A method for preparing PRP separation gel and application thereof

Abstract

The application relates to a preparation method of PRP separation glue and application thereof, and comprises the following steps: adding N-(hydroxymethyl) acrylamide, hydroxyethyl methacrylate, lauryl methacrylate and / or polyethylene glycol into water, stirring uniformly, then adding ammonium persulfate and N, N, N', N'-tetramethyl ethylenediamine, stirring uniformly, obtaining a prepolymer mixture; placing the prepolymer mixture until a gel is formed; and cleaning the gel to obtain PRP separation glue. The N-(hydroxymethyl) acrylamide and the hydroxyethyl methacrylate used in the application are monomers commonly used in the biomedical field, the lauryl methacrylate is a long-chain alkyl acrylate monomer, plays a role in regulating the density of the PRP separation glue, and improves the thixotropy of the main glue material. The polyethylene glycol can be used as a plasticizer and a compatibility regulator in the separation glue system. The above raw materials have good biological affinity, and the preparation method is simple and the conditions are mild.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer materials for PRP separation, specifically to a highly selective whole blood cell removal type PRP separating gel and its preparation method. Background Technology

[0002] PRP separating gel is a material with good physiological inertness, sealing properties, and thixotropy. Utilizing its density difference, it separates erythrocytes and other cells from platelets, enabling rapid and safe extraction of platelets-rich plasma. The quality of PRP preparation directly determines its clinical efficacy. Residual impurity cells (erythrocytes, leukocytes, lymphocytes, etc.) can severely impact the therapeutic effect and safety of PRP: the presence of erythrocytes may trigger local hemolysis, and the released hemoglobin can interfere with the activity of growth factors; excessive accumulation of leukocytes, especially neutrophils, can release large amounts of inflammatory factors, exacerbating local inflammation, counteracting the anti-inflammatory and repairing effects of PRP, and potentially even inhibiting tissue regeneration; residual lymphocytes may increase the risk of immune responses, hindering the stability of the repair microenvironment. Therefore, developing a separation technology that can efficiently enrich platelets and precisely remove impurity cells is a core technological challenge in the current field of PRP preparation.

[0003] CN106366426 discloses a separating gel system for the extraction and purification of platelet-rich plasma, comprising an inert separating gel (polyethylene butylene and chlorinated paraffin-52), nano-porous silica, and modified silica. However, the recovery rate is low, approximately 50%, and the purity is less than 90%. CN118307911A discloses a platelet-rich plasma separating gel prepared by a method comprising, by weight, 330-400 parts of polyvinyl acetate, 110-150 parts of dioctyl adipate, 30-40 parts of specific gravity adjuster nano-silica, and 2-3 parts of organic thixotropic agent hydrogenated castor oil. CN 118794765A discloses a modified PRP separating gel, which involves introducing a hydrolysis-resistant monomer, epoxidized soybean oil acrylate, into the polyacrylate substrate of the separating gel for modification. Anthraquinone, polymerization inhibitors, styrene, catalysts, and other components are further added to this separating gel, which is then mixed uniformly with a thixotropic agent and a plasticizer, resulting in a complex composition. CN 121775009 A discloses a method for preparing a separating gel by mixing acrylate resin, surface-modified nano-silica composite material, polyether-modified polysiloxane, and polyvinylpyrrolidone under high vacuum conditions to obtain a PRP separating gel. The above preparation methods generally involve a large number of components and complex process conditions. CN116715801A discloses a low-cytotoxic PRP separating gel, which is prepared by using hydroxyethyl methacrylate, 1-dodecene, and terminal vinyl silicone oil as monomers to obtain a polymer, which is then dispersed and formulated into a PRP separating gel by adding nano-silica. CN107513126A discloses a method for preparing a separating gel in a heptane solvent system, using styrene, acrylates, and a silane coupling agent as monomers, and dimethyl azobisisobutyrate as an initiator to initiate polymerization into a polymer resin, which is then used as a raw material to prepare a serum separating gel. Most of these preparation methods require the removal of organic solvents, which is quite complicated. In addition, the introduction of other additives such as silica or silane coupling agents may affect the purity or application of the separated platelets.

[0004] Current PRP separating gels still have many technical problems that need improvement: First, the separation selectivity is poor. Most separating gels can only achieve preliminary separation of red blood cells and platelets, and the retention effect on white blood cells and lymphocytes, whose density is closer to that of platelets, is not good. Second, there are potential biocompatibility issues. Although the monomer raw materials used in some separating gels are stable after polymerization, residual monomers or organic solvents are not easy to completely remove, which may interact with blood components, affect platelet activity, or cause potential biotoxicity. Third, the performance stability is insufficient. The density gradient of some separating gels is easily affected by environmental factors such as centrifugation speed and temperature, resulting in fluctuations in separation effect and making it difficult to achieve standardized preparation. Summary of the Invention

[0005] To address the problems existing in the prior art, one objective of this invention is to provide a method for preparing PRP separating gel, comprising the following steps:

[0006] 1) Prepare a prepolymer mixture in which the mass-volume percentage of N-(hydroxymethyl)acrylamide is 6-10%, the mass-volume percentage of hydroxyethyl methacrylate is 8-12%, the mass-volume percentage of lauryl methacrylate is 0.5-5%, the mass-volume percentage of polyethylene glycol is 0-0.5%, the mass-volume percentage of ammonium persulfate is 0.04-0.08%, the mass-volume percentage of N,N,N',N'-tetramethylethylenediamine is 0.08-0.16%, and the remainder is water;

[0007] 2) Allow the prepolymer mixture to stand until a gel forms; wash the gel to obtain the PRP separating gel.

[0008] In some embodiments, the N-(hydroxymethyl)acrylamide has a density of 1.074 g / mL, the hydroxyethyl methacrylate has a density of 1.073 g / mL, and the lauryl methacrylate has a density of 0.868 g / mL; the PRP separating gel prepared from N-(hydroxymethyl)acrylamide, hydroxyethyl methacrylate, lauryl methacrylate, and / or polyethylene glycol has a density range of 1.04-1.06 g / mL.

[0009] In some embodiments, step 1) of preparing the prepolymer mixture includes adding N-(hydroxymethyl)acrylamide, hydroxyethyl methacrylate, lauryl methacrylate and / or polyethylene glycol to water and vortexing them, then adding ammonium persulfate and N,N,N',N'-tetramethylethylenediamine and mixing them to obtain the prepolymer mixture;

[0010] In some embodiments, step 1) of preparing the prepolymer mixture includes: preparing a 20-40% (w / v) N-(hydroxymethyl)acrylamide aqueous solution, adding hydroxyethyl methacrylate, lauryl methacrylate, and / or a 2.5-10% (w / v) polyethylene glycol aqueous solution, vortexing and mixing, adding ammonium persulfate and N,N,N',N'-tetramethylethylenediamine, diluting with water, and mixing to obtain the prepolymer mixture.

[0011] In some embodiments, step 1) involves adding 0.1-0.5% (w / v) ammonium persulfate aqueous solution and 10-30% (v / v) N,N,N',N'-tetramethylethylenediamine aqueous solution. Ammonium persulfate acts as a free radical initiator, forming a redox system with the reducing agent (tetramethylethylenediamine TEMED). Free radicals are generated in the aqueous phase at room temperature, initiating the polymerization reaction.

[0012] In some embodiments, step 2) involves allowing the mixture to stand for 6-24 hours to form a gel.

[0013] This invention uses N-(hydroxymethyl)acrylamide, hydroxyethyl methacrylate, and lauryl methacrylate as core monomers to prepare a PRP separating gel in an aqueous phase through a simple room-temperature static setting method. N-(hydroxymethyl)acrylamide and hydroxyethyl methacrylate are both commonly used hydrophilic monomers in the biomedical field, possessing excellent copolymerization activity and biocompatibility, which can enhance the hydrophilicity of the PRP separating gel and reduce non-specific adsorption to platelets. Lauryl methacrylate, as a long-chain alkyl acrylate monomer, not only regulates the density of the PRP separating gel but also improves the thixotropy of the main gel material. Polyethylene glycol, as a classic biocompatible polymer, can act as a plasticizer and compatibility regulator in the separating gel system. In some embodiments, the molecular weight of the polyethylene glycol is 1000-2000 Da. Polyethylene glycol, as an optional component, enhances the thixotropy of the separating gel and improves its gelling properties. All the above raw materials are widely available and inexpensive.

[0014] Another object of this invention application is to provide a method for obtaining platelet-rich plasma from whole blood, comprising the following steps:

[0015] a) Prepare a prepolymer mixture containing 6-10% by weight (w / v) N-(hydroxymethyl)acrylamide, 8-12% by weight (w / v) hydroxyethyl methacrylate, 0.5-5% by weight (w / v) lauryl methacrylate, 0-0.5% by weight (w / v) polyethylene glycol; 0.04-0.08% by weight (w / v) ammonium persulfate, and 0.08-0.16% by weight (w / v) N,N,N',N'-tetramethylethylenediamine; allow the prepolymer mixture to stand until a gel forms, and wash the gel.

[0016] b) Add the gel to whole blood and centrifuge at 3000-5000 rpm for 5-10 minutes. The supernatant is platelet-rich plasma.

[0017] In some embodiments, step b) includes adding the gel to whole blood and centrifuging at 3000 rpm for 9 minutes. Finally, a certain volume of PPP is extracted, and the PRP tube is inverted to completely detach the white film on the separating gel and suspend it in the remaining plasma to obtain platelet-rich plasma.

[0018] This invention application further provides a plasma separation blood collection tube, which includes the PRP separation gel.

[0019] Platelet separation from whole blood using the PRP separating gel is achieved with significantly lower levels of cellular impurities such as red blood cells, white blood cells, and lymphocytes compared to most existing similar products, while also greatly improving platelet recovery rate. Compared to existing technologies, this invention offers the following beneficial technical advantages:

[0020] 1) The monomers N-(hydroxymethyl)acrylamide, hydroxyethyl methacrylate, lauryl methacrylate and polyethylene glycol used in the PRP separating gel provided by the present invention are commonly used in the field of biomedical engineering, and the polymers prepared by them have high cell compatibility and high blood compatibility.

[0021] 2) The PRP separating gel provided by this invention has both a high platelet recovery rate and a high clearance effect of impurity cells (red blood cells, white blood cells and lymphocytes), with an overall platelet recovery rate of over 85%.

[0022] 3) The PRP separating gel preparation method provided by this invention is simple and low-cost. The preparation process requires no organic solvents, can be carried out at room temperature, does not require extreme reaction conditions, and only requires conventional production equipment. The clearly defined monomer ratios and process parameters significantly improve the stability of the production process and result in good product performance uniformity. Existing blood separating gel preparation processes require the use of organic solvents, necessitating subsequent solvent removal steps, and residual organic matter may still affect platelet quality. The separating gel of this invention can rapidly crosslink in an aqueous phase at room temperature, greatly simplifying the preparation process and being more environmentally friendly. Attached Figure Description

[0023] Figure 1 The stratification of the copolymer of N-(hydroxymethyl)acrylamide and hydroxyethyl methacrylate in whole blood.

[0024] Figure 2 Photos of the actual products after standing and gelling in Examples 1 and 2.

[0025] Figure 3 The separation effect of the separating gels prepared in Example 2 and Comparative Examples 1-3 in whole blood.

[0026] Figure 4 Shear thinning test results of the PRP separating gels prepared in Examples 1 and 2.

[0027] Figure 5 Macroscopic image of platelet separation from anticoagulated porcine whole blood using the PRP separating gel prepared in Example 1.

[0028] Figure 6 Macroscopic comparison images of platelet separation using the PRP separating gel prepared in Example 1 and commercial PRP separating gel.

[0029] Figure 7Results of hemolysis rate test of the PRP separating gel prepared in Example 1.

[0030] Figure 8 The staining fluorescence observation image of the PRP separating gel prepared in Example 1 for cell compatibility testing. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise stated, the reagents used in the embodiments are all commercially available products.

[0032] Example 1

[0033] To achieve the separation of platelets and blood cells from whole blood, the specific gravity of the PRP separating gel should be in the range of 1.04-1.06 g / cm³. 3 It exhibits a certain degree of thixotropy. The density of N-hydroxymethylacrylamide (NMA) is between 1.07 and 1.08 g / cm³. 3 In polymers containing NMA, cross-linking reactions can be easily triggered by heating or adding an acid catalyst, allowing for the flexible synthesis of desired polymers and precise control over their structures. This example first investigated the density of acrylamide polymers and their copolymers in whole blood. Different monomer concentrations of NMA aqueous solutions were prepared, photoinitiated into gels, and then an equal volume of whole blood was added and centrifuged. The results are shown in Table 1 below.

[0034] Table 1

[0035]

[0036] Preliminary analysis showed that the density of NMA polymers with a solid content of 8%-10% after adding whole blood was close to the range of the separating gel's function. Considering the high hydrophilicity of NMA polymers, other hydrophobic polymeric monomers were introduced into the NMA polymers to improve stability and separation ability, thereby adjusting the density and separation performance of the gel after formation.

[0037] Different types of copolymer monomers, as shown in Table 2, were tested. The method involved adding 20 μL of the copolymer monomer to an 8% NMA monomer solution and initiating the gelation reaction using two different methods. The colloid was then added to whole blood to observe its density and mechanical properties, determining its suitability as a separating gel. The experiments revealed that while some copolymer monomers and NMA could form gels after crosslinking, their moduli were either too low or too high, making them unsuitable as separating gels. Furthermore, a comparison of the two initiation methods showed that ammonium persulfate as the initiator resulted in a gel with better gelation performance, and the density of the gel was close to the suitable density range for a separating gel.

[0038] Table 2

[0039]

[0040] After initial screening, the copolymer of N-(hydroxymethyl)acrylamide and hydroxyethyl methacrylate exhibited suitable properties and met the density requirements well. Through reproduction and scaling up of experiments, it was confirmed that it can indeed achieve a certain effect in separating blood (e.g., Figure 1 As shown in the figure, the experiment was repeated multiple times, but the colloidal shear thinning energy was not ideal, and the composition needs to be further adjusted so that it can exhibit good flowability or thixotropy under high shear force.

[0041] Based on this, in this embodiment, silica, lauryl methacrylate (LMA), and PEG (molecular weight 2000 Da) were further added to the main material. The results are shown in Table 3. According to the results in Table 3, the addition of LMA and PEG both help to improve the mechanical properties and blood separation performance of the main adhesive.

[0042] This invention also found that the amounts of the polymerization initiators ammonium persulfate and N,N,N',N'-tetramethylethylenediamine have a crucial impact on gel formation and properties. Too low an initiator concentration fails to effectively form a gel network with a certain modulus and viscosity. While too high an initiator concentration accelerates the reaction rate, it may also lead to shorter polymer chains, affecting the final gel properties. Experimental verification showed that a concentration of 0.04-0.08 wt% ammonium persulfate and 0.08-0.16% N,N,N',N'-tetramethylethylenediamine prepared a gel that met the performance requirements of a separating gel and could be gelled in an aqueous phase at room temperature.

[0043] Table 3

[0044]

[0045] Based on the above experiments, this embodiment prepares a PRP separation gel using N-(hydroxymethyl)acrylamide, hydroxyethyl methacrylate, and lauryl methacrylate as monomers.

[0046] The preparation method of the PRP separating gel in this embodiment includes the following steps:

[0047] 1) Prepare 25% (w / v) N-(hydroxymethyl)acrylamide aqueous solution, 0.25% (w / v) ammonium persulfate aqueous solution and 20% (v / v) N,N,N',N'-tetramethylethylenediamine aqueous solution respectively, and dissolve them by ultrasonic vibration.

[0048] 2) Add 240 μL of N-(hydroxymethyl)acrylamide aqueous solution, 80 μL of hydroxyethyl methacrylate, and 10 μL of lauryl methacrylate to a centrifuge tube, and vortex quickly to mix them thoroughly.

[0049] 3) Add 320 μL of ammonium persulfate aqueous solution and 8 μL of N,N,N',N'-tetramethylethylenediamine aqueous solution to a centrifuge tube, then add 212 μL of deionized water to make the total gel volume 1 mL. Mix thoroughly and let stand at room temperature for 6-24 h to form a milky white, opaque colloid. See the image below for the gel solidification. Figure 2 As shown. The colloid can be easily removed from the container.

[0050] 4) Wash the obtained PRP separating gel multiple times to remove residual initiator and unreacted monomers.

[0051] Example 2

[0052] The PRP separating gel prepared in this embodiment includes the following steps:

[0053] 1) Prepare a 25% (w / v) N-(hydroxymethyl)acrylamide aqueous solution, a 2.5% (w / v) polyethylene glycol aqueous solution, a 0.25% (w / v) ammonium persulfate aqueous solution and a 20% (v / v) N,N,N',N'-tetramethylethylenediamine aqueous solution, and dissolve by ultrasonic vibration.

[0054] 2) Add 240 μL of N-(hydroxymethyl)acrylamide aqueous solution, 80 μL of hydroxyethyl methacrylate, 10 μL of lauryl methacrylate and 120 μL of polyethylene glycol aqueous solution to a centrifuge tube, and vortex quickly to mix them thoroughly.

[0055] 3) Add 320 μL of ammonium persulfate aqueous solution and 8 μL of N,N,N',N'-tetramethylethylenediamine aqueous solution to a centrifuge tube. Add deionized water to make the total gel system 1 mL. Mix thoroughly and let stand at room temperature for 6-24 h to form a gel (see actual image). Figure 2 (As shown).

[0056] 4) Wash the obtained PRP separating gel multiple times to remove residual initiator and unreacted monomers.

[0057] Example 3

[0058] This embodiment uses the method of Example 2 to prepare the separating gel, except that the amount of polyethylene glycol added is 200 μL (0.5%).

[0059] Example 4

[0060] This embodiment uses the method of Example 2 to prepare the separating gel. The difference is that in step 2), 320 μL of N-(hydroxymethyl)acrylamide aqueous solution, 80 μL of hydroxyethyl methacrylate, 20 μL of lauryl methacrylate and 40 μL of polyethylene glycol aqueous solution are added to a centrifuge tube and vortexed rapidly to mix them thoroughly.

[0061] Example 5

[0062] This embodiment uses the method of Example 2 to prepare the separating gel. The difference is that in step 2), 240 μL of N-(hydroxymethyl)acrylamide aqueous solution, 80 μL of hydroxyethyl methacrylate, 5 μL of lauryl methacrylate and 120 μL of polyethylene glycol aqueous solution are added to a centrifuge tube and vortexed rapidly to mix them thoroughly.

[0063] Example 6

[0064] This embodiment uses the method of Example 2 to prepare the separating gel. The difference is that in step 2), 240 μL of N-(hydroxymethyl)acrylamide aqueous solution, 100 μL of hydroxyethyl methacrylate, 20 μL of lauryl methacrylate and 100 μL of polyethylene glycol aqueous solution are added to a centrifuge tube and vortexed rapidly to mix them thoroughly.

[0065] Example 7

[0066] This embodiment uses the method of Example 2 to prepare the separating gel. The difference is that in step 2), 240 μL of N-(hydroxymethyl)acrylamide aqueous solution, 120 μL of hydroxyethyl methacrylate, 5 μL of lauryl methacrylate and 80 μL of polyethylene glycol aqueous solution are added to a centrifuge tube and vortexed rapidly to mix them thoroughly.

[0067] Testing revealed that the PRP separating gels prepared in Examples 1-7 had densities between 1.04 and 1.06 g / mL. Furthermore, after centrifugation with whole blood, they exhibited a certain degree of separation. Specifically, the PRP separating gel of Example 1 had a density of 1.040 g / mL, while the PRP separating gel of Example 2 had a density of 1.052 g / mL. The colloidal densities of Examples 2-7 ranged from 1.05 to 1.06 g / mL, all falling between the densities of erythrocytes and platelets, with a viscosity of 45-55 Pa·s. Mechanical property testing showed that they exhibited thixotropy and, after centrifugation in whole blood, effectively separated plasma, thus qualifying them as blood separating gels.

[0068] Comparative Example 1

[0069] This comparative example uses the method of Example 2 to prepare the separating gel, the difference being that in step 2), 240 μL of N-(hydroxymethyl)acrylamide aqueous solution, 40 μL of hydroxyethyl methacrylate, 10 μL of lauryl methacrylate, and 120 μL of polyethylene glycol aqueous solution are added to a centrifuge tube and rapidly vortexed to mix thoroughly. In this comparative example, the gel density formed by the prepolymer solution is too low (e.g., ...). Figure 3 (As shown).

[0070] Comparative Example 2

[0071] This comparative example uses the method of Example 2 to prepare the separating gel, except that in step 2), 200 μL of N-(hydroxymethyl)acrylamide aqueous solution, 40 μL of hydroxyethyl methacrylate, 10 μL of lauryl methacrylate, and 120 μL of polyethylene glycol aqueous solution are added to a centrifuge tube and rapidly vortexed to mix thoroughly. In this comparative example, the prepolymer solution was left to stand at room temperature for 24 hours, resulting in poor gelation and an inability to obtain a gel suitable for use as a separating gel (e.g., ...). Figure 3 (As shown).

[0072] Comparative Example 3

[0073] This comparative example uses the method of Example 2 to prepare the separating gel, except that in step 2), 320 μL of N-(hydroxymethyl)acrylamide aqueous solution, 40 μL of hydroxyethyl methacrylate, 10 μL of lauryl methacrylate, and 120 μL of polyethylene glycol aqueous solution are added to a centrifuge tube and rapidly vortexed to mix thoroughly. In this comparative example, the gel density formed after the prepolymer solution is allowed to stand at room temperature is relatively low (e.g., ...). Figure 3 (As shown).

[0074] Example of effect 1

[0075] Rheological tests were performed on the PRP separating gels prepared in Examples 1 and 2. Figure 4 As shown, the viscosity of the two PRP separating adhesives is 45-55 Pa·s, and both have excellent shear thinning ability. As the shear rate increases, the viscosity of the PRP separating adhesive continuously decreases.

[0076] PRP separating gels were prepared in batches using molds, and their stability was tested. Prepolymer solutions were prepared according to the methods in Examples 1-7, and the materials were added to 7*7 molds. The molds had a diameter of 16 mm, a depth of 30 mm, and a total of 49 holes. The mixtures were thoroughly mixed and allowed to stand at room temperature for 6-24 hours to form a gel. The obtained PRP separating gels were randomly removed and washed repeatedly to remove residual initiators and unreacted monomers. The prepared separating gels were sealed in centrifuge tubes and allowed to stand at room temperature and in a refrigerator at 4 degrees Celsius, respectively. The density and thixotropy of the separating gels were tested at one-month intervals. The experimental results showed that the separating gel remained stable for two weeks at room temperature, and the separating gel in a 4-degree Celsius refrigerator maintained stable appearance, density, and thixotropy for six months.

[0077] Example 2

[0078] The PRP separating gels prepared in Examples 1 and 2 were subjected to whole blood platelet separation tests (e.g.) Figure 5 (As shown). Anticoagulated porcine whole blood (purchased from Henan Juwu Biotechnology Co., Ltd.) was sequentially placed into tubes containing PRP as described in Examples 1 and 2 above, and centrifuged at 3000 rpm for 9 minutes. Finally, a certain volume of PPP was extracted, and the PRP tube was inverted to completely detach the white film on the separating gel and suspend it in the remaining plasma, thus obtaining PRP. A commercial PRP separating gel product was used as a comparison to verify the separation effect. The method of use was in accordance with the instructions of the commercial product. The commercial separating gels used in this effect example are:

[0079] Kangshifei: Product Specifications: 8 ml; Product Name: Disposable Vacuum Blood Collection Tube, Sodium Citrate + Separating Gel Blood Collection Tube; Product Registration Certificate Number: Lu Medical Device Registration Certificate 20172220312; Production License Number: Lu Food and Drug Administration Medical Device Production Permit 20120084.

[0080] Sanli: Product Specifications: 8 mL; Product Name and Model: Disposable Human Venous Blood Sample Collection Container, Sodium Citrate + Separating Gel; Product Registration Certificate Number: Xiangxiezhuzhun 20152410045; Production License Number: Xiangshiyaojianxiechanshengxu 20150049.

[0081] BD (USA): Product Specifications: 8 mL; Product Name and Model: PRP Extraction Blood Collection Tube, BD 362761; Product Registration Certificate Number: National Medical Device Registration Certificate 20152223659.

[0082] Xinling: Product Specifications: 10 mL; Product Name: Disposable Vacuum Blood Collection Device, PET Sterile PRP Blood Collection Tube; Product Registration Certificate Number: Wanxiezhuzhun 20162220206; Production License Number: Wanshiyaojianxiechanshengxu 20150063.

[0083] Experimental macroscopic observation diagram as shown Figure 6As shown in Table 4 below, the test results of the PRP prepared by the separating gel are as follows. Observation shows that the supernatant plasma color is consistent between the examples and the comparative examples of this invention. Characterization results of the PRP confirm that the platelet separation efficiency of the PRP separating gel prepared by this invention is comparable to or better than that of commercial products, demonstrating its potential for commercial application.

[0084] Table 4. Platelet recovery rate and purity of PRP separating gels prepared in Examples 1 and 2 (experimental groups) compared with commercial PRP separating gels.

[0085]

[0086] As shown in Table 4 above, the PRP separating gel prepared in this invention can effectively recover platelets from whole blood compared to commercially available PRP separating gels. In particular, the platelet recovery rate in this embodiment is over 85%, and the content of white blood cells and lymphocytes in the PRP is extremely low, resulting in high-quality platelet-rich plasma. The separating gel prepared in this invention efficiently enriches platelets and precisely removes impurities such as red blood cells, white blood cells, and lymphocytes, showing promising applications in the medical field.

[0087] The hemolysis rate of the PRP separating gel prepared in Example 1 was tested according to GB / T 16886.4-2022 standard. Figure 7 As shown, the hemolysis rates of PRP separating gel extracts at different concentrations (6.25, 12.5, 25, 50, and 100 mg / mL) were comparable to those of the negative control group, approximately 0. This indicates that the PRP separating gel prepared in this invention has good blood compatibility.

[0088] The PRP separating gel prepared in Example 1 was subjected to cell compatibility testing. The cytotoxicity of the material was assessed using the calcein / propidium iodide double staining method. 100 mg / mL of PRP separating gel extract was added to adherent mesenchymal stem cells, and after incubation for 24 h, live / dead staining was performed. Figure 8 As shown, all mesenchymal stem cells exhibited bright green fluorescence, and no positive red fluorescent cells were observed, indicating no cell death. Further observation of cell morphology revealed intact cell membranes, well-extended pseudopodia, and active adherent growth, without signs of shrinkage, detachment, or other toxic reactions. This demonstrates that the PRP separating gel prepared in this invention possesses excellent biocompatibility.

[0089] The above embodiments are merely illustrative and explanatory of the technical solutions of the present invention or are only preferred implementation schemes, and are not intended to limit the technical solutions of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing PRP separating gel, comprising the following steps: 1) Prepare a prepolymer mixture in which N-(hydroxymethyl)acrylamide has a mass-volume percentage of 6-10%, hydroxyethyl methacrylate has a volume percentage of 8-12%, lauryl methacrylate has a volume percentage of 0.5-5%, polyethylene glycol has a mass-volume percentage of 0-0.5%, ammonium persulfate has a mass-volume percentage of 0.04-0.08%, N,N,N',N'-tetramethylethylenediamine has a volume percentage of 0.08-0.16%, and the remainder is water; 2) Allow the prepolymer mixture to stand until a gel forms; wash the gel to obtain PRP separating gel.

2. The method as described in claim 1, characterized in that, The density range of the PRP separating gel is 1.04-1.06 g / mL.

3. The method as described in claim 1 or 2, characterized in that, The molecular weight of the polyethylene glycol is 1000-2000 Da.

4. The method as described in claim 1 or 2, characterized in that, The preparation method of the prepolymer mixture in step 1) includes: preparing an aqueous solution of N-(hydroxymethyl)acrylamide with a mass-volume percentage of 20-40%, adding hydroxyethyl methacrylate, lauryl methacrylate, and / or an aqueous solution of polyethylene glycol with a mass-volume percentage of 2.5-10%, vortexing and mixing, then adding ammonium persulfate and N,N,N',N'-tetramethylethylenediamine, and diluting and mixing with water.

5. The method as described in claim 4, characterized in that, In step 1), add 0.1-0.5% by weight of ammonium persulfate aqueous solution and 10-30% by volume of N,N,N',N'-tetramethylethylenediamine aqueous solution.

6. The method as described in claim 1 or 2, characterized in that, In step 2), let it stand for 6-24 hours to form a gel.

7. A method for obtaining platelet-rich plasma from whole blood, comprising the following steps: a) The method according to any one of claims 1-6 is used to prepare PRP separating gel; b) Add the separating gel to whole blood and centrifuge at 3000-5000 rpm for 5-10 minutes. The supernatant is platelet-rich plasma.

8. The method as described in claim 7, characterized in that, In step b), the PRP separating gel is added to whole blood and centrifuged at 3000 rpm for 8-10 minutes.

9. The PRP separating gel prepared by the method according to any one of claims 1-6.

10. A plasma separation blood collection tube, comprising the PRP separation gel prepared by the method of any one of claims 1-6.

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