Method for constructing TMAO-derived zwitterionic polymer PTMAO coating on surface of polyurethane stent

Abstract

The invention discloses a method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent, and the coating is formed by gradually grafting 1, 6-hexamethylene diisocyanate (HMDI), polyethyleneimine (PEI) and a TMAO-derived polymer (PTMAO) on the surface of the polyurethane stent. According to the method, firstly, a TMAO-derived zwitterionic polymer PTMAO is prepared, the melting enthalpy of the PTMAO is extremely low, a PTMAO coating support (PTMAO-s) constructed by the PTMAO can be strongly combined with water in a solution, and it is proved through an AFM test that the PTMAO-s can generate a high-strength hydration layer in the aqueous solution; proved by an anti-protein adsorption experiment and an anti-bacterial adhesion experiment, PTMAO-s has excellent anti-protein adsorption capacity and anti-bacterial adhesion capacity; in-vitro and in-vivo anti-crusting experiments prove that the PTMAO-s shows excellent anti-crusting performance; the cytotoxicity of the coated stent is detected through an MTT experiment and a living / dead cell staining experiment, and results show that after the PTMAO-s and HK-2 cells are co-cultured, the cell activity is not obviously reduced, and the PTMAO-s shows relatively good biological safety.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterial preparation, and in particular to a method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent. Background Art

[0002] Ureteral stents are important medical devices for the treatment of urinary system diseases (such as urinary stones and ureteral stenosis), which can relieve obstruction and maintain urinary drainage. However, stent crusting often occurs in clinical applications. It is reported that the crusting rate of polyurethane ureteral stents is 26.8% within 6 weeks, 56.9% between 6 and 12 weeks, and 75.9% after 12 weeks. Stent crusting not only significantly shortens the service life of the stent, but also increases the risk of ureteral re-obstruction and urosepsis, seriously damaging kidney function. Therefore, preventing the formation of ureteral stent crusting is of great clinical significance. The formation of ureteral stent crusting is closely related to the interaction between proteins, bacteria and inorganic salt ions in urine and the stent surface. In order to inhibit the formation of stent crusting, it is crucial to construct a superhydrophilic coating on the stent surface to form a high-strength hydration layer to improve the lubricity of the stent surface and reduce the interaction between proteins, bacteria and multivalent ions in urine and the stent surface, thereby inhibiting the formation of stent crusting. Summary of the invention

[0003] The purpose of the present invention is to solve the shortcomings existing in the prior art, and proposes a method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent. The method successfully constructs a TMAO-derived zwitterionic polymer (PTMAO) coating on the surface of a polyurethane stent through a layer-by-layer branching amplification strategy.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent, wherein the coating is gradually grafted onto the surface of the polyurethane stent by 1,6-hexamethylene diisocyanate (HMDI), polyethyleneimine (PEI) and a TMAO-derived polymer (PTMAO); The specific steps include: Step 1: polymerize dimethylaminopropyl acrylamide (DMAPA) monomer and glycidyl methacrylate (GMA) monomer in a ratio of 20:2 through a traditional free radical reaction to generate a tertiary amine polymer PDMG; Step 2: PDMG is then further oxidized using 3-chloroperoxybenzoic acid (mCPBA) to obtain TMAO-derived zwitterionic polymer PTMAO; Step 3: After PTMAO is successfully synthesized, the secondary amine groups on the surface of the polyurethane scaffold are used to react with the double bonds of HMDI to graft HMDI onto the surface of the scaffold to obtain isocyanate groups; Step 4: The HMDI-modified scaffold is then further reacted with PEI, and the amino group of PEI reacts with isocyanate to further connect PEI to the surface of the scaffold; Step 5: Finally, PTMAO is reacted with the PEI-modified stent, and the amino groups on the PEI surface further react with the epoxy groups in the polymer molecules to graft PTMAO onto the stent surface to obtain a PTMAO-coated stent (PTMAO-s).

[0005] Preferably, in step three, after the synthesis of PTMAO, the surface of the polyurethane stent needs to be modified.

[0006] Preferably, in step 4, the molecular weight of PEI participating in the reaction is 10,000.

[0007] Preferably, in step 4, the reaction temperature is 45° C. and the reaction time is 24 h.

[0008] Preferably, in step five, the reaction temperature is 60° C. and the reaction time is 72 h.

[0009] Preferably, in step five, the contact angle of the prepared coated stent PTMAO-s is 5.2°, showing super hydrophilicity.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. The PTMAO polymer prepared by the present invention has an extremely low melting enthalpy. The low melting enthalpy indicates a high content of bound water in the polymer, which shows that PTMAO-s has an excellent ability to bind water; 2. AFM test observed that the hydrophilic probe can generate an adsorption force of about 11.04nN when it contacts the surface of PTMAO-s. This phenomenon proves that PTMAO-s can generate a high-strength hydration layer in aqueous solution to interact with the hydrophilic probe; 3. Through the anti-protein adsorption experiment and anti-bacterial adhesion experiment, PTMAO-s has excellent anti-protein adsorption and anti-bacterial adhesion capabilities; 4. Through in vitro and in vivo anti-crusting experiments, PTMAO-s showed excellent anti-crusting performance; 5. The cytotoxicity of the coated scaffolds was detected by MTT experiment and live / dead cell staining experiment. The results showed that the cell activity of PTMAO-s was not significantly reduced after co-culture with HK-2 cells, showing good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more specifically and intuitively illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0012] Figure 1 This is a schematic diagram of the mode proposed by the present invention. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0014] Reference Figure 1 A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent, wherein the coating is gradually grafted onto the surface of the polyurethane stent by 1,6-hexamethylene diisocyanate (HMDI), polyethyleneimine (PEI) and a TMAO-derived polymer (PTMAO); The specific steps include: Step 1: polymerize dimethylaminopropyl acrylamide (DMAPA) monomer and glycidyl methacrylate (GMA) monomer in a ratio of 20:2 through a traditional free radical reaction to generate a tertiary amine polymer PDMG; Step 2: PDMG is then further oxidized using 3-chloroperoxybenzoic acid (mCPBA) to obtain TMAO-derived zwitterionic polymer PTMAO; Step 3: After PTMAO is successfully synthesized, the surface of the polyurethane stent is modified by using the secondary amine groups on the surface of the polyurethane stent to react with the double bonds of HMDI to graft HMDI onto the surface of the stent to obtain isocyanate groups; Step 4: The HMDI-modified scaffold is then further reacted with PEI, the molecular weight of the PEI involved in the reaction is 10,000, the amino group of PEI reacts with isocyanate, and the PEI is further connected to the surface of the scaffold, the reaction temperature is 45°C, and the reaction time is 24 hours; Step 5: Finally, PTMAO was reacted with the PEI-modified stent. The amino groups on the PEI surface further reacted with the epoxy groups in the polymer molecules to graft PTMAO onto the stent surface to obtain a PTMAO-coated stent (PTMAO-s). The reaction temperature was 60°C and the reaction time was 72h.

[0015] The contact angle of the prepared coated stent PTMAO-s was about 5.2°, showing superhydrophilic properties.

[0016] The DSC test results show that the melting enthalpy of PTMAO polymer is extremely low, about 4.16 J / kg. The low melting enthalpy indicates that the bound water content in the polymer is high, which shows that PTMAO has excellent ability to bind water.

[0017] Through AFM testing, it was observed that the hydrophilic probe can generate an adsorption force of about 11.04nN when contacting the PTMAO-s surface. This phenomenon proves that PTMAO-s can produce a high-strength hydration layer in aqueous solution to interact with the hydrophilic probe.

[0018] In addition, ITC experiments found that the hydration layer formed by PTMAO adsorbing water in salt solution can effectively isolate the interaction with inorganic salt ions.

[0019] In the anti-protein adsorption experiment, the protein adsorption amount of PTMAO-s was approximately 22.9% of that of PU and 32.7% of that of Bard.

[0020] In the antibacterial adhesion experiment, the Escherichia coli colonies on the surface of PTMAO-s were approximately 12.6% of those on PU and 34.2% of those on Bard, and the Proteus mirabilis colonies on the surface of PTMAO-s were approximately 10.3% of those on PU and 28.5% of those on Bard.

[0021] The above experimental results confirm that PTMAO-s has excellent anti-protein adsorption and anti-bacterial adhesion capabilities.

[0022] Subsequently, in vitro anti-crusting experiments showed that the crusting weight on the surface of PTMAO-s was approximately 13.8% of that of PU and 27.5% of that of Bard, showing the best anti-crusting performance.

[0023] Then, we tested the cytotoxicity of the coated scaffolds. The results of the MTT experiment and live / dead cell staining experiment showed that the cell activity of HK-2 cells was not significantly reduced after PTMAO-s was co-cultured with them.

[0024] In the in vivo experiment, we found that the colonies of Proteus mirabilis on the surface of PTMAO-s were about 8.6% of PU and 19.8% of Bard, and the weight of PTMAO-s crust was about 17.6% of PU and 30.0% of Bard. Finally, we performed H&E staining on the bladder and kidney tissues of rats, and the results showed that PTMAO-s did not increase the risk of urinary tract infection.

[0025] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent, characterized in that: The coating is gradually grafted onto the surface of the polyurethane stent by 1,6-hexamethylene diisocyanate HMDI, polyethyleneimine PEI and TMAO-derived polymer PTMAO; The specific steps include: Step 1: polymerize dimethylaminopropyl acrylamide (DMAPA) monomer and glycidyl methacrylate (GMA) monomer in a ratio of 20:2 through a traditional free radical reaction to generate a tertiary amine polymer PDMG; Step 2: PDMG is then further oxidized using 3-chloroperoxybenzoic acid (mCPBA) to obtain a TMAO-derived zwitterionic polymer, PTMAO; Step 3: After PTMAO is successfully synthesized, the secondary amine groups on the surface of the polyurethane scaffold are used to react with the double bonds of HMDI to graft HMDI onto the surface of the scaffold to obtain isocyanate groups; Step 4: The HMDI-modified scaffold is then further reacted with PEI, and the amino group of PEI reacts with isocyanate to further connect PEI to the surface of the scaffold; Step 5: Finally, PTMAO is reacted with the PEI-modified stent, and the amino groups on the PEI surface further react with the epoxy groups in the polymer molecules to graft PTMAO onto the stent surface to obtain a PTMAO-coated stent (PTMAO-s).

2. A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent according to claim 1, characterized in that: In step three, after the synthesis of PTMAO, the surface of the polyurethane scaffold needs to be modified.

3. A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent according to claim 2, characterized in that: In step 4, the molecular weight of PEI involved in the reaction is 10,000.

4. A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent according to claim 3, characterized in that: In step 4, the reaction temperature is 45°C and the reaction time is 24h.

5. A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent according to claim 4, characterized in that: In step 5, the reaction temperature is 60°C and the reaction time is 72h.

6. A method for constructing a TMAO-derived zwitterionic polymer PTMAO coating on the surface of a polyurethane stent according to claim 5, characterized in that: In step five, the surface water contact angle of the prepared coated stent PTMAO-s was 5.2°.

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