Method for preparing structurally intact red blood cell membrane and use thereof
Patent Information
- Application Number
- PCT/CN2025/102766
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-02-19
- Filing Date
- 2025-06-23
- Publication Date
- 2026-08-27
Smart Images
Figure CN2025102766_27082026_PF_FP_ABST
Abstract
Description
A method for preparing and applying a structurally intact erythrocyte membrane.
[0001] This application claims priority to Chinese Patent Application No. CN202510179568.2, filed on February 19, 2025, entitled "A structurally complete red blood cell membrane and its preparation method and application", the entire contents of which are incorporated herein by reference. Technical Field
[0002] This application belongs to the fields of cell engineering and biochemical detection technology, specifically relating to a method for preparing and applying a structurally intact red blood cell membrane. Background Technology
[0003] Red blood cell reagents are indispensable core chemical reagents in hematological testing, widely used in various clinical diagnostic procedures such as blood typing, antibody titer determination, neonatal hemolytic disease screening, and platelet antibody detection. They effectively identify blood group antigens and antibodies in the blood, helping medical personnel determine a patient's blood type compatibility, detect potential immune responses, and provide rapid diagnostic evidence in emergencies, playing a crucial role, especially in key areas such as transfusion medicine and perinatal medicine. However, existing red blood cell reagents require very stringent storage conditions, typically cold chain storage, and have a short shelf life, usually only 3–6 months. Furthermore, the antigenicity of the red blood cell membrane gradually decreases with increasing storage time, affecting the accuracy of test results. Moreover, the cellular components in red blood cell reagents are relatively fragile, and hemolysis and denaturation problems frequently occur during storage, further shortening their lifespan.
[0004] To address the aforementioned issues, existing technologies have proposed strategies that use engineered red blood cells to replace fresh red blood cell reagents. Examples include Chinese patents CN200510017117.1, CN200910158168.4, and CN201220684879.2, which employ different types of nanoparticles or solid microspheres as carriers to coat red blood cell membrane antigens onto their surfaces, thus mimicking red blood cell membrane antigens. However, these technologies typically involve fragmenting the red blood cell membrane to fit the surface coating of the nanoparticles. While this fragmentation process can preserve some of the activity of the red blood cell membrane antigens, it also introduces a series of potential problems. First, the mechanical forces such as ultrasound or lysis reagents used to prepare fragmented red blood cell membranes often cause irreversible damage, resulting in the loss of red blood cell integrity and the quantity of blood group antigens. Furthermore, the spatial structure may be altered, leading to reduced antigen activity and affecting the sensitivity and accuracy of blood group antibody detection, especially potentially causing misidentification of blood types in weakly agglutinated samples. Summary of the Invention
[0005] The purpose of this application is to overcome the shortcomings of the existing technology, maintain the integrity of the red blood cell membrane structure while retaining the key blood type antigens on its surface, avoid the problem of reduced antigenicity caused by red blood cell membrane fragmentation, and prepare an engineered red blood cell biomembrane that makes the agglutination reaction under antibody induction visible, has high sensitivity for blood type reverse typing detection, and can be preserved for a long time at room temperature.
[0006] To achieve the above objectives, this application provides a structurally intact erythrocyte membrane, which is obtained by lysing erythrocytes with a release solution and then separating the membrane; the release solution comprises 15-20 mM Ca(NO3)2 and 2-4% v / v fetal bovine serum.
[0007] Preferably, the release solution further includes 10 mM KHCO3 and / or 0.1 mM Na2EDTA.
[0008] Preferably, the pH value of the release solution is 7.2 to 7.4; and the solvent of the release solution is water.
[0009] This application also provides a method for preparing the red blood cell membrane described in the above technical solution, comprising the following steps: mixing the release solution and red blood cells evenly, lysing for 5-10 minutes, separating the solid and liquid, collecting the precipitate and washing it to obtain the red blood cell membrane.
[0010] Preferably, the number-to-volume ratio of the red blood cells to the released fluid is 1×10⁻⁶. 10 1 mL.
[0011] Preferably, the solid-liquid separation includes centrifugation, wherein the centrifugation speed is 7000 rpm and the time is 10 min;
[0012] The washing process uses an aqueous solution containing 2% v / v fetal bovine serum; the washing is performed three times, with the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the erythrocyte release solution being 1:1 for each wash.
[0013] This application also provides an engineered erythrocyte biomembrane, comprising an erythrocyte membrane and nanoparticles coupled to the surface of the erythrocyte membrane; the erythrocyte membrane is an erythrocyte membrane prepared using the preparation method described in the above technical solution.
[0014] Preferably, the mass ratio of the red blood cell membrane to the nanoparticles is 1:5.
[0015] This application also provides the application of the engineered erythrocyte biomembrane described above in one or more of the following:
[0016] (1) Preparation of blood typing reverse typing test products;
[0017] (2) Preparation of platelet antibody detection products;
[0018] (3) Prepare low-titer O-type whole blood detection products;
[0019] (4) Preparation of irregular antibody detection products;
[0020] (5) Prepare detection products for IgM anti-A and anti-B antibodies in whole blood and / or plasma;
[0021] (6) Prepare detection products for IgG anti-A and anti-B antibodies in whole blood and / or plasma;
[0022] (7) Prepare products for detecting neonatal hemolysis;
[0023] (8) Prepare blood type antibody adsorption products.
[0024] This application also provides a method for preserving the engineered erythrocyte biomembrane described in the above technical solution, comprising the following steps:
[0025] The engineered red blood cell biomembrane was kept at a constant temperature of 4.0℃ under vacuum of 0.0 Pa for 10 min to obtain the first treated sample.
[0026] The first treated sample was kept at a constant temperature of 0.0 Pa and -50.0 °C for 60 min to obtain the second treated sample.
[0027] The second treated sample was kept at a constant temperature of 0.0 Pa and -30.0 °C for 60 min to obtain the third treated sample.
[0028] The third-treated sample was kept at a constant temperature of 0.0 Pa and -45.0 °C for 120 min to obtain the fourth-treated sample.
[0029] The fourth treated sample was kept at a constant temperature of 12.0 Pa and -45.0 °C for 360 min to obtain the fifth treated sample.
[0030] Under a vacuum of 8.0 Pa for 60 min, the temperature of the fifth treated sample was raised to -30.0 °C, and then kept at a constant temperature for 360 min under a vacuum of 8.0 Pa and a temperature of -30.0 °C to obtain the sixth treated sample.
[0031] Under a vacuum of 5.0 Pa for 30 min, the temperature of the sixth treated sample was raised to -20.0 °C, and then kept at a constant temperature of 5.0 Pa and -20.0 °C for 60 min to obtain the seventh treated sample.
[0032] Under a vacuum of 5.0 Pa for 10 min, the temperature of the seventh treated sample was raised to -10.0 °C, and then kept at a constant temperature of 5.0 Pa and -10.0 °C for 30 min to obtain the eighth treated sample.
[0033] Under a vacuum of 5.0 Pa for 10 min, the temperature of the eighth processed sample was raised to 0.0 °C, and then kept at a constant temperature of 0.0 °C for 30 min under a vacuum of 5.0 Pa to obtain the ninth processed sample.
[0034] Under a vacuum of 3.0 Pa for 10 min, the temperature of the ninth processed sample was raised to 100.0 °C, and then kept at a constant temperature of 10.0 °C for 30 min under a vacuum of 3.0 Pa to obtain the tenth processed sample.
[0035] Under a vacuum of 5.0 Pa for 20 min, the temperature of the tenth treated sample was lowered to 0.0 °C, and then kept at a constant temperature of 0.0 °C for 40 min under a vacuum of 5.0 Pa to obtain the eleventh treated sample.
[0036] Under a vacuum of 3.0 Pa for 20 min, the temperature of the eleventh treated sample was raised to 10.0 °C, and then kept at a constant temperature of 10.0 °C for 40 min under a vacuum of 3.0 Pa to obtain the twelfth treated sample.
[0037] Under a vacuum of 3.0 Pa for 10 min, the temperature of the twelfth processed sample was raised to 25°C and kept at a constant temperature of 25.0°C for 60 min under a vacuum of 3.0 Pa to obtain the thirteenth processed sample.
[0038] The thirteenth-treatment sample was kept at a constant temperature of 25.0℃ for 60 minutes under vacuum of 2.0 Pa to obtain the fourteenth-treatment sample.
[0039] The fourteenth-stage sample was kept at a constant temperature of 25.0℃ under vacuum conditions of 0.0 Pa for 240 min. Beneficial effects:
[0040] This application utilizes a release solution to lyse red blood cells and separate the red blood cell membrane; the release solution comprises 15–20 mM Ca(NO3)2 and 2–4% v / v fetal bovine serum. By limiting the components of the release solution and using mild lysis conditions, this application can release protein components such as hemoglobin from the red blood cells, removing their cellular properties while preserving the integrity of their internal skeletal structure and the activity of membrane surface antigens. This maintains the integrity of the red blood cell membrane structure while retaining key blood group antigens on its surface, avoiding the problem of reduced antigenicity caused by red blood cell membrane fragmentation.
[0041] Furthermore, this application couples nanoparticles to the surface of the erythrocyte membrane, enabling visualization of the agglutination reaction of the erythrocyte membrane under antibody induction, thus obtaining an engineered erythrocyte biomembrane. Subsequently, the engineered erythrocyte biomembrane obtained through a specific preservation technique is preserved, which significantly reduces intracellular water content, effectively preventing damage to the engineered erythrocyte biomembrane from ice crystal formation, ensuring that the antigenicity of the erythrocyte biomembrane remains unaffected, and allowing for long-term preservation at room temperature, extending the shelf life to up to two years. It maintains its basic functions of reverse blood typing and one-step determination of blood type antibody titers, greatly improving its feasibility and practicality in clinical and laboratory applications. Attached Figure Description
[0042] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0043] Figure 1 shows the effect of different erythrocyte release solutions on the aggregation intensity of engineered erythrocyte biomembranes;
[0044] Figure 2 shows RBC and RBC. bm and RBC bm @Fluo's actual images under bright field and ultraviolet fluorescence;
[0045] Figure 3 shows RBC and RBC. bm and RBC bm @Fluo's cytoskeleton results under a confocal microscope;
[0046] Figure 4 shows RBC and RBC. bm and RBC bm @Fluo's image showing the expression of phospholipid bilayer and CD47 molecules on the surface of erythrocytes under a confocal microscope;
[0047] Figure 5 shows the results of reverse blood typing test.
[0048] Figure 6 shows the results of blood type antibody titer detection.
[0049] Figure 7 shows RBCs in liquid, lyophilized, and rehydrated states. bm @Fluo's actual images under bright field and ultraviolet fluorescence;
[0050] Figure 8 shows RBCs in liquid and lyophilized states. bm @Fluo graph showing the change in coagulation effect over storage time;
[0051] Figure 9 shows the platelet antibody detection results for negative samples N1-N3;
[0052] Figure 10 shows the platelet antibody detection results of positive samples P1 to P6;
[0053] Figure 11 shows the detection limit for P1 platelet antibody detection in positive samples;
[0054] Figure 12 shows the repeatability results of platelet antibody detection in negative sample N1 and positive sample P1. Detailed Implementation
[0055] This application provides a structurally intact erythrocyte membrane, which is obtained by lysing erythrocytes with a release solution and then separating it; the release solution includes 15-20 mM Ca(NO3)2 and 2-4% v / v fetal bovine serum.
[0056] In one embodiment, the concentration of Ca(NO3)2 in the release solution described in this application is 15 mM. In another embodiment, the concentration of fetal bovine serum in the release solution described in this application is 2% v / v. In one embodiment, the release solution described in this application further includes 10 mM KHCO3 and / or 0.1 mM Na2EDTA; in another embodiment, the release solution described in this application further includes 10 mM KHCO3 and 0.1 mM Na2EDTA. In one embodiment, the pH value of the release solution described in this application is 7.2–7.4; in another embodiment, the pH value of the release solution described in this application is 7.2. In one embodiment, the solvent of the release solution described in this application is water. Improvements to the release solution formulation, i.e., other formulations obtained by non-inventive adjustments, improvements, or changes to the formulation, should also be considered as protected content of this application. These improvements or changes, which optimize or adjust the formulation or its application field without changing the basic technical concept of this application, are also within the scope of protection of this application.
[0057] This application provides a method for preparing the red blood cell membrane described in the above technical solution, comprising the following steps: mixing the release solution and red blood cells evenly, lysing for 5-10 minutes, separating the solid and liquid, collecting the precipitate, washing it, and obtaining the red blood cell membrane.
[0058] In one embodiment, the quantity-to-volume ratio of red blood cells to the release fluid described in this application is 1×10⁻⁶. 10 Quantity: 1 mL. As one embodiment, the solid-liquid separation described in this application includes centrifugation at 7000 rpm for 10 min. As one embodiment, the washing described in this application uses an aqueous solution containing 2% v / v fetal bovine serum. As one embodiment, the washing is performed three times, with the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the release solution being 1:1 for each wash. As one embodiment, this application uses a vortex mixer to thoroughly mix the release solution and red blood cells.
[0059] The release solution described in this application has a mild composition. Using this solution for lysis releases hemoglobin and other protein components from inside red blood cells, removing their cellular properties while preserving the integrity of their internal skeletal structure and the activity of membrane surface antigens, thus preparing a red blood cell biomembrane. The red blood cell release solution described in this application maintains the integrity of the red blood cell membrane structure while retaining crucial blood group antigens on its surface, avoiding the problem of reduced antigenicity caused by red blood cell membrane fragmentation.
[0060] This application also provides an engineered erythrocyte biomembrane, comprising an erythrocyte membrane and nanoparticles coupled to the surface of the erythrocyte membrane; the erythrocyte membrane is the erythrocyte membrane prepared by the preparation method described in the above technical solution.
[0061] In one embodiment, the mass ratio of the red blood cell membrane to the nanoparticles in this application is 1:5. In one embodiment, the nanoparticles in this application include one or more of fluorescent nanoparticles, photosensitizer nanoparticles, latex nanoparticles, colloidal gold, (superpara)magnetic iron oxide nanoparticles, and aggregation-induced emission nanoparticles; in another embodiment, the nanoparticles in this application are fluorescent nanoparticles, photosensitizer nanoparticles, latex nanoparticles, colloidal gold, (superpara)magnetic iron oxide nanoparticles, or aggregation-induced emission nanoparticles. In one embodiment, the fluorescent nanoparticles in this application are Fluo. In one embodiment, the aggregation-induced emission nanoparticles in this application are AIE. In one embodiment, the latex nanoparticles in this application include one or more of red latex nanoparticles (RLNP), black latex nanoparticles (BLNP), and purple latex nanoparticles (PLNP); in another embodiment, the latex nanoparticles in this application include red latex nanoparticles (RLNP), black latex nanoparticles (BLNP), or purple latex nanoparticles (PLNP). As one embodiment, the (superpara)magnetic iron oxide nanoparticles described in this application include Fe3O4 magnetic nanoparticles.
[0062] This application couples nanoparticles to the surface of the erythrocyte membrane, ensuring stable binding between the nanoparticles and the erythrocyte membrane, enabling it to emit a detectable signal. The labeled erythrocyte membrane can be effectively tracked and used for subsequent agglutination reaction monitoring. This application does not impose strict requirements on the method of coupling nanoparticles to the surface of the erythrocyte membrane; conventional methods in the art, such as click chemistry, can be used.
[0063] In view of the advantages of the engineered erythrocyte biomembrane, its application in one or more of the following is also within the scope of protection of this application: (1) preparation of blood typing reverse typing products; (2) preparation of platelet antibody detection products; (3) preparation of low-titer O-type whole blood detection products; (4) preparation of irregular antibody detection products; (5) preparation of IgM anti-A and anti-B antibody detection products in whole blood and / or plasma; (6) preparation of IgG anti-A and anti-B antibody detection products in whole blood and / or plasma; (7) preparation of neonatal hemolysis detection products; (8) preparation of blood type antibody adsorption products. As one embodiment, the products described in this application include reagents.
[0064] This application uses the preparation of human erythrocyte membranes as an example in the embodiments, but it should not be construed as covering the entire scope of protection of this application. It also applies to the preparation of erythrocyte membranes of animals such as cats, dogs, cattle, and monkeys.
[0065] This application also provides a method for preserving the engineered erythrocyte biomembrane described in the above technical solution, comprising the following steps:
[0066] The engineered red blood cell biomembrane was kept at a constant temperature of 4.0℃ under vacuum of 0.0 Pa for 10 min to obtain the first treated sample.
[0067] The first treated sample was kept at a constant temperature of 0.0 Pa and -50.0 °C for 60 min to obtain the second treated sample.
[0068] The second treated sample was kept at a constant temperature of 0.0 Pa and -30.0 °C for 60 min to obtain the third treated sample.
[0069] The third-treated sample was kept at a constant temperature of 0.0 Pa and -45.0 °C for 120 min to obtain the fourth-treated sample.
[0070] The fourth treated sample was kept at a constant temperature of 12.0 Pa and -45.0 °C for 360 min to obtain the fifth treated sample.
[0071] Under a vacuum of 8.0 Pa for 60 min, the temperature of the fifth treated sample was raised to -30.0 °C, and then kept at a constant temperature for 360 min under a vacuum of 8.0 Pa and a temperature of -30.0 °C to obtain the sixth treated sample.
[0072] Under a vacuum of 5.0 Pa for 30 min, the temperature of the sixth treated sample was raised to -20.0 °C, and then kept at a constant temperature of 5.0 Pa and -20.0 °C for 60 min to obtain the seventh treated sample.
[0073] Under a vacuum of 5.0 Pa for 10 min, the temperature of the seventh treated sample was raised to -10.0 °C, and then kept at a constant temperature of 5.0 Pa and -10.0 °C for 30 min to obtain the eighth treated sample.
[0074] Under a vacuum of 5.0 Pa for 10 min, the temperature of the eighth processed sample was raised to 0.0 °C, and then kept at a constant temperature of 0.0 °C for 30 min under a vacuum of 5.0 Pa to obtain the ninth processed sample.
[0075] Under a vacuum of 3.0 Pa for 10 min, the temperature of the ninth processed sample was raised to 100.0 °C, and then kept at a constant temperature of 10.0 °C for 30 min under a vacuum of 3.0 Pa to obtain the tenth processed sample.
[0076] Under a vacuum of 5.0 Pa for 20 min, the temperature of the tenth treated sample was lowered to 0.0 °C, and then kept at a constant temperature of 0.0 °C for 40 min under a vacuum of 5.0 Pa to obtain the eleventh treated sample.
[0077] Under a vacuum of 3.0 Pa for 20 min, the temperature of the eleventh treated sample was raised to 10.0 °C, and then kept at a constant temperature of 10.0 °C for 40 min under a vacuum of 3.0 Pa to obtain the twelfth treated sample.
[0078] Under a vacuum of 3.0 Pa for 10 min, the temperature of the twelfth processed sample was raised to 25°C and kept at a constant temperature of 25.0°C for 60 min under a vacuum of 3.0 Pa to obtain the thirteenth processed sample.
[0079] The thirteenth-treatment sample was kept at a constant temperature of 25.0℃ for 60 minutes under vacuum of 2.0 Pa to obtain the fourteenth-treatment sample.
[0080] The fourteenth-stage sample was kept at a constant temperature of 25.0℃ under vacuum conditions of 0.0 Pa for 240 min.
[0081] The preservation method provided in this application employs a specific procedure that significantly reduces intracellular water content, effectively preventing ice crystal formation and damage to engineered erythrocyte biomembranes, thus ensuring that the antigenicity of the membrane remains unaffected. Engineered erythrocyte biomembranes treated using this method can be stored at room temperature for extended periods without the need for complex cold chain transportation, making them suitable for large-scale production and long-distance transport. The shelf life can be extended to two years, maintaining their basic functions of reverse blood typing and one-step determination of blood type antibody titers, greatly enhancing their feasibility and practicality in clinical and laboratory applications.
[0082] To further illustrate this application, the following will describe in detail, with reference to the accompanying drawings and embodiments, a method for preparing a structurally intact red blood cell membrane and its application.
[0083] Example 1
[0084] A red blood cell release solution consisting of 15 mM Ca(NO3)2, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH of 7.2.
[0085] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2 and 20mL FBS, dissolve them in 850mL H2O, adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL, and store at 4℃.
[0086] Example 2
[0087] A red blood cell release solution consisting of 15 mM Ca(NO3)2, 10 mM KHCO3, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH of 7.2.
[0088] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2, 1.0012g KHCO3 and 20mL LFBS, dissolve them in 850mL H2O, adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL, and store at 4℃.
[0089] Example 3
[0090] A red blood cell release solution consisting of 15 mM Ca(NO3)2, 0.1 mM Na2EDTA, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH of 7.2.
[0091] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2, 0.0336g Na2EDTA and 20mL FBS, dissolve them in 850mL H2O, adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL, and store at 4℃.
[0092] Example 4
[0093] A red blood cell release solution consisting of 15 mM Ca(NO3)2, 10 mM KHCO3, 0.1 mM Na2EDTA, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH of 7.2.
[0094] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2, 1.0012g KHCO3, 0.0336g Na2EDTA and 20mL LFBS, dissolve them in 850mL H2O, adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL, and store at 4℃.
[0095] Comparative Example 1
[0096] A red blood cell release solution consisting of 15 mM Ca(NO3)2 and the remainder water, with a pH of 7.2.
[0097] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2 and dissolve it in 850mL H2O. Adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL and store at 4℃.
[0098] Comparative Example 2
[0099] A red blood cell release solution, consisting of 15 mM Ca(NO3)2, 10 mM KHCO3 and the balance water, with a pH of 7.2.
[0100] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2 and 1.0012g KHCO3, dissolve them in 850mL H2O, adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL, and store at 4℃.
[0101] Comparative Example 3
[0102] A red blood cell release solution, consisting of 15 mM Ca(NO3)2, 0.1 mM Na2EDTA and the balance water, with a pH of 7.2.
[0103] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2 and 0.0336g Na2EDTA, dissolve them in 850mL H2O, adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL, and store at 4℃.
[0104] Comparative Example 4
[0105] A red blood cell release solution, consisting of 15 mM Ca(NO3)2, 10 mM KHCO3, 0.1 mM Na2EDTA and the balance water, with a pH of 7.2.
[0106] Taking the preparation of 1000mL of the red blood cell release solution as an example, the specific preparation method is as follows: Weigh 1.6650g Ca(NO3)2, 1.0012g KHCO3 and 0.0336g Na2EDTA, dissolve them in 850mL H2O, adjust the pH value to 7.2 using K2CO3, then add H2O to 1000mL, and store at 4℃.
[0107] Example 5
[0108] Preparation of Fluo nanoparticle-labeled erythrocyte biomembranes
[0109] 1. Separation and washing of red blood cells
[0110] Red blood cells were isolated from type A / B / O whole blood samples. The whole blood was centrifuged at 1000 rpm for 5 min to obtain concentrated red blood cells. The concentrated red blood cells were washed three times with physiological saline, the supernatant was discarded, and the red blood cell pellet was retained for later use.
[0111] 2. Lysis of red blood cells
[0112] Using the erythrocyte release solutions from Examples 1-4 and Comparative Examples 1-4 respectively, the erythrocyte precipitate obtained in step 1 was mixed with the erythrocyte release solution at a ratio of 1×10⁻⁶. 10 Mix 1 mL of the sample with a volume ratio of 1 mL, thoroughly mix using a vortex mixer, centrifuge at 7000 rpm for 10 min, discard the supernatant, and collect the red blood cell membrane precipitate (RBC).
[0113] 3. Preparation of erythrocyte biomembranes
[0114] The erythrocyte membrane precipitate obtained in step 2 was mixed with purified water containing 2% v / v fetal bovine serum (FBS); the volume ratio of the purified water containing 2% v / v fetal bovine serum (FBS) to the erythrocyte release solution from step 2 was 1:1. The mixture was washed repeatedly three times to further remove residual free hemoglobin, ultimately preparing a complete erythrocyte biomembrane (RBC). bm ).
[0115] 4. Preparation of nanoparticle-labeled erythrocyte biomembranes
[0116] The erythrocyte biomembrane prepared in step 3 was mixed with fluorescent nanoparticles (Fluorescent Nanoparticles, Fluo, particle size 200 nm, excitation / emission wavelength: Ex / Em(nm) 365 / 615) at a ratio of 1:5. The nanoparticles were then coupled to the erythrocyte membrane surface using click chemistry to obtain a nanoparticle-labeled erythrocyte biomembrane, i.e., an engineered erythrocyte biomembrane (RBC). bm @Fluo).
[0117] Test Example 1
[0118] Red blood cell membrane aggregation strength test
[0119] Using engineered erythrocyte biofilms obtained in step 4 of Example 5 with different erythrocyte release solutions as test samples, different test samples were reacted with different titers (1:256, 1:128, 1:64, 1:32, 1:16, 1:8, and 1:4) of anti-A / B IgM antibodies. The reaction results for each titer were scored according to the erythrocyte agglutination scoring criteria in the AABB Technical Manual. The agglutination scores corresponding to the seven titers were added together to obtain the final agglutination score. The agglutination score reflects the intensity of the antigen-antibody reaction: the higher the score, the more obvious the agglutination reaction, the stronger the antigen-antibody binding, and thus the stronger the erythrocyte membrane antigen. The specific agglutination scoring criteria are as follows, and the scoring results are shown in Figure 1 and Table 1.
[0120] 0 points: No agglutination, the solution is homogeneous, and there are no visible red blood cell clumps or agglutination.
[0121] 3 points: Suspected agglomeration, extremely weak reaction, only a small number of tiny particles or uncertain agglomeration phenomena are seen in the solution;
[0122] 5 points: Weak agglutination, small and sparse red blood cell agglutination clusters are visible in the solution, and the background is relatively turbid;
[0123] 8 points: Moderate agglutination, medium-sized red blood cell agglutination clusters are visible in the solution, and the background is relatively clear.
[0124] 10 points: Strong agglutination, large red blood cell clumps are visible in the solution, with a clear background and no free red blood cells.
[0125] 12 points: Extremely strong agglutination, red blood cells are completely agglutinated into a large clump, the solution is clear and there are no free red blood cells.
[0126] Table 1 Aggregation intensity of engineered erythrocyte biomembranes by different erythrocyte release fluids Note: Compared with Comparative Example 1, * in the table indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.
[0127] As shown in Figure 1 and Table 1, Comparative Example 1, using a single reagent (Ca(NO3)2), had a low agglutination score, with a mean of 33.00, indicating that it might have a negative impact on the agglutination results during erythrocyte lysis. While the agglutination score improved somewhat after introducing other reagents (such as KHCO3 or Na2EDTA), the difference was not significant, and the mean remained around 32.80. This indicates that adding these reagents alone has a limited promoting effect on the agglutination reaction. In contrast, the addition of FBS significantly improved the agglutination score of erythrocytes. Examples 1 (Ca(NO3)2 + FBS), 2 (Ca(NO3)2 + KHCO3 + FBS), and 3 (Ca(NO3)2 + Na2EDTA + FBS) all showed significant enhancement of the agglutination reaction, with mean values reaching 49.40, 52.20, and 53.20, respectively. Example 4 (Ca(NO3)2+KHCO3+Na2EDTA+FBS) showed the most outstanding performance, with a mean agglutination score as high as 59.60 and a standard deviation of only 0.89, demonstrating high stability and consistency in the agglutination effect. Therefore, the introduction of FBS played a key role in promoting the agglutination reaction of erythrocyte membranes, especially when used in combination with other chemical reagents (such as KHCO3 and Na2EDTA). The synergistic effect of the four components in Example 4 may have significantly enhanced the stability of erythrocyte membranes and optimized the conditions for the agglutination reaction, thereby achieving the best agglutination effect.
[0128] Test Example 2
[0129] Integrity Detection
[0130] 1. Take the erythrocyte membrane precipitate (RBC) and intact erythrocyte biomembrane (RBC) obtained in Example 5 using the erythrocyte release solution from Example 4. bm ) and engineered red blood cell biomembranes (RBCs) bm @Fluo), detected under bright field (BF) and ultraviolet fluorescence (UVFL), respectively, the results showed that the engineered erythrocyte biomembrane (RBC) bm @Fluo emits bright red fluorescence under ultraviolet light, and the fluorescence is evenly distributed (Figure 2).
[0131] 2. Take the erythrocyte membrane precipitate (RBC) and intact erythrocyte biomembrane (RBC) obtained in Example 5 using the erythrocyte release solution from Example 4. bm ) and engineered red blood cell biomembranes (RBCs) bm @Fluo), under confocal microscopy, red represents actin, green represents tubulin, and yellow represents a combined red and green image. The results indicate that RBCs and RBCs... bm and RBCbm @Fluo's erythrocyte membranes remained intact, and the cytoskeleton structure was partially preserved (Figure 3).
[0132] 3. Take the erythrocyte membrane precipitate (RBC) and intact erythrocyte biomembrane (RBC) obtained in Example 5 using the erythrocyte release solution from Example 4. bm ) and engineered red blood cell biomembranes (RBCs) bm Using conventional methods, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DIL) was used to label the phospholipid bilayer, and erythrocytes were labeled with PE-labeled anti-human CD47 antibody. Confocal microscopy was used to detect the expression of CD47 molecules on the phospholipid bilayer (PBL) and the erythrocyte surface. The results showed that the labeling process had little effect on the phospholipid bilayer, and the erythrocyte membrane remained intact and was not fragmented (Figure 4). Based on the conclusions of step 2, it can be determined that the engineered erythrocyte biomembrane obtained in this application can maintain the integrity of erythrocyte membrane antigens.
[0133] Test Example 3
[0134] Performance testing
[0135] 1. The engineered erythrocyte biomembrane (RBC) obtained in Example 5 was used. bm @Fluo) and erythrocyte membrane precipitation (RBC) were used for blood typing using human ABO reverse typing erythrocyte reagent (purchased from Shanghai Blood Biopharmaceutical Co., Ltd., denoted as RBC) according to the product instructions. The results are shown in Figure 5. As can be seen from Figure 5, RBC and RBC... bm The agglutination phenomenon caused by blood group antigen-antibody reactions is similar between @Fluo.
[0136] 2. Using the B-type engineered erythrocyte biofilm and B-type erythrocyte membrane precipitate obtained in Example 5, and with anti-AIgM antibody as a negative control, B-type erythrocytes (denoted as RBCs) and B-type RBCbm@Fluo (denoted as RBCs) were detected. bm The agglutination results of @Fluo with different titers (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) of anti-B IgM antibody are shown in Figure 6; where (-) in Figure 6 is the negative control (anti-AIgM antibody).
[0137] As can be seen from Figure 6, RBC and RBC bm @Fluo have consistent agglutination scores caused by blood group antigen-antibody reactions.
[0138] 3. Using bio-layer interferometry (BLI), the B-type erythrocyte membrane precipitate (RBC) and intact B-type erythrocyte biomembrane (RBC) obtained from the erythrocyte release solution of Example 4 in Example 5 were measured. bm ) and type B engineered erythrocyte biomembrane (RBC) bm The dissociation rate constant Kon, binding rate constant Koff, and equilibrium dissociation constant (affinity) KD of the antigen-antibody reaction between @Fluo and anti-B IgM antibody (purchased from Shanghai Blood Biopharmaceutical Co., Ltd.) are calculated. The results are shown in Table 2.
[0139] Table 2. Results of Antigen-Antibody Reaction Detection
[0140] As can be seen from Table 2, RBC, RBC bm、 RBC bm @Fluo's equilibrium dissociation constants are all around 10. -8 Order of magnitude, RBC bm The preparation and Fluo labeling process do not affect the affinity of the antigen-antibody reaction.
[0141] Example 6
[0142] freeze-drying preservation
[0143] The engineered erythrocyte biofilm (RBC) obtained in Example 5 was processed according to the procedure in Table 3. bm @Fluo) performed freeze-drying. The freeze-dried RBCs bm @Fluo appears as a white, uniform, loose powder, with an average moisture content reduced to (4.37±0.32)%, meeting the requirements for effective freeze-drying. The freeze-dried RBCs were gently shaken in sterile deionized water. bm @Fluo performs rehydration, and the lyophilized formulation dissolves into a clear liquid without any unexpected coagulation. The rehydration time is approximately 10 seconds.
[0144] Table 3 Freeze-drying procedures and temperature control steps for freeze-drying preservation.
[0145] Test Example 4
[0146] Performance testing
[0147] 1. Take the engineered erythrocyte biomembrane (RBC) from Example 6 (i.e., before lyophilization), lyophilized and rehydrated. bm@Fluo), and detected under bright field (BF) and ultraviolet fluorescence (UVFL), respectively. The results showed that engineered erythrocyte biomembranes in different states showed uniform fluorescence under ultraviolet light (Figure 7).
[0148] 2. Take the engineered erythrocyte biomembrane (RBC) from Example 6 in liquid state (i.e., before freeze-drying). bm @Fluo) was stored at 4°C, and the lyophilized engineered red blood cell biomembrane (RBC) from Example 6 was taken. bm @Fluo), RBCs were stored at room temperature and 4°C, and their different states were monitored during the storage process. bm The agglutination of @Fluo samples is shown in Figure 8. As can be seen from Figure 8, samples stored in liquid form at 4°C showed a decrease in agglutination score of approximately 20% within 3 months, and a significant decrease of 70% by 6 months, essentially losing their detection function. Further monitoring was discontinued after 8 months. In contrast, samples stored in lyophilized form at room temperature and reconstituted within a two-year storage period showed no significant change in agglutination score compared to before lyophilization.
[0149] Test Example 5
[0150] Blood type reverse typing test clinical trial
[0151] Using the clinical microcolumn gelation method as a comparative method, RBCs rehydrated after lyophilization for 7 days as described in Example 6 were used. bm @Fluo tested 641 clinical plasma samples, and the results are shown in Tables 4 and 5.
[0152] Table 4. Qualitative results of blood typing for blood type 641
[0153] Table 5. Clinical trial results of blood type reverse typing test
[0154] As can be seen from Tables 4 and 5, using RBC bm @Fluo testing revealed 188 cases of type A, 143 cases of type B, 48 cases of type AB, and 262 cases of type O, with a 100% concordance rate. The experimental results indicate that RBC... bm @Fluo demonstrated good accuracy in reverse blood typing.
[0155] Example 7
[0156] Preparation of AIE nanoparticle-labeled IgG-sensitized erythrocyte biomembranes
[0157] 1. Preparation of IgG-sensitized red blood cells
[0158] Add 2 drops of RhD(+) hematocrit cells and 2 drops of IgG anti-D blood typing reagent to a clean test tube, mix well, and incubate at 37°C for 30 min, mixing continuously during incubation. Wash 6–8 times with physiological saline (1000×g, 1 min). After the final centrifugation, discard the supernatant and blot away any remaining liquid from the tube opening with absorbent paper to prepare sensitized hematocrit cells. In another clean test tube, add 1 mL of physiological saline and then 30 μL of sensitized hematocrit cells to prepare a 3% IgG antibody-sensitized hematocrit cell saline suspension.
[0159] 2. Release of IgG-sensitized erythrocyte biomembranes
[0160] Using the erythrocyte release solution from Example 4, the 3% IgG antibody-sensitized erythrocyte saline suspension obtained in step 1 was mixed with the erythrocyte release solution at a ratio of 1×10⁻⁶. 10 One red blood cell: 1 mL of the mixture was mixed by volume, thoroughly mixed by vortexing, centrifuged at 7000 rpm for 10 min, the supernatant was discarded and the precipitate was collected;
[0161] The precipitate was mixed with purified water containing 2% v / v fetal bovine serum (FBS); the volume ratio of purified water containing 2% v / v fetal bovine serum (FBS) to erythrocyte release solution was 1:1. The mixture was washed three times to further remove residual free hemoglobin, ultimately preparing a complete erythrocyte biofilm (RBC). bm ).
[0162] 3. Preparation of AIE nanoparticle-labeled IgG-sensitized erythrocyte biomembranes
[0163] The erythrocyte biomembrane prepared in step 2 was mixed with aggregation-induced emission (AIE) nanoparticles (purchased from the Institute for Advanced Study, Shenzhen University) at a ratio of 1:5. The nanoparticles were then coupled to the erythrocyte membrane surface using click chemistry to obtain a nanoparticle-labeled erythrocyte biomembrane, namely, an AIE nanoparticle-labeled IgG-sensitized erythrocyte biomembrane (AIE@RBC). bm ).
[0164] Test Example 6
[0165] Platelet antibody test
[0166] 1. The experimental sample information is shown in Table 6. The experimental environment was: temperature 26℃, relative humidity 47%.
[0167] Table 6 Experimental Sample Information
[0168] 2. Platelet antibody detection verification steps
[0169] 1) Remove microplate strips from the pouch as needed. Unused microplate strips can be stored in the pouch containing desiccant. 2) Add one drop (50 μL) of platelet antigen screening solution to each well (patient serum well) using a plastic pipette. Perform positive control (positive control well, PC) and negative control (negative control well, NC) tests on each microplate strip. 3) Centrifuge at 50 rf for 5 min to fix the platelets onto the microplate surface (centrifuge continuously). 4) Wash the plate three times manually with 150 μL of 0.005% PBS / Tween to remove unbound platelets. Add the washing buffer dropwise using a multichannel pipette and let stand for 10 seconds. After each wash, slowly pour and gently tap to remove the PBS / Tween. 5) Add 2 drops (100 μL) of platelet antibody detection solution (LISS solution) to each well. 6) Add 1 drop (50 μL) of positive or negative control from the platelet antibody test kit to the appropriate well. The positive control checks whether the donor platelets are sufficient. 7) Add 1 drop (50 μL) of patient serum to the remaining wells. 8) Incubate the microplate at 37°C for 30 min (sealing the wells during incubation). 9) Wash the plate manually 5 times with 150 μL of PBS / Tween, following step 4). 10) Immediately after washing, add 1 drop of anti-IgG reagent from the platelet antibody test kit to each well. 11) Add 1 drop (50 μL) of indicator red blood cells from the platelet antibody test kit and AIE@RBCbm obtained in Example 7 to each well, and gently vortex. 12) Centrifuge the microplate at 200 rcf for 5 min (continuous centrifugation). 13) Interpret the results visually or using a plate reader.
[0170] 3. Reference sample conformity test
[0171] (1) Using the three negative samples N1, N2 and N3 in Table 6 as negative references, the tests were performed in the manner of step 2. The results showed that the detection results of the three negative samples N1, N2 and N3 were all negative, and the compliance rate of the negative references was 100% (Figure 9).
[0172] (2) The three strong positive samples P1, P2 and P3 and the three weak positive samples P4, P5 and P6 in Table 6 were used as positive references. Among them, P1, P3 and P6 all contained HLA+HPA antibodies. They were tested according to the method in step 2. The results showed that the experimental results of P1, P3 and P6 were all positive, and the positive reference concordance rate was 100% (Figure 10).
[0173] 4. Limit of detection
[0174] Using the P1 sample containing HLA+HPA antibodies as a positive reference, serial dilutions were performed, and the test was conducted according to step 2. The results showed that the P1 sample was still positive after a 32-fold dilution (Figure 11).
[0175] 5. Repeatability testing
[0176] Using P1 samples containing HLA+HPA antibodies as positive references and N1 samples as negative references, the tests were repeated 10 times according to step 2. The experiment showed that the test results of N1 samples were all negative, and the test results of P1 samples were all positive, with consistent intensity (Figure 12).
[0177] As can be seen from the above, the AIE@RBC obtained in Example 7 bm It can replace indicator red blood cells for platelet antibody detection, ensuring the accuracy of platelet antibody testing.
[0178] Example 8
[0179] Preparation of RLNP nanoparticle-labeled type A erythrocyte biomembranes
[0180] 1. Separation and washing of red blood cells
[0181] Red blood cells were separated from type A whole blood samples. The whole blood was centrifuged at 1000 rpm for 5 min to obtain concentrated red blood cells. The concentrated red blood cells were washed three times with physiological saline, the supernatant was discarded, and the red blood cell precipitate was retained for later use.
[0182] 2. Release of type A erythrocyte biomembrane
[0183] Using the erythrocyte release solution from Example 4, the erythrocyte precipitate obtained in step 1 was mixed with the erythrocyte release solution at a ratio of 1×10⁻⁶. 10 One red blood cell: 1 mL of the mixture was mixed by volume, thoroughly mixed by vortexing, centrifuged at 7000 rpm for 10 min, the supernatant was discarded and the precipitate was collected;
[0184] The precipitate was mixed with purified water containing 2% v / v fetal bovine serum (FBS); the volume ratio of purified water containing 2% v / v fetal bovine serum (FBS) to erythrocyte release solution was 1:1. The mixture was washed three times to further remove residual free hemoglobin, ultimately preparing a complete type A erythrocyte biofilm (RBC). bm ).
[0185] 3. Preparation of nanoparticle-labeled type A erythrocyte biomembranes
[0186] The type A erythrocyte biomembrane prepared in step 2 was mixed with red latex nanoparticles (RLNPs) at a ratio of 1:5. The nanoparticles were then coupled to the erythrocyte membrane surface using click chemistry to obtain a nanoparticle-labeled erythrocyte biomembrane, i.e., an engineered erythrocyte biomembrane (A-RBC). bm @RLNP).
[0187] Example 9
[0188] Preparation of BLNP nanoparticle-labeled type B erythrocyte biomembranes
[0189] 1. Separation and washing of red blood cells
[0190] Red blood cells were separated from type B whole blood samples. The whole blood was centrifuged at 1000 rpm for 5 min to obtain concentrated red blood cells. The concentrated red blood cells were washed three times with physiological saline, the supernatant was discarded, and the red blood cell pellet was retained for later use.
[0191] 2. Release of B-type erythrocyte biomembrane
[0192] Using the erythrocyte release solution from Example 4, the erythrocyte precipitate obtained in step 1 was mixed with the erythrocyte release solution at a ratio of 1×10⁻⁶. 10 One red blood cell: 1 mL of the mixture was mixed by volume, thoroughly mixed by vortexing, centrifuged at 7000 rpm for 10 min, the supernatant was discarded and the precipitate was collected;
[0193] The precipitate was mixed with purified water containing 2% v / v fetal bovine serum (FBS); the volume ratio of purified water containing 2% v / v fetal bovine serum (FBS) to erythrocyte release solution was 1:1. The mixture was washed three times to further remove residual free hemoglobin, ultimately preparing a complete type B erythrocyte biofilm (RBC). bm ).
[0194] 3. Preparation of nanoparticle-labeled type B erythrocyte biomembranes
[0195] The B-type erythrocyte biomembrane prepared in step 2 was mixed with black latex nanoparticles (BLNP) at a ratio of 1:5. The nanoparticles were then coupled to the erythrocyte membrane surface using click chemistry to obtain a nanoparticle-labeled erythrocyte biomembrane, i.e., an engineered erythrocyte biomembrane (B-RBC). bm @BLNP).
[0196] Test Example 7
[0197] Low-titer O whole blood (LTOWB) testing
[0198] (1) Based on the US low-titer type O whole blood (LTOWB) titer standard (1:128), the A-RBCs obtained in Example 8 were used. bm @RLNP was used to replace type A red blood cells in the detection of plasma titers. The results showed that using A-RBCs... bm When @RLNP was used to replace type A red blood cells in plasma titer testing, the results were highly consistent with those of traditional type A red blood cell testing. Visible agglutination was observed at titers higher than 1:128; however, no agglutination was observed at titers lower than 1:128. This indicates that A-RBCs... bm @RLNP has demonstrated good substitution performance in blood group antibody testing, providing a reliable and effective solution for blood group antibody testing.
[0199] (2) Based on the US low-titer type O whole blood (LTOWB) titer standard (1:128), the B-RBCs obtained in Example 9 were used. bm @BLNP was used to replace type B red blood cells in the detection of plasma titers. The results showed that using B-RBCs... bm When @RLNP was used to replace type A red blood cells in plasma titer testing, the results were highly consistent with those of traditional type B red blood cell testing. Visible agglutination was observed at titers higher than 1:128; however, no agglutination was observed at titers lower than 1:128. This indicates that B-RBCs... bm @RLNP has demonstrated good substitution performance in blood group antibody testing, providing a reliable and effective solution for blood group antibody testing.
[0200] Example 10
[0201] Preparation of PLNP nanoparticle-labeled erythrocyte biomembranes
[0202] 1. Separation and washing of red blood cells
[0203] Red blood cells were isolated from type A / B / O whole blood samples. The whole blood was centrifuged at 1000 rpm for 5 min to obtain concentrated red blood cells. The concentrated red blood cells were washed three times with physiological saline, the supernatant was discarded, and the red blood cell pellet was retained for later use.
[0204] 2. Release of erythrocyte biomembrane
[0205] Using the erythrocyte release solution from Example 4, the erythrocyte precipitate obtained in step 1 was mixed with the erythrocyte release solution at a ratio of 1×10⁻⁶. 10One red blood cell: 1 mL of the mixture was mixed by volume, thoroughly mixed by vortexing, centrifuged at 7000 rpm for 10 min, the supernatant was discarded and the precipitate was collected;
[0206] The precipitate was mixed with purified water containing 2% v / v fetal bovine serum (FBS); the volume ratio of purified water containing 2% v / v fetal bovine serum (FBS) to erythrocyte release solution was 1:1. The mixture was washed three times to further remove residual free hemoglobin, ultimately preparing a complete erythrocyte biofilm (RBC). bm ).
[0207] 3. Preparation of nanoparticle-labeled erythrocyte biomembranes
[0208] The erythrocyte biomembrane prepared in step 2 was mixed with purple latex nanoparticles (PLNP) at a ratio of 1:5. The nanoparticles were then coupled to the erythrocyte membrane surface using click chemistry to obtain a nanoparticle-labeled erythrocyte biomembrane, i.e., an engineered erythrocyte biomembrane (PLNP@RBC). bm ).
[0209] Test Example 8
[0210] Irregular antibody detection
[0211] Using the PLNP@RBC obtained in Example 10 bm This reagent replaces traditional anti-red blood cell screening reagents for the detection of irregular antibodies and is compared with conventional anti-red blood cell screening reagents. The antibodies detected include, but are not limited to, anti-D, anti-C, anti-E, anti-c, anti-e, and anti-JK antibodies. a JK b M, N, S, s, Fy a 、Fy b , K, k, Kp a Kp b Le a Le b P1, Xg a Lu a Lu b Di a The results of the positive antibody standards are shown in Tables 7 and 8.
[0212] Table 7 Results of routine detection of irregular antibodies against screening red blood cells (human red blood cells) Note: In the table, "+" indicates positive and "-" indicates negative, and the same applies below.
[0213] Table 8 PLNP@RBC bm Irregular antibody test results
[0214] As can be seen from Tables 7 and 8, PLNP@RBC bm -I, II, III (PI, P-II, P-III) still retain D, C, E, c, e, JK a JK b M, N, S, s, Fy a 、Fy b , K, k, Kp a Kp b Le a Le b P1, Xg a Lu a Lu b Di a The activity of the blood group antigen was confirmed, and its agglutination reaction was consistent with that of the conventional anti-red blood cell screening reagent, thus verifying its feasibility as an alternative reagent in the detection of irregular antibodies.
[0215] Example 11
[0216] Preparation of MNP nanoparticle-labeled erythrocyte biomembranes
[0217] 1. Separation and washing of red blood cells
[0218] Red blood cells were separated from type A / B whole blood samples. The whole blood was centrifuged at 1000 rpm for 5 min to obtain concentrated red blood cells. The concentrated red blood cells were washed three times with physiological saline, the supernatant was discarded, and the red blood cell pellet was retained for later use.
[0219] 2. Release of erythrocyte biomembrane
[0220] Using the erythrocyte release solution from Example 4, the erythrocyte precipitate obtained in step 1 was mixed with the erythrocyte release solution at a ratio of 1×10⁻⁶. 10 One red blood cell: 1 mL of the mixture was mixed by volume, thoroughly mixed by vortexing, centrifuged at 7000 rpm for 10 min, the supernatant was discarded and the precipitate was collected;
[0221] The precipitate was mixed with purified water containing 2% v / v fetal bovine serum (FBS); the volume ratio of purified water containing 2% v / v fetal bovine serum (FBS) to erythrocyte release solution was 1:1. The mixture was washed three times to further remove residual free hemoglobin, ultimately preparing a complete erythrocyte biofilm (RBC). bm ).
[0222] 3. Preparation of nanoparticle-labeled erythrocyte biomembranes
[0223] The erythrocyte biomembrane prepared in step 2 was mixed with Fe3O4 magnetic nanoparticles (MNPs) at a ratio of 1:5. The nanoparticles were then coupled to the surface of the erythrocyte membrane using click chemistry to obtain a nanoparticle-labeled erythrocyte biomembrane, i.e., an engineered erythrocyte biomembrane A-RBC. bm @MNP and B-RBC bm @MNP, A-RBC bm @MNP and B-RBC bm @MNPs were mixed at a 1:1 mass ratio to obtain engineered erythrocyte biomembranes (RBCs). bm @MNP.
[0224] Test Example 9
[0225] IgM / IgG anti-A and anti-B antibody titers detection
[0226] 2 mg of RBC obtained in Example 11 bm @MNP is incubated with 2 mL of whole blood or plasma at room temperature for 5 min to form blood type antibodies and RBCs. bm The specific binding of the MNP surface antigen was achieved. During this process, IgM / IgG anti-A and anti-B antibodies were effectively removed by magnetic adsorption. Then, the titers of IgM / IgG anti-A and anti-B antibodies in whole blood and plasma before and after adsorption were detected using the microcolumn gel electrophoresis method. The results are shown in Table 9.
[0227] Table 9. Titers of IgM / IgG anti-A and anti-B antibodies in whole blood and plasma.
[0228] As shown in Table 9, before adsorption, the titers of IgM / IgG anti-A and anti-B antibodies in the 6 plasma samples were all higher than 1:128; the titers of IgM / IgG anti-A and anti-B antibodies in the 6 whole blood samples were also higher than 1:128. (This information was obtained through RBC analysis.) bm After immunoadsorption treatment with @MNP, the titers of IgM / IgG anti-A and anti-B antibodies in all samples decreased significantly, falling below 1:32, indicating that this method can effectively remove blood type antibodies.
[0229] Test Case 10
[0230] Neonatal hemolysis test
[0231] Anti-human globulin test card (detection of incomplete antibodies against neonatal hemolytic disease of the fetus / infant), add A-RBCs to wells 1 and 4 respectively. bm @MNP erythrocyte membrane suspension 50 μL each; add B-RBCs to wells 2 and 5 respectively.bm @MNP erythrocyte membrane suspension 50 μL each; add O-RBCs to wells 3 and 6 respectively. bm @MNP erythrocyte membrane suspension, 50 μL each. Add 50 μL neonatal plasma to wells 1-3, and 50 μL neonatal erythrocyte eluate to wells 4-6. Incubate the test cards at 37°C for 15 min. Centrifuge using a microcolumn gel card centrifuge for 5 min (900 rpm for 2 min, 1500 rpm for 3 min), remove the test cards, and interpret the results. If RBC... bm @MNP agglutination blocks located on the gel surface or within the gel are considered positive, indicating the presence of antibodies corresponding to the antigen in the subject's serum or that the blood cells have been sensitized; if RBCs are present... bm If the MNP completely settles to the bottom of the gel and forms a red blood cell knot at the bottom of the gel tube, it is negative, indicating that no antibody induced by the corresponding antigen was detected in the serum, or that the blood cells were not sensitized by the antibody.
[0232] As can be seen from the above, the technical solution provided in this application can maintain the integrity of the red blood cell membrane structure, retain the key blood type antigens on its surface, avoid the problem of reduced antigenicity caused by red blood cell membrane fragmentation, and the engineered red blood cell biomembrane prepared can visualize the agglutination reaction induced by antibodies and can be stored for a long time at room temperature.
[0233] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative improvement, and these embodiments all fall within the protection scope of this application.
Claims
1. A method for preparing a structurally intact erythrocyte membrane, characterized in that, The process includes the following steps: mixing the release solution and red blood cells, lysing for 5-10 minutes, separating the solid and liquid phases, collecting the precipitate, washing it, and obtaining a structurally intact red blood cell membrane; The release solution comprises 15–20 mM Ca(NO3)2 and 2–4% v / v fetal bovine serum.
2. The preparation method according to claim 1, characterized in that, The release solution also includes 10 mM KHCO3 and / or 0.1 mM Na2EDTA.
3. The preparation method according to claim 1 or 2, characterized in that, The pH value of the release solution is 7.2 to 7.4; the solvent of the release solution is water.
4. The preparation method according to claim 1, characterized in that, The ratio of the number of red blood cells to the volume of the released fluid is 1×10. 10 1 mL.
5. The preparation method according to claim 1, characterized in that, The solid-liquid separation includes centrifugation, wherein the centrifugation speed is 7000 rpm and the time is 10 min; The washing process uses an aqueous solution containing 2% v / v fetal bovine serum; the washing is performed three times, and the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the release liquid is 1:1 for each wash.
6. An engineered erythrocyte biomembrane, characterized in that, It includes a red blood cell membrane and nanoparticles coupled to the surface of the red blood cell membrane; the red blood cell membrane is a structurally intact red blood cell membrane prepared by the preparation method according to any one of claims 1 to 5.
7. The engineered erythrocyte biomembrane according to claim 6, characterized in that, The mass ratio of the red blood cell membrane to the nanoparticles is 1:
5.
8. The engineered erythrocyte biomembrane according to claim 6 or 7, characterized in that, The nanoparticles include one or more of the following: fluorescent nanoparticles, photosensitizer nanoparticles, latex nanoparticles, colloidal gold nanoparticles, magnetic iron oxide nanoparticles, and aggregation-induced emission nanoparticles.
9. The engineered erythrocyte biomembrane according to claim 8, characterized in that, The fluorescent nanoparticles are Fluo nanoparticles; The aggregation-induced emission nanoparticles are AIE nanoparticles; The latex nanoparticles include one or more of red latex nanoparticles, black latex nanoparticles, and purple latex nanoparticles; The magnetic iron oxide nanoparticles include Fe3O4 magnetic nanoparticles.
10. The use of the engineered erythrocyte biomembrane according to any one of claims 6 to 9 in one or more of the following: (1) Preparation of blood typing reverse typing test products; (2) Preparation of platelet antibody detection products; (3) Prepare low-titer O-type whole blood detection products; (4) Preparation of irregular antibody detection products; (5) Prepare detection products for IgM anti-A and anti-B antibodies in whole blood and / or plasma; (6) Prepare detection products for IgG anti-A and anti-B antibodies in whole blood and / or plasma; (7) Prepare products for detecting neonatal hemolysis; (8) Prepare blood type antibody adsorption products.
11. A method for preparing the lyophilized formulation of the engineered erythrocyte biomembrane according to any one of claims 6 to 9, characterized in that, Includes the following steps: The engineered red blood cell biomembrane was kept at a constant temperature of 4.0℃ under vacuum of 0.0 Pa for 10 min to obtain the first treated sample. The first treated sample was kept at a constant temperature of 0.0 Pa and -50.0 °C for 60 min to obtain the second treated sample. The second treated sample was kept at a constant temperature of 0.0 Pa and -30.0 °C for 60 min to obtain the third treated sample. The third-treated sample was kept at a constant temperature of 0.0 Pa and -45.0 °C for 120 min to obtain the fourth-treated sample. The fourth treated sample was kept at a constant temperature of 12.0 Pa and -45.0 °C for 360 min to obtain the fifth treated sample. Under a vacuum of 8.0 Pa for 60 min, the temperature of the fifth treated sample was raised to -30.0 °C, and then kept at a constant temperature for 360 min under a vacuum of 8.0 Pa and a temperature of -30.0 °C to obtain the sixth treated sample. Under a vacuum of 5.0 Pa for 30 min, the temperature of the sixth treated sample was raised to -20.0 °C, and then kept at a constant temperature of 5.0 Pa and -20.0 °C for 60 min to obtain the seventh treated sample. Under a vacuum of 5.0 Pa for 10 min, the temperature of the seventh treated sample was raised to -10.0 °C, and then kept at a constant temperature of 5.0 Pa and -10.0 °C for 30 min to obtain the eighth treated sample. Under a vacuum of 5.0 Pa for 10 min, the temperature of the eighth processed sample was raised to 0.0 °C, and then kept at a constant temperature of 0.0 °C for 30 min under a vacuum of 5.0 Pa to obtain the ninth processed sample. Under a vacuum of 3.0 Pa for 10 min, the temperature of the ninth processed sample was raised to 100.0 °C, and then kept at a constant temperature of 10.0 °C for 30 min under a vacuum of 3.0 Pa to obtain the tenth processed sample. Under a vacuum of 5.0 Pa for 20 min, the temperature of the tenth treated sample was lowered to 0.0 °C, and then kept at a constant temperature of 0.0 °C for 40 min under a vacuum of 5.0 Pa to obtain the eleventh treated sample. Under a vacuum of 3.0 Pa for 20 min, the temperature of the eleventh treated sample was raised to 10.0 °C, and then kept at a constant temperature of 10.0 °C for 40 min under a vacuum of 3.0 Pa to obtain the twelfth treated sample. Under a vacuum of 3.0 Pa for 10 min, the temperature of the twelfth processed sample was raised to 25°C and kept at a constant temperature of 25.0°C for 60 min under a vacuum of 3.0 Pa to obtain the thirteenth processed sample. The thirteenth-treatment sample was kept at a constant temperature of 25.0℃ for 60 minutes under vacuum of 2.0 Pa to obtain the fourteenth-treatment sample. The fourteenth-treatment sample was kept at a constant temperature of 25.0℃ for 240 min under vacuum of 0.0 Pa to obtain the lyophilized formulation of the engineered erythrocyte biomembrane.