the amino acid sequence His-Trp-Lys-Ala-Val-Asn-Trp-Leu-Lys-Pro-Trp-Thr (PEP-1), Ser-Ser-Phe-Phe-His-Leu-Arg-His-His-Ala-His-Leu (PEP-2), Gly-Leu-Ser-Arg-Tyr-Lys-Pro-Gly-Arg-Ser-Lys-Leu (PEP-3), Trp-Arg-Ser-Ala-Gly-Glu-Ser-His-Phe-Gly-Leu-Thr (PEP-4) or

[0012] Cys-Pro-Arg-Thr-Leu-Ala-Ser-Arg-Ser-Asn-Cys-Tyr(PEP-5);

[0013] The coupling unit used to link the polymer to the endotoxin-affinity peptide is glycidyl methacrylate (GMA), which reacts with the terminal amino or amino side group of the endotoxin-affinity peptide via an epoxy ring-opening reaction.

[0014] Secondly, another object of the present invention is to provide the use of the endotoxin-specific binding polymer in blood purification.

[0015] Thirdly, the endotoxin-specific binding polymer described in this invention has high specificity and affinity for endotoxins, and also exhibits good anti-fouling properties, anti-interference properties, and blood compatibility.

[0016] The features and beneficial effects of this invention are as follows: The endotoxin-specific binding polymer of this invention has high affinity for endotoxins, high specificity, and good anti-pollution performance, and can achieve precise separation of endotoxins in the blood. It has certain guiding significance for the purification of blood in severe cases, especially for the treatment of sepsis.

[0017] The aforementioned functional polymer exhibits high specificity and affinity for endotoxins, while also possessing excellent anti-fouling, anti-interference, and blood compatibility properties. It can achieve precise removal of endotoxins from the blood and has broad applications in various fields of biomedicine, such as bioseparation, biodetection, blood purification, and disease diagnosis and treatment. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram illustrating the synthesis of poly(PEGMEA-co-PEP-1) using reversible addition-fragmentation chain transfer polymerization.

[0020] Figure 2 This is a schematic diagram of the synthesis of poly(PEGMEA-co-PEP-2) using conventional free radical random copolymerization.

[0021] Figure 3 This is a schematic diagram of the preparation of SiO2@poly(PEGMEA-co-PEP-1) microspheres.

[0022] Figure 4 The endotoxin removal performance of SiO2@poly(PEGMEA-co-PEP-1) microspheres in aqueous solution.

[0023] Figure 5 The endotoxin removal performance of SiO2@poly(PEGMEA-co-PEP-1) microspheres in protein solution.

[0024] Figure 6 The QCM adsorption kinetics curves of poly(PEGMEA-co-PEP-1) for endotoxins or plasma proteins are shown.

[0025] Figure 7 The amount of endotoxin or plasma protein adsorbed on the surface of a gold chip modified with poly(PEGMEA-co-PEP-1).

[0026] Figure 8 The endotoxin removal performance of SiO2@poly(PEGMEA-co-PEP-1) microspheres in whole blood (in vitro experiment).

[0027] Figure 9 The endotoxin removal performance of PES / poly(PEGMEA-co-PEP-1) microsphere blood perfusion column in blood (animal experiment). Detailed Implementation

[0028] Information of SEQ ID No.1

[0029] (a) Sequence characteristics

[0030] *Length: 12 amino acids

[0031] *Type: Amino acid

[0032] *Chain type: Single chain

[0033] (b) Molecular type: protein

[0034] Sequence description:

[0035] SEQ ID No. 1: His-Trp-Lys-Ala-Val-Asn-Trp-Leu-Lys-Pro-Trp-Thr (abbreviated as: PEP-1);

[0036] Information of SEQ ID No.2

[0037] (a) Sequence characteristics

[0038] *Length: 12 amino acids

[0039] *Type: Amino acid

[0040] *Chain type: Single chain

[0041] (b) Molecular type: protein

[0042] Sequence description:

[0043] SEQ ID No. 2:

[0044] Ser-Ser-Phe-Phe-His-Leu-Arg-His-His-Ala-His-Leu (abbreviated as: PEP-2);

[0045] Information from SEQ ID No. 3

[0046] (a) Sequence characteristics

[0047] *Length: 12 amino acids

[0048] *Type: Amino acid

[0049] *Chain type: Single chain

[0050] (b) Molecular type: protein

[0051] Sequence description:

[0052] SEQ ID No. 3:

[0053] Gly-Leu-Ser-Arg-Tyr-Lys-Pro-Gly-Arg-Ser-Lys-Leu (abbreviated as: PEP-3);

[0054] Information from SEQ ID No. 4

[0055] (a) Sequence characteristics

[0056] *Length: 12 amino acids

[0057] *Type: Amino acid

[0058] *Chain type: Single chain

[0059] (b) Molecular type: protein

[0060] Sequence description:

[0061] SEQ ID No. 4: Trp-Arg-Ser-Ala-Gly-Glu-Ser-His-Phe-Gly-Leu-Thr (abbreviated as: PEP-4);

[0062] Information from SEQ ID No. 5

[0063] (a) Sequence characteristics

[0064] *Length: 12 amino acids

[0065] *Type: Amino acid

[0066] *Chain type: Single chain

[0067] (b) Molecular type: protein

[0068] Sequence description:

[0069] SEQ ID No. 5: Cys-Pro-Arg-Thr-Leu-Ala-Ser-Arg-Ser-Asn-Cys-Tyr (abbreviated as: PEP-5) The above 5 endotoxin affinity peptides were all synthesized by Nanjing Jietai Biotechnology Co., Ltd.

[0070] Example 1:

[0071] Synthesis of functional polymers targeting endotoxins using reversible addition-fragmentation chain transfer polymerization (FLTRP) Figure 1 )

[0072] (1) Synthesis of poly(PEGMEA-co-GMA): The polymerization method used was reversible addition-fragmentation chain transfer (RAFT) polymerization. The initiator used was azobisisobutyronitrile (AIBN, CAS No. 78-67-1), the chain transfer agent used was 4-cyano-4-(thiobenzoyl)valerate (CPPA, CAS No. 201611-92-9), and the monomers used were polyethylene glycol monomethyl ether acrylate (PEGMEA, CAS No. 32171-39-4, degree of polymerization: n=9, Sigma-Aldrich reagent) and glycidyl methacrylate (GMA, CAS No. 106-91-2, Shanghai Aladdin reagent). The specific synthesis steps are as follows: PEGMEA (4.8 g, 10 mmol), GMA (1.14 g, 10 mmol), AIBN (20.5 mg, 0.125 mmol), and CPPA (70 mg, 0.25 mmol) were dissolved in 100 mL of N,N-dimethylformamide (DMF). After three cycles of "freezing-vacuuming-nitrogen purging-thawing" (freezing temperature -80℃, time 20 min; thawing temperature at room temperature, time 20 min; vacuum degree 100 Pa; nitrogen purging pressure 100 kPa), the reaction was magnetically stirred at 65℃ for 24 h. The reactants were purified by dialyzing in ultrapure water for 3 days (dialysis bag molecular weight cutoff: 3500 Da). The solution in the dialysis bag was then freeze-dried under vacuum (vacuum degree: 100 Pa, temperature -40℃, time 48 h) to obtain 2.0 g of a light pink viscous liquid product, poly(PEGMEA-co-GMA). The successful synthesis of poly(PEGMEA-co-GMA) was confirmed by NMR, IR, and gel permeation chromatography (structure shown in [link to structure]). Figure 1, x=30~50y=30~50). Number average molecular weight=24920Da, polydispersity (PDI)=1.9. (2) Synthesis of poly(PEGMEA-co-PEP-1) by modification of endotoxin affinity peptide: Based on the epoxy ring-opening reaction between the epoxy group of GMA molecule in the poly(PEGMEA-co-GMA) polymer chain and the terminal amino group of endotoxin affinity peptide, endotoxin affinity peptide PEP-1 (all 5 peptides involved in this invention were synthesized by Nanjing Jietai Biotechnology Co., Ltd.) was modified onto poly(PEGMEA-co-GMA) polymer to obtain a functional polymer [poly(PEGMEA-co-PEP-1)] that can target endotoxin. The specific synthesis steps are as follows: PEP-1 (20mg) and poly(PEGMEA-co-GMA) (20mg) were dissolved in 2mL of ultrapure water and magnetically stirred at 40℃ for 48h. The reactants were purified by dialyzing in ultrapure water within a dialysis bag (dialysis bag molecular weight cutoff: 3500 Da). The solution inside the dialysis bag was then freeze-dried under vacuum (vacuum: 100 Pa, temperature: -40℃, time: 48 h) to obtain 25 mg of a light pink viscous liquid product, poly(PEGMEA-co-PEP-1). The successful synthesis of poly(PEGMEA-co-PEP-1) was confirmed by NMR and gel permeation chromatography (structure shown in [link to structure]). Figure 1 (x = 30–50, y = 30–50), two-dimensional NMR results show that the peptide binds to the poly(PEGMEA-co-GMA) polymer via an epoxy ring-opening reaction between the GMA unit in poly(PEGMEA-co-GMA) and the terminal amino group (histidine amino group) or amino side group (lysine amino side group) of the endotoxin-affinity peptide PEP-1 (the product linkage structure at the reaction site is -CH(OH)-NH-). Number average molecular weight = 31220 Da, PDI = 2.3.

[0073] Example 2:

[0074] Synthesis of functional polymers targeting endotoxins using conventional free radical random copolymerization Figure 2 )

[0075] (1) Synthesis of poly(PEGMEA-co-GMA): The polymerization method used was free radical random copolymerization. The initiator used was benzoyl peroxide (BPO, CAS No. 94-36-0, Shanghai Aladdin Reagent). The monomers used were polyethylene glycol monomethyl ether acrylate (PEGMEA, CAS No. 32171-39-4, degree of polymerization: n=9, Sigma-Aldrich Reagent) and glycidyl methacrylate (GMA, CAS No. 106-91-2, Shanghai Aladdin Reagent). The specific synthesis steps are as follows: PEGMEA (4.8g, 10mmol), GMA (1.14g, 10mmol), and BPO (1.06mg, 12.5μmol) were dissolved in 100mL of N,N-dimethylformamide (DMF) and reacted with magnetic stirring at 65℃ for 24h. The reactants were purified by dialyzing in ultrapure water for 3 days in a dialysis bag (dialysis bag molecular weight cutoff: 3500 Da). The solution in the dialysis bag was then freeze-dried under vacuum (vacuum: 100 Pa, temperature: -40℃, time: 48 h) to obtain 3.4 g of the viscous liquid product poly(PEGMEA-co-GMA). The successful synthesis of poly(PEGMEA-co-GMA) was confirmed by NMR, IR, and gel permeation chromatography (structure shown in [link to structure]). Figure 2 , x=300~400y=300~400). Number average molecular weight=204kDa, polydispersity (PDI)=2.9. (2) Synthesis of poly(PEGMEA-co-PEP-2) by modifying endotoxin affinity peptide: Based on the epoxy ring-opening reaction between the epoxy group of GMA molecule in the poly(PEGMEA-co-GMA) polymer chain and the terminal amino group of endotoxin affinity peptide, endotoxin affinity peptide PEP-2 (synthesized by Nanjing Jietai Biotechnology Co., Ltd.) was modified onto poly(PEGMEA-co-GMA) polymer to obtain a functional polymer [poly(PEGMEA-co-PEP-2)] that can target endotoxin. The specific synthesis steps are as follows: PEP-2 (20mg) and poly(PEGMEA-co-GMA) (20mg) were dissolved in 2mL of ultrapure water and magnetically stirred at 40℃ for 48h. The reactants were purified by dialyzing in ultrapure water within a dialysis bag (dialysis bag molecular weight cutoff: 3500 Da). The solution inside the dialysis bag was then freeze-dried under vacuum (vacuum: 100 Pa, temperature: -40℃, time: 48 h) to obtain 26 mg of the viscous liquid product poly(PEGMEA-co-PEP-2). The successful synthesis of poly(PEGMEA-co-PEP-2) was confirmed by NMR and gel permeation chromatography (structure shown in [link to structure]). Figure 2(x = 300–400, y = 300–400), 2D NMR results show that the peptide binds to the poly(PEGMEA-co-GMA) polymer via an epoxy ring-opening reaction between the GMA unit in poly(PEGMEA-co-GMA) and the terminal amino group (serine amino group) of the endotoxin-affinity peptide PEP-2 (the product linkage structure at the reaction site is -CH(OH)-NH-). Number average molecular weight = 271 kDa, PDI = 3.5.

[0076] Example 3

[0077] Preparation of SiO2@poly(PEGMEA-co-PEP-1) Figure 3 )

[0078] First, poly(PEGMEA-co-GMA) was grafted onto the surface of silica microspheres via reversible addition-fragmentation chain transfer polymerization. Then, the endotoxin-affinity peptide was modified onto the polymer through a ring-opening reaction between the epoxy groups of GMA and the terminal amino groups of the endotoxin-affinity peptide. The specific reaction steps are as follows:

[0079] (1) Preparation of SiO2@CTA: First, 4-cyano-4-(thiobenzoyl)valerate (CPPA, 100 mg, 0.35 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 333 mg, 1.61 mmol), and N-hydroxysuccinimide (NHS, 192 mg, 1.67 mmol) were dissolved in 35 mL of DMF. After stirring at 30 °C for 2 h, 300 mg of aminosilicone spheres (particle size: 5 μm, pore size: NH2SPS300-5 (FUJISILYSIA CHEMICAL LTD, Japan) was added to the above reaction solution, and the reaction was continued at 30°C for 48 h to obtain chain transfer agent modified silica microspheres (SiO2@CTA). The product was collected by centrifugation (8000 rpm, 10 min), washed successively with DMF and ultrapure water, and then lyophilized to obtain product SiO2@CTA, 280 mg.

[0080] (2) Preparation of SiO2@poly(PEGMEA-co-GMA): GMA (142 mg, 1 mmol), PEGMEA (480 mg, 1 mmol), and AIBN (0.41 mg, 2.5 μmol) were dissolved in 50 mL of DMF. 50 mg of SiO2@CTA was added, and the mixture underwent three cycles of "freezing-vacuuming-nitrogen purging-thawing" (freezing temperature -80℃, time 20 min; thawing temperature at room temperature, time 20 min; vacuum degree 100 Pa; nitrogen purging pressure 100 kPa). The mixture was then reacted at 65℃ for 24 h under magnetic stirring to obtain poly(PEGMEA-co-GMA) modified silica microspheres. The product was collected by centrifugation (8000 rpm, 10 min) and washed sequentially with DMF and ultrapure water. The product, SiO2@poly(PEGMEA-co-GMA), 48 mg, was then lyophilized.

[0081] (3) Preparation of SiO2@poly(PEGMEA-co-PEP-1): 20 mg SiO2@poly(PEGMEA-co-GMA) was dispersed in 5 mL of an aqueous solution of endotoxin-affinity peptide PEP-1 (1 mg / mL). The mixture was reacted at 40 °C for 48 h under magnetic stirring to obtain poly(PEGMEA-co-PEP-1) modified silica microspheres. The product was collected by centrifugation (8000 rpm, 10 min) and washed successively with DMF and ultrapure water. Then, the product SiO2@poly(PEGMEA-co-PEP-1), 19 mg, was obtained by lyophilization.

[0082] Example 4

[0083] Endotoxin removal effect of SiO2@poly(PEGMEA-co-PEP-1)

[0084] To investigate the endotoxin scavenging performance of the functional polymer, 2 mg of SiO2@poly(PEGMEA-co-PEP-1) microspheres (prepared according to Example 3, particle size: 5 μm) were added to 2 mL of Tris buffered saline solution (TBS, pH = 7.4) (concentration 0.1 μg / mL) containing E. coli endotoxin, and incubated with shaking at room temperature. After incubation for 0 h, 0.5 h, 1 h, and 1.5 h, 100 μL of the incubated sample was taken, centrifuged (8000 rpm, 10 min) to separate the microspheres, and 50 μL of the supernatant was collected. The endotoxin level in the solution was detected using a dynamic turbidimetric assay with Limulus amebocyte lysate (LAL) reagent (KT-125, Zhanjiang Andus Biotechnology Co., Ltd.) according to the product instructions. All experiments used pyrogen-free vials and endotoxin detection water (Zhanjiang Andus Biotechnology Co., Ltd.). The test results are as follows: Figure 4As shown, this result indicates that SiO2@poly(PEGMEA-co-PEP-1) can rapidly remove more than 99% of endotoxins from the solution within 0.5 h, demonstrating good endotoxin removal ability.

[0085] To further investigate the adsorption specificity and anti-interference performance of SiO2@poly(PEGMEA-co-PEP-1) for endotoxin, we also tested the material's endotoxin scavenging effect in protein solutions. The specific experimental steps are as follows: Human serum albumin (HSA) was added to a TBS solution (Tris Buffered Saline, pH = 7.4) containing 0.1 μg / mL of E. coli endotoxin at a mass ratio of 1:1, 1:10, or 1:100 to obtain endotoxin / HSA mixed solutions. Then, 2 mg of SiO2@poly(PEGMEA-co-PEP-1) microspheres were added to 2 mL of each of these endotoxin / HSA mixed solutions, and the solutions were incubated with shaking at room temperature. After 3 hours of incubation, the endotoxin level in the solution was detected using an endotoxin detector according to the product instructions and the dynamic turbidimetric method with Limulus Amebocyte Lysate (LAL) reagent (KT-125, Zhanjiang Andus Biotechnology Co., Ltd.). The above experiments used pyrogen-free vials and endotoxin testing water (Zhanjiang Andus Biotechnology Co., Ltd.) throughout the entire process. The test results are as follows: Figure 5 As shown, this result indicates that SiO2@poly(PEGMEA-co-PEP-1) microspheres can still maintain about 80% of their endotoxin clearance capacity even under HSA protein interference at a ratio of 1:1000.

[0086] Example 5

[0087] Quartz crystal dissipative microbalance (QCM-D) adsorption experiment

[0088] First, a poly(PEGMEA-co-PEP) modified quartz crystal microbalance (QCM) chip was prepared: (i) a QCM chip (frequency: 4.95MHz±50kHz, diameter: 14mm, RQ5MTAP, Shenzhen Renlu Crystal Co., Ltd.) was soaked in 2mL of 1mM ethanol solution of β-mercaptoethylamine for 12h to obtain a β-mercaptoethylamine modified QCM chip. (ii) 4-Cyano-4-(thiobenzoyl)valerate (CPPA, 100 mg, 0.35 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 333 mg, 1.61 mmol) and N-hydroxysuccinimide (NHS, 192 mg, 1.67 mmol) were dissolved in 35 mL of DMF. After stirring at 30 °C for 2 h, a QCM chip modified with β-mercaptoethylamine was added to 2 mL of the above reaction solution. The reaction was continued at 30 °C for 48 h. Then, the chip was washed three times with DMF and ultrapure water to obtain the QCM chip modified with chain transfer agent. (iii) GMA (142 mg, 1 mmol), PEGMEA (480 mg, 1 mmol), and AIBN (0.41 mg, 2.5 μmol) were dissolved in 50 mL of DMF, and the mixture was subjected to three cycles of "freezing-vacuuming-nitrogen purging-thawing" (freezing temperature -80 °C, time 20 min; thawing temperature at room temperature, time 20 min; vacuum degree 100 Pa; nitrogen purging pressure 100 kPa) to obtain the reaction solution. Subsequently, a QCM chip modified with chain transfer agent (4-cyano-4-(thiobenzoyl)valerate) was added to 2 mL of the above reaction solution and reacted at 65 °C for 24 h. Then, it was washed three times with DMF and ultrapure water to obtain a poly(PEGMEA-co-GMA) modified QCM chip. (iv) A single poly(PEGMEA-co-GMA) modified QCM chip was added to 5 mL of an aqueous solution of endotoxin affinity peptide PEP-1 (synthesized by Nanjing Jietai Biotechnology Co., Ltd.) (1 mg / mL), and reacted at 40 °C for 48 h. The chip was then washed with DMF and ultrapure water to obtain a poly(PEGMEA-co-PEP-1) modified QCM chip.

[0089] QCM-D testing was performed at 20°C using a Q-Sense E4 system (Biolin Scientific). First, the QCM channels and tubing were thoroughly rinsed with ultrapure water and dried with nitrogen. Then, the poly(PEGMEA-co-PEP-1) modified QCM chip was loaded into the test cell. After rinsing with ultrapure water and TBS until the baseline signal stabilized, a TBS solution (5 mg / mL, pH 7.4) containing endotoxin (LPS), human serum albumin (HSA), immunoglobulin G (IgG), or transferrin (TRF) was pumped in at a rate of 100 L / min. The frequency and dissipation changes over time were recorded using Q-Sense software. Figure 6 The adsorption amount was calculated using the Sauerbrey equation. Figure 7 ):

[0090]

[0091] Δm: Chip mass change; Δf: Oscillation frequency change; C: Constant, for a 5MHz crystal, C = 17.7 ng / (cm³) 2 ·Hz); n: odd multiple of frequency. For the gold chip used in this experiment, n=3.

[0092] like Figures 6-7 As shown, after the endotoxin solution was pumped in, a significant decrease in chip frequency was observed (Δf = -23.6 Hz), indicating that endotoxin binds to the surface of the poly(PEGMEA-co-PEP) polymer. Conversely, no significant change in chip frequency was observed when HSA, IgG, or TRF solutions were pumped in, indicating that the poly(PEGMEA-co-PEP) polymer has good selectivity for endotoxin. According to the Sauerbrey equation, the adsorption capacity of endotoxin on the polymer surface is 139.1 ng / cm³. 2 The adsorption capacity was significantly higher than that of plasma proteins (HAS: 13.7 ng / cm³). 2 IgG: <1 ng / cm 2 TRF: 44.0 ng / cm 2 The experimental results indicate that poly(PEGMEA-co-PEP) can specifically bind to E. coli endotoxins and has good anti-pollution properties against plasma proteins.

[0093] Example 6

[0094] 10 mg of the SiO2@poly(PEGMEA-co-PEP-1) microspheres prepared in Example 3 were added to 2 mL of fresh whole blood (from healthy male volunteers aged 25-30 years) containing 0.1 mg / mL endotoxin, and incubated with shaking at 37°C for 3 h. After incubation for 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 3 h, respectively, the endotoxin level in the blood was monitored using a dynamic turbidimetric method with Limulus amebocyte lysate (LAL) reagent (KT-125, Zhanjiang Andus Biotechnology Co., Ltd.), according to the product instructions. Pyrogen-free vials and endotoxin detection water (Zhanjiang Andus Biotechnology Co., Ltd.) were used throughout the detection process. Figure 8 As shown, after co-incubation with SiO2@poly(PEGMEA-co-PEP-1) microspheres for 3 hours, the endotoxin concentration in the blood decreased from 149.1±14.0 EU / mL to 35.3±3.4 EU / mL, indicating that SiO2@poly(PEGMEA-co-PEP-1) microspheres still exhibit good endotoxin clearance ability in the blood and can effectively remove endotoxins from whole blood.

[0095] Example 7

[0096] Preparation of PES / poly(PEGMEA-co-PEP-1) microspheres: (i) Preparation of PES / poly(PEGMEA-co-PEP-1) droplet stock solution: Polyethersulfone (PES, weight average molecular weight 51kDa, Ultrason E6020P, BASF Europe), poly(PEGMEA-co-PEP-1) prepared in Example 1, and dimethylacetamide (DMF) were mixed at a mass ratio of 12:6:88. After complete dissolution by stirring, the mixture was allowed to stand for 48 hours at 4°C to obtain the PES / poly(PEGMEA-co-PEP-1) droplet stock solution; (ii) Preparation of PES / poly(PEGMEA-co-PEP-1) microspheres: The microspheres were extruded through the inner hole of a needle using a metering pump (needle type: 26G, needle diameter: 0.45mm, Zhejiang). Jiangoujian Medical Equipment Co., Ltd.), the droplet rate of PES / poly(PEGMEA-co-PEP-1) droplet stock solution is 30-40 drops / min; the initial droplets are dropped vertically into a water / DMF mixed bath (volume ratio 1:1) after traveling 15cm in the air to solidify and form PES / poly(PEGMEA-co-PEP-1) microspheres; (iii) Post-treatment of PES / poly(PEGMEA-co-PEP-1) microspheres: microspheres (particle size 1-3mm) are soaked in ultrapure water at 4℃ for 72h to remove residual solvent.

[0097] Example 8

[0098] Animal experiment on blood perfusion with PES / poly(PEGMEA-co-PEP-1) microspheres (i) 10 mL of PES / poly(PEGMEA-co-PEP-1) microspheres from Example 7 were loaded into a perfusion apparatus (HC-0120-01, Beijing Ruida Heng Hui Technology Development Co., Ltd.), and the apparatus and tubing were pre-flushed with 125 IU / mL heparinized saline. Subsequently, a perfusion system was established in a sepsis animal model anesthetized with 1.5% sodium pentobarbital (1.5 mL / kg) (sepsis modeling: male New Zealand rabbits, injected with E. coli endotoxin via the marginal ear vein at a dose of 500 μg / kg, and blood perfusion experiment performed 1 h after injection) by cannulating the left femoral artery-left femoral vein. (Blood was drawn from or introduced into the blood vessel using a disposable blood collection needle, which was connected to the perfusion apparatus via a disposable medical tubing). Perfusion rate: 5 mL / min; perfusion time: 2 h. During the perfusion process, 1 mL of blood was collected from the left femoral artery every 0.5 hours. After centrifugation at 3500 rpm for 15 minutes, the supernatant plasma was collected. The endotoxin level in the blood was measured using a dynamic turbidimetric assay with Limulus amebocyte lysate (LAL) reagent (KT-125, Zhanjiang Andus Biotechnology Co., Ltd.), according to the product instructions. Figure 9 As shown, after 2 hours of perfusion using a PES / poly(PEGMEA-co-PEP-1) microsphere perfusion column, the endotoxin level in the blood decreased from 2.63±0.01 EU / mL to 0.78±0.05 EU / mL, with an endotoxin clearance rate exceeding 70%. Compared with the empty perfusion column control group (blood perfusion performed using a perfusion device without adsorbent material) and the PES microsphere perfusion group (blood perfusion performed using a perfusion device filled with PES microspheres obtained in Example 7), the PES / poly(PEGMEA-co-PEP-1) microsphere perfusion column exhibited better endotoxin clearance efficiency. These results indicate that the PES / poly(PEGMEA-co-PEP-1) microsphere perfusion column possesses excellent blood purification capabilities, effectively reducing endotoxin levels in the blood and playing a positive role in the treatment of sepsis.

[0099] Matters not covered in this invention are common knowledge.

[0100] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A functional polymer targeting endotoxins, characterized in that: The endotoxin-targeting functional polymer consists of an antifouling unit, an endotoxin-binding unit, and a coupling unit for connecting the endotoxin-binding unit, as shown in the following molecular structure diagram: The antifouling unit is derived from polyethylene glycol monomethyl ether acrylate (PEGMEA), where x is an integer from 10 to 10000 and n is an integer from 1 to 50; the endotoxin-binding unit is an endotoxin-affinity polypeptide (PEP); the coupling unit for connecting the endotoxin-binding unit is derived from glycidyl methacrylate (GMA), which reacts with one or more terminal amino groups or amino side groups of the endotoxin-affinity polypeptide through an epoxy ring-opening reaction, where y is an integer from 10 to 10000, PEGMEA and GMA are random copolymers, and the xy part is randomly linked. The polymer chain ends are composed of one or more initiators or chain transfer agents among the residues after participating in the polymerization reaction. ； The endotoxin-affinity polypeptide has one or more amino acid sequences listed in SEQ ID NO: 1-5; its amino acid sequence is one or more of the following: His-Trp-Lys-Ala-Val-Asn-Trp-Leu-Lys-Pro-Trp-Thr (PEP-1), Ser-Ser-Phe-Phe-His-Leu-Arg-His-His-Ala-His-Leu (PEP-2), Gly-Leu-Ser-Arg-Tyr-Lys-Pro-Gly-Arg-Ser-Lys-Leu (PEP-3), Trp-Arg-Ser-Ala-Gly-Glu-Ser-His-Phe-Gly-Leu-Thr (PEP-4) or Cys-Pro-Arg-Thr-Leu-Ala-Ser-Arg-Ser-Asn-Cys-Tyr (PEP-5).

2. The functional polymer targeting endotoxins according to claim 1, characterized in that: x is an integer from 10 to 1000, n is an integer from 5 to 15, and y is an integer from 10 to 1000.

3. A method for preparing the functional polymer targeting endotoxin according to claim 1, characterized in that: The specific process is as follows: (i) Poly(polyethylene glycol monomethyl ether acrylate) co Glycidyl methacrylate) [poly(PEGMEA- co Synthesis: Initiator, chain transfer agent, and / or chain transfer agent modified onto the surface of a solid material, polyethylene glycol monomethyl ether acrylate, and glycidyl methacrylate were dissolved in a reaction solvent at a molar ratio of 1:(0~2.5):(10~10000):(10~10000) via free radical polymerization. After 3~5 cycles of freezing, vacuuming, nitrogen purging, and thawing, the reaction mixture was magnetically stirred at 50~80℃ for 12~48 h. The reactants were purified by dialysis and then freeze-dried under vacuum to obtain a light pink viscous liquid product, poly(PEGMEA- co -GMA); (ii) Poly(polyethylene glycol monomethyl ether acrylate) co - Endotoxin affinity peptide) [poly(PEGMEA- co Synthesis: This involves combining one or more endotoxin-affinity peptides with poly(PEGMEA-)[…]. co Poly(PEGMEA-GMA) was dissolved in water at a mass ratio of 1:1 to 1:20, and 20 mg of endotoxin-affinity peptide was dissolved in 1-20 mL of water. The mixture was magnetically stirred at 30-45 °C for 12-48 h. After dialysis purification, the reactants were freeze-dried under vacuum to obtain a light pink viscous liquid product, poly(PEGMEA-GMA). co -PEP).

4. The preparation method according to claim 3, characterized in that: The initiator is one or more of azobisisobutyronitrile, benzoyl peroxide, or potassium persulfate. The chain transfer agent is one or more of 4-cyano-4-(thiobenzoyl)valerate, 2-mercapto-S-thiobenzoylacetic acid or 2-phenylpropane thiobenzoic acid ester. The molar ratio of initiator, chain transfer agent, polyethylene glycol monomethyl ether acrylate, and glycidyl methacrylate is 1:(1~2.5):(100~10000):(100~10000). The monomer solid content is 1~10 wt%, the initiator and chain transfer agent solid content is 0.001~0.1 wt%, the vacuum degree is 0.1~10 Pa, and the nitrogen pressure is 0~1 MPa.

5. The use of the functional polymer for targeting endotoxins as described in claim 1 or 2 in the preparation of endotoxin separation or analytical materials.

6. An endotoxin separation material, characterized in that: It consists of a carrier and a functional polymer targeting endotoxins as described in any one of claims 1-2; wherein the carrier is polyethersulfone microspheres.

7. A method for preparing the endotoxin separation material according to claim 6, characterized in that: This method includes two steps: preparation of the droplet stock solution and preparation of microspheres using a liquid-liquid phase separation method. Step 1: Preparation of droplet stock solution First, combine polyethersulfone (PES), solvent, and the poly(PEGMEA-) as described in any one of claims 1-2. co After mixing thoroughly, poly(PEGMEA-PEP) is obtained. co -PEP) modified polyethersulfone solution, after being left to mature, is used to obtain modified polyethersulfone droplet stock solution; Step 2: Preparation of modified polyethersulfone microspheres via liquid-liquid phase separation method The original solution for the droplets is extruded through the inner hole of a needle using a metering pump. The initial droplets travel a distance of 5-30 cm in the air before being dropped into a coagulation bath to form modified polyethersulfone microspheres. The coagulation bath is water, or a mixture of water and one or more organic solvents selected from DMF, DMAc, or DMSO at a volume ratio of 1:0.5 to 1:1.

5. Subsequently, the modified polyethersulfone microspheres are soaked in water at a temperature of 2-10℃ for 24-72 h to remove residual solvent.

8. The method for preparing the endotoxin separation material according to claim 7, characterized in that: The modified polyethersulfone microspheres were soaked in water at a temperature of 2-5℃ for 48-72 h.

9. The method for preparing the endotoxin separation material according to claim 8, characterized in that: The solvent is N,N-dimethylacetamide, DMAc; Polyethersulfone has the structure shown below, with a molecular weight of 10,000~100,000 and a mass concentration of 8~20 wt% in solvent; poly(PEGMEA- co -PEP) at a mass concentration of 4~8 wt% in solvent; ； The maturation conditions are 2~10℃ and standing for 24~72 h.

10. The method for preparing the endotoxin separation material according to claim 9, characterized in that: Polyethersulfone molecular weight: 50,000~100,000; polyethersulfone mass concentration in solvent: 10~15 wt%; poly(PEGMEA- co -PEP) at a mass concentration of 6~8 wt% in solvent; The maturation conditions are 2~5℃ and standing for 48~72 h.

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