A zwitterionic polymer precursor material for coating an implantable sensor and a method of making the same

Abstract

The application provides an amphoteric ion polymer precursor material for coating an implantable sensor and a preparation method thereof. The amphoteric ion polymer precursor material is prepared by copolymerizing siloxane monomers, amphoteric ion compound precursors, monomers for anchoring a probe surface and soft monomers, so that the polymer softness and hardness can be controlled, the coating has better bending resistance and is not easy to fall off, the oxygen permeability and selective permeability of the coating can be improved, the biocompatibility is better, the amphoteric ion polymer precursor coating is coated on the surface of the implantable biosensor to form a dense film layer, the detection sensitivity of the implantable biosensor can be significantly improved, and the effective detection time of the implantable biosensor can be effectively prolonged.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biocompatible permselective membrane technology, in particular, to a surface chemical coating treatment of an implantable biosensor, and more particularly to a zwitterionic polymer precursor material for coating implantable sensors and a method of making the same. BACKGROUND

[0002] An implantable biosensor refers to a sensor device that can be partially or wholly implanted into a human body, and can continuously measure important physiological and pathological parameters such as blood oxygen, blood glucose, virus antibodies, lactate, cholesterol, bilirubin, and amino acids, which change over time, and more directly reflect changes in the signs of the measured object due to environmental changes. At present, the interaction between the implantable sensor and the implanted tissue causes a large number of protein molecules to be deposited on the surface of the sensor due to heterogenic rejection after the sensor is implanted into the body, which blocks the transmission path of glucose molecules, hinders the molecular penetration and exchange between the sensor and the tissue fluid, and makes it difficult to obtain accurate and reliable signals for the sensor to detect the concentration of the analyte, which may even endanger the patient's life in severe cases.

[0003] In the prior art, a glucose sensor generally includes a high-molecular material inner membrane layer, an enzyme membrane layer, and a high-molecular outer membrane layer, such as patents CN101530328 / CN201398971 / CN102243208A / CN101530327, etc. However, the high-molecular diffusion layer of these sensors mostly uses polyvinyl alcohol, polyethylene glycol, polyurethane, or polytetrafluoroethylene, etc. However, these materials have poor bending resistance in the body, the membrane layer is easy to fall off, and there are problems of biocompatibility, etc., which are the main reasons why the sensor is difficult to achieve an effective detection time longer than 14 days. Therefore, how to improve the bending resistance of the membrane layer, make it more flexible, not easy to fall off, and reduce the heterogenic rejection reaction, and improve the biocompatibility of the sensor, is the key to improving the detection accuracy and service life of the sensor.

[0004] Bending resistance, biocompatibility, cytotoxicity, appropriate permeation and diffusion performance for the target analyte and barrier performance for potential interferents, as well as resistance to hydrolysis, heat, and other degradation mechanisms, are major challenges faced by high-molecular outer layer materials. In addition, due to the requirements of production, transportation, and storage, the material also needs to have a stable chemical molecular structure to maintain stable properties for a long time before being used.

[0005] At present, the selection of biocompatible permeable membranes is very limited. Therefore, developing a biocompatible high-molecular membrane material with good bending resistance, not easy to fall off, and excellent selective permeation performance is of great significance for improving the detection sensitivity of biosensors and prolonging the effective monitoring time of biosensors. SUMMARY

[0006] The present application aims at the deficiencies of the prior art, and provides a zwitterionic polymer precursor material for coating an implantable sensor and a preparation method thereof, which is prepared by copolymerizing siloxane monomers, zwitterionic compound precursors, monomers for anchoring a probe surface and soft monomers into the zwitterionic polymer precursor material, so that the polymer softness and hardness can be controlled, the coating has better bending resistance and is not easy to fall off, the oxygen permeability and selective permeability of the coating can be improved, the biocompatibility is better, the zwitterionic polymer precursor coating is coated on the surface of the implantable biosensor to form a dense film layer, the detection sensitivity of the implantable biosensor can be significantly improved, and the effective detection time of the implantable biosensor can be effectively prolonged.

[0007] The zwitterionic polymer precursor material is a precursor material specially developed for improving the stability of the zwitterionic polymer, facilitating long-time stability before use, and facilitating production, transportation and storage; the precursor material only needs to be added with ethanol for hydrolysis when used, and can also be hydrolyzed after coating, so that the use process is very convenient.

[0008] In one aspect, the present application provides a zwitterionic polymer precursor material, which is polymerized from siloxane monomers, zwitterionic compound precursors, monomers for anchoring a probe surface and soft monomers, and the soft monomers are any one or more selected from lauryl acrylate, ethyl acrylate, n-butyl acrylate, 2-ethylhexyl acrylate, lauryl methacrylate and n-butyl methacrylate.

[0009] The existing zwitterionic polymers for coating implantable biosensors generally have the problems of difficult adjustment of softness and hardness, poor bending resistance, detection sensitivity decline after long-term use, and even film layer falling off.

[0010] The zwitterionic polymer precursor material provided by the present application is polymerized from siloxane monomers, zwitterionic compound precursors, monomers for anchoring a probe surface and soft monomers; the siloxane part can improve the oxygen permeability of the coating and selectively permeate oxygen and glucose molecules; the zwitterionic compound precursor can hydrolyze to generate a zwitterionic structure, provide good biocompatibility and strong anti-protein non-specific adsorption characteristics; the soft monomer ratio is changed to regulate the flexibility of the polymer; the monomers for anchoring a probe surface mainly crosslink and fix the zwitterionic coating and the sensor surface layer through chemical bonds, the polymer material is bonded to the surface of the sensor through chemical bonds to form a dense and stable polymer film layer. The zwitterionic polymer coating material can significantly improve the accuracy and biocompatibility of the sensor while realizing stable transmission of electrochemical signals, and prolong the effective detection time of the sensor.

[0011] The present application creatively adds soft monomers capable of adjusting the softness and hardness of the film layer to the zwitterionic polymer precursor material, which can effectively improve the flexibility of the zwitterionic polymer, and after coating the implantable sensor, can significantly improve the bending resistance during long-term use, and can effectively prevent the film layer from falling off, thereby effectively prolonging the effective detection time of the implantable biosensor, and can significantly improve the detection accuracy and ensure the detection sensitivity during long-term use.

[0012] Further, the siloxane monomer is any one or more selected from the group consisting of methacryloyloxypropyl tris(trimethylsiloxy)silane, methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane, methyl-vinylsiloxane, methyl-vinylpolysiloxane, polymethylsiloxane, amino-terminated siloxane, and epoxy-terminated siloxane.

[0013] Silicones have good air permeability, softness, stability and biocompatibility, and are a common biomedical material, which can be used as medical catheters, contact lenses, and skin dressings.

[0014] In some modes, the present application uses methacryloyloxypropyl tris(trimethylsiloxy)silane (TRIS) as a silicon-containing monomer, which has good molecular chain flexibility and a main chain containing a large (CH3)3SiO- group, and has particularly excellent oxygen permeability.

[0015] In some modes, the methacryloyloxypropyl tris(trimethylsiloxy)silane (TRIS) can also be a polysiloxane monomer, or a siloxane compound grafted onto the polymer by grafting or post-grafting. The siloxane can also be grafted into the zwitterionic polymer containing active functional groups, which can be replaced by polydimethylsiloxane and its derivatives, commonly used polydimethylsiloxane, cyclo-methylsiloxane, amino-siloxane, poly-methylphenylsiloxane and polyether polysiloxane copolymer, wherein n is 10-1000, and X is amino, epoxy or alkane substituent.

[0016]

[0017] In some modes, the methacryloyloxypropyl tris(trimethylsiloxy)silane (TRIS) can also be replaced by methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane or methyl-vinyl(siloxane and polysiloxane), all of which can improve the oxygen permeability of the coating to a certain extent.

[0018] Further, the zwitterionic compound precursor is ethyl methacryloyloxyethyl methylaminopropyl acrylate (CBE).

[0019] Zwitterionic polymers are a class of macromolecular materials that are overall electrically neutral and contain equal amounts of cationic and anionic groups on the side chains. Due to their strong hydration ability and good biocompatibility, zwitterionic polymers have been widely studied and applied in the fields of biological medicine and the like. Zwitterionic polymers have excellent anti-protein non-specific adsorption performance and biocompatibility, and are widely used in the fields of antifouling coatings, protein modification, drug delivery, membrane separation materials and the like.

[0020] Since ethyl methacryloyloxyethyl methylaminopropyl acrylate (CBE) is more suitable for use with the solvent anhydrous tetrahydrofuran, and has better stability, the present application preferably uses ethyl methacryloyloxyethyl methylaminopropyl acrylate (CBE) as the zwitterionic compound precursor.

[0021] Further, the soft monomer is lauryl acrylate; and the siloxane monomer is methacryloylpropyl tris(trimethylsiloxy)silane.

[0022] By using different soft monomers and adjusting the proportion of the amount, a zwitterionic polymer precursor material with suitable flexibility and bending resistance can be obtained. When lauryl acrylate is used as the soft monomer, the zwitterionic polymer precursor material prepared has not only good flexibility and bending resistance, but also is more stable, is not easy to deteriorate during long-term storage, and can further prolong the effective detection time of the implanted biosensor after coating the implanted biosensor, and can significantly improve the detection accuracy and ensure the detection sensitivity during long-term use.

[0023] Further, the monomer for anchoring the probe surface is a monomer containing an active functional group capable of being combined with the probe surface; the monomer containing the active functional group capable of being combined with the probe surface is selected from any one or more of a silane coupling agent, a hydroxyl-containing compound, an amino-containing compound or a compound capable of exposing amino groups after hydrolysis, a carboxyl-containing compound or a compound capable of exposing carboxyl groups after hydrolysis; and the zwitterionic polymer precursor material has a structural formula as shown in formula (5):

[0024]

[0025] In the formula, FG is an active functional group; (n, m, l, p total number is 5000-100000), [n / (n+m+l+p)]×100%≈25%;

[0026] [m / (n+m+l+p)] x 100% ≈ 25%; [l / (n+m+l+p)] x 100% ≈ 25%; [p / (n+m+l+p)] x 100% ≈ 25%.

[0027] Further, the silane coupling agent is any one or more of methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltri(2-methoxyethoxy)silane; the hydroxyl-containing compound is any one or more of polyethylene glycol methacrylate, hydroxyethyl methacrylate, polyol, polyether, polyester amide, castor oil.

[0028] In some ways, the monomer anchored to the probe surface can be a silane coupling agent with a vinyl group copolymerized with other monomers, or first grafted to the probe surface through a silane coupling agent containing an organic group (such as amino, epoxy, double bond, etc.) at one end, and then grafted to the probe surface by reacting the alkoxyl group with the hydroxyl group. The silane coupling agent is used to connect the zwitterionic polymer to the probe surface, including one or more of methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltri(2-methoxyethoxy)silane, etc.

[0029]

[0030] In some ways, in addition to the above-mentioned method of using a silane coupling agent, the monomer anchored to the probe surface also includes polyethylene glycol methacrylate (PEGMA), hydroxyl-containing compounds such as hydroxyethyl methacrylate, and active hydroxyl-containing compounds such as polyol, polyether, polyester amide, castor oil, and other compounds containing amino or carboxyl groups that can be exposed by hydrolysis.

[0031] In some ways, the monomer anchored to the probe surface is polyethylene glycol methacrylate (PEGMA).

[0032] PEGMA mainly crosslinks and fixes the zwitterionic coating layer to the surface layer of the sensor through chemical bonds. The polymer is bonded to the surface of the sensor through chemical bonds, forming a dense and stable polymer film layer. The coating material improves the accuracy and biocompatibility of the sensor while achieving stable transmission of electrochemical signals, prolonging the effective detection time of the sensor.

[0033] Different kinds of monomers anchored to the surface of the probe can be anchored to different implanted sensor inner layer enzymes, and different cross-linking agents for different anchor groups are slightly different. Protein groups that can be coupled under mild conditions include: amino, carboxyl, cysteine thiol, histidine imidazole, tyrosine phenol, serine and threonine hydroxyl. For example, amino and imidazole can use cross-linking agents such as PEGDGE (polyethylene glycol diglycidyl ether) and the like, and thiol and hydroxyl can use isocyanate and the like for connection. The group participating in covalent binding with the carrier cannot be a group necessary for enzyme activity. Therefore, different monomers anchored to the surface of the probe can be selected according to the coating material of the implanted sensor inner layer enzyme and the like.

[0034] Polyethylene glycol methacrylate (PEGMA) is suitable for coating materials of implanted sensors containing cysteine (thiol) and threonine (hydroxyl) fragments.

[0035] In some ways, when the prepared zwitterionic polymer precursor material is coated on the implanted sensor, a cross-linking agent also needs to be added, and different anchor groups to the surface of the probe can select different cross-linking agents.

[0036] In some ways, the cross-linking agent can use diisocyanate including toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), diphenylmethane diisocyanate (MDI), dicyclohexylmethane diisocyanate (HMDI), hexamethylene diisocyanate (HDI), lysine diisocyanate (LDI) and the like, and the high activity unsaturated bond in the isocyanate structure is used to react with active hydroxyl compounds to solidify and cross-link the zwitterionic polymer precursor material on the polyurethane surface, or triethylene glycol dimethacrylate (TEGDMA), ethylene glycol dimethacrylate (EDMA) trimethylolpropane trimethacrylate (TMPTMA) and the like are cross-linked to the polyurethane coating on the outer surface of the probe with organic peroxide under heat, light and irradiation.

[0037] Further, the structure formula has formula (1), (2), (3) or (4) as shown:

[0038]

[0039]

[0040] Wherein a = 5-20, (n, m, l, p total number is 5000-100000), [n / (n+m+l+p)]x100% ≈ 25%; [m / (n+m+l+p)]x100% ≈ 25%; [l / (n+m+l+p)]x100% ≈ 25%; [p / (n+m+l+p)]x100% ≈ 25%.

[0041] In some embodiments, the number average molecular weight of the zwitterionic polymer precursor material is between 10,000 and 10,000,000. If the molecular weight is less than 10,000, other molecules such as protein molecules and other interfering substances will also pass through the polymer film layer, and the selectivity will be poor. If the molecular weight is greater than 10,000,000, the permeability of the polymer film layer will be poor, the glucose molecules will be blocked outside the layer, the sensor will be difficult to truly reflect the real glucose concentration in the body, and in addition, the strength of the polymer film layer will be large but brittle, and the film layer will be difficult to stably exist on the surface of the probe.

[0042] In some embodiments, the content of each monomer in the zwitterionic polymer precursor material is as follows: methacryloyloxyethyl methyl amino acrylate is 1-30%; methacryloyloxypropyl tris(trimethylsiloxy)silane is 0.5-45%; polyethylene glycol methacrylate (PEGMA) is 0.5-40%; and lauryl acrylate is 1-25%.

[0043] In another aspect, the present application provides a preparation method of a zwitterionic polymer precursor material, which comprises the following steps:

[0044] (1) preparing a zwitterionic compound precursor;

[0045] (2) mixing the zwitterionic compound precursor, a siloxane monomer, a monomer for anchoring the surface of the probe, a soft monomer, an initiator and a solvent to perform a copolymerization reaction.

[0046] Further, the zwitterionic compound precursor is methacryloyloxyethyl methyl amino acrylate, the siloxane monomer is methacryloyloxypropyl tris(trimethylsiloxy)silane, and the soft monomer is lauryl acrylate; wherein the content of methacryloyloxyethyl methyl amino acrylate is 1-30%, the content of lauryl acrylate is 1-25%, the content of the monomer for anchoring the surface of the probe is 0.5-40%, and the content of methacryloyloxypropyl tris(trimethylsiloxy)silane is 0.5-45%.

[0047] Further, the method further comprises step (3): introducing nitrogen to remove air in the reaction system, and placing the reaction system in an oil bath at 62°C for 12 hours; after the reaction is completed, most of the organic solvent is removed by distillation under reduced pressure, and then the reaction system is precipitated, centrifuged and washed to remove small molecules of un-polymerized polymers in the reaction system, the precipitate is collected by suction filtration, and the precipitate is placed at 50°C under high vacuum for 2 days.

[0048] In some embodiments, the solvent is a polymerization solvent of the hydrolysis precursor polymer, in addition to tetrahydrofuran, it can also be methanol, ethanol, DMSO and other organic solvents; the solvent in the monomer synthesis process can also be anhydrous dichloromethane, anhydrous dimethyl sulfoxide, anhydrous acetonitrile and other organic solvents in addition to anhydrous tetrahydrofuran.

[0049] In some embodiments, the solvent 2 is a solvent used for precipitation, in addition to n-hexane, it can also be diethyl ether, petroleum ether and other organic solvents with lower polarity.

[0050] In some embodiments, the initiator of the polymerization process can be azo compounds (such as azobisisoheptyl nitrile and dimethyl azobisisobutyrate initiator, etc.), peroxides (such as hydrogen peroxide, ammonium persulfate, potassium persulfate, benzoyl peroxide, tert-butyl benzoyl peroxide, methyl ethyl ketone peroxide, etc.), complex initiation systems (such as azobisisobutyronitrile and azobisisoheptyl nitrile ABVN complex initiation system, dibenzoyl peroxide BPO and tert-butyl peroxybenzoate complex initiation system, etc.) in addition to azobisisobutyronitrile (AIBN).

[0051] In some embodiments, the initiator is azobisisobutyronitrile, and the solvent is anhydrous tetrahydrofuran.

[0052] Further, the preparation method of the zwitterionic compound precursor in step (1) is as follows: 50.06 g (0.5 mol) of ethyl acrylate is added to a 500 mL round-bottom flask, 2-methylaminoethanol 37.56 g (0.5 mol) is slowly added dropwise under ice bath conditions with a constant pressure separatory funnel, stirring for 12 h to obtain colorless transparent viscous liquid 2-hydroxyethyl methylamino ethyl acrylate. To the 2-hydroxyethyl methylamino ethyl acrylate, add triethylamine 90 mL, 2,6-di-tert-butyl-p-cresol 125 mg, anhydrous tetrahydrofuran 50 mL, and slowly add dropwise a mixed solution of 58 mL of methyl methacryloyl chloride in 50 mL of tetrahydrofuran under ice bath conditions with a constant pressure separatory funnel, and stop the reaction after stirring for 12 h. The product solution is filtered through a sand core funnel to remove the generated triethylamine hydrochloride, the filtrate is rotary evaporated to remove the solvent therein, and finally extracted and purified, and the solvent is removed by reduced pressure distillation to obtain colorless oily liquid, i.e. methyl methacryloyloxyethyl methylamino ethyl acrylate.

[0053]

[0054] In some embodiments, R1 of the above 2-methylaminoethanol can be substituted by ethyl, tert-butyl and the like in addition to being substituted by methyl, such as being substituted by ethyl to obtain 2-ethylaminoethanol, and being substituted by tert-butyl to obtain 2-tert-butylaminoethanol.

[0055]

[0056] In some embodiments, the R2 moiety of the ethyl acrylate described above can be substituted with a methyl group, a tert-butyl group, or the like, i.e., methyl acrylate, tert-butyl acrylate, or the like, in addition to being substituted with an ethyl group.

[0057] In some embodiments, the ethyl acrylate described above can be attached to the 2-methylaminoethanol via a Michael addition of a tertiary amine to an ethyl acrylate containing a double bond, or via a nucleophilic substitution reaction of a carbon-halogen bond, and R3 can be a methyl group, a tert-butyl group, or the like, in addition to being an ethyl group, i.e., methyl methacrylate, ethyl methacrylate, tert-butyl methacrylate, or the like, can be hydrolyzed to produce the carboxylate functional group of the carboxybetaine analog.

[0058] In some embodiments, the length of the tertiary amine and the ethyl acrylate segment is not adjustable, and the distance between the methacryloxy group and the tertiary amine in the methacryloxyethyl methylamino ethyl acrylate can be adjusted by modifying the compound. The hydrolysis rate of the zwitterionic polymer precursor can be achieved by changing the length of the ester group of the carboxylate.

[0059] In another aspect, the present application provides a use of a soft monomer for preparing a zwitterionic polymer precursor material for coating an implantable sensor, the soft monomer being any one or more selected from the group consisting of lauryl acrylate, ethyl acrylate, n-butyl acrylate, 2-ethylhexyl acrylate, lauryl methacrylate, and n-butyl methacrylate.

[0060] Further, the present application provides a use of a soft monomer for preparing a zwitterionic polymer precursor material for coating an implantable sensor, the soft monomer being capable of adjusting the softness and hardness of the zwitterionic polymer precursor material, thereby effectively prolonging the detection time and improving the detection sensitivity.

[0061] The present application has the following advantages:

[0062] 1. By copolymerizing a siloxane monomer, a zwitterionic compound precursor, a monomer for anchoring a probe surface, and a soft monomer into a zwitterionic polymer precursor material, a polymer having a controllable softness and hardness is prepared, and the coating has better bending resistance and is less likely to fall off.

[0063] 2. The oxygen permeability and selective permeability of the coating are improved, and the coating has better biocompatibility.

[0064] 3. While improving the oxygen permeability of the outer membrane of a biosensor, the influence of proteins adsorbed on the surface of the sensor on the accurate transmission of electrochemical signals and the like is reduced.

[0065] 4. The detection sensitivity of an implantable biosensor is significantly improved, and the effective detection time of the implantable biosensor is effectively prolonged. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1Synthesis reaction formula of methacryloyloxyethyl methylamino acrylate in Example 1;

[0067] Figure 2 Structural formula of polymer 1 in Example 2;

[0068] Figure 3 Structural formula of polymer 2 in Example 3;

[0069] Figure 4 Structural formula of polymer 3 in Example 4;

[0070] Figure 5 Structural formula of polymer 4 in Example 5;

[0071] Figure 6 Fluctuation curve of current signal of implantable biosensor with different membrane layer modification with respect to glucose concentration in Example 8;

[0072] Figure 7 Detection result diagram of relative protein adsorption amount of implantable biosensor with different membrane layer modification in Example 9;

[0073] Figure 8 Cell toxicity test result diagram of different membrane layers in Example 10;

[0074] Figure 9 Platelet adhesion experiment result diagram of different membrane layers in Example 11. DETAILED DESCRIPTION

[0075] The application will be further described below in conjunction with examples. It should be noted that the following examples are intended to facilitate the understanding of the application and do not limit the application in any way.

[0076] Example 1 Synthesis of methacryloyloxyethyl methylamino acrylate (CBE)

[0077] The synthesis reaction formula of methacryloyloxyethyl methylamino acrylate provided in the example is as follows: Figure 1The main synthesis step is shown in the following: 50.06 g (0.5 mol) of ethyl acrylate was added into a 500 mL round bottom flask, 37.56 g (0.5 mol) of 2-methylaminoethanol was slowly added dropwise into the flask using a constant pressure dropping funnel under ice bath condition, and the reaction was stirred for 12 hours to obtain colorless transparent viscous liquid 2-hydroxyethyl methylamino acrylate. Then, 90 mL of triethylamine, 125 mg of 2,6-di-tert-butyl-p-cresol, and 50 mL of anhydrous tetrahydrofuran were added into the 2-hydroxyethyl methylamino acrylate, and a mixed solution of 58 mL of methyl acryloyl chloride and 50 mL of tetrahydrofuran was slowly added dropwise into the flask using a constant pressure dropping funnel under ice bath condition. The reaction was stopped after being stirred for 12 hours. The product solution was filtered through a sand core funnel to remove the generated triethylamine hydrochloride, the solvent in the filtrate was removed by rotary evaporation, and finally the product was extracted and purified, and the solvent was removed by distillation under reduced pressure to obtain colorless oily liquid, i.e., methyl acryloyloxyethyl methylamino acrylate.

[0078] Example 2 Synthesis and application of polymer 1

[0079] The structural formula of polymer 1 provided in this example is shown in the following: Figure 2 The parameters of polymer 1 are: [l / (n+m+l+p)]x100% ≈ 25%; [m / (n+m+l+p)]x100% ≈ 25%; [o / (n+m+l+p)]x100% ≈ 25%; [p / (n+m+l+p)]x100% ≈ 25%.

[0080] The polymerization process of polymer 1 is as follows: 25.6 g of methyl acryloyloxyethyl methylamino acrylate CBE, 15 mL of polyethylene glycol methacrylate PEGMA, 54.7 g of methyl acryloyloxypropyl tris(trimethylsiloxy)silane TRIS, 21 g of lauryl acrylate (soft monomer), 0.88 g of azobisisobutyronitrile AIBN, and 1 L of anhydrous tetrahydrofuran were added into a reaction bottle, and after being fully stirred and dissolved, nitrogen was introduced to remove air in the reaction system, and finally the reaction bottle was placed in a 62°C oil bath for reaction for 12 hours. After the reaction was completed, most of the organic solvent was removed by distillation under reduced pressure, and then the reaction system was precipitated, centrifuged, and washed with n-hexane to remove small molecule polymers. The precipitate was collected by suction filtration, and was placed at 50°C under high vacuum for 2 days.

[0081] The process of coating the implantable sensor with polymer 1 is as follows: polymer 1 is dissolved in ethanol (to hydrolyze the zwitterionic polymer) and after shaking to dissolve, a polymer mixture solution of 0.5 g / L is prepared, and 30 mg of hexamethylene diisocyanate HDI (about 0.55 times the molar amount of hydroxyl groups in the copolymer) is added thereto, and the probe portion of the implantable biosensor is immediately immersed in the solution after mixing uniformly, and after immersion coating, the probe is placed in a dry environment for curing, and then washed with anhydrous ethanol to remove residual components and polymers that are not bonded to the surface of the probe, and placed in a dry environment to volatilize and remove the ethanol solvent, thereby obtaining a probe modified with a zwitterionic polymer.

[0082] Example 3 Synthesis and application of polymer 2

[0083] The structural formula of polymer 2 provided in this example is as shown in Figure 3 . Among them, the parameters of polymer 2 are: [l / (n+m+l+p)]x100%≈25%; [m / (n+m+l+p)]x100%≈25%; [o / (n+m+l+p)]x100%≈25%; [p / (n+m+l+p)]x100%≈25%.

[0084] The polymerization preparation process of polymer 2 is as follows: methyl methacryloyloxyethyl methyl aminopropyl acrylate CBE (3.4 g), tert-butyl aminoethyl methacrylate (1.2 g), methyl methacryloyloxypropyl tris(trimethylsiloxy)silane TRIS (5.5 g), lauryl acrylate (1.3 g), azobisisobutyronitrile AIBN (125 mg) and anhydrous tetrahydrofuran (100 mL) are added to a reaction bottle, and after fully stirring and dissolving, nitrogen is introduced to remove air in the reaction system, and finally the reaction bottle is placed in a 62°C oil bath for reaction for 12 hours. After the reaction is completed, most of the organic solvent is removed by distillation under reduced pressure, and then the precipitate is centrifuged and washed to remove small molecule polymers in the reaction system that have not polymerized, and the precipitate is collected by suction filtration, and placed in a high vacuum at 50°C for 2 days, thereby obtaining polymer 2 with a structural formula as shown in Figure 3 .

[0085] The process of coating the implantable sensor with polymer 2 is as follows: polymer 2 is dissolved in anhydrous ethanol, and after being dissolved by shaking, a polymer mixed solution of 0.5 g / L is prepared. 0.08 g of polyethylene glycol diglycidyl ether (Sigma-Aldrich, number average molecular weight 500) is added, and the probe is immediately immersed in the polymer mixed film solution. Then, 0.03% of a trace amount of triethylamine accelerator is added to promote the ring opening of the epoxy group. After a stable film layer is formed on the surface, the probe is removed, and the epoxy groups at both ends of the polyethylene glycol diglycidyl ether are used to crosslink the secondary amine in the zwitterionic polymer film layer and the hydroxyl group on the surface of the sensor probe, respectively. Then, the unbound polymer is removed by ethanol cleaning, and the probe is dried in a high-vacuum drying oven to form a stable antibiofilm layer on the outer surface of the probe.

[0086] Example 4: Synthesis and application of polymer 3

[0087] The structural formula of polymer 3 provided in this example is shown in Figure 4 . In the polymer 3, the parameters are: [l / (n+m+l+p)]x100% ≈ 25%; [m / (n+m+l+p)]x100% ≈ 25%; [o / (n+m+l+p)]x100% ≈ 25%; and [p / (n+m+l+p)]x100% ≈ 25%.

[0088] The polymerization process of polymer 3 is as follows: methyl methacryloyloxyethyl methylaminopropyl acrylate CBE (3.4 g), 2-aminoethyl methacrylate (1.8 g), methyl methacryloyloxypropyl tris(trimethylsiloxy)silane TRIS (5.5 g), lauryl acrylate (1.3 g), azobisisobutyronitrile AIBN (125 mg), and anhydrous tetrahydrofuran (100 mL) are added to a reaction bottle, and after being fully stirred and dissolved, nitrogen is introduced to remove air in the reaction system. Finally, the reaction bottle is placed in a 62°C oil bath for 12 hours. After the reaction is completed, most of the organic solvent is removed by distillation under reduced pressure, and then the reaction system is precipitated, centrifuged, and washed to remove small molecule polymers that have not been polymerized. The precipitate is collected by suction filtration and placed in a high-vacuum drying oven at 50°C for 2 days to obtain polymer 3 with the structural formula shown in Figure 4 .

[0089] The process of coating the implantable sensor with polymer 3 is as follows: polymer 3 is dissolved in anhydrous ethanol, and after being shaken to dissolve, a mixed solution of polymer 2 is configured at 0.5 g / L, 0.08 g of maleic anhydride is added, and acylation reaction occurs between maleic anhydride and the amino group of polymer 3, so as to introduce reactive double bonds into the zwitterionic polymer. In addition, maleic anhydride and its derivatives are difficult to self-polymerize, so no additional polymerization inhibitor is needed during the acylation process. Then the probe of the sensor is immersed in the mixed film solution of polymer 3, and the remaining double bonds after acylation of maleic anhydride are used to connect the zwitterionic polymer to the surface of the probe. After a stable film layer is formed on the surface, the probe is taken out, washed and dried.

[0090] In this embodiment, polyethylene glycol diglycidyl ether can also be used instead of maleic anhydride as a crosslinking agent. The epoxy groups at both ends of the polyethylene glycol diglycidyl ether crosslink the primary amine in the zwitterionic polymer film layer and the hydroxyl group on the surface of the sensor probe, respectively. Then, the unbound polymer is removed by ethanol washing, dried in a high-vacuum drying oven, and fixed on the outer surface of the probe to form a stable antibiofilm layer.

[0091] In this embodiment, maleic anhydride is preferably used as a crosslinking agent to connect the zwitterionic polymer to the surface of the probe.

[0092] Example 5: Synthesis and application of polymer 4

[0093] The structural formula of polymer 4 provided in this embodiment is as shown in Figure 5 . In the formula, the parameters of polymer 4 are: [l / (n+m+l+p)]x100%≈25%; [m / (n+m+l+p)]x100%≈25%; [o / (n+m+l+p)]x100%≈25%; [p / (n+m+l+p)]x100%≈25%.

[0094] The polymerization process of polymer 4 is as follows: polymer methyl methacryloyloxyethyl methylamino acrylate CBE (3.4 g), lysine methacrylic monomer LysMA (1.2 g), methyl methacryloyloxypropyl tris(trimethylsiloxy)silane TRIS (5.5 g), lauryl acrylate (1.3 g), azobisisobutyronitrile AIBN (125 mg), and anhydrous tetrahydrofuran (100 mL) are added to a reaction bottle, stirred to dissolve, and then nitrogen is introduced to remove air in the reaction system. Finally, the reaction bottle is placed in a 62°C oil bath for 12 hours. After the reaction is completed, most of the organic solvent is removed by distillation under reduced pressure, and then the reaction system is precipitated, centrifuged and washed with n-hexane to remove un-polymerized small molecules. The precipitate is collected by suction filtration and placed in a high-vacuum drying oven at 50°C for 2 days to obtain polymer 4 with the structural formula as shown in Figure 5 .

[0095] Example 6: Preparation of a polymer without soft monomers

[0096] The method provided in Example 2 was used to prepare the polymer, and the specific preparation process was as follows: methacryloyloxyethyl methylaminopropyl acrylate CBE (12.8 g), polyethylene glycol methacrylate PEGMA (30 mL), methacryloylpropyl tris (trimethylsiloxy) silane TRIS (54.7 g), azobisisobutyronitrile AIBN (0.88 g) and anhydrous tetrahydrofuran (1 L) were added to a reaction bottle, and after being fully stirred and dissolved, nitrogen was introduced to remove air in the reaction system, and finally the reaction bottle was placed in a 62°C oil bath for reaction for 12 hours. After the reaction was completed, most of the organic solvent was removed by distillation under reduced pressure, then the small molecule polymer in the reaction system was removed by precipitation, centrifugation and washing with n-hexane, the precipitate was collected by suction filtration, and was placed at 50°C under high vacuum for 2 days.

[0097] Example 7: Zwitterionic polymer precursor materials prepared using different soft monomers

[0098] The method provided in Example 2 was used to prepare the polymer, and the specific preparation process was as follows: methacryloyloxyethyl methylaminopropyl acrylate CBE (12.8 g), polyethylene glycol methacrylate PEGMA (30 mL), methacryloylpropyl tris (trimethylsiloxy) silane TRIS (54.7 g), azobisisobutyronitrile AIBN (0.88 g) and anhydrous tetrahydrofuran (1 L) were added to a reaction bottle, and after being fully stirred and dissolved, nitrogen was introduced to remove air in the reaction system, and finally the reaction bottle was placed in a 62°C oil bath for reaction for 12 hours. After the reaction was completed, most of the organic solvent was removed by distillation under reduced pressure, then the small molecule polymer in the reaction system was removed by precipitation, centrifugation and washing with n-hexane, the precipitate was collected by suction filtration, and was placed at 50°C under high vacuum for 2 days.

[0099] Table 1: Effect of different soft monomers on the preparation of zwitterionic polymer precursor materials

[0100]

[0101] As can be seen from Table 1, when no soft monomer is contained, the prepared polymer has poor stability, weak flexibility and bending resistance, is prone to falling off after coating, and has a short effective detection time and low detection sensitivity; when the soft monomer is added, the polymer stability can be obviously improved, long-term storage and convenient transportation are suitable, the flexibility and bending resistance of the sensor after coating can be improved, falling off is not prone to occur, and the detection time and detection sensitivity can be significantly improved; among them, when lauryl acrylate is used as the soft monomer, the performance is more stable, the comprehensive effect is better, and the detection sensitivity is also higher.

[0102] Example 8 Influence of different contents of soft monomers on preparation of zwitterionic polymer precursor materials

[0103] In this example, the polymer was prepared by the preparation method of Polymer 1 provided in Example 2, wherein lauryl acrylate was used as the soft monomer, and 1%, 5%, 10%, 15%, 20%, 25% and 30% of lauryl acrylate were added respectively to prepare seven different polymers. The seven polymers were used to coat implantable biosensors respectively, and the stability, flexibility, bending resistance, non-falling-off property, effective detection time and long-term detection sensitivity of the seven implantable biosensors were investigated. The detection method of stability was to observe the properties after being placed in a 50°C high-temperature environment for 30 days. The detection method of flexibility and bending resistance was to judge the fold mark on the surface of the polymer film after being bent by 180° and whether the fold mark recovered. The detection method of falling-off property was to implant the coated implantable biosensor into a mouse and maintain it for two months, and then observe whether the film on the surface of the sensor peeled off, protruded or swelled, and the coating film of each polymer was repeated five times. The detection method of effective detection time was to immerse the coated electrode in PBS buffer solution, take out the electrode every day for electrochemical detection of glucose gradient concentration, continue to immerse the electrode in PBS buffer solution after detection, and the detection method of long-term detection sensitivity was to compare the electrodes after the first test and continuous immersion for 20 days, and then detect the glucose detection sensitivity again (herein, only the sensitivity more than 90% was regarded as the effective detection time). The results are shown in Table 2.

[0104] Table 2, Influence of different contents of soft monomers on preparation of zwitterionic polymer precursor materials

[0105]

[0106] As can be seen from Table 2, when the content of soft monomer lauryl acrylate is less than 5%, the stability of the prepared polymer is slightly poor, the flexibility and bending resistance are both low, the coating may fall off, and the effective detection time is short and the detection sensitivity is not high; when the content of soft monomer is 10-25%, the stability of the zwitterionic polymer can be obviously improved, long-term storage and convenient transportation are suitable, the flexibility and bending resistance of the sensor after coating can be improved, and the sensor is not easy to fall off, and the detection time and detection sensitivity can be significantly improved; among them, especially when the content of soft monomer lauryl acrylate is 20%, the performance is more stable, the comprehensive effect is better, and the detection sensitivity is higher; but when the content of soft monomer is too high, the detection sensitivity decreases; therefore, the content of soft monomer is most preferably 20%.

[0107] Example 8 Glucose permeability performance test of the zwitterionic polymer precursor material provided by the application

[0108] In this example, the zwitterionic polymer precursor material prepared in Example 2 is coated on an implantable sensor, and at the same time, a polyurethane (PU) and a Nafion film coated implantable sensor are used as a comparison; the film thickness is controlled to be consistent (about 25 μm), and 0, 5, 10, 15, 20, 25, 30, and 40 mM glucose PBS solutions are configured, respectively, electrochemical test is carried out by using a multi-channel electrochemical workstation CHI660, amperometric i-t curve is selected, voltage is set to 0.55 V, sensitivity (A / V) is set to 10-6, and current-time curve (i-t) is drawn, after the baseline is stable, the glucose PBS solution is replaced according to the concentration of 0, 5, 10, 15, 20, 25, 30, and 40 mM in turn; finally, the data is exported to draw the current-time curve, and the fluctuation of the current signal and the trend of the change with the glucose concentration are observed, and the results are shown in Figure 6 .

[0109] Figure 6 As shown in the figure, the electrochemical signal of the probe modified by the zwitterionic polymer is stable and orderly, the unmodified probe is well fitted below 15 mM, the electrochemical signal decreases when the concentration is higher than 15 mM, and the probe modified by the zwitterionic polymer is better than the case of using polyurethane as the outer film layer, and shows better signal transmission, which shows that the coating can smoothly permeate glucose and oxygen molecules and be successfully converted into electrochemical signals.

[0110] Example 9 Anti-non-specific protein adsorption performance test of the zwitterionic polymer precursor material provided by the application

[0111] The non-specific protein adsorption resistance of the sensor probe coated with the polymer film was determined by a modified enzyme-linked immunosorbent assay (ELISA). First, the circular film pieces prepared from the zwitterionic polymer precursor material of Example 2 and the same-sized circular polyurethane film pieces were placed in a 48-well plate, 1 mL of pure water was added to each well, and the plate was placed on a horizontal shaker for 5 min. The pure water was then aspirated, and 1 mL of 1x PBS buffer solution was added to each well, and the plate was placed on a horizontal shaker for two washes. The buffer solution was aspirated, and 1 mL of Anti-IgG HRP conjugate solution was added to each well, and the plate was incubated at 37°C on a horizontal shaker for 75 min. After the incubation, the conjugate solution was aspirated, and 1 mL of PBS buffer solution was added to each well. The PU film pieces were removed and placed in new wells with 1 mL of PBS buffer solution on a horizontal shaker for 5 min, and the process was repeated twice. Finally, the same solution was added to another empty well on a horizontal shaker for 5 min as a blank control. The PBS buffer solution was removed, and 500 μL of TMB color developing solution was added to each well, and the plate was color developed at 37°C on a horizontal shaker for 15 min. To avoid cross-contamination, the tweezers were cleaned with pure water in a timely manner when different film pieces were clamped. Then, 500 μL of hydrochloric acid stop solution was added to each well, and the plate was shaken well. 200 μL of the liquid was added to a 96-well plate, and the absorbance was measured at 450 nm and 620 nm wavelengths after the enzyme-labeled instrument was shaken at a medium speed for 5 s. The test results were expressed as 100% adsorption of the protein adsorbed on TCPS. The formula for the relative protein adsorption amount is as follows:

[0112]

[0113] where Asample is the absorbance of the sample to be tested; Ablank is the absorbance of the blank well; and Acontrol is the absorbance of the TCPS well. Three parallel samples were set for each group. The detection results of the relative protein adsorption amount are shown in Table 1. Figure 7 As can be seen from Table 1, the zwitterionic polymer modified probe surface adsorbed a very small amount of protein, and the protein adsorption amount was reduced by 99% compared with the unmodified bare needle. Figure 7

[0114] Example 10 Cell toxicity test of the zwitterionic polymer precursor material provided by the application

[0115] ​The cytotoxicity assay uses CCK-8, a rapid and highly sensitive assay kit widely used for cell proliferation and cytotoxicity. The intensity of the color is inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450 nm using an ELISA reader, which indirectly reflects the number of live cells.

[0116] HUVEC cells were first seeded at 5 × 10⁴ / mL in sterile 96-well plates, 100 μL per well, and cultured in a cell culture incubator for 24 h. Then, the culture medium was replaced with DMEM medium containing 0.2 g / mL of the probe extract before and after modification (using either the zwitterionic polymer precursor material prepared in Example 2 or polyurethane modification, respectively). Blank and experimental controls were also included, and the cells were cultured for another 24 h. After culture, the culture medium was removed, and the cells were washed twice with DMEM medium. Then, 100 μL of DMEM medium containing 100 μL / mL CCK-8 was added, and the cells were cultured for another 0.5 h. After culture, the absorbance was measured using a microplate reader at a detection wavelength of 450 nm and a reference wavelength of 620 nm. Figure 8 Both the polyurethane membrane and the zwitterionic polymer membrane exhibited cell viability exceeding 95%, indicating good material safety.

[0117] Example 11 Platelet adhesion experiment of the zwitterionic polymer precursor material provided by the present invention

[0118] Platelet adhesion assay: Membranes approximately 0.5 cm in diameter were fabricated from polyurethane and the zwitterionic polymer precursor material prepared in Example 2, respectively, and fixed to the bottom of a 96-well plate. 100 μL of platelet-rich plasma (PRP) was added to each well, and the plates were incubated at 37°C for static adsorption for 2 h. After adsorption, the membranes were slowly rinsed twice with sterile PBS buffer solution, and then fixed in PBS solution containing 1 mL of 2.5% (w / v) glutaraldehyde at 4°C for 45 min. After fixation, the membranes were slowly rinsed with pure water, and 200 μL of pure water was added to centrifuge tubes. The membranes were then placed in the centrifuge tubes, flash-frozen in liquid nitrogen, and then freeze-dried for approximately 24 h. The microstructure and morphology of the membrane surface were observed using a field emission scanning electron microscope (SEM) SU-8010. The results are as follows: Figure 9 As shown.

[0119] Figure 9 It is evident that a large number of platelets are adsorbed on the surface of the polyurethane membrane, while platelets are basically not adhered to the surface of the zwitterionic polymer membrane. This shows that the zwitterionic polymer precursor material provided by the present invention can prevent platelet adhesion.

[0120] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. The use of a soft monomer in preparing zwitterionic polymer precursor materials for coating implantable sensors, and in improving the effective detection time and detection sensitivity of implantable sensors, characterized in that, The soft monomer is lauryl acrylate, and the zwitterionic polymer precursor material is polymerized from a siloxane monomer, a zwitterionic compound precursor, a monomer on the anchoring probe surface, and a soft monomer; the siloxane monomer is selected from any one or more of methacryloyloxypropyltris(trimethylsiloxane), methacryloyloxypropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane, methyl-vinylsiloxane, and methyl-vinylpolysiloxane; the zwitterionic compound precursor is... The monomer on the surface of the anchoring probe is a monomer containing active functional groups that can bind to the probe surface.

2. A zwitterionic polymer precursor material, characterized in that, It is polymerized from a siloxane monomer, a zwitterionic compound precursor, a monomer on the surface of an anchoring probe, and a soft monomer, wherein the soft monomer is lauryl acrylate; the siloxane monomer is selected from any one or more of methacryloyloxypropyltris(trimethylsiloxane), methacryloyloxypropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane, methyl-vinylsiloxane, and methyl-vinylpolysiloxane; the zwitterionic compound precursor is... The monomer on the surface of the anchoring probe is a monomer containing active functional groups that can bind to the probe surface.

3. The zwitterionic polymer precursor material as described in claim 2, characterized in that, The siloxane monomer is methacryloyloxypropyltris(trimethylsiloxane)silane.

4. The zwitterionic polymer precursor material as described in claim 3, characterized in that, The monomer containing an active functional group capable of binding to the probe surface is selected from any one or more of the following: silane coupling agents, hydroxyl-containing compounds, compounds containing amino groups or compounds that can expose amino groups upon hydrolysis, and compounds containing carboxyl groups or compounds that can expose carboxyl groups upon hydrolysis; the zwitterionic polymer precursor material has the structural formula shown in formula (5): Equation (5); FG is an active functional group; The total number of n, m, l and p is 5000-100000; [n / (n+m+l+p)]×100%=25%; [m / (n+m+l+p)]×100%=25%; [l / (n+m+l+p)]×100%=25%; [p / (n+m+l+p)]×100%=25%.

5. The zwitterionic polymer precursor material as described in claim 4, characterized in that, The silane coupling agent is any one or more of methacryloyloxypropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, and vinyltri(2-methoxyethoxy)silane; the hydroxyl-containing compound is any one or more of polyethylene glycol methacrylate and hydroxyethyl methacrylate; the amino-containing compound is tert-butylaminoethyl methacrylate, 2-aminoethyl methacrylate, and lysine methacrylate monomer LysMA.

6. The zwitterionic polymer precursor material as described in claim 5, characterized in that, It has a structural formula as shown in equation (2), (3) or (4): Equation (2); Equation (3); Equation (4); Where a = 5 to 20, and the total number of n, m, l, and p is 5000-100000; [n / (n+m+l+p)]×100%=25%; [m / (n+m+l+p)]×100%=25%; [l / (n+m+l+p)]×100%=25%; [p / (n+m+l+p)]×100%=25%.

7. The method for preparing the zwitterionic polymer precursor material according to any one of claims 2 to 6, characterized in that, Includes the following steps: (1) Preparation of zwitterionic compound precursors; (2) The zwitterionic precursor, siloxane monomer, monomer on the surface of the anchoring probe, soft monomer, initiator and solvent are mixed to undergo copolymerization reaction.

8. The method as described in claim 7, characterized in that, The zwitterionic compound precursor is The siloxane monomer is methacryloyloxypropyltris(trimethylsiloxane)silane, and the soft monomer is lauryl acrylate; wherein, the content of the zwitterionic compound precursor is 1-30%, the content of lauryl acrylate is 1-25%, the content of the monomer on the anchoring probe surface is 0.5-40%, and the content of methacryloyloxypropyltris(trimethylsiloxane)silane is 0.5-45%; the initiator is azobisisobutyronitrile, and the solvent is anhydrous tetrahydrofuran.

Images