Modified materials that reduce unintended activation of platelets and methods of making and using the same

Abstract

The application discloses modified materials capable of reducing unexpected activation of platelets and a preparation method and application thereof, and belongs to the technical field of high polymer modified materials. The application solves the problem of easy adhesion, deformation, aggregation and unexpected activation of platelets during preparation of PRP. The application comprises a zwitterionic polymer modified by a reactive functional group, which is obtained by substitution and polymerization of a zwitterionic monomer through a reactive functional group, the zwitterionic monomer is one or more of a phosphorylcholine monomer, a sulfobetaine monomer and a carboxybetaine monomer, and the reactive functional group is one or more of an isocyanate group, an epoxy group, a silane group and a polymerizable double bond; the substitution degree of the reactive functional group is 5% to 20%, the weight average molecular weight is 50,000 to 300,000, and the elastic modulus is 0.13 to 0.35 MPa. The application can reduce protein-mediated platelet adhesion and contact activation, weaken mechanical stimulation of a high-rigidity tube wall on platelets, and reduce unexpected activation of platelets.

Description

Technical Field

[0001] This invention belongs to the field of polymer modified materials technology, specifically relating to modified materials that can reduce unintended platelet activation, their preparation methods, and applications. Background Technology

[0002] Platelet-rich plasma (PRP) is a plasma preparation obtained by centrifuging autologous whole blood, with a platelet concentration significantly higher than physiological levels (typically 3–6 times that of whole blood). It is widely used in orthopedics, sports medicine, dermatology, and wound repair. Its therapeutic effect primarily relies on the release of various growth factors after platelet activation, including platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF). These factors synergistically promote cell proliferation, angiogenesis, and tissue regeneration. However, the clinical efficacy of PRP is highly dependent on the maintenance of platelet activity during collection, storage, and processing, and unintended activation is the core bottleneck leading to decreased therapeutic efficacy.

[0003] Platelet-rich plasma (PRP) preparation utilizes disposable vacuum blood collection tubes, with centrifugation performed under transfer-free, fully enclosed conditions. Currently, most mainstream blood collection tubes use polyethylene terephthalate (PET) plastic as the tube material, and their inner walls are generally siliconized to reduce surface energy and decrease blood cell adhesion. Although this process improves centrifugation efficiency, the inherent moderate surface energy and lipophilicity of PET material still easily induce non-specific adsorption of plasma proteins (especially fibrinogen). According to the blood-material interface interaction mechanism, adsorbed fibrinogen undergoes conformational changes on hydrophobic or polarity-mismatched surfaces, triggering platelet adhesion, activation, and degranulation. This process occurs even without exogenous activators (such as thrombin or calcium chloride), and is termed "unintended activation." This phenomenon leads to premature release of growth factors in PRP before application, premature formation of the fibrin network, ultimately reducing the effective therapeutic concentration and increasing the risk of local inflammation. Meanwhile, existing conventional PRP blood collection tubes are mostly made of polymer materials, and the surface of the tube wall usually has a high elastic modulus and strong rigidity, which makes it difficult to simulate the soft hydration microenvironment of the extracellular matrix or blood contact interface. Under the action of centrifugal force, they are prone to mechanical stimulation of platelets, promoting their adhesion, deformation and non-specific activation.

[0004] To suppress this unintended activation, existing technologies attempt to introduce functional coatings into the inner wall of the container, mainly including:

[0005] Heparin coating: Heparin molecules are fixed by covalent or physical adsorption to enhance the activity of anticoagulant factors. However, it is easy to fall off during long-term storage or repeated centrifugation, and it cannot block the direct adhesion pathway of platelets. Systemic anticoagulants are still required, which increases the risk of bleeding.

[0006] Polyethylene glycol (PEG) coating: Utilizing the high hydrophilicity and anti-protein adsorption properties of PEG to form a hydration layer, reducing plasma protein adsorption, but it is easily competitively replaced by serum proteins in the blood environment, has poor stability, and is prone to failure under mechanical shear force.

[0007] In summary, existing technologies for improving the surface compatibility of blood contact materials mainly focus on reducing protein adsorption and cell adhesion by constructing hydrophilic antifouling interfaces, low-fouling interfaces, or other biocompatible coatings. However, the application scenario of blood collection tubes has certain unique characteristics: after blood collection, they are simultaneously affected by centrifugal force, blood component sedimentation, tube wall collision, and interfacial shear during a short centrifugation process. Platelets are more prone to adhesion, deformation, aggregation, and undesirable activation on the surface of high-rigidity, hydrophobic polymer tubes. Therefore, conventional blood compatibility modification strategies are difficult to effectively regulate the dynamic interfacial stimulation during PRP preparation. Summary of the Invention

[0008] To address the problem that platelets tend to adhere, deform, aggregate, and undergo unintended activation on the surface of high-rigidity, hydrophobic polymer tubes during the preparation of PRP using existing technologies, this invention provides modified materials that can reduce unintended platelet activation, their preparation methods, and applications.

[0009] The technical solution adopted in this invention is as follows:

[0010] A modified material that can reduce unintended platelet activation is a zwitterionic polymer modified with reactive functional groups. The zwitterionic polymer is obtained by substitution and polymerization of zwitterionic monomers through reactive functional group substitution. The zwitterionic monomers are one or more of phosphorylcholine monomers, sulfobetaine monomers, and carboxybetaine monomers. The reactive functional groups are introduced through reactive monomers, and the reactive monomers are one or more of glycidyl methacrylate, 2-isocyanate ethyl methacrylate, 3-methacryloyloxypropyltrimethoxysilane, and methacrylic acid.

[0011] The degree of substitution of reactive functional groups in zwitterionic polymers modified with reactive functional groups is 5%–20%;

[0012] The weight-average molecular weight of the zwitterionic polymer modified with reactive functional groups is 50,000 to 300,000, and the elastic modulus is 0.13 to 0.35 MPa.

[0013] After adopting this technical solution, conventional blood collection tubes typically have high elastic modulus and strong rigidity, which can easily cause abnormal mechanical stimulation to platelets during centrifugation, further promoting platelet deformation, adhesion, and undesirable activation. PRP contains a large number of platelets and plasma proteins. During centrifugation, platelets easily come into contact with the tube wall, and plasma proteins are also easily adsorbed on the tube wall and undergo conformational changes, further inducing platelet adhesion, spreading, and activation. To solve this problem, this invention uses reactive functional groups to undergo a partial substitution reaction with zwitterionic monomers, while the zwitterionic monomers undergo a polymerization reaction, ultimately obtaining zwitterionic polymers modified with reactive functional groups. The zwitterionic polymers modified with reactive functional groups can form a cell membrane-inspired zwitterionic interface on the inner wall of PRP blood collection tubes. Zwitterionic polymers contain both positive and negative charged groups, and are a cell membrane-inspired polymer that can form a highly hydrated polymer layer on the tube wall surface, reducing the effective rigidity and interfacial friction of the tube wall surface, and constructing a low-modulus hydrated microenvironment that is closer to the physiological soft interface. This hydrated zwitterionic interface reduces the non-specific adsorption of plasma proteins such as fibrinogen on the tube wall surface, decreases protein-mediated platelet adhesion and contact activation, and weakens the mechanical stimulation of platelets by the high-rigidity tube wall, thereby helping to maintain the activity and functional integrity of platelets in PRP. After introducing reactive functional groups, the polymer can form covalent bonds with the surface of the blood collection tube, improving the stability of the coating during centrifugation, washing, and storage. Because the zwitterionic polymer modified with reactive functional groups can fully bond to the inner wall of the PRP blood collection tube, the elastic modulus of the inner wall of the PRP blood collection tube reaches a suitable range, effectively improving the modification effect.

[0014] The degree of substitution of reactive functional groups in zwitterionic polymers modified with reactive functional groups is 5%–20%. If the substitution is too low, there are insufficient reaction sites between the polymer and the tube wall, resulting in weak coating adhesion and easy detachment during centrifugation or washing, thus affecting the modification effect. If the substitution is too high, excessive reactive groups will disrupt the original charge balance structure of the zwitterionic polymer, reducing its ability to inhibit plasma protein adsorption and platelet adhesion; it may also cause excessive cross-linking, coating hardening, or an increase in residual active groups, thereby increasing the risk of platelet activation.

[0015] The zwitterionic polymer modified with reactive functional groups has a weight-average molecular weight of 50,000 to 300,000 and an elastic modulus of 0.13 to 0.35 MPa. More preferably, the elastic modulus is 0.15 to 0.35 MPa. The preferred weight-average molecular weight of the zwitterionic polymer modified with reactive functional groups is 50,000 to 300,000, which balances film-forming properties, coating uniformity, and interfacial stability. If the molecular weight is too low, the polymer chain length is insufficient, making it difficult to form a continuous and stable modified layer, resulting in poor erosion and centrifugal stability. If the molecular weight is too high, the polymer solution viscosity is high, which is not conducive to uniform coating on the inner wall of the blood collection tube and may also reduce the contact efficiency between reactive groups and surface groups on the tube wall, leading to uneven coating or decreased bonding efficiency. The surface modulus of the modified PRP blood collection tube inner wall is 0.13 to 0.35 MPa. In comparison, the elastic modulus of conventional polycarbonate materials is typically about 2.0 to 2.5 GPa, and the elastic modulus of glass materials is typically about 60 to 75 GPa. Therefore, the modified inner wall surface formed by this invention has a significantly lower modulus than conventional rigid tube wall materials such as polycarbonate or glass, and more closely resembles the low-modulus mechanical characteristics of the human blood vessel wall and blood contact interface. This allows for the formation of a relatively soft, biomimetic contact microenvironment on the inner wall of the PRP blood collection tube. This low-modulus surface helps to reduce the abnormal mechanical stimulation of platelets by the high-rigidity tube wall during centrifugation, and reduces unintended activation of platelets caused by contact, collision, adhesion, and spreading, thereby helping to maintain the activity and functional integrity of platelets in PRP.

[0016] Preferably, the reactive functional group modified zwitterionic polymer is one or more of poly(2-methacryloyloxyethyl phosphorylcholine), polysulfobetaine methacrylate, polycarboxybetaine methacrylate, and polytrimethylamine oxide derivative type zwitterionic polymers.

[0017] Preferably, the zwitterionic polymer modified with reactive functional groups is obtained by reactive functional group substitution and polymerization of epoxy group-containing monomers and phosphorylcholine monomers. The epoxy group-containing monomers are glycidyl methacrylate or glycidyl acrylate, and the phosphorylcholine monomers are 2-methacryloyloxyethyl phosphorylcholine.

[0018] A method for preparing a modified material that can reduce unintended platelet activation includes the following steps:

[0019] Step 1: Mix the zwitterionic monomer, reactive monomer, and solvent until the zwitterionic monomer and reactive monomer dissolve to obtain a mixed system;

[0020] Step 2: Add an initiator to the resulting mixture and react at 50–80°C for 10–15 h to obtain a reaction solution;

[0021] Step 3: Add the reaction solution to acetone to precipitate a solid, thus obtaining a modified material that can reduce unintended platelet activation.

[0022] Preferably, the initiator is one of ammonium persulfate and azobisisobutyronitrile.

[0023] Preferably, the molar ratio of reactive monomer to zwitterionic monomer in step 1 is 1:4.

[0024] The application of a modified material that reduces unintended platelet activation, used to form a modified coating on the contact surface between a container holding platelet-rich plasma and the platelet-rich plasma.

[0025] Preferably, the container for holding platelet-rich plasma is a blood collection tube, and the specific steps for forming a modified coating on the inner wall of the blood collection tube using a modified material that can reduce unintended platelet activation include:

[0026] S1: The inner wall of the blood collection tube is activated, and the activation treatment is one or more of oxygen plasma activation treatment, ultraviolet / ozone treatment and chemical oxidation treatment;

[0027] S2: Prepare a modified solution by modifying the zwitterionic polymer with reactive functional groups, coat the inner wall of the blood collection tube with the modified solution or immerse the blood collection tube in the modified solution, so that the zwitterionic polymer modified with reactive functional groups forms a connection with the blood collection tube, and obtain a modified coating.

[0028] Preferably, after the activation treatment of S1, the process further includes initial modification with a surface modifier, followed by S2. The surface modifier is a silane coupling agent with an amino group at the end, a polymerizable double bond at the end, or a thiol group at the end.

[0029] Furthermore, the surface modifier is one or more of the following: aminosilane coupling agent, epoxysilane coupling agent, mercaptosilane coupling agent, and silane coupling agent with polymeric double bonds.

[0030] By adopting this technical solution, the surface modifier can introduce active sites such as amino, epoxy, mercapto, hydroxyl or polymerizable double bonds on the surface of the blood collection tube, so that the reactive zwitterionic polymer can better form a covalent bond with the tube wall.

[0031] Preferably, zwitterionic polymers modified with reactive functional groups are applied to blood collection tubes via surface grafting polymerization, dip coating curing, self-assembly, or composite deposition.

[0032] Preferably, the zwitterionic polymer modified with reactive functional groups is constructed in situ as a polymer molecular brush layer or a surface graft polymer layer through surface graft polymerization.

[0033] Preferably, the surface grafting polymerization is one or more of the following: surface-initiated free radical polymerization, surface-initiated atom transfer radical polymerization, surface-initiated reversible addition-fragmentation chain transfer polymerization, plasma-induced grafting polymerization, or photo-induced grafting polymerization.

[0034] Preferably, the blood collection tube is made of a polymer substrate or a glass substrate.

[0035] Preferably, the polymer substrate is selected from one or more of polypropylene, polycarbonate and polyethylene terephthalate; the glass substrate is selected from one or more of borosilicate glass, soda-lime glass and quartz glass.

[0036] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0037] In this invention, reactive monomers are introduced into the polymer backbone through polymerizable double bonds, while reactive groups are retained on the side chains. These reactive groups can covalently react with amino, hydroxyl, thiol, or other nucleophilic groups on the surface of the surface-modified blood collection tube, thereby enhancing the bonding stability of the zwitterionic polymer on the blood collection tube surface. The zwitterionic polymer modified with reactive functional groups can form a cell membrane-inspired zwitterionic interface on the inner wall of the PRP blood collection tube. The zwitterionic polymer contains both positively and negatively charged groups, making it a cell membrane-inspired polymer that can form a highly hydrated polymer layer on the tube wall surface, reducing the effective rigidity and interfacial friction of the tube wall surface and constructing a low-modulus hydrated microenvironment that more closely resembles a physiological soft interface. This hydrated zwitterionic interface can reduce the non-specific adsorption of plasma proteins such as fibrinogen on the tube wall surface, reduce protein-mediated platelet adhesion and contact activation, and weaken the mechanical stimulation of platelets by the high-rigidity tube wall, thus helping to maintain the activity and functional integrity of platelets in PRP. After introducing reactive functional groups, the polymer can form covalent bonds with the surface of the blood collection tube, improving the stability of the coating during centrifugation, cleaning, and storage. Since the zwitterionic polymer modified by the reactive functional groups can be fully bonded to the inner wall of the PRP blood collection tube, the elastic modulus of the inner wall of the PRP blood collection tube reaches a suitable range, effectively improving the modification effect. Attached Figure Description

[0038] Figure 1 The images show physical images of the zwitterionic polymers modified with reactive functional groups obtained in Examples 1-6.

[0039] Figure 2 This is a photograph of the modified control tube 1.

[0040] Figure 3 This is a photograph of the modified control tube 2.

[0041] Figure 4This is a diagram showing the inner wall of the modified control tube 2. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with the description in the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0043] Example 1

[0044] Preparation of modified materials that can reduce unintended platelet activation:

[0045] Step 1: Take 0.12g GMA (glycidyl methacrylate) and 1g MPC (2-methacryloyloxyethyl phosphorylcholine) and mix them to make the molar ratio of GMA to MPC 1:4. Add the mixture to a mixed solvent of 2 mL methanol and 6 mL deionized water and stir until dissolved to obtain a mixed system.

[0046] Step 2: Add 0.018 g APS (ammonium persulfate) to the mixture and stir continuously at 60°C for 12 h to obtain the reaction solution;

[0047] Step 3: Slowly add the reaction solution dropwise to 100 mL of anhydrous acetone to precipitate a solid. Collect the precipitate by centrifugation or filtration, wash the solid product twice with deionized water, add 5 mL of deionized water to allow it to swell, and then freeze at -20°C for 12 hours. After freezing, grind to obtain a modified material powder that can reduce unintended platelet activation (the modified material powder that can reduce unintended platelet activation obtained in this example is as follows). Figure 1 (As shown).

[0048] Application of modified materials that can reduce unintended platelet activation:

[0049] Modifying blood collection tubes with materials that reduce unintended platelet activation involves the following steps:

[0050] S1: Take blood collection tubes (PRP BP-I from Zhejiang Paulman Biotechnology Co., Ltd.), and wash them three times each with deionized water and ethanol to remove surface contaminants. Then, dry them in a 60℃ oven for 10 min for later use. Place the pretreated blood collection tubes in an oxygen plasma cleaner under the following conditions: 70 W, 30 sccm O2, 50 Pa, for 5 min, to introduce active groups on their surface. Then, prepare a silane coupling agent solution: mix 1 mL of aminopropyltriethoxysilane, 50 mL of ethanol, and 1 mL of deionized water evenly. Immerse the blood collection tubes treated with oxygen plasma in the silane coupling agent solution and react at 60℃ for 2 h. After the reaction, wash them three times each with deionized water and ethanol, and dry them in a 60℃ oven for 20 min to obtain activated blood collection tubes.

[0051] S2: Weigh 1 g of the modified material that can reduce unintended platelet activation, add it to 50 mL of deionized water, stir to dissolve, and then add triethylamine to adjust the pH to 8.5 to prepare a modified solution with a mass fraction of 2%. Immerse the blood collection tubes obtained in S1 in the solution and react at 60°C for 3 h to fix the zwitterionic polymer modified with reactive functional groups on the surface of the blood collection tubes. After the reaction, wash the tubes three times each with deionized water and ethanol, and dry them at 60°C for 10 min to obtain blood collection tubes with a modified material coating that can reduce unintended platelet activation on the surface.

[0052] Example 2

[0053] Preparation of modified materials that can reduce unintended platelet activation:

[0054] Step 1: Take 0.12g GMA (glycidyl methacrylate) and 1g SBMA (sulfobetaine methacrylate) and mix them to make the molar ratio of GMA to SBMA 1:4. Add the mixture to a mixed solvent of 2 mL methanol and 6 mL deionized water and stir until dissolved to obtain a mixed system.

[0055] Step 2: Add 0.018 g APS (ammonium persulfate) to the mixture and stir continuously at 60°C for 12 h to obtain the reaction solution;

[0056] Step 3: Slowly add the reaction solution dropwise to 100 mL of anhydrous acetone to precipitate a solid. Collect the precipitate by centrifugation or filtration, wash the solid product twice with deionized water, add 5 mL of deionized water to allow it to swell, and then freeze at -20°C for 12 hours. After freezing, grind to obtain a modified material powder that can reduce unintended platelet activation (the modified material powder that can reduce unintended platelet activation obtained in this example is as follows). Figure 1 (As shown).

[0057] Application of modified materials that can reduce unintended platelet activation:

[0058] S1: Same as S1 in Example 1;

[0059] S2: Weigh 1 g of the modified material powder that can reduce unintended platelet activation, add it to 50 mL of deionized water, stir to dissolve, and then add triethylamine to adjust the pH to 8.5 to prepare a modified solution with a mass fraction of 2%. Immerse the blood collection tubes obtained in S1 in the solution and react at 60°C for 3 h to fix the zwitterionic polymer modified by reactive functional groups on the surface of the blood collection tubes. After the reaction, wash the tubes three times each with deionized water and ethanol, and dry them at 60°C for 10 min to obtain blood collection tubes with a modified material coating that can reduce unintended platelet activation on the surface.

[0060] Example 3

[0061] Preparation of modified materials that can reduce unintended platelet activation:

[0062] Step 1: Take 0.16g GMA (glycidyl methacrylate) and 1g CBMA (carboxybetaine methacrylate), making the molar ratio of GMA to CBMA 1:4, add them to a mixed solvent of 2 mL methanol and 6 mL deionized water and stir until dissolved to obtain a mixed system;

[0063] Step 2: Add 0.018 g APS (ammonium persulfate) to the mixture and stir continuously at 60°C for 12 h to obtain the reaction solution;

[0064] Step 3: Slowly add the reaction solution dropwise to 100 mL of anhydrous acetone to precipitate a solid. Collect the precipitate by centrifugation or filtration, wash the solid product twice with deionized water, add 5 mL of deionized water to allow it to swell, and then freeze at -20°C for 12 hours. After freezing, grind to obtain a modified material powder that can reduce unintended platelet activation (the modified material powder that can reduce unintended platelet activation obtained in this example is as follows). Figure 1 (As shown).

[0065] Application of modified materials that can reduce unintended platelet activation:

[0066] S1: Same as S1 in Example 1;

[0067] S2: Weigh 1 g of the modified material powder that can reduce unintended platelet activation, add it to 50 mL of deionized water, stir to dissolve, and then add triethylamine to adjust the pH to 8.5 to prepare a 2% (w / w) solution of the modified material that can reduce unintended platelet activation. Immerse the blood collection tubes obtained in S1 in the solution and react at 60°C for 3 h to fix the zwitterionic polymer modified by reactive functional groups onto the surface of the blood collection tubes. After the reaction, wash the tubes three times each with deionized water and ethanol, and dry them at 60°C for 10 min to obtain blood collection tubes with a coating of the modified material that can reduce unintended platelet activation on the surface.

[0068] Example 4

[0069] Preparation of modified materials that can reduce unintended platelet activation:

[0070] Step 1: Take 0.13 g of 2-isocyanate ethyl methacrylate and 1.00 g of MPC and mix them to make a molar ratio of 1:4. Add the mixture to 8 mL of anhydrous dimethyl sulfoxide (DMSO) and stir until completely dissolved to obtain a mixed system.

[0071] Step 2: Add 0.018 g of azobisisobutyronitrile to the mixed system and react at 60 °C for 12 h under nitrogen protection to allow the methacrylate double bonds in the two monomers to undergo free radical copolymerization, thus obtaining the reaction solution;

[0072] Step 3: Slowly add the reaction solution dropwise to 100 mL of anhydrous acetone to precipitate the material. Collect the precipitate by centrifugation or filtration, and wash it 2-3 times with anhydrous acetone. Then dry it under vacuum to obtain a modified material powder that can reduce unintended platelet activation (the modified material powder that can reduce unintended platelet activation obtained in this example is as follows). Figure 1 (As shown).

[0073] Application of modified materials that can reduce unintended platelet activation:

[0074] S1: Same as S1 in Example 1;

[0075] S2: Weigh 1.00 g of the modified material powder that can reduce unintended platelet activation, add it to 50 mL of anhydrous DMSO, stir to dissolve, and prepare a 2% (w / w) solution of the modified material that can reduce unintended platelet activation; immerse the blood collection tube obtained in S1 in the solution and react at 60°C for 3 h to fix the zwitterionic polymer modified by reactive functional groups onto the surface of the blood collection tube; after the reaction, wash with deionized water and ethanol three times each, and dry at 60°C for 10 min to obtain a blood collection tube with a modified material coating that can reduce unintended platelet activation on the surface.

[0076] Example 5

[0077] Preparation of modified materials that can reduce unintended platelet activation:

[0078] Step 1: Take 0.21g of 3-methacryloxypropyltrimethoxysilane and 1g of MPC (2-methacryloxyethylphosphorylcholine) and mix them to make the molar ratio of 3-methacryloxypropyltrimethoxysilane to MPC 1:4. Add both to 8mL of anhydrous dimethyl sulfoxide to dissolve them and obtain a mixed system.

[0079] Step 2: Add 0.018 g of azobisisobutyronitrile to the mixed system and react at 60°C for 12 h under nitrogen protection to obtain the reaction solution;

[0080] Step 3: Slowly add the reaction solution dropwise to 100 mL of anhydrous acetone to precipitate a solid. Collect the precipitate by centrifugation or filtration, wash the solid product twice with deionized water, add 5 mL of deionized water to allow it to swell, and then freeze at -20°C for 12 hours. After freezing, grind to obtain a modified material powder that can reduce unintended platelet activation (the modified material powder that can reduce unintended platelet activation obtained in this example is as follows). Figure 1 (As shown).

[0081] Application of modified materials that can reduce unintended platelet activation:

[0082] S1: Take blood collection tubes (PRP BP-I from Zhejiang Paulman Biotechnology Co., Ltd.), and wash them three times each with deionized water and ethanol to remove surface contaminants. Then dry them in a 60℃ oven for 10 min for later use. Place the pretreated blood collection tubes in an oxygen plasma cleaner with the following conditions: 70 W, 30 sccm O2, 50 Pa, 5 min, to introduce active groups on their surface.

[0083] S2: Weigh 1 g of the modified material powder that reduces unintended platelet activation, and add it to 50 mL of a mixed solution of anhydrous ethanol and water in a ratio of 1:4 (volume). Adjust the pH to 4.5–5.5 to allow the silane groups to hydrolyze appropriately to generate silanol groups, thus preparing a 2% (w / w) solution of the modified material that reduces unintended platelet activation. Then, immerse the blood collection tubes obtained in S1 after oxygen plasma treatment in this solution and react at 60°C for 3 h. This allows the silanol groups on the polymer side chains to condense with the hydroxyl groups on the surface of the blood collection tubes, forming Si–O–C or Si–O–Si bonds, thereby fixing the zwitterionic polymer onto the surface of the blood collection tubes. After the reaction, wash the tubes three times each with deionized water and ethanol, and dry them at 60°C for 10 min to obtain blood collection tubes with a surface coating of the modified material that reduces unintended platelet activation.

[0084] Example 6

[0085] Preparation of modified materials that can reduce unintended platelet activation:

[0086] Step 1: Take 0.120 g GMA (glycidyl methacrylate) and 1.000 g MPC (2-methacryloyloxyethyl phosphorylcholine) and mix them to make the molar ratio of GMA to MPC 1:4. Add the mixture to a mixed solvent of 2 mL methanol and 6 mL deionized water to obtain a mixed system.

[0087] Step 2: Add 0.018 g APS (ammonium persulfate) to the mixed system and react at 60 ℃ for 12 h to obtain the reaction solution;

[0088] Step 3: Slowly add the reaction solution dropwise to 100 mL of anhydrous acetone to precipitate a solid. Collect the precipitate by centrifugation or filtration, wash the solid product twice with deionized water, add 5 mL of deionized water to swell it, and then freeze it at -20℃ for 12 h. After freezing, grind to obtain P(MPC-co-GMA) copolymer powder. Weigh 1.000 g of the above P(MPC-co-GMA) copolymer powder, dissolve it in 20 mL of anhydrous DMSO (dimethyl sulfoxide), add 0.078 g of methacrylic acid and 10 μL of triethylamine, and react at 50 ℃ in the dark for 10 h to allow the carboxyl groups in the methacrylic acid to undergo a ring-opening reaction with the epoxy groups on the polymer side chain, introducing polymerizable double bonds. Add the obtained reaction solution dropwise to 200 mL of anhydrous acetone to precipitate, and wash the precipitate three times with 30 mL of anhydrous acetone each time, followed by vacuum drying at 40 ℃ for 24 hours. h, to obtain a modified material powder that can reduce unintended platelet activation (the modified material powder that can reduce unintended platelet activation obtained in this embodiment is as follows) Figure 1 (As shown).

[0089] Application of modified materials that can reduce unintended platelet activation:

[0090] S1: Take blood collection tubes (PRP BP-I from Zhejiang Paulman Biotechnology Co., Ltd.), and wash them three times each with deionized water and ethanol to remove surface contaminants. Then, dry them in a 60℃ oven for 10 min for later use. Place the pretreated blood collection tubes in an oxygen plasma cleaner under the following conditions: 70 W, 30 sccm O2, 50 Pa, for 5 min, to introduce active groups on their surface. Then, prepare a silane coupling agent solution: mix 1 mL of 3-methacryloyloxypropyltrimethoxysilane, 50 mL of ethanol, and 1 mL of deionized water evenly. Immerse the blood collection tubes treated with oxygen plasma in the silane coupling agent solution and react at 60℃ for 2 h. After the reaction, wash them three times each with deionized water and ethanol, and dry them in a 60℃ oven for 20 min to obtain activated blood collection tubes.

[0091] S2: Take 1.00 g of the modified material powder that reduces unintended platelet activation, add it to 50 mL of deionized water, stir for 1 h until completely dissolved, then add 0.025 g of I2959 as a photoinitiator, stir in the dark for 30 min to obtain a 2% (w / w) solution of the modified material that reduces unintended platelet activation; immerse the blood collection tubes treated with S1 and containing double bonds on the surface in this solution, immerse in the dark for 30 min, and then expose to 365 nm ultraviolet light at 10 mW / cm². 2 Irradiation under certain conditions for 10 min allows the polymer side chain double bonds to undergo free radical grafting or cross-linking reactions with the double bonds on the surface of the blood collection tube. The tube is then washed three times with 10 mL of deionized water each time, and then soaked and washed with deionized water or PBS for 12 h to obtain a blood collection tube with a modified material coating on the surface that can reduce the unexpected activation of platelets.

[0092] Example 7

[0093] This embodiment is basically the same as Example 1, except that: no silane coupling agent is used for modification.

[0094] This invention also includes comparative examples, which are basically the same as those in Example 1, with the differences shown in Table 1. The performance test results of the modified materials that reduce unintended platelet activation obtained from each example and comparative example, and the elastic modulus results of the modified PRP tubes are shown in Table 1 (elastic modulus was measured using atomic force microscopy, with the test environment temperature kept constant at 25°C. During the measurement process, the blood collection tubes were always immersed in physiological saline. Indentation tests were performed using a spherical probe, and the maximum loading force was set to 10 nN).

[0095] Table 1

[0096]

[0097] To verify the effect of the cell membrane biomimetic coating of the present invention on reducing platelet activation, all blood collection tubes obtained in Examples 1-7 and Comparative Examples 1-7 were designated as control tube 1. These are blood collection tubes suitable for low-to-medium speed centrifugation, and their inner wall elastic modulus is 71.5 GPa before modification. A physical image of control tube 1 obtained after modification using the method described in Example 1 is shown below. Figure 2 As shown; medical borosilicate glass blood collection tubes suitable for high-speed centrifugation were modified using the same method as in Examples 1-7 and Comparative Examples 1-7. The elastic modulus of the inner wall of the unmodified medical borosilicate glass blood collection tube was 2.3 GPa. All the modified medical borosilicate glass blood collection tubes were designated as control tubes 2. A physical image of the control tubes 2 obtained after modification using the method in Example 1 is shown below. Figure 3 and Figure 4 As shown.

[0098] Fresh whole blood from the same donor was added to separate blood collection tubes and centrifuged using a dedicated PRP tube centrifuge under the same centrifugation mode to obtain platelet-rich plasma. All control tubes (1) were centrifuged at a medium-low speed (15 cm radius, 2000 r / min, 10 min), corresponding to a relative centrifugal force of approximately 670 × g. All control tubes (2) were centrifuged at a high speed (15 cm radius, 3500 r / min, 10 min), corresponding to a relative centrifugal force of approximately 2050 × g. After centrifugation, platelet-rich plasma was collected from each blood collection tube, and the levels of platelet factor 4 (PF4) and β-thromboglobulin (β-TG) were measured using ELISA to evaluate the effect of different inner surfaces of the blood collection tubes on platelet activation during centrifugation. Using the corresponding uncoated blank blood collection tubes as a reference, the reduction rates of PF4 and β-TG were calculated, and the average results from three blood donors were taken to obtain the average reduction rates of PF4 and β-TG in platelet-rich plasma in each blood collection tube. PF4 and β-TG are both marker proteins released during platelet activation. The results for each control tube 1 are shown in Table 2, and the results for each control tube 2 are shown in Table 3.

[0099] Table 2

[0100]

[0101] Table 3

[0102]

[0103] As shown in Tables 2 and 3, the PF4 reduction rate and β-TG reduction rate of control tubes 1 and 2 were calculated using uncoated blank blood collection tubes as controls, according to the formula (Cblank − Ccoating) / Cblank × 100%, where Cblank and Ccoating represent the concentrations of the corresponding activation markers in the blank and modified blood collection tubes, respectively. Table 2 shows that the PF4 reduction rate of control tube 1 modified using the methods of Examples 1-7 was 11.7%–14.8%, and the β-TG reduction rate was 12.9%–19.2%; while the PF4 reduction rate of control tube 1 modified using the methods of Comparative Examples 1-7 was only 0.9%–4.5%, and the β-TG reduction rate was only 1.6%–5.1%. These results indicate that, compared to the comparative modification methods, the modification method of this invention can more effectively reduce the unintended platelet activation induced by medium- and low-speed blood collection tubes during PRP preparation. As shown in Table 3, the PF4 reduction rate of control tube 2 modified using the methods of Examples 1-7 was 11.4%–18.2%, and the β-TG reduction rate was 13.9%–20.3%; while the PF4 reduction rate of control tube 2 modified using the methods of Comparative Examples 1-7 was only 0.5%–3.7%, and the β-TG reduction rate was only 1.1%–4.2%. These results indicate that the modification method of the present invention is applicable not only to blood collection tubes that can be centrifuged at medium and low speeds, but also to blood collection tubes that can be centrifuged at high speeds, and can exhibit good inhibitory effects on platelet activation under different centrifugation modes.

[0104] As can be seen from Tables 2 and 3, the modification methods of Examples 1-7 significantly reduced the release of PF4 and β-TG during centrifugation, and the overall effect was significantly better than that of the comparative examples. PF4 and β-TG are typical markers of platelet activation and release; their reduced release levels indicate that the unintended activation of platelets during PRP preparation by centrifugation was mitigated. Therefore, this invention can effectively improve the blood compatibility of the inner surface of commercially available blood collection tubes, which helps to improve the stability of platelet status during PRP preparation. This is because, after introducing a zwitterionic polymer modified with reactive functional groups onto the inner surface of the PRP blood collection tube, a cell membrane-inspired zwitterionic hydration interface can be formed on the tube wall surface. This interface can bind a large number of water molecules through ionic solvation, forming a stable and dense hydration layer, thereby reducing plasma protein adsorption and platelet adhesion, and reducing direct contact and frictional stimulation between platelets and the tube wall during centrifugation. Furthermore, the elastic modulus of the modified blood collection tube inner wall surface is controlled at 0.13–0.35 MPa, significantly lower than the GPa level elastic modulus of conventional polycarbonate tube wall materials and the tens of GPa level elastic modulus of glass materials. This allows for the creation of a low-modulus hydration contact environment on the inner surface of the tube wall that more closely resembles the soft interface between the blood and the endothelium of a natural blood vessel. This low-modulus hydration interface can reduce the abnormal mechanical stimulation of platelets by the high-rigidity tube wall during centrifugation, decreasing unintended activation of platelets caused by contact, collision, adhesion, and spreading. Consequently, the release of activation marker proteins such as PF4 and β-TG is correspondingly reduced, helping to maintain the activity and functional integrity of platelets in PRP.

[0105] Therefore, the modified material developed in this invention can reduce the unexpected activation of platelets in different types of commercially available PRP tubes, and has good applicability and stability. It is of positive significance for improving the blood compatibility of the inner surface of PRP tubes and improving the quality of PRP preparation.

[0106] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. The application of a modified material that can reduce unintended platelet activation, characterized in that: This is used to form a modified coating on the contact surface between the blood collection tube and platelet-rich plasma; the blood collection tube is used to hold fresh whole blood, which is then centrifuged to obtain platelet-rich plasma. The modified material that can reduce unintended platelet activation is a zwitterionic polymer modified with reactive functional groups, and its preparation method includes the following steps: Step 1: Mix the zwitterionic monomer, reactive monomer, and solvent until the zwitterionic monomer and reactive monomer dissolve to obtain a mixed system; Step 2: Add an initiator to the resulting mixture and react at 50–80°C for 10–15 h to obtain a reaction solution; Step 3: Add the reaction solution to acetone to precipitate a solid, and obtain a modified material that can reduce unintended platelet activation; The zwitterionic monomer is one or more of phosphorylcholine monomers, sulfobetaine monomers, and carboxybetaine monomers, and the reactive monomer is one or more of glycidyl methacrylate, 2-isocyanate ethyl methacrylate, and 3-methacryloyloxypropyltrimethoxysilane. The degree of substitution of reactive functional groups in zwitterionic polymers modified with reactive functional groups is 5%–20%; The weight-average molecular weight of the zwitterionic polymer modified with reactive functional groups is 50,000 to 300,000, and the elastic modulus is 0.13 to 0.35 MPa. The method for forming the modified coating specifically includes the following steps: S1: The inner wall of the blood collection tube is activated, and the activation treatment is one or more of oxygen plasma activation treatment, ultraviolet / ozone treatment and chemical oxidation treatment; after the activation treatment of S1, the tube is first modified with a surface modifier, and then S2 is performed. The surface modifier is a silane coupling agent with an amino group at the end or a mercapto group at the end. S2: Prepare a modified solution by modifying the zwitterionic polymer with reactive functional groups, coat the inner wall of the blood collection tube with the modified solution or immerse the blood collection tube in the modified solution, so that the zwitterionic polymer modified with reactive functional groups forms a connection with the blood collection tube, and obtain a modified coating.

2. The application according to claim 1, characterized in that: The modified material that can reduce unintended platelet activation is obtained by polymerization of epoxy group-containing monomers and phosphorylcholine monomers. The epoxy group-containing monomer is glycidyl methacrylate, and the phosphorylcholine monomer is 2-methacryloyloxyethyl phosphorylcholine.

3. The application according to claim 1, characterized in that: The initiator is one of ammonium persulfate and azobisisobutyronitrile.

4. The application according to claim 1, characterized in that: In step 1, the molar ratio of reactive monomer to zwitterionic monomer is 1:4.

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