Fluorine-containing polyamphiphilic copolymer nanoparticles with stable protease action, preparation method and application

Abstract

The present application provides a fluorine-containing polyampholyte copolymer nanoparticle with stable protease action, a preparation method and application, the fluorine-containing polyampholyte copolymer nanoparticle includes one or more fluorine-containing polyampholyte copolymer molecules, the monomer of the fluorine-containing polyampholyte copolymer includes amphoteric alkyl (methyl) acrylate and fluorinated alkyl (methyl) acrylate, and the molar ratio of the amphoteric alkyl (methyl) acrylate and the fluorinated alkyl (methyl) acrylate is 10:90-90:10.The fluorine-containing polyampholyte copolymer nanoparticle provided by the present application is stable in structure, can be used as a stabilizer for protease, effectively improves the room temperature solution storage efficiency of protease, avoids the inhibition of enzyme activity caused by conventional enzyme stabilization strategies such as surface chemical modification and immobilization, and even leads to complete inactivation.

Description

Technical Field

[0001] This invention belongs to the field of functional polymer nanomaterials, and specifically relates to fluorinated zwitterionic copolymer nanoparticles with stabilizing protease activity, their preparation method and application. Background Technology

[0002] Since Eisenberg et al. systematically reported the self-assembly behavior of block copolymers in solution in the mid-1990s, the field of macromolecular self-assembly research has developed rapidly. A wide variety of assembly structures with diverse morphologies, including micelles, vesicles, and complex hierarchical ordered structures, can be achieved (see Jiang Ming, A. Eisenberg, Liu Guojun, Zhang Xi, *Macromolecular Self-Assembly*, Science Press, 2006; Liu Shiyong, *A New Compilation of Macromolecular Self-Assembly*, Science Press, 2018). In the assembly research based on amphiphilic block copolymers, fluorinated blocks, through their self-phase-forming effect unlike typical hydrophilic or hydrophobic segments, endow the corresponding assembly systems with stronger assembly driving forces and better control over assembly morphology. The research group of Professor Yuan Jinying at Tsinghua University prepared a fluorinated amphiphilic block copolymer PDMA-bP(BzMA-co-FMA) using reversible addition-fragmentation chain transfer (RAFT) polymerization. The hydrophilic segment is poly(dimethylamine ethyl methacrylate) PDMA, while the P(BzMA-co-FMA) block, which is a random copolymer of benzyl methacrylate (BzMA) and perfluorooctyl methacrylate (FMA), is hydrophobic. Under the premise of a fixed ratio of hydrophilic and hydrophobic blocks, the size of polymer vesicles can be controlled by changing the content of solubilizing PFMA segments, and various morphologies such as large composite micelles, vesicles, hexagonal stacked hollow structures, and nanoporous spheres can be obtained (see M.Huo, G.Song, J.Zhang, Y.Wei, J.Yuan, ACS Macro Lett., 2018, 7, 956-961; M.Huo, M.Zeng, D.Wu, Y.Wei, J.Yuan, Polym. Chem., 2018, 9, 912-919; F.Lv, Z.An, P.Wu, Nature Commun., 2019, 10, 1397-1403).

[0003] Compared to micron-scale self-assembly systems based on block copolymers, nanoscale assemblies formed by differential phases of interactions between side groups with different properties in amphiphilic random copolymers, which have been developed in recent years, have more precise structures and offer different control methods than block copolymers. They are of great significance in fields such as selective catalysis and molecular recognition (see M. Grzelczak, LMLiz-Marzan, R. Klajn, Chem. Soc. Rev., 2019, 48, 1342-1361; B. Zhang, Y. Zhao, X. Sun, X. Fei, W. Wei, X. Li, X. Liu, J. Ind. Eng. Chem., 2020, 83, 224-234). The introduction of fluorinated functional side groups into amphiphilic random copolymers can effectively promote the formation of quasi-nanoscale structures based on single-chain or oligochain assemblies. Japanese researchers, including Sawamoto et al., reported a process using atom transfer controlled radical polymerization (ATRP) to simultaneously introduce hydrophilic polyethylene glycol side groups (-PEG). 8.5 ) and hydrophobic n-dodecane chain side group (-C 12 H 25 The controlled synthesis of the amphiphilic random copolymer PEGMA / DMA was achieved by systematically investigating the effects of three structural parameters on assembly: polymer molecular weight, the ratio of hydrophilic to lipophilic side groups, and the overall hydrophilicity / hydrophobicity of the polymer, including the polymer carbon backbone. This allowed for the preparation of single-chain nanoparticles with uniform and controllable size, narrow particle size distribution, and stable cavities (see M. Shibata, M. Matsumoto, Y. Hirai, M. Takenaka, M. Sawamoto, T. Terashima, Macromolecules, 2018, 51, 3738-3745; M. Matsumoto, M. Sawamoto, T. Terashima, ACS Macro Lett., 2019, 8, 320-325; Y. Ommura, S. Imai, M. Takenaka, M. Ouchi, T. Terashima, ACS Macro Lett., 2020, 9, 426-430).

[0004] The polyethylene glycol (PEG) used in the aforementioned literature is the most commonly used hydrophilic segment selection in the assembly system to date, and it is also a biomedical polymer with excellent biocompatibility that has long been regarded as the "gold standard". However, clinical trial results have shown that PEG-modified medical materials, such as PEG-surface-modified implantable medical devices, can produce physiologically toxic substances that induce immune responses when left in the human body for extended periods. Furthermore, a recent report in the authoritative journal *The New England Journal of Medicine* revealed that a PEG-modified mRNA vaccine developed for SARS-CoV-2 caused severe allergic reactions primarily due to PEG coating modification (see NH Park, W. Cheng, F. Lai, C. Yang, PFSessions, B. Periaswamy, CW Chu, S. Bianco, S. Liu, S. Venkataraman, Q. Chen, Y. Yang, J. Hedrick, J. Am. Chem. Soc., 2018, 140, 4244-4252; MC Castells, EJ Phillips, N. Engl. J. Med. 2020, 384, 643-649). Therefore, the biocompatibility, tolerance, and cellular metabolic behavior of PEG materials in complex physiological environments require further in-depth investigation.

[0005] On the other hand, the simultaneous introduction of both positive and negative ion groups into the structure of zwitterionic compounds results in overall electroneutrality, exhibiting not only excellent hydrophilicity and chemical stability but also superior biocompatibility. The research group of Shao-Yi Jiang at the University of Washington has prepared a superhydrophilic biomimetic polymer material, PTMAO, mimicking the structure of trimethylamine nitride (TMAO), a zwitterionic permeate found in deep-sea fish. Even in complex physiological environments, PTMAO demonstrates excellent biocompatibility and "stealth" characteristics, resisting non-specific protein adsorption and cell adhesion, providing a widely applicable material option for solving the intractable problems caused by biofouling. Therefore, zwitterionic polymers are a promising alternative to PEG materials and an ideal choice to avoid some of the aforementioned adverse reactions in clinical applications (see Y. Han, Z. Yuan, P. Zhang, S. Jiang, Chem. Sci., 2018, 9, 8561-8566; B. Li, P. Jain, J. Ma, JK Smith, Z. Yuan, H. Hung, Y. He, X. Lin, K. Wu, J. Pfaendtner, S. Jiang, Sci. Adv., 2019, 5, eaaw9562; LD Blackman, P. A. Gunatillake, P. Cass, K. ESL ocock, Chem. Soc. Rev., 2019, 48, 757-770).

[0006] Based on the aforementioned non-patent literature reports and patent literature in related fields, there are only a few reports on zwitterionic side-group homopolymers and copolymers with fluorinated side groups and hydrophilic side groups of polyethylene glycol (PEG). Therefore, there is still a desire to provide new zwitterionic copolymers.

[0007] In addition, common methods for maintaining enzyme protein activity include: storage in solid form (dry powder or crystals), or in solutions under a low-temperature (-70°C) cold chain environment supplemented with organic preservatives (such as 2-mercaptoethanol) that affect its activity. Although strategies such as enzyme surface chemical modification and enzyme immobilization engineering have shown the ability to improve enzyme structural stability, it is difficult to achieve the storage goal of simultaneously satisfying both protease structural stability and activity (see AMKlibanov, Nature, 2001, 409, 241-246; L. Lancaster, W. Abdallah, S. Banta, I. Wheeldon, Chem. Soc. Rev., 2018, 47, 5177-5186). Summary of the Invention

[0008] In view of this, the purpose of this invention is to address the technical problems existing in the prior art by providing fluorinated polyzwitterionic copolymer nanoparticles with protease stabilizing effects, preparation methods, and applications. The fluorinated polyzwitterionic copolymer nanoparticles provided by this invention have a stable structure and can be used as a stabilizer for proteases, effectively improving the storage efficiency of proteases in room temperature solutions and avoiding the problems of inhibition or even complete inactivation of enzyme activity caused by conventional enzyme stabilization strategies such as surface chemical modification and immobilization.

[0009] The objective of this invention is achieved through the following technical solutions.

[0010] In a first aspect, the present invention provides fluorinated zwitterionic copolymer nanoparticles with stabilizing protease activity, wherein the fluorinated zwitterionic copolymer nanoparticles comprise one or more fluorinated zwitterionic copolymer molecules.

[0011] In this invention, the monomers of the fluorinated zwitterionic copolymer include zwitterionic alkyl (meth)acrylates and fluorinated alkyl (meth)acrylates, and the molar ratio of the zwitterionic alkyl (meth)acrylates to the fluorinated alkyl (meth)acrylates is 10:90 to 90:10.

[0012] The inventors of this application have discovered that by copolymerizing zwitterionic alkyl (meth)acrylates and fluorinated alkyl (meth)acrylates as comonomers, fluorinated zwitterionic random copolymers can be obtained. These random copolymers simultaneously introduce bis-hydrophobic (hydrophobic and oleophobic) fluorinated alkyl groups and zwitterionic side groups with good hydrophilicity and biocompatibility. Fluorinated zwitterionic copolymer nanoparticles can be prepared through solution assembly. The fluorinated zwitterionic copolymer nanoparticles of this invention have a stable structure and can be used as stabilizers for proteases, effectively improving the room temperature solution storage efficiency of proteases. This avoids the problems of inhibition or even complete inactivation of enzyme activity caused by conventional enzyme stabilization strategies such as surface chemical modification and immobilization, providing a novel technical route for achieving stable room temperature storage of highly active proteases, such as horseradish peroxidase (HRP).

[0013] In this invention, fluorinated zwitterionic copolymers generally involve random copolymers of zwitterionic alkyl (meth)acrylates and fluorinated alkyl (meth)acrylates. Therefore, the terms "fluorinated zwitterionic copolymer" and "fluorinated zwitterionic random copolymer" can be used interchangeably.

[0014] In this invention, the term "(meth)acrylate" refers to acrylates and / or methacrylates.

[0015] The fluorinated zwitterionic copolymer nanoparticles provided by the present invention are prepared via a reversible addition-fragmentation chain transfer (RAFT) polymerization reaction. In this invention, the fluorinated zwitterionic copolymer can be controlled using RAFT polymerization, resulting in nanoparticles with uniform and controllable size. Furthermore, the surface and core properties can be controllably designed by adjusting the structural units and degree of polymerization of the fluorinated zwitterionic copolymer, thereby providing stabilizing effects on proteases with different properties, sizes, and biological activities.

[0016] The fluorinated zwitterionic copolymer nanoparticles provided by the present invention wherein the anion in the zwitterionic alkyl (meth)acrylate can be a carboxylate or a sulfonate, and the cation can be a quaternary ammonium cation. Further, the alkyl group in the zwitterionic alkyl (meth)acrylate can be a C1-C6 alkyl group, for example, methylene, ethylene, or propylene.

[0017] Examples of zwitterionic alkyl (meth)acrylates suitable for use in this invention include, but are not limited to, methacryloyl ethylidene sulfobetaine (SBMA) and 3-[[2-(methacryloyloxy)ethylene]dimethylammonium]propionate (CBMA). In some embodiments, the zwitterionic alkyl (meth)acrylate is methacryloyl ethylidene sulfobetaine (SBMA).

[0018] The fluorinated zwitterionic copolymer nanoparticles provided by the present invention, wherein the fluorinated alkyl chain in the fluorinated alkyl (meth)acrylate can be a fluorinated C1-C12 alkyl chain, preferably a fluorinated C4-C10 alkyl chain.

[0019] The fluorinated alkyl chain can be a perfluorinated alkyl chain or a partially fluorinated alkyl chain. It is believed that, in order to provide sufficient hydrophobicity and fluorine effect, the fluorinated alkyl chain typically has at least three fluorine atoms. In some embodiments, the fluorinated alkyl chain is a 3- to 20-fluoroalkyl chain, and in some embodiments it is a 6- to 17-fluoroalkyl chain.

[0020] Examples of fluorinated alkyl (meth)acrylates suitable for use in this invention include, but are not limited to: hexafluorobutyl methacrylate (F6MA), 2-(perfluorobutyl)ethylene methacrylate (F9MA), and heptadecafluorodecyl methacrylate (F9MA). 17 MA). In some preferred embodiments, the fluorinated alkyl (meth)acrylate is hexafluorobutyl methacrylate (F6MA).

[0021] The fluorinated zwitterionic copolymer nanoparticles provided by the present invention wherein the molar ratio of the zwitterionic alkyl (meth)acrylate to the fluorinated alkyl (meth)acrylate is 20:80 to 80:20, preferably 60:40 to 80:20, for example, 70:30.

[0022] The fluorinated polyzwitterionic copolymer nanoparticles provided by the present invention have a designed degree of polymerization of 50 to 500, preferably 100 to 400, and more preferably 270 to 320.

[0023] In this invention, the terms "design degree of polymerization," "theoretical degree of polymerization," and "target degree of polymerization" can be used interchangeably, referring to the molar ratio of monomer to chain transfer agent in RAFT polymerization.

[0024] Secondly, the present invention provides a method for preparing fluorinated polyzombie copolymer nanoparticles with stabilizing protease activity, wherein the preparation method includes the following steps:

[0025] S100. In the presence of an initiator and a chain transfer agent, zwitterionic alkyl (meth)acrylates and fluorinated alkyl (meth)acrylates undergo RAFT polymerization in a reaction solvent to obtain a fluorinated polyzwitterionic copolymer.

[0026] S200: Dissolve the fluorinated zwitterionic copolymer obtained in step S100 in a mixed solvent of trifluoroethanol and water to prepare an assembly solution of fluorinated zwitterionic copolymer nanoparticles; wherein, the volume ratio of trifluoroethanol to water in the mixed solvent of trifluoroethanol and water is 0-3:0-3 and the volumes of both trifluoroethanol and water are not 0, and the concentration of the fluorinated zwitterionic copolymer in the assembly solution is 0.1-10 mg / mL.

[0027] In this invention, fluorinated zwitterionic copolymers are prepared using RAFT polymerization, achieving controllable preparation of these copolymers. The resulting nanoparticles exhibit uniform and controllable size, and their amphiphilicity and fluorination effects can be adjusted by modifying the monomer types and ratios, providing a feasible solution for specifically maintaining the bioactivity of proteases. Furthermore, by employing a specific solvent system and an appropriate concentration of the fluorinated zwitterionic copolymer in the assembly solution, and under the multiple assembly driving forces of the random zwitterionic copolymer, including its solubility, ionic interactions, and fluorination effects, precise self-folding assembly of the single chains of the random zwitterionic copolymer is achieved, yielding nanoparticles with a particle size of approximately 10 nm. Of course, multi-chain assembly of the random zwitterionic copolymer can also be performed to obtain particles with relatively larger particle sizes.

[0028] According to the preparation method provided by the present invention, in step S100, the molar ratio of zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent is 10-270:10-270:0.1-0.5:0.2-1.

[0029] This invention does not have special requirements on the amount of initiator used; conventional amounts used in the art can be adopted as needed. In this invention, the target degree of polymerization is controlled by adjusting the ratio of zwitterionic alkyl (meth)acrylates and fluorinated alkyl (meth)acrylates to the chain transfer agent.

[0030] In some embodiments, the molar ratio of zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent is 10–270:10–270:0.25–0.5:1; in some embodiments it is 30–240:30–240:0.25–0.5:1; in some embodiments it is 180–240:120–60:0.25–0.5:1; and in some embodiments it is 210:90:0.5:1.

[0031] According to the preparation method provided by the present invention, the initiator may be an azo initiator or an organic peroxide initiator, preferably an azo initiator.

[0032] Examples of azo initiators suitable for use in this invention include, but are not limited to, azobisisobutyronitrile (AIBN). Examples of organic peroxide initiators suitable for use in this invention include, but are not limited to, benzoyl peroxide (BPO).

[0033] According to the preparation method provided by the present invention, the chain transfer agent is 2-cyanopropyl-2-ylbenzodisulfide (also known as "isobutyronitrile dithiobenzoate", CPDB).

[0034] According to the preparation method provided by the present invention, the reaction solvent in step S100 is selected from one or more of toluene, 1,4-dioxane, trifluoroethanol, methanol, water and dimethyl sulfoxide.

[0035] In some embodiments, the reaction solvent in step S100 is a mixture of water and methanol or trifluoroethanol. The volume ratio of water to methanol in the water and methanol mixture is 0–7:0–7, preferably 7:3–1:3.

[0036] According to the preparation method provided by the present invention, step S100 includes the following steps:

[0037] S101. Add zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent to the reaction solvent, mix, introduce inert gas to remove oxygen, seal, and obtain the reaction system.

[0038] S102. Under stirring conditions and at a temperature of 60–90°C, the reaction system obtained in step S101 undergoes a polymerization reaction to obtain the reaction product.

[0039] S103. Dialyze the reaction product obtained in step S102 and dry it to obtain a fluorinated zwitterionic copolymer.

[0040] According to the preparation method provided by the present invention, in step S101, the reaction system is mixed by stirring to make it homogeneous. In the present invention, nitrogen or argon can be used as the inert gas in step S101. In addition, oxygen can be removed by purging with inert gas once or more.

[0041] According to the preparation method provided by the present invention, the stirring speed in step S102 is 800-3000 rpm, preferably 1500-2000 rpm.

[0042] According to the preparation method provided by the present invention, the polymerization reaction in step S102 is carried out at a temperature of 60–90°C for a time of 30–360 min. In some specific embodiments, the polymerization reaction in step S102 is carried out at a temperature of 80°C for a time of 120 min.

[0043] According to the preparation method provided by the present invention, in step S103, unreacted monomers (zwitterionic alkyl (meth)acrylates, fluoroalkyl (meth)acrylates), undecomposed initiators (e.g., AIBN) and / or low molecular weight polymers are removed by dialysis.

[0044] In some specific implementations, the dialysis conditions in step S103 are as follows: the solvent is trifluoroethanol, the molecular weight cutoff of the dialysis membrane is at least 1 kDa, and the dialysis time is preferably at least 48 hours, for example, 72 hours.

[0045] According to the preparation method provided by the present invention, the drying operation in step S103 includes: rotary evaporation of the retentate solution in the dialysis bag obtained by dialysis to obtain the rotary evaporation product; and vacuum drying the rotary evaporation product at 30-40°C.

[0046] According to the preparation method provided by the present invention, in step S200, the volume ratio of trifluoroethanol to water in the trifluoroethanol and water mixed solvent is 1 to 2:1, for example, 3:2.

[0047] In this invention, the water in the trifluoroethanol and water mixed solvent is ultrapure water.

[0048] According to the preparation method provided by the present invention, in step S200, the concentration of the fluorinated polyzwitterionic copolymer in the assembly solution is controlled to be below 4 mg / mL, which can yield fluorinated polyzwitterionic copolymer nanoparticles formed from single chains. However, when the concentration of the fluorinated polyzwitterionic copolymer is too low, the solvent consumption is large and the efficiency is low. In some embodiments, the concentration of the fluorinated polyzwitterionic copolymer in the assembly solution is 1 to 4 mg / mL, and in some preferred embodiments it is 2 mg / mL.

[0049] Thirdly, this invention provides the application of fluorinated zwitterionic copolymer nanoparticles or the preparation method described above in stabilizing proteases. In this invention, using fluorinated zwitterionic copolymer nanoparticles as a stabilizer can improve the room temperature solution storage efficiency of proteases.

[0050] According to the application provided by the present invention, the application includes the following steps:

[0051] S300 provides a PBS buffer solution containing 0.05–0.5 mg / mL of protease;

[0052] S400. The PBS buffer solution of the protease is mixed with a solution of fluorinated zwitterionic copolymer nanoparticles, wherein the concentration of the fluorinated zwitterionic copolymer in the solution of the fluorinated zwitterionic copolymer nanoparticles is 1-4 mg / mL, preferably 2 mg / mL; and the volume ratio of the solution of the fluorinated zwitterionic copolymer nanoparticles to the PBS buffer solution of the protease is 1-5:1-40.

[0053] According to the application provided by the present invention, the PBS buffer solution for the protease in step S300 is typically neutral or weakly alkaline. In some embodiments, the pH of the PBS buffer solution for the protease is less than 7.5, particularly 7.2 to 7.4.

[0054] According to the application provided by the present invention, the concentration of the PBS buffer solution of the protease in step S300 can be 0.1 to 0.3 mg / mL, preferably 0.2 mg / mL.

[0055] According to the application provided by the present invention, in step S400, the volume ratio of the solution of the fluorinated zwitterionic copolymer nanoparticles to the PBS buffer solution of the protease is 1:2 to 5.

[0056] According to the application provided by the present invention, the protease is horseradish peroxidase (HRP).

[0057] In the fourth and fifth aspects, the present invention also provides the above-mentioned fluorinated zwitterionic copolymer and its preparation method.

[0058] This invention has the following advantages:

[0059] (1) In current research on macromolecular self-assembly, polyethylene glycol (PEG) is the most commonly used hydrophilic segment in the assembly system to date, while oil-soluble polymers such as polymethyl methacrylate (PMMA) are the most widely studied hydrophobic segment. The amphiphilic block copolymer PEG-b-PMMA, obtained by covalently linking the above two polymers through covalent bonds, is one of the most classic research objects of macromolecular self-assembly. In contrast, in this invention, zwitterionic alkyl (meth)acrylate (ZMA) and fluorinated alkyl (meth)acrylate (FMA) are used as comonomers, and controlled random copolymerization is carried out by RAFT polymerization to prepare a new amphiphilic random copolymer material based on zwitterionic side groups and fluorinated alkyl groups—fluorinated polyzwitterionic copolymer P(ZMA-r-FMA).

[0060] (2) In current research on macromolecular self-assembly, solution assembly based on the hydrophilicity and hydrophobicity of conventional block copolymers can yield micron-sized micelles, worms, vesicles, and other assembled structures. The fluorinated zwitterionic random copolymer P(ZMA-r-FMA) of this invention possesses multiple assembly driving forces, including ionic interactions and fluorine effects, in addition to conventional hydrophobic interactions. It effectively undergoes microphase separation single-chain self-assembly behavior in a trifluoroethanol and water mixed solvent system, producing quasi-nanoscale functional assemblies, which cannot be achieved by solution assembly based on amphiphilic block copolymers.

[0061] (3) The chemical nature of biological reagents such as enzymes and antibodies is protein. Their natural three-dimensional structure is easily altered by physical (high temperature, light) and chemical (strong acid, strong alkali) environmental factors, resulting in denaturation and loss of catalytic and clinical therapeutic functions. Stabilization techniques that preserve enzyme protein activity are usually stored in solid form as dry powder or crystals, or in solution form under a low-temperature (-70℃) cold chain environment supplemented with organic preservatives (such as 2-mercaptoethanol) that affect their activity. In addition, researchers have developed strategies such as enzyme surface chemical modification, enzyme immobilization engineering, and artificial synthesis of molecular chaperones to improve enzyme structural stability, but it is still difficult to meet the storage goal of both stable protease structure and activity. In the polymer nanoparticles obtained by assembling fluorinated zwitterionic copolymer solutions according to the present invention, zwitterionic alkyl groups provide excellent hydrophilicity, chemical stability, and biocompatibility, while fluorinated alkyl groups have a unique fluorination effect and a positive effect promoting protease stabilization, achieving the functional goal of stable storage of highly active proteases, such as horseradish peroxidase (HRP), at room temperature, which is of great application value.

[0062] In addition, the fluorinated zwitterionic copolymer nanoparticles of the present invention can avoid the problems of inhibition of enzyme activity or even complete inactivation caused by conventional enzyme stabilization strategies such as surface chemical modification and immobilization.

[0063] (4) In the preparation method of the present invention, the polymerization reaction is carried out under normal laboratory conditions without the need for special operating instruments (such as glove box), and the solvents and other chemical reagents used are used directly without further purification. The reaction can still proceed efficiently, which is more conducive to the simple, environmentally friendly and efficient synthesis of novel fluorinated zwitterionic copolymers, the preparation of polymer nanoparticles by solution assembly, and the stable storage of highly active proteases in room temperature solution. Attached Figure Description

[0064] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0065] Figure 1 The graph shows the relationship between the conversion rate of SBMA and F6MA and polymerization time in the preparation of fluorinated zwitterionic copolymers using methacryloyl ethyl sulfobetaine (SBMA) and hexafluorobutyl methacrylate (F6MA) monomers.

[0066] Figure 2 The present invention is a fluorinated zwitterionic copolymer P(SBMA) 70 -r-F6MA 30 )of 1 1H NMR spectrum;

[0067] Figure 3 This is a transmission electron microscope (TEM) image of the fluorinated zwitterionic copolymer nanoparticles of the present invention.

[0068] Figure 4 This is a dynamic light scattering DLS analysis diagram of the fluorinated zwitterionic copolymer nanoparticles of the present invention.

[0069] Figure 5 The images are TEM images of horseradish peroxidase stabilized by the fluorinated zwitterionic copolymer nanoparticles of the present invention, wherein Figure (a) is a control image without the addition of copolymer nanoparticle stabilizer. Detailed Implementation

[0070] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0071] The raw materials used in this invention are shown in Table 1.

[0072] Table 1 Raw Materials

[0073]

[0074]

[0075] Molecular weight and molecular weight distribution M w / M n

[0076] 1. Oil-soluble homopolymer PFMA

[0077] Number average molecular weight (Mn) of oil-soluble homopolymer PFMA n,GPC ) and molecular weight distribution (M w / M nThe molecular weight was determined using a Viscotek TDAmax gel permeation chromatography system (GPC, Malvern, UK), equipped with a TDA305 differential refractive index detector, a TGard guard column, and two test columns, T4000 and T2500. The measurable molecular weight range is 5 × 10⁻⁶. 2 ~5×10 5 g / mol. Tetrahydrofuran (THF) was used as the mobile phase during the test at 35℃ and a flow rate of 1.0 mL / min. Samples were injected using a Malvern autosampler, and the relative molecular weight was determined using polystyrene (PSt) standards.

[0078] 2. Water-soluble polymer PZMA

[0079] Number-average molecular weight (M) of water-soluble polymer PZMA n,GPC ) and molecular weight distribution (M w / M n The molecular weight was determined using an Agilent gel permeation chromatography system (model: PL-GPC50), equipped with a differential refractive index detector, one PL aquagel-OH Gard guard column (8μm, 50×7.5mm), and two test columns: PL aquagel-OH 30 (8μm, 300×7.5mm) and PL aquagel-OH 40 (8μm, 300×7.5mm). The measurable molecular weight range is 5×10⁻⁶. 2 ~5×10 5 g / mol. The mobile phase was ultrapure water (0.1 M NaNO3), the temperature was 35℃, and the flow rate was 1.0 mL / min. The relative molecular weight was determined using polyethylene glycol (PEG) standards.

[0080] Conversion rate

[0081] The monomer conversion rate was measured using nuclear magnetic resonance (NMR). 1 H and 13 C10 NMR measurements were performed on a 400 MHz NMR spectrometer (model: AVANCE AV III, Bruker GmbH, Germany), using CDCl3, D2O, or CF3COOD as solvents and tetramethylsilane (TMS, δ=0) as an internal standard reference.

[0082] Structural characterization of fluorinated zwitterionic random copolymer P(ZMA-r-FMA)

[0083] The structure of the fluorinated zwitterionic random copolymer P(ZMA-r-FMA) was characterized using nuclear magnetic resonance (NMR), in which... 1 H and 13C10 NMR measurements were performed on a 400 MHz NMR spectrometer (model: AVANCE AV III, Bruker GmbH, Germany), using CDCl3, D2O, or CF3COOD as solvents and tetramethylsilane (TMS, δ=0) as an internal standard reference.

[0084] TEM

[0085] Transmission electron microscopy (TEM) tests were performed on a JEOL JEM-1100 transmission electron microscope (operating voltage: 100kV).

[0086] Unless otherwise specified, the following experiments and examples were conducted in accordance with the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all common products that can be purchased from legitimate channels.

[0087] Experiment 1

[0088] Polymethacryloyl ethyl sulfobetaine (PSBMA) with different degrees of polymerization was prepared by RAFT polymerization in a water and methanol mixed solvent system.

[0089] (1) According to the initial feeding ratio of [SBMA]0:[AIBN]0:[CPDB]0=100:0.5:1, methacryloyl ethyl sulfobetaine (SBMA, 0.28g, 1.0mmol) and AIBN (0.82g, 5×10) were added. -3 mmol) and CPDB (2.2 mg, 0.01 mmol) were added to a mixture of 1 mL of water and methanol (V) 水 :V 甲醇 Add a stir bar to an ampoule with a ratio of 1:3, purge with nitrogen to remove oxygen, and seal.

[0090] (2) Transfer the ampoule to a magnetic stirrer with a set speed of 1800 rpm, heat it in an oil bath, and carry out the RAFT polymerization reaction at 80°C. After the predetermined time is reached, take out and open the sealed ampoule, dilute the polymerization system with trifluoroethanol (2 mL), and pour it into a large amount of methanol (200 mL) to precipitate. Filter the mixture, and place the filter cake in a vacuum oven at 35°C to dry to constant weight to obtain the desired polymer.

[0091] By changing the initial feed ratio in step (1) and the polymerization reaction time in step (2), the conversion rate, molecular weight, and molecular weight distribution M of each obtained polymer were measured. w / M n The results are shown in Table 2.

[0092] Table 2. Reaction conditions and properties of polymethacryloyl ethyl sulfobetaine (PSBMA)

[0093]

[0094] Given the hydrophilic properties of the sulfonate betaine group (SB) in SBMA and the strong polarity of the polymer polymethacryloyl ethyl sulfobetaine (PSBMA), in Experiment 1, a mixed solvent of water and methanol was used as the polymerization solvent system, and PSBMA with different target degrees of polymerization were synthesized in a controlled manner by adjusting the initial feed ratio.

[0095] As shown in Table 2, when the designed degree of polymerization (i.e., [SBMA]0:[CPDB]0) was 50 (Experiment 1.1) and 100 (Experiment 1.2), respectively, relatively ideal polymerization results were obtained. The number-average molecular weight of the polymer measured by aqueous phase GPC showed a high degree of agreement with the theoretical molecular weight, and the molecular weight distribution was within 1.2. This demonstrates the excellent controllability of RAFT polymerization over SBMA polymerization, and that PSBMA with different target degrees of polymerization can be prepared in a controlled manner.

[0096] Experiment 2

[0097] Poly(3-[[2-(methacryloyloxy)ethylene]dimethylammonium]propionate (PCBMA) was prepared by RAFT polymerization in a water and methanol mixed solvent system.

[0098] (1) According to the initial feed ratio of [CBMA]0:[AIBN]0:[CPDB]0 = 50:0.5:1, 3-[[2-(methacryloyloxy)ethylene]dimethylammonium]propionate (CBMA, 0.23 g, 1.0 mmol), AIBN (1.64 g, 0.01 mmol), and CPDB (4.4 mg, 0.02 mmol) were added to a 1 mL water and methanol mixed solvent system (V 水 :V 甲醇 Add a stir bar to an ampoule with a ratio of 1:3, purge with nitrogen to remove oxygen, and seal.

[0099] (2) Transfer the ampoule to a magnetic stirrer with a set speed of 1800 rpm, heat it in an oil bath, and carry out the RAFT polymerization reaction at 80°C. After the predetermined time is reached, take out and open the sealed ampoule, dilute the polymerization system with trifluoroethanol (2 mL), and pour it into a large amount of methanol (200 mL) to precipitate. Filter the mixture, and place the filter cake in a vacuum oven at 35°C to dry to constant weight to obtain the desired polymer.

[0100] The measured polymer conversion rate, molecular weight, and molecular weight distribution M w / M n The results are shown in Table 3.

[0101] Table 3 Properties of different polyzwitterionic polymers PZMA

[0102]

[0103] Table 3 shows that the RAFT polymerization of both SBMA and CBMA monomers yielded ideal polymerization results. In particular, when the RAFT solution polymerization of CBMA was carried out at 80℃ (Experiment 2.3), the monomer conversion rate reached as high as 94.8% after 60 minutes of reaction. The number-average molecular weight (10.3 kDa) of the polymerized product, measured by aqueous phase GPC, was in excellent agreement with the theoretical molecular weight (10.9 kDa), and the molecular weight distribution was narrow (M... w / M n =1.18).

[0104] In addition, the results in Table 3 also show that RAFT polymerization has excellent tolerance to zwitterionic alkyl (meth)acrylates, which provides a basis for subsequent adjustment of the amphiphilicity, fluorination effect and other properties of fluorinated zwitterionic copolymers through monomer structure design.

[0105] Experiment 3

[0106] In Experiment 3, polymethacrylamide ethyl sulfonated betaine (PSBMA) was prepared by RAFT polymerization using different solvent systems. Specifically, PSBMA was prepared using a method that was basically the same as in Experiment 1.2, with the only differences being: (a) the solvent systems listed in Table 4 were used; and (b) the polymerization time in step (2) was 300 min.

[0107] The conversion rate, molecular weight, and molecular weight distribution M of the obtained polymer were measured. w / M n The results are shown in Table 4.

[0108] Table 4. Properties of polymethacryloyl ethylidene sulfonate betaine (PSBMA) prepared under different solvent systems

[0109]

[0110] Table 3 shows that when pure water is used as the reaction solvent (Experiment 3.1), the molecular weight and monomer conversion rate of the polymerized product are relatively low. When a mixed solvent of water and methanol is used as the reaction solvent system (Experiments 3.2 and 3.3), the molecular weight of the polymerized product is relatively high, but the monomer conversion rate is still relatively low. However, when trifluoroethanol is used as the reaction solvent (Experiment 3.4), both the molecular weight and monomer conversion rate of the polymerized product are relatively high. To avoid being limited by theory, it is generally believed that preventing polymer precipitation during RAFT polymerization is a key factor in improving polymerization efficiency. The polarity of the solvent may affect the initiation efficiency, reaction rate, and side reactions such as free radical termination in RAFT polymerization. PSBMA is hydrophilic, water is a good solvent, while methanol is a poor solvent (it can be used as a precipitant for refining the polymer after polymerization). Therefore, combined with Experiment 1 (Table 2), when a mixed solvent of water and methanol is used as the reaction solvent, a volume ratio of water to methanol of 7:3 to 1:3 results in a relatively high conversion rate and a high molecular weight. Furthermore, it is believed that the trifluoromethyl group in trifluoroethanol, which has a strong electron-withdrawing effect, can form hydrogen bonds with the sulfonate group in SBMA to promote the efficient occurrence of the polymerization reaction.

[0111] Experiment 4

[0112] Experiment 4 used different reaction temperatures to prepare polymethacrylamide ethyl sulfonated betaine (PSBMA) via RAFT polymerization. Specifically, PSBMA was prepared using a method that was basically the same as in Experiment 1.2, with the only differences being: (a) trifluoroethanol was used as the reaction solvent; (b) the polymerization temperature in step (2) is shown in Table 5; and (c) the polymerization time in step (2) was 300 min.

[0113] The conversion rate, molecular weight, and molecular weight distribution M of the obtained polymer were measured. w / M n The results are shown in Table 5.

[0114] Table 5. Properties of polymethacryloyl ethyl sulfonate betaine (SBMA) prepared at different reaction temperatures.

[0115] 4.1 60 34.6 9.9 8.7 1.12 4.2 70 54.6 15.5 14.7 1.16 4.3 (Same as 3.4) 80 60.3 17.1 15.9 1.15 4.4 90 61.1 17.3 16.0 1.17

[0116] Table 5 shows that reaction temperature has a significant impact on the decomposition efficiency of the free radical thermal initiator AIBN and the concentration of free radicals in the polymerization system, thus affecting the RAFT polymerization process of methacryloyl ethyl sulfobetaine (SBMA) monomer. Specifically, within the same reaction time, the monomer conversion rate of SBMA increases with increasing polymerization temperature, which is consistent with the free radical reaction mechanism of RAFT polymerization. When the reaction temperature increases, the decomposition rate of the free radical thermal initiator AIBN increases, leading to an increase in the concentration of free radicals in the reaction system. Therefore, within the same reaction time, the monomer conversion rate of SBMA increases significantly with increasing reaction temperature. When the reaction temperature increases from 60℃ (Experiment 4.1) to 90℃ (Experiment 4.4), the monomer conversion rate increases from 34.6% to 61.1%, and the number-average molecular weight of the polymer PSBMA also shows an increasing trend with increasing monomer conversion rate, while the corresponding molecular weight distribution remains consistently below 1.2.

[0117] Furthermore, Experiment 4 further demonstrates the excellent controllability of RAFT polymerization over SBMA polymerization and the “active” / controllable characteristics of RAFT polymerization.

[0118] Experiment 5

[0119] Experiment 5 used different solvent systems to prepare polyheptafluorodecyl methacrylate (PF6) by RAFT polymerization. 17 MA).

[0120] (1) According to [F 17 The initial feed ratio of MA]0:[AIBN]0:[CPDB]0 = 100:0.5:1 was used to feed heptadecafluorodecyl methacrylate (F... 17 MA, 0.53g, 1.0mmol), AIBN (0.82g, 5×10 -3 Add 1 mmol) and CPDB (2.2 mg, 0.01 mmol) to an ampoule containing 1 mL of solvent, add a stir bar, purge with nitrogen to remove oxygen, and seal.

[0121] (2) Transfer the ampoule to a magnetic stirrer with a set speed of 1800 rpm, heat it in an oil bath, and carry out the RAFT polymerization reaction at 80°C for 300 min. Then, take out and open the sealed ampoule, add trifluoroethanol (2 mL) to dilute the polymerization reaction system, dialyze it in a large amount of trifluoroethanol (with a molecular weight cutoff of 1 kDa) for 72 hours, evaporate it to dryness by rotary evaporation, and then place it in a vacuum oven at 35°C to dry to constant weight to obtain the desired polymer.

[0122] The conversion rate of the obtained polymer was measured, and the results are shown in Table 6.

[0123] Table 6 Polyhexadecanoacrylate (PFA) prepared under different solvent systems17 MA performance

[0124]

[0125] Note: In Table 6, "a" indicates that the polymerized product is insoluble in tetrahydrofuran, and the molecular weight and molecular weight distribution of GPC were not determined.

[0126] As shown in Table 6, F 17 When MA undergoes high-temperature RAFT polymerization in common organic solvents, such as toluene (Experiment 5.1), dimethyl sulfoxide (Experiment 5.2), tetrahydrofuran (Experiment 5.3), and 1,4-dioxane (Experiment 5.4), polymerization proceeds smoothly, but monomer conversion is low. However, when trifluoroethanol (Experiment 5.5) is used as the reaction solvent, a relatively high monomer conversion can be achieved smoothly. However, due to the polymer PF... 17 The high fluorine content in MA makes the product insoluble in tetrahydrofuran for GPC characterization, which is consistent with the low monomer conversion rate observed when tetrahydrofuran is used as a reaction solvent.

[0127] Furthermore, in conjunction with Experiment 3, it was surprisingly found that trifluoroethanol is also a good solvent for the RAFT polymerization of ZMA monomers, and trifluoroethanol is a good solvent for the preparation of fluorinated zwitterionic copolymers in this invention.

[0128] Experiment 6

[0129] Polyheptafluorodecyl methacrylate (PF6) with different degrees of polymerization was prepared by RAFT polymerization in a trifluoroethanol solvent system. 17 MA). Specifically, polyhexadecafluorodecyl methacrylate (PFMA) was prepared using a method essentially the same as in Experiment 5.5. 17 The only difference is that the initial feed ratio listed in Table 7 is used.

[0130] The conversion rate of the obtained polymer was measured, and the results are shown in Table 7.

[0131] Table 7. Polyhexadecanoacrylate (F) prepared using different initial feed ratios 17 MA) performance

[0132]

[0133] Note: In Table 7, "a" indicates that the polymerized product is insoluble in tetrahydrofuran, and the molecular weight and molecular weight distribution of GPC were not determined.

[0134] As shown in Table 7, when the design aggregation degree ([F 17When the monomer conversion ratios were 25 (Experiment 6.1), 50 (Experiment 6.2), and 100 (Experiment 6.3), respectively, the polymerization reaction achieved relatively high monomer conversion rates. However, due to the polymer PF 17 The excessively high fluorine content in MA prevented the product from dissolving in tetrahydrofuran for GPC characterization. Achieving a polymerized product PFMA with an actual molecular weight matching the theoretical molecular weight by controlling the initial feed ratio of the polymerization reaction is an important indicator for verifying the controllability of RAFT polymerization over FMA polymerization and its ability to design the primary structure of the polymer. Although the GPC molecular weight and molecular weight distribution were not measured, the high monomer conversion rate of PFMA... 17 The successful preparation of MA also demonstrates the excellent polymer synthesis capability of RAFT polymerization for FMA.

[0135] Experiment 7

[0136] Fluorinated polymethyl methacrylate (PFMA) was prepared by RAFT polymerization.

[0137] (1) According to the initial feeding ratio of [F6MA]0:[AIBN]0:[CPDB]0=50:0.5:1, add hexafluorobutyl methacrylate (F6MA, 0.25g, 1.0mmol), AIBN (1.64g, 0.01mmol) and CPDB (4.4mg, 0.02mmol) into an ampoule containing 1mL of trifluoroethanol, add a stir bar, purge with nitrogen to remove oxygen, and seal.

[0138] (2) Transfer the ampoule to a magnetic stirrer with a set speed of 1800 rpm, heat it in an oil bath, and carry out the RAFT polymerization reaction at 80°C for 300 min. Then, take out and open the sealed ampoule, add trifluoroethanol (2 mL) to dilute the polymerization reaction system, dialyze it in a large amount of trifluoroethanol (with a molecular weight cutoff of 1 kDa) for 72 hours, evaporate it to dryness by rotary evaporation, and then place it in a vacuum oven at 35°C to dry to constant weight to obtain the desired polymer.

[0139] The conversion rate, molecular weight, and molecular weight distribution M of the obtained polymer were measured. w / M n The results are shown in Table 8.

[0140] Table 8 Properties of Fluorinated Polymethyl Methacrylate (PFMA)

[0141]

[0142] Note: In Table 8, "a" indicates that the polymerized product is insoluble in tetrahydrofuran, and the molecular weight and molecular weight distribution of GPC were not determined.

[0143] Table 8 shows that hexafluorobutyl methacrylate F6MA (Test 7.1), 2-(perfluorobutyl)ethylene methacrylate F9MA (Test 7.2), and heptadecafluorodecyl methacrylate F... 17 The RAFT polymerization of MA (Experiment 7.3) yielded very satisfactory experimental results. In particular, PF9MA and PF6MA, prepared by RAFT polymerization of the shorter fluoroalkanes F9MA and F6MA, both exhibited high monomer conversion rates and considerably high number-average molecular weights (M). n,GPC ) and narrow molecular weight distribution (M w / M n ).

[0144] Experiment 8

[0145] In Experiment 8, different reaction temperatures were used to prepare poly(hexafluorobutyl methacrylate) (F6MA) via RAFT polymerization. Specifically, poly(hexafluorobutyl methacrylate) (F6MA) was prepared using a method essentially the same as that in Experiment 7.1, with the only differences being: (a) in step (1) [F6MA]0:[AIBN]0:[CPDB]0 = 100:0.5:1; and (b) the polymerization temperature in step (2) is shown in Table 9.

[0146] The conversion rate, molecular weight, and molecular weight distribution M of the obtained polymer were measured. w / M n The results are shown in Table 9.

[0147] Table 9 Properties of polyhexafluorobutyl methacrylate (PF6MA) prepared at different polymerization temperatures

[0148]

[0149] As shown in Table 9, the monomer conversion rate of F6MA increases with increasing polymerization temperature within the same reaction time, which is consistent with the free radical reaction mechanism of RAFT controlled polymerization. When the temperature increases, the decomposition rate of the free radical thermal initiator AIBN increases, leading to an increase in the free radical concentration in the reaction system. Therefore, within the same polymerization time, the monomer conversion rate of F6MA increases significantly with increasing reaction temperature. When the reaction temperature increases from 60℃ (Experiment 8.1) to 80℃ (Experiment 8.3), the monomer conversion rate increases from 29.6% to 63.2%, and the number-average molecular weight of the polymerized product PF6MA also shows an increasing trend with increasing monomer conversion rate, while the corresponding molecular weight distribution remains consistently below 1.2. When the reaction temperature is further increased to 90℃ (Experiment 8.4), the monomer conversion rate decreases, and the corresponding number-average molecular weight of PF6MA also decreases slightly. This may be because the excessively high reaction temperature leads to an excessively rapid decomposition rate of AIBN, making it impossible to maintain a relatively constant free radical concentration in the later stages of polymerization, thus resulting in a slight decrease in monomer conversion rate.

[0150] Furthermore, combined with Experiment 4, it was surprisingly found that the polymerization reaction temperature of 80°C is also the optimal reaction temperature for RAFT polymerization of ZMA monomers such as SBMA.

[0151] Example 1

[0152] Fluorinated zwitterionic random copolymer P(SBMA) was prepared by RAFT polymerization using two monomers, methacryloyl ethyl sulfobetaine (SBMA) and hexafluorobutyl methacrylate (F6MA). 30 -r-F6MA 70 ).

[0153] (1) According to the initial feeding ratio of [SBMA]0:[F6MA]0:[AIBN]0:[CPDB]0=30:70:0.5:1, add methacryloyl ethyl sulfobetaine (SBMA, 0.17g, 0.6mmol), hexafluorobutyl methacrylate (F6MA, 0.35g, 1.4mmol), AIBN (1.64mg, 0.01mmol) and CPDB (4.4mg, 0.02mmol) into an ampoule containing 1mL of trifluoroethanol, add a stir bar, purge with nitrogen to remove oxygen, and seal.

[0154] (2) Transfer the ampoule to a magnetic stirrer with a set speed of 1800 rpm, heat it in an oil bath, and carry out the RAFT polymerization reaction at 80°C. After the predetermined time is reached, take out and open the sealed ampoule, add trifluoroethanol (2 mL) to dilute the reaction system, and dialyze it in a large amount of trifluoroethanol (with a molecular weight cutoff of 1 kDa) for 72 hours. Then, evaporate it to dryness by rotary evaporation and place it in a vacuum oven at 35°C to dry to constant weight to obtain the desired polymer P (SBMA-r-F6MA).

[0155] By changing the initial feed ratio of the two monomers in step (1) ([SBMA]0:[F6MA]0) and the RAFT polymerization reaction time in step (2), the conversion rate was measured, and the results are shown in […]. Figure 1 ,in, Figure 1 In (a) and (b), the total amount of SBMA and F6MA is 2 mmol, and the total designed degree of polymerization DP total That is, ([SBMA]0+[F6MA]0):[CPDB]0 is 100, and ([SBMA]0:[F6MA]0) are 30:70 and 70:30 respectively; Figure 1 In (c) and (d), the total amount of SBMA and F6MA is 2 mmol, and the total designed degree of polymerization DP totalThat is, ([SBMA]0+[F6MA]0):[CPDB]0 is 300, and ([SBMA]0:[F6MA]0) are 90:210 and 210:90 respectively.

[0156] Depend on Figure 1 It can be seen that changing the initial feed ratio of the two monomers ([SBMA]0:[F6MA]0) has no significant effect on the polymerization behavior, and ideal polymerization yields are obtained in both cases. This indicates that SBMA and F6MA exhibit a competitive polymerization relationship during copolymerization, which is determined solely by the feed ratio, and the segments of the two different side groups are randomly distributed in the polymer chain.

[0157] The fluorinated zwitterionic copolymer P(SBMA) prepared in Example 1 was analyzed by nuclear magnetic resonance. 30 -r-F6MA 70 The values ​​30 and 70 in the lower right corner represent the target degree of polymerization of the two monomers, SBMA and F6MA, respectively.

[0158] Figure 2 Showing Figure 1 (a) Fluorinated zwitterionic copolymer P(SBMA) prepared with a reaction time of 120 min 30 -r-F6MA 70 ) NMR 1 1H NMR spectrum. Figure 2 The clear assignment of the proton peak indicates that the expected fluorinated zwitterionic copolymer P(SBMA) was successfully prepared. 30 -r-F6MA 70 ) and the high retention of RAFT reagents at the polymer chain ends.

[0159] NMR of other copolymers prepared in Example 1 1 The 1H NMR spectrum also clearly verified the correctness of its polymer structure and the high retention of the RAFT reagent at the polymer chain ends.

[0160] Example 1 demonstrates that the preparation method of the present invention, especially using preferred polymerization conditions, can very efficiently prepare a novel amphiphilic random copolymer material with the target structure—a fluorinated zwitterionic copolymer P (ZMA-r-FMA).

[0161] Example 2

[0162] Fluorinated zwitterionic copolymer P(ZMA-r-FMA) prepared in Example 1 was used to prepare fluorinated zwitterionic copolymer nanoparticles.

[0163] (1) Weigh 10 mg of the fluorinated zwitterionic copolymer P(SBMA) prepared in Example 1 with a polymerization reaction time of 120 min.210 -r-F6MA 90 Dissolve the polymer in 3 mL of trifluoroethanol, add a stir bar, and stir overnight at room temperature to ensure that the polymer is fully dissolved, thus obtaining a polymer solution.

[0164] (2) Add 2 mL of ultrapure water to the polymer solution prepared in step (1) to obtain the assembly solution. Then, use a Finnpipette pipette to transfer 20 μL of the sample solution onto a carbon film copper grid. After drying at room temperature for 24 hours, perform TEM characterization. The results are shown in the figure. Figure 3 .

[0165] Change the fluorinated zwitterionic copolymer P(SBMA) in step (1) 210 -r-F6MA 90 Different concentrations of assembly solutions were prepared by adjusting the dosage of [specific ingredient], and TEM characterization was performed. The results are shown in [Figure number missing]. Figure 3 ,in, Figure 3 The concentrations of the assembly solutions used in (a) to (e) were 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL.

[0166] Meanwhile, each assembly solution was characterized using DLS. Figure 4 DLS results for assembled solutions at concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL are shown.

[0167] like Figure 3 (a) and Figure 4 As shown, when the concentration of the assembly solution is 1 mg / mL, P(SBMA) 210 -r-F6MA 90 Assembly has been successfully achieved, forming fluorinated zwitterionic copolymer nanoparticles (D...). h =14.5 nm), with good particle size distribution uniformity (PDI = 0.050), and the nanoparticle distribution density within the field of view is much higher than that of typical single-chain nanoparticle assemblies reported in the literature [see T. Terashima et al. Macromolecules 2018, 51, 3738]. When the assembly solution concentration is gradually increased from 2 mg / mL to 4 mg / mL (e.g. Figure 3 (b)~(d)) shows a significant increasing trend in the distribution density of nanoparticles within the field of view; when the concentration is further increased to 5 mg / mL ( Figure 3 (e)) large-sized aggregates (D) appeared. h =98.0nm). Furthermore, it can be seen that the corresponding DLS test results and TEM observation results have a fairly high degree of consistency.

[0168] Comparison of TEM test results of the assembly system at different concentrations shows that the concentration of the assembly solution has a significant impact on the size of the assembled particles. The single-chain or multi-chain assembly of fluorinated zwitterionic copolymer P(ZMA-r-FMA) and the structural indicators such as nanoparticle concentration and particle size can be controlled by adjusting the concentration of the assembly solution.

[0169] Example 3

[0170] Stabilization of horseradish peroxidase (HRP) was achieved using a solution of fluorinated zwitterionic copolymer nanoparticles.

[0171] (1) Prepare PBS buffer (0.1 mL, pH 7.2-7.4) with a concentration of 0.2 mg / mL horseradish peroxidase by adjusting the volume.

[0172] (2) Add horseradish peroxidase in PBS buffer to the solution of fluorinated zwitterionic copolymer nanoparticles prepared in Example 2—P(SBMA) 210 -r-F6MA 90 The solution was added to the assembly solution (2 mg / mL), shaken thoroughly, and then 20 μL of the solution was transferred using a Finnpipette pipette and dropped onto a carbon film copper grid. After drying at room temperature for 24 hours, TEM characterization was performed. The results are as follows. Figure 5 As shown, (a) is a TEM image of horseradish peroxidase in PBS buffer (control image); (b) and (c) are TEM images of horseradish peroxidase in PBS buffer at a volume of 100 μL and assembly solution at a volume of 20 μL; and (d) to (f) are TEM images of horseradish peroxidase in PBS buffer at a volume of 100 μL and assembly solution at a volume of 40 μL.

[0173] Depend on Figure 5 (a) It can be seen that, due to the dynamic conformational changes of HRP, structurally stabilized HRP cannot be observed by TEM characterization in PBS buffer solution (pH 7.2–7.4) without polymer nanoparticle stabilizers. Figure 5 (b) and (c) show a well-defined assembly morphology with a particle size of approximately 700 nm, indicating that the fluorinated zwitterionic copolymer nanoparticles can stabilize the HRP protease. When the volume of the polymer nanomicelle solution is further increased to 40 μL ( Figure 5 (d) The distribution density of HRP protease stabilized by fluorinated zwitterionic copolymer nanoparticles increased, and the particle size of the complex was stabilized at 150 nm, which is consistent with the structural information of the stabilized single HRP molecule reported in the literature (see R. Imamura, H. Mori, ACS Omega, 2019, 4, 18234-18247). Figure 5(e) and (f) are respectively Figure 5 (d) is a magnified view of the part marked in red.

[0174] From the above, it can be seen that: (1) In systems where fluorinated zwitterionic copolymer nanoparticles and protease solutions are mixed at different concentrations, there are two types of particles with particle sizes of about 150 nm and 1 μm, which represent HRP protease in stable monomolecular and multimolecular aggregate states, respectively; (2) When the amount of fluorinated zwitterionic copolymer nanoparticles is increased ( Figure 5 (d)~(f)) Fluorinated zwitterionic copolymer nanoparticles can better achieve HRP stabilization and the composite particle size is more uniform.

[0175] Therefore, the systematic characterization by TEM fully demonstrates that by adjusting the ratio of fluorinated zwitterionic copolymer nanoparticles to proteases, room temperature solution stabilization and storage of highly active proteases modeled by HRP with uniform size and stable structure can be successfully achieved.

[0176] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing fluorinated zwitterionic copolymer nanoparticles with protease-stabilizing properties, wherein, The fluorinated zwitterionic copolymer nanoparticles comprise one or more fluorinated zwitterionic copolymer molecules; wherein the monomers of the fluorinated zwitterionic copolymer comprise zwitterionic alkyl (meth)acrylates and fluorinated alkyl (meth)acrylates, the molar ratio of the zwitterionic alkyl (meth)acrylates to the fluorinated alkyl (meth)acrylates is 10:90 to 90:10, and the fluorinated alkyl (meth)acrylates contain 6 to 17 fluorinated alkyl groups; The preparation method includes the following steps: S100. In the presence of an initiator and a chain transfer agent, zwitterionic alkyl (meth)acrylates and fluorinated alkyl (meth)acrylates undergo RAFT polymerization in a reaction solvent to obtain a fluorinated polyzwitterionic copolymer. S200: Dissolve the fluorinated zwitterionic copolymer obtained in step S100 in a mixed solvent of trifluoroethanol and water to prepare an assembly solution of fluorinated zwitterionic copolymer nanoparticles; wherein, the volume ratio of trifluoroethanol to water in the mixed solvent of trifluoroethanol and water is 0~3:0~3, and the volumes of both trifluoroethanol and water are not 0, and the concentration of the fluorinated zwitterionic copolymer in the assembly solution is 0.1~4 mg / mL.

2. The preparation method according to claim 1, wherein, The zwitterionic alkyl (meth)acrylate is methacryloyl ethylidene sulfobetaine and / or 3-[[2-(methacryloyloxy)ethylidene]dimethylammonium]propionate; And / or, the fluoroalkyl (meth)acrylate is selected from one or more of hexafluorobutyl methacrylate, 2-(perfluorobutyl)ethylidene methacrylate and heptadecafluorodecyl methacrylate; And / or, the molar ratio of the zwitterionic alkyl (meth)acrylate to the fluoroalkyl (meth)acrylate is 20:80 to 80:20; And / or, the theoretical degree of polymerization of the fluorinated zwitterionic copolymer is 50 to 500.

3. The preparation method according to claim 2, wherein, The zwitterionic alkyl (meth)acrylate is methacryloyl ethyl sulfobetaine; And / or, the fluoroalkyl (meth)acrylate is hexafluorobutyl methacrylate; And / or, the molar ratio of the zwitterionic alkyl (meth)acrylate to the fluoroalkyl (meth)acrylate is 60:40 to 80:20; And / or, the theoretical degree of polymerization of the fluorinated zwitterionic copolymer is 100 to 400.

4. The preparation method according to claim 3, wherein, The molar ratio of the zwitterionic alkyl (meth) acrylate to the fluoroalkyl (meth) acrylate is 70:

30. And / or, the theoretical degree of polymerization of the fluorinated zwitterionic copolymer is 270~320.

5. The preparation method according to claim 1, wherein, In step S100, the molar ratio of zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent is 10~270:10~270:0.1~0.5:0.2~1.

6. The preparation method according to claim 1, wherein, In step S100, the molar ratio of zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent is 10~270:10~270:0.25~0.5:

1.

7. The preparation method according to claim 1, wherein, In step S100, the molar ratio of zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent is 30~240:30~240:0.25~0.5:

1.

8. The preparation method according to claim 1, wherein, In step S100, the molar ratio of zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent is 180~240:120~60:0.25~0.5:

1.

9. The preparation method according to claim 1, wherein, In step S100, the molar ratio of zwitterionic alkyl (meth)acrylate, fluorinated alkyl (meth)acrylate, initiator and chain transfer agent is 210:90:0.5:

1.

10. The preparation method according to any one of claims 1 to 9, wherein, The reaction solvent is selected from one or more of toluene, 1,4-dioxane, trifluoroethanol, methanol, water, and dimethyl sulfoxide; And / or, the initiator is azobisisobutyronitrile; And / or, the chain transfer agent is 2-cyanopropyl-2-ylbenzodisulfide.

11. The preparation method according to claim 10, wherein, The reaction solvent is a mixture of water and methanol or trifluoroethanol, wherein the volume ratio of water to methanol in the mixture of water and methanol is 0~7:0~7.

12. The preparation method according to claim 10, wherein, The reaction solvent is a mixture of water and methanol or trifluoroethanol, wherein the volume ratio of water to methanol in the mixture of water and methanol is 7:3 to 1:

3.

13. The preparation method according to any one of claims 1 to 9, wherein, Step S100 includes the following steps: S101. Add zwitterionic alkyl (meth) acrylate, fluorinated alkyl (meth) acrylate, initiator and chain transfer agent to the reaction solvent, mix, introduce inert gas to remove oxygen, seal, and obtain the reaction system. S102. Under stirring conditions and at a temperature of 60~90℃, the reaction system obtained in step S101 undergoes a polymerization reaction to obtain the reaction product. S103. Dialyze the reaction product obtained in step S102 and dry it to obtain a fluorinated zwitterionic copolymer.

14. The preparation method according to claim 13, wherein, In step S102, the stirring speed is 800~3000 rpm; And / or, the polymerization reaction time in step S102 is 30~360 min.

15. The preparation method according to claim 13, wherein, In step S102, the stirring speed is 1500~2000 rpm; And / or, the polymerization reaction in step S102 is carried out at a temperature of 80°C for a time of 120 min.

16. The preparation method according to any one of claims 1 to 9, wherein, In step S200, the volume ratio of trifluoroethanol to water in the trifluoroethanol and water mixed solvent is 1~2:1; And / or, the concentration of the fluorinated zwitterionic copolymer in the assembly solution is 1~4 mg / mL.

17. The preparation method according to any one of claims 1 to 9, wherein, In step S200, the volume ratio of trifluoroethanol to water in the trifluoroethanol and water mixed solvent is 3:2; And / or, the concentration of the fluorinated zwitterionic copolymer in the assembly solution is 2 mg / mL.

18. The application of fluorinated zwitterionic copolymer nanoparticles prepared by the preparation method according to any one of claims 1 to 17 in stabilizing proteases.

19. The application according to claim 18, wherein, The application includes the following steps: S300 provides a PBS buffer solution containing 0.05~0.5 mg / mL of protease; S400. The PBS buffer solution of the protease is mixed with a solution of fluorinated zwitterionic copolymer nanoparticles, wherein the concentration of the fluorinated zwitterionic copolymer in the solution of the fluorinated zwitterionic copolymer nanoparticles is 1~4 mg / mL; and the volume ratio of the solution of the fluorinated zwitterionic copolymer nanoparticles to the PBS buffer solution of the protease is 1~5:1~40.

20. The application according to claim 19, wherein, The concentration of the fluorinated zwitterionic copolymer in the solution of the fluorinated zwitterionic copolymer nanoparticles is 2 mg / mL.

21. The application according to claim 19, wherein, In step S300, the pH of the PBS buffer solution for the protease is 7.2-7.4; And / or, in step S400, the volume ratio of the solution of the fluorinated zwitterionic copolymer nanoparticles to the PBS buffer solution of the protease is 1:2~5; And / or, the protease is horseradish peroxidase.

22. The application according to claim 19, wherein, In step S300, the concentration of the PBS buffer solution containing the protease is 0.1~0.3 mg / mL.

23. The application according to claim 19, wherein, In step S300, the concentration of the PBS buffer solution containing the protease is 0.2 mg / mL.

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