Synthetic method and application of artificial erythrocyte
By combining fluorinated liquid crystal block copolymer with stabilizers with different topologies in artificial erythrocyte synthesis for polymerization-induced self-assembly, the problem of difficulty in mimicking red blood cell morphology and oxygen binding ability in the prior art is solved, and efficient and low-cost erythrocyte-like vesicle synthesis is achieved.
Patent Information
- Application Number
- CN202411936923.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-04-18
AI Technical Summary
The prior art is difficult to successfully simulate the double concave disc morphology of red blood cells and have oxygen binding capabilities, and the synthesis process of artificial red blood cells is complex and costly.
The vesicle formation in the biconcave disc morphology is achieved by combining fluorinated liquid crystal block copolymers with stabilizers with different topological structures for polymerization-induced self-assembly (PISA).
The morphology and oxygen binding capacity of red blood cells were successfully simulated, the solubility of fluorinated block polymers in traditional self-assembly process was overcome, and the complexity and cost of the synthesis process were reduced.
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Figure CN119837830A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedical materials, and in particular to a method for synthesizing artificial red blood cells and applications thereof. Background Art
[0002] Erythrocytes, characterized by their biconcave disc shape, are specialized cells responsible for oxygen transport in the blood. Artificial oxygen carriers (AOCs), commonly known as artificial red blood cells, have been designed to address critical issues such as blood supply shortages, blood type mismatches, and the risk of spreading infectious diseases. In addition, AOCs are widely used to increase oxygen levels in the local microenvironment, thereby improving the efficacy of photodynamic therapy in anti-tumor or anti-bacterial treatment.
[0003] In the past few decades, research on artificial red blood cells has made significant progress. Initially, the focus of research was on hemoglobin-based oxygen carriers (HBOCs) using purified hemoglobin. However, early HBOCs caused hypertension and nephrotoxicity when used, mainly due to oxidative stress caused by free hemoglobin. To address these problems, researchers have introduced various improvements, including PEG modification, cross-linking, and polymerization of hemoglobin. In addition, significant progress has been made in encapsulating hemoglobin in liposomes or polymer nanoparticles to improve its stability and biocompatibility. However, the above-mentioned processes of isolating, purifying, and modifying hemoglobin are relatively complicated and costly. Therefore, researchers have also developed alternative non-hemoglobin-based AOCs, which are inspired by the structure and function of natural hemoglobin and constructed by synthesizing and self-assembling metalloporphyrins. For example, Kano et al. studied a myoglobin model system composed of β-cyclodextrin dimers and iron porphyrins with reversible dioxygen binding. Shen et al. developed composite micelles as a model of hemoglobin through the hierarchical assembly of diblock copolymers, β-cyclodextrin, and iron porphyrin.
[0004] Fluorocarbons are widely used in the development of AOCs due to their ability to dissolve large amounts of oxygen, chemical inertness, and biocompatibility. For example, Gao et al. reported the use of perfluorinated compounds encapsulated in erythrocyte membranes as nanoscale artificial erythrocytes to alleviate tumor hypoxia and improve the efficacy of cancer radiotherapy. However, the use of fluorocarbons to replicate the biconcave disc-like morphology of erythrocytes has not yet been achieved. Although some existing studies have successfully produced erythrocyte-like structures, these materials lack the ability to load oxygen. Moreover, few cases of successfully mimicking the morphology and function of erythrocytes have been reported. Summary of the invention
[0005] In order to solve the problems existing in the above background technology, the present invention provides a method for synthesizing artificial red blood cells, which combines a fluorinated liquid crystal block copolymer with a stabilizer block having different topological structures for polymerization-induced self-assembly (PISA), and the stabilizer having different topological structures induces microphase separation, resulting in uneven surface tension of the vesicle membrane, thereby transforming the vesicle into a concave red blood cell-like morphology. In addition, the present invention also provides the use of the artificial red blood cells obtained by the above synthesis method as oxygen carriers or as carriers carrying functional molecules such as photosensitizers.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A first aspect of the present invention provides a method for synthesizing artificial red blood cells, comprising the following steps:
[0008] S1, two stabilizers A and B with different topological structures are subjected to reversible addition-fragmentation chain transfer polymerization (RAFT) with fluorocarbon monomers to synthesize fluorinated liquid crystal block copolymers C and D respectively;
[0009] S2, fluorinated liquid crystal block copolymers C and D are self-assembled in situ together, different topological structures induce microphase separation, and the surface tension of the vesicle membrane formed by self-assembly is uneven, resulting in biconcave disc-shaped vesicles.
[0010] Furthermore, in step S1, stabilizer A is PEG113-CTA, and stabilizer B is PDMAEMA30-CTA.
[0011] PEG113-CTA and PDMAEMA30-CTA were synthesized using existing technology. Specifically, PEG113-CTA is a linear polymer with a number average molecular weight (Mn) of 5.2 kDa and a polydispersity coefficient of is 1.05; PDMAEMA30-CTA is a bottle brush polymer with a number average molecular weight (Mn) of 5.0 kDa and a polydispersity coefficient is 1.12.
[0012] Furthermore, the fluorocarbon monomer is 2-(perfluorooctyl)ethyl methacrylate.
[0013] Furthermore, the molar ratio of the stabilizer A, the stabilizer B, and the fluorocarbon monomer is 1:(1-3):(35-60).
[0014] Preferably, the molar ratio of the stabilizer A, the stabilizer B, and the fluorocarbon monomer is 1:(1-2):(35-45).
[0015] Furthermore, the reversible addition-fragmentation chain transfer polymerization reaction in step S1 and the in-situ self-assembly in step S2 are carried out in an ethanol system.
[0016] The second aspect of the present invention provides an application of artificial red blood cells produced by the above-mentioned synthesis method, wherein the artificial red blood cells are directly used as oxygen carriers.
[0017] The third aspect of the present invention provides an application of artificial red blood cells prepared by the above-mentioned synthesis method, in which functional molecules are loaded in situ during the polymerization-induced self-assembly process to obtain artificial red blood cells carrying functional molecules, which are applied in the biomedical field.
[0018] Furthermore, the artificial red blood cells carry photosensitizers. The artificial red blood cells can not only be loaded with photosensitizers in situ, but also retain their red blood cell-like morphology. The artificial red blood cells loaded with photosensitizers can enhance photodynamic therapy by increasing the oxygen level in the surrounding microenvironment.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The inventors of this application proposed a concept based on previous experimental research topics: whether it is possible to use fluorinated liquid crystal block copolymers to prepare biconcave discoid vesicles to realize artificial red blood cells. However, in actual experimental operations, it was found that direct self-assembly of fluorinated liquid crystal block copolymers usually produces spherical vesicles. After extensive research, the inventors found that uneven interfacial tension in the vesicle membrane can cause vesicle deformation, and local composition changes in the vesicle membrane structure are the key factor in changing the interfacial tension.
[0021] The present application has determined the synthesis scheme of artificial red blood cells through a large number of experiments: using a fluorinated polymer containing at least 7 fluorine-carbon bonds to form an ordered lamellar liquid crystal phase, providing a strong driving force for the phase separation between incompatible blocks, and achieving vesicle deformation by self-assembling fluorinated block polymers using stabilizers with different topological structures. Different topological structures will induce microphase separation, resulting in uneven surface tension on the vesicle membrane, thereby causing the vesicle to deform and form a concave red blood cell-like morphology. However, due to the high hydrophobicity of fluorinated block polymers, the traditional self-assembly process is more difficult. Therefore, the present application chooses to use the polymerization-induced self-assembly (PISA) method, which can overcome the solubility problem of fluorinated block polymers and successfully synthesize fluorinated nanoparticles. Finally, the present application combines fluorinated block copolymers with stabilizer blocks of different topological structures for PISA to obtain biconcave disc-shaped vesicles, which successfully simulate the morphology and oxygen binding ability of red blood cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0023] Figure 1Schematic diagram of the synthesis of biconcave disc-shaped vesicles by polymerization-induced self-assembly (PISA) in the present invention.
[0024] Figure 2 The following are scanning electron microscope (SEM) images of the products synthesized in Examples 1-5.
[0025] Figure 3 Detailed characteristic photos of the synthetic product of Example 2; 3A-C: transmission electron microscopy (TEM) images; 3D: atomic force microscopy (AFM) image; 3E: AFM height profile; 3F and 3G: transmission electron microscopy-energy dispersive spectroscopy (TEM-EDS) fluorine element distribution maps.
[0026] Figure 4 The scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the products synthesized in Examples 6 and 7 are shown.
[0027] Figure 5 The following are scanning electron microscope (SEM) images of the products synthesized in Examples 8-10. DETAILED DESCRIPTION
[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0029] The present invention is further described below by way of examples. Unless otherwise specified, the various reagents and raw materials used in the present invention are all commercially available products or products that can be prepared by known methods.
[0030] A method for synthesizing artificial red blood cells, such as Figure 1 As shown, the following steps are included:
[0031] S1. Two stabilizers PEG113-CTA and PDMAEMA30-CTA with different topological structures are subjected to reversible addition-fragmentation chain transfer polymerization reaction with monomer FMA (2-(perfluorooctyl)ethyl methacrylate) and free radical initiator AIBN in an ethanol system. During the reaction, the solid content in the ethanol system is 100 mg / mL, the reaction time is 12 hours, and the reaction temperature is 70°C. Fluorinated liquid crystal block copolymers C and D are synthesized accordingly. One end group of the fluorinated liquid crystal block copolymer C is -PEG113, and one end group of the fluorinated liquid crystal block copolymer D is -PDMAEMA30.
[0032] PEG113-CTA and PDMAEMA30-CTA also act as macromolecular chain transfer agents in this reaction system. PEG113-CTA is a linear polymer with a number average molecular weight (Mn) of 5.2 kDa and a polydispersity coefficient of is 1.05; PDMAEMA30-CTA is a bottle brush polymer with a number average molecular weight (Mn) of 5.0 kDa and a polydispersity coefficient is 1.12.
[0033] S2. Fluorinated liquid crystal block copolymers C and D were self-assembled in situ, and different topological structures induced microphase separation. The surface tension of the vesicle membrane formed by self-assembly was uneven, and biconcave disc-shaped vesicles were obtained.
[0034] Examples 1-5
[0035] Examples 1-5 all adopt the following synthesis method:
[0036] S1. Two stabilizers PEG113-CTA and PDMAEMA30-CTA with different topological structures are subjected to reversible addition-fragmentation chain transfer polymerization reaction with monomer FMA (2-(perfluorooctyl)ethyl methacrylate) and free radical initiator AIBN in an ethanol system. During the reaction, the solid content in the ethanol system is 100 mg / mL, the reaction time is 12 hours, and the reaction temperature is 70°C. Fluorinated liquid crystal block copolymers C and D are synthesized accordingly. One end group of the fluorinated liquid crystal block copolymer C is -PEG113, and one end group of the fluorinated liquid crystal block copolymer D is -PDMAEMA30.
[0037] PEG113-CTA is a linear polymer with a number average molecular weight (Mn) of 5.2 kDa and a polydispersity coefficient of is 1.05; PDMAEMA30-CTA is a bottle brush polymer with a number average molecular weight (Mn) of 5.0 kDa and a polydispersity coefficient is 1.12.
[0038] S2. Fluorinated liquid crystal block copolymers C and D were self-assembled in situ, and different topological structures induced microphase separation. The surface tension of the vesicle membrane formed by self-assembly was uneven, and biconcave disc-shaped vesicles were obtained.
[0039] In Examples 1-5, the molar ratio of PEG113-CTA to PDMAEMA30-CTA was fixed at 1:2, and the molar ratio of FMA was varied from 24 to 90. Specifically, in Example 1, the molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D) to monomer FMA (F) was 1:2:24, and in Example 2, the molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D) to monomer FMA (F) was 1: 2:40; in Example 3, the feeding molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D) and monomer FMA (F) is 1:2:45; in Example 4, the feeding molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D) and monomer FMA (F) is 1:2:60; in Example 5, the feeding molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D) and monomer FMA (F) is 1:2:90.
[0040] The artificial red blood cells prepared in Examples 1-5 were observed by scanning electron microscopy (SEM) to obtain the morphology. The test results are shown in Figure 2 .like Figure 2 As shown in A, when the FMA feed ratio is 24, spherical nanoparticles with an average diameter of 66nm are obtained. Figure 2 As shown in Figure 2, when the FMA feed ratio was increased to 40, uniform red blood cell-like particles were obtained, which were concave in shape. The diameter of these particles was about 500 nm and the thickness was about 130 nm, which was very close to the diameter-to-thickness ratio of natural red blood cells. Additional SEM images of this sample ( Figure 2 C) clearly reveals the rupture of the vesicle wall, confirming that these particles are indeed vesicles rather than solid particles. This shows that the present invention uses mixed stabilizers with different topological structures to change the uniformity of the vesicle membrane surface, thereby promoting the feasibility of constructing a concave erythrocyte-like morphology. Figure 2 As shown in D, when the FMA feed ratio increases to 45, the main morphology of the obtained particles is still red blood cell-like, but the size uniformity decreases and irregular spheres begin to appear. Figure 2 As shown in E, when the FMA feed ratio was increased to 60, a higher proportion of irregular spheres was observed, but particles with red blood cell-like structures still existed. Figure 2 As shown in Figure F, when the ratio of FMA increases to 90, the erythrocyte-like structure completely disappears and solid particles become the main morphology. The above results show that stabilizer molecules with different topological structures play an important role in regulating the erythrocyte-like morphology. In addition, the addition ratio of FMA is also a key factor.
[0041] The product prepared in Example 2 was analyzed using transmission electron microscopy (TEM) and atomic force microscopy (AFM) to further characterize the artificial red blood cell-like morphology. The test results are shown in Figure 3 . Figure 3 TEM images of A, 3B, and 3C clearly show the erythrocyte-like morphology in different orientations (“face up” and “edge up”), an observation that further confirms that the concave shape on the vesicles is formed during the assembly process rather than due to the collapse of the vesicles due to water loss during sample drying. In addition, the cavity and vesicle wall can be clearly observed, further confirming that these product particles are vesicles. Figure 3 In B, a vesicle with the "edge facing up" was selected and its dimensions were measured. The vesicle thickness was about 140nm and the wall thickness was about 18nm. Figure 3 In C, the diameter of the "edge-up" vesicle was measured to be approximately 442 nm, and the TEM data agreed well with the SEM data. Atomic force microscopy (AFM) was also used to further characterize the erythrocyte-like morphology. Figure 3 A clear biconcave erythrocyte-like particle was observed in D. Figure 3 E height distribution shows that the diameter of the erythrocyte-like vesicle is about 500nm, the thickness is about 140nm, and the depth of the central depression is about 20-30nm. These measurement results are in good agreement with the SEM and TEM data. In addition, the TEM-EDS spectrum shows that the fluorine element is evenly distributed in the erythrocyte-like vesicle, such as Figure 3 F, as shown in 3G.
[0042] Embodiment 6, 7
[0043] In order to further study the effect of the ratio of different topological stabilizers on the structural morphology of the product, Examples 6 and 7 were set up. The synthesis methods of Examples 6 and 7 were the same as those of Examples 1-5, with the only difference being:
[0044] In Example 6, the molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D) and monomer FMA (F) is 1:1:40; in Example 7, the molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D) and monomer FMA (F) is 0.1:0.9:20.
[0045] The morphology of the products in Examples 6 and 7 was observed using a scanning electron microscope (SEM) and a transmission electron microscope (TEM). The test results are shown in Figure 4 Compared with Example 2, Example 6 reduced the content of bottle brush polymer PDMAEMA30-CTA, and both SEM and TEM images showed that the erythrocyte-like morphology was still formed ( Figure 4A). In Example 7, the content of bottle brush polymer PDMAEMA30-CTA increased significantly. Figure 4 The SEM image of B showed no obvious erythrocyte morphology, and most of the particle structures were spherical, and the TEM image confirmed that they were spherical vesicles. The above results indicate that the stabilizer blocks with different topological structures play a key role in regulating the erythrocyte-like morphology. In addition, when one stabilizer block is absolutely dominant, the non-uniform interfacial tension required for vesicle membrane deformation cannot be achieved, thus preventing the vesicle from deforming to form a concave erythrocyte-like morphology.
[0046] In addition to being an oxygen carrier, the artificial red blood cells produced by the present invention can also be designed to carry various functional molecules, thereby greatly expanding their potential biomedical applications. For example, artificial red blood cells loaded with photosensitizers can enhance photodynamic therapy by increasing the oxygen level in the surrounding microenvironment.
[0047] Next, it is further studied whether the erythrocyte-like nanostructures prepared by the present invention can be loaded with photosensitizers in situ and whether the erythrocyte-like morphology can still be retained.
[0048] Examples 8-10
[0049] Porphyrin molecules were selected as photosensitizers, and the effects of porphyrin molecules on the morphology of red blood cell-like particles during polymerization-induced self-assembly were studied.
[0050] Examples 8-10 all adopt the following synthesis method:
[0051] S1. Two stabilizers with different topological structures, PEG113-CTA and PDMAEMA30-CTA, are subjected to a reversible addition-fragmentation chain transfer polymerization reaction with the monomer FMA (2-(perfluorooctyl)ethyl methacrylate) in an ethanol system. Porphyrin is also added to the ethanol system to synthesize fluorinated liquid crystal block copolymers C and D accordingly. The end group of the fluorinated liquid crystal block copolymer C is -PEG113, and the end group of the fluorinated liquid crystal block copolymer D is -PDMAEMA30.
[0052] PEG113-CTA is a linear polymer with a number average molecular weight (Mn) of 5.2 kDa and a polydispersity coefficient of is 1.05; PDMAEMA30-CTA is a bottle brush polymer with a number average molecular weight (Mn) of 5.0 kDa and a polydispersity coefficient is 1.12.
[0053] S2, fluorinated liquid crystal block copolymers C and D are self-assembled in situ together, different topological structures induce microphase separation, and the surface tension of the vesicle membrane formed by self-assembly is uneven, resulting in biconcave disc-shaped vesicles.
[0054] In Example 8, the molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D), monomer FMA (F), and porphyrin (P) is 1:2:40:0.01; in Example 9, the molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D), monomer FMA (F), and porphyrin (P) is 1:2:40:0.02; in Example 10, the molar ratio of PEG113-CTA (G), PDMAEMA30-CTA (D), monomer FMA (F), and porphyrin (P) is 1:2:40:0.04.
[0055] The morphology of the products in Examples 8-10 was observed using a scanning electron microscope (SEM). The test results are shown in Figure 5 .like Figure 5 As shown in A and B, when the porphyrin feeding ratio was 0.01 and 0.02, erythrocyte-like nanostructures were still formed, but the size was significantly reduced, especially when the feeding ratio was 0.01, the size of the product particles was most significantly reduced. When the porphyrin feeding ratio increased to 0.04, the erythrocyte-like structure was replaced by a trumpet-like morphology ( Figure 5 C).
[0056] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for synthesizing artificial red blood cells, characterized in that: The steps include: S1, conducting a reversible addition-fragmentation chain transfer polymerization reaction between two stabilizers A and B having different topological structures and a fluorocarbon monomer to synthesize fluorinated liquid crystal block copolymers C and D respectively; S2, fluorinated liquid crystal block copolymers C and D are self-assembled in situ together, different topological structures induce microphase separation, and the surface tension of the vesicle membrane formed by self-assembly is uneven, resulting in biconcave disc-shaped vesicles.
2. The method for synthesizing artificial red blood cells according to claim 1, characterized in that: In the step S1, stabilizer A is PEG113-CTA, and stabilizer B is PDMAEMA30-CTA.
3. The method for synthesizing artificial red blood cells according to claim 2, characterized in that: The fluorocarbon compound monomer is 2-(perfluorooctyl)ethyl methacrylate.
4. The method for synthesizing artificial red blood cells according to claim 3, characterized in that: The molar ratio of the stabilizer A, the stabilizer B and the fluorocarbon compound monomer is 1:(1-3):(35-60).
5. The method for synthesizing artificial red blood cells according to claim 4, characterized in that: The molar ratio of the stabilizer A, the stabilizer B and the fluorocarbon compound monomer is 1:(1-2):(35-45).
6. The method for synthesizing artificial red blood cells according to claim 2, characterized in that: PEG113-CTA is a linear polymer with a number average molecular weight of 5.2 kDa and a polydispersity coefficient of 1.05; PDMAEMA30-CTA is a bottle brush polymer with a number average molecular weight of 5.0 kDa and a polydispersity coefficient of 1.
12.
7. The method for synthesizing artificial red blood cells according to claim 1, characterized in that: The reversible addition-fragmentation chain transfer polymerization reaction in step S1 and the in-situ self-assembly in step S2 are both carried out in an ethanol system.
8. Use of artificial red blood cells prepared by the synthesis method according to any one of claims 1 to 7, characterized in that: The artificial red blood cells directly serve as oxygen carriers.
9. Use of artificial red blood cells prepared by the synthesis method according to any one of claims 1 to 7, characterized in that: Functional molecules are loaded in situ during the polymerization-induced self-assembly process to obtain artificial red blood cells carrying functional molecules for use in the biomedical field.
10. The use of artificial red blood cells according to claim 9, characterized in that: The functional molecule carried by the artificial red blood cells is a photosensitizer.