Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an ultrathin single-layer phosphorylcholine modified material. The inventors have surprisingly found that a monolayer phosphorylcholine coating not only enables a uniform, stable presence of the coating on the surface of medical biomaterials, but also enables a good maintenance of the basic structure for substrates/devices with micro-nano and porous type basic structures. The coating performance aspect also achieves biocompatibility and blood compatibility comparable to the existing polymer coating. In addition, the preparation process of the single-layer phosphorylcholine is simpler, and the high-efficiency modification of various modeling and special structure instruments is facilitated.
Solution for solving the problem
The present invention relates to a medical article. Comprises a substrate surface with a micropore structure or a micro-nano topological foundation structure characteristic;
The monolayer coating material has a phosphorylcholine structure and forms a functional surface layer, wherein the phosphorylcholine structure contains a reactive site and is bonded to the substrate through the reactive site;
The monolayer coating material is configured to resist protein adsorption and antithrombotic effects and to cause maintenance of greater than 80% of the substrate surface infrastructure, as measured by SEM.
When the substrate surface is provided with a micro-nano topological structure, the monolayer coating material causes the change rate of the micro-pore quantity or area in the unit area to be not more than 5%, and when the substrate surface is provided with a micro-nano topological structure, the monolayer coating material causes the change rate of the thickness to be not more than 1% compared with the substrate.
Wherein the functional skin layer has a thickness of no more than 10nm a, more preferably the functional skin layer has a thickness of less than 5 a nm a or even less.
Wherein the functional surface layer has a phosphorus atom content of 0.01 to 2 atomic% relative to the total atomic content excluding hydrogen atoms, as measured by X-ray photoelectron spectroscopy (XPS), and more preferably has a phosphorus atom content of 0.01 to 1 atomic% relative to the total atomic content excluding hydrogen atoms, as measured by X-ray photoelectron spectroscopy (XPS).
Wherein the functional skin layer can reduce protein adsorption by at least 20% or more, preferably the functional skin layer can reduce protein adsorption by at least 30% or more, and more preferably the functional skin layer can reduce protein adsorption by at least 40% or more.
In the invention, the compound with phosphorylcholine structure in the molecule has a general formula shown in the following formula I: Wherein R2 represents a C1-C10 alkylene group, the terminal end of the R1 structure contains a functionalized reactive site, and the compound of the formula has a molecular weight of 1000 or less.
Wherein, the compound with phosphorylcholine structure in the molecule is functionalized from raw materials with the general formula shown in the following formula II or III: Wherein R2 represents a C1-C10 alkylene group.
The medical product further comprises a transition layer between the base material and the functional surface layer, wherein the transition layer is obtained by surface activation treatment and coupling agent treatment, the surface activation treatment comprises acid treatment, alkali treatment, plasma treatment, chemical reagent treatment and the like, and the coupling agent treatment comprises catechol substance treatment, silane coupling agent treatment, isocyanate treatment and the like.
Wherein the substrate is a substrate having fibers, pores, filaments, microspheres, or a combination thereof.
The invention also provides a preparation method of the medical product, which is characterized by comprising the steps of (1) providing a substrate with a micropore structure or a micro-nano topological foundation structure characteristic surface, (2) coating a dispersion liquid containing a compound represented by the following chemical formula I on the substrate surface, and (3) forming a monolayer coating material on the substrate surface by at least one treatment of chemical grafting, solvent volatilization, thermal curing, photo curing and radiation curing on the coated substrate.Wherein R2 represents a C1-C10 alkylene group, the terminal end of the R1 structure contains a functionalized reactive site, and the compound of the formula has a molecular weight of 1000 or less.
Further, the concentration of the compound in the dispersion liquid is 0.01 mg/mL-50 mg/mL.
Further, the dispersion comprises a solvent selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, water, ethylene glycol, diethylene glycol, polyethylene glycol, glycerol, propylene glycol, pentaerythritol, vinyl alcohol, polyvinyl alcohol, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate, toluene, chloroform, methylene chloride, and combinations thereof.
Further, the coating mode is at least one selected from dip coating, spray coating, bar coating, brush coating, spin coating, electrospray and combinations thereof.
ADVANTAGEOUS EFFECTS OF INVENTION
The ultrathin single-molecule phosphorylcholine layer can keep the surface structure of a base material, cannot cause obvious change of the surface of a microporous structure, a micro-nano topological structure or a weaving structure during SEM observation, or can generate the accumulation condition of a coating, and cannot damage the basic performance of the base material. And the polymer has no internal crosslinking site in a covalent connection mode of the terminal and the substrate, and has better stability compared with a copolymer containing an intramolecular crosslinking site. Despite the ultra-thin coating, it is still capable of achieving coating functions similar to polymeric materials, such as having substantially comparable protein adhesion resistance and antithrombotic capacity to polymeric coatings. And thus can be widely applied to medical equipment and medical appliances.
Description of the embodiments
Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, well known methods, procedures, means, equipment and steps have not been described in detail so as not to obscure the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present specification, the numerical range indicated by "numerical values A to B" means a range including the end point numerical values A, B.
In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
It should be understood that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Reference in the specification to "one or more particular/preferred embodiments/aspects," "another or other particular/preferred embodiments/aspects," "one or another embodiment/aspect," "one or another technical aspect," etc., means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.
The term "comprising" and any variations thereof in the description of the invention and in the claims is intended to cover a non-exclusive inclusion. For example, a process, method, or system, article, or apparatus that comprises a list of steps or elements is not limited to only those listed but may optionally include additional steps or elements not listed or inherent to such process, method, article, or apparatus.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise. In the description of the present invention, the meaning of "several" means at least one, such as one, two, etc., unless specifically defined otherwise.
The term "functionalized" and related terms in the present invention include processes that treat a material to alter its surface properties to meet specific requirements for a particular application, or processes that provide functions that it does not typically have by adding groups to a chemical.
The medical article of the invention is characterized by a substrate surface having microporous or micro-nano topological infrastructure features, and a monolayer coating material covalently bonded to at least a portion of the substrate, the monolayer coating material having a phosphorylcholine structure and comprising a functional skin layer, the phosphorylcholine structure containing reactive sites and being bonded to the substrate through the reactive sites, the monolayer coating material being configured to resist protein adsorption and antithrombotic effects, and the monolayer coating material being configured to cause greater than 80% of the maintenance of the substrate surface infrastructure as measured by SEM. Still further, the monolayer coating material is configured to cause greater than 85% of the substrate surface infrastructure to remain, and more preferably, the monolayer coating material is configured to cause greater than 90% of the substrate surface infrastructure to remain.
The substrate is an organic material or an inorganic material, and is generally a polymer material or a metal material. In the present invention, the material of the base material is not particularly limited, and examples of the polymer material include polyester, polytetrafluoroethylene, polyurethane, polyether polyurethane, polyamide, vinyl chloride, polycarbonate, polystyrene, polyethylene, polypropylene, polymethylpentene, polymethyl methacrylate, and various synthetic fibers, and examples of the metal material include stainless steel, titanium and alloy, cobalt-based alloy, magnesium alloy, and shape memory alloy.
The characteristic of the micro-porous structure or micro-nano topological foundation structure refers to the characteristic of the substrate as a micro-size, such as nano-microspheres, micro-fluidic devices, high-precision sensors, chips and the like, or the micro-nano topological structure of the substrate surface, such as micro-nano structural patterning or surface roughening treatment, or the material surface with very small pores, such as porous expanded polytetrafluoroethylene, a dense mesh bracket formed by monofilaments, multifilaments and the like, or a fabric structure of warp yarns and weft yarns, and the like, formed by weaving, expanding, overlapping and the like. In general, such substrates having a microporous structure or micro-nano topology, conventional polymer coatings, while imparting some functionality to the surface, can be very disadvantageous to the device at the expense of or damage to the surface microstructure, such as the polymer coating meshing or stacking at the overlap of the micropores/braid wires.
The monolayer coating material is a coating material or a nano film with a single-layer molecular thickness order, has a phosphorylcholine structure and is covalently bonded on the surface of the substrate. The phosphorylcholine structure has the characteristic of imitating cell membranes, so that the phosphorylcholine structure can endow the substrate with the functions of protein adsorption resistance, bacterial adhesion resistance, thrombus resistance, better blood compatibility and the like, and can meet the purposes of the substrate as an instrument material for contact treatment with human tissues and body fluids, in-vitro detection and the like.
The phosphorylcholine structure contains reactive groups including, but not limited to, aldehyde groups, mercapto groups, hydroxyl groups, amino groups, carboxyl groups, azido groups, isocyanate groups, alkynyl groups, double bonds, chlorinated hydrocarbons, and the like. The reactive groups can be chemically bonded to the substrate surface to form a stable and firm functional surface layer.
The monolayer coating material has a surprising beneficial effect in bonding to a substrate. The thickness is small enough so as not to obscure or cover the original microporous structure or micro-nano basic structural features of the substrate or to create a coating build-up condition. In the present invention, the monolayer coating material is configured to result in a maintenance of greater than 80% of the substrate surface infrastructure, as measured by SEM. Still further, the monolayer coating material is configured to cause greater than 85% of the substrate surface infrastructure to remain, and more preferably, the monolayer coating material is configured to cause greater than 90% of the substrate surface infrastructure to remain.
Further, when the substrate surface is of a micro-porous structure, the monolayer coating material causes a change rate of the number or area of micro-pores per unit area of not more than 5%, and when the substrate surface is of a micro-nano topological structure, the monolayer coating material causes a change rate of thickness of not more than 1% compared with the substrate itself.
The coating of the invention can achieve ultra-thin coating thickness, which can reach at least 10nm or even 5 nm or lower in theory, because it forms a monolayer by chemical bonding to the substrate surface through a single molecular structure and has no internal crosslinking sites. Preferably, in the present invention, the functional skin layer has a thickness of not more than 10nm a, more preferably, the functional skin layer has a thickness of less than 5a nm a or even less. More preferably, the functional skin layer has a thickness of no more than 2 nm a, and even more preferably, the functional skin layer has a thickness of no more than 1 nm a.
In the present invention, the functional surface layer has a phosphorus atom content of 0.01 to 2 atomic% relative to the total atomic content excluding hydrogen atoms, as measured by X-ray photoelectron spectroscopy (XPS), and more preferably has a phosphorus atom content of 0.01 to 1 atomic% relative to the total atomic content excluding hydrogen atoms, as measured by X-ray photoelectron spectroscopy (XPS).
Despite the ultra-thin coating, it is still capable of achieving coating functions similar to polymeric materials, such as having substantially comparable protein adhesion resistance and antithrombotic capacity to polymeric coatings. The terminal connection mode has no internal crosslinking site, has better stability compared with a copolymer containing an intramolecular crosslinking site, can better expose a phosphorylcholine structure and plays a role. In the present invention, the functional surface layer is capable of reducing protein adsorption by at least 20% or more, more preferably by at least 30% or more, and even more preferably by at least 40% or more, as compared to the untreated substrate surface.
The anti-protein adsorption test can be performed by means of enzyme-linked reaction detection, isotope detection and the like, and can be, for example, fibrinogen adsorption test, albumin adsorption test and the like.
Further, the compound having a phosphorylcholine structure in the molecule has a general formula I as shown below: Wherein R2 represents a C1-C10 alkylene group. More preferably C1 to C5 alkylene. More preferably C2-C3 alkylene. Most preferred is a C2 alkylene group. The R1 structure terminal contains a functionalized reactive site, and the molecular weight of the compound of formula (I) is 1000 or less. Further, R1 is a linear or branched structure with a functionality greater than 1. Preferably a linear group having a functionality of 1 to 3 carbon atoms, more preferably a functionality of 1 to 2.
The end-functional reactive sites include, but are not limited to, aldehyde groups, thiol groups, hydroxyl groups, amino groups, carboxyl groups, azide groups, isocyanate groups, alkyne groups, double bonds, chlorinated hydrocarbons, acrylate groups, methacrylate groups, and the like. It is within the scope of the present invention as long as it can create covalent bonding with the substrate surface.
The compound with phosphorylcholine structure in the molecule can be one or a mixture of several of the compounds in the general formula.
In some embodiments of the present invention, it is further preferred that suitable compounds having a phosphorylcholine structure within the molecule according to the present invention include one or more of the following structures: Further, the compound having a phosphorylcholine structure in the molecule can be functionalized from a raw material of the general formula shown in the following formula II or formula III: Wherein R2 represents a C1-C10 alkylene group. More preferably C1 to C5 alkylene. More preferably C2-C3 alkylene. Most preferred is a C2 alkylene group.
In the present invention, a transition layer between the substrate and the functional skin layer is also included. The presence of the transition layer may provide reactive sites for certain substrates that do not have reactive functional groups, or may provide more uniform and dense reactive sites for the substrate surface, facilitating reactive covalent bonding of subsequent monolayer coating materials. The transition layer is obtained by surface activation treatment and coupling agent treatment. The surface activation treatment comprises acid treatment, alkali treatment, chemical reagent treatment, plasma treatment, corona discharge treatment, radiation irradiation treatment, heating treatment and the like, and the coupling agent treatment comprises catechol substance treatment, silane coupling agent treatment, isocyanate treatment and the like.
The invention also provides a preparation method of the medical product. The method comprises the steps of (1) providing a substrate with a surface having a microporous structure or a micro-monomolecular topological foundation structure, 2) coating the surface of the substrate with a dispersion containing a compound represented by the following chemical formula I, and (3) forming a monolayer coating material on the surface of the substrate by at least one treatment selected from the group consisting of chemical grafting, solvent evaporation, thermal curing, photo curing and radiation curing.Wherein R2 represents a C1-C10 alkylene group, more preferably a C1-C5 alkylene group. More preferably C2-C3 alkylene. Most preferred is a C2 alkylene group. The R1 structure terminal contains a functionalized reactive site, and the molecular weight of the compound of formula (I) is 1000 or less. Further, R1 is a linear or branched structure with a functionality greater than 1. Preferably a linear group having a functionality of 1 to3 carbon atoms, more preferably a functionality of 1 to 2. Higher functionality allows more reactive groups to be obtained, the higher the chemical grafting efficiency
Such functionalized reactive sites include, but are not limited to, aldehyde groups, thiol groups, hydroxyl groups, amino groups, carboxyl groups, azide groups, isocyanate groups, alkyne groups, double bonds, chlorinated hydrocarbons, acrylate groups, methacrylate groups, and the like. It is within the scope of the present invention as long as it can create covalent bonding with the substrate surface.
The compound with phosphorylcholine structure in the molecule can be one or a mixture of several of the compounds in the general formula.
In the present invention, the substrate having a microstructured or nano-based feature surface may be some medical article. In one aspect, the substrate itself may have a micro-scale or fine structure, such as a nano-microsphere, microfluidic device, small caliber vascular device, etc., or the substrate surface may have a micro-nano scale topology, such as a micro-nano structure patterned surface, or the substrate surface may be woven, expanded, overlapped, etc., to form a material surface having very small pores, such as porous expanded polytetrafluoroethylene, a dense mesh scaffold made of monofilaments, multifilaments, etc., or a fabric structure of warp and weft yarns, etc.
Of course, the ultra-thin single molecule phosphorylcholine layer of the present invention may also be used to coat virtually any medical article, such as a medical device, for which it is desirable to provide a stable functional coating on its surface. Exemplary medical articles include drug delivery vascular stents, other vascular devices (e.g., grafts, catheters, valves, artificial hearts, heart assist devices), implantable defibrillators, blood oxygenator devices (e.g., tubing, membranes), surgical devices, cell culture apparatus, biosensors, wound treatment devices, endoscopic devices, orthopedic devices, dental instruments, urological instruments, colostomy pocket attachment devices, ophthalmic devices, intraocular lenses, dialysis-required devices, and the like.
In the present invention, the curing manner of the coating layer is not limited. Including but not limited to chemical grafting in solution, solvent evaporation, thermal curing, photo-curing, radiation curing, and the like.
In a preferred mode of the invention, the concentration of the compound in the dispersion is 0.01 mg/mL-50 mg/mL. When the concentration of the compound is too low, the amount of coating due to covalent bonding with the substrate is reduced, and thus it is difficult to obtain high anti-protein adsorption properties and high antithrombotic properties, preferably 0.1mg/mL or more. On the other hand, the upper limit of the compound concentration is not excessively limited, and is preferably 20 mg/mL or less, more preferably 10 mg/mL or less.
In a preferred mode of the present invention, the dispersion is selected without excessive limitation, including but not limited to solvents of methanol, ethanol, isopropanol, n-butanol, water, ethylene glycol, diethylene glycol, polyethylene glycol, glycerol, propylene glycol, pentaerythritol, vinyl alcohol, polyvinyl alcohol, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate, toluene, chloroform, dichloromethane, and combinations thereof.
In a preferred mode of the present invention, the coating mode is adaptable according to the processing requirements or process of the actual sample, and the like, and is selected from at least one of dip coating, spray coating, bar coating, brush coating, spin coating, electrospray, and combinations thereof.
Examples
With reference to the foregoing embodiments, the technical solutions of the present application will be illustrated for the sake of more specific clarity and understanding, but it should be noted that the present application is not limited to the following examples.
Examples
The hollow dialysis fiber is selected as a sample and is subjected to cleaning treatment, and then the sample is completely immersed in KH-550 aqueous solution with the concentration of 1% (v%) after being subjected to plasma treatment for 5min, and the sample is subjected to reaction for 2h at 70 ℃ and then is cleaned and dried to obtain the surface moisture. The sample was then immersed in 10 mg/ml of 2-methacryloyloxyethyl phosphorylcholine in methanol and cured at 60 ℃ for 24 hours, after which the surface was rinsed and dried.
Examples
And selecting a PDMS material with a microstructure on the surface as a sample and performing cleaning treatment, wherein the microstructure can be prepared by one or more of etching, stamping, reverse molding and stamping. The PDMS material of the sample is a cylindrical groove structure with the surface formed by an inverted film method. Then, after the sample is treated by an ozone generator for 10min, the sample is immersed in KH-550 aqueous solution with the concentration of 1% (v%) for reaction at 70 ℃ for 2h, then the sample is cleaned to remove residual solution on the surface of the sample, and the surface moisture is dried at 70 ℃ to obtain the pretreated PDMS material.
The aldehyde phosphorylcholine was prepared by dissolving 1.8 g glycerophosphorylcholine, 3.0. 3.0 g sodium periodate in 60 mL purified water, dropwise adding 0.6 g ethylene glycol at 5 ℃, and stirring for a further 15h. And after the reaction is finished, removing deionized water in the reaction product by freeze drying, and obtaining the aldehyde phosphorylcholine. The aldehyde phosphorylcholine is prepared into 200 ml of 0.2 mg/ml aqueous solution, and 8 mg sodium borohydride is added to be uniformly mixed. The pretreated PDMS material was then immersed in the aldehyde phosphorylcholine solution described above, cured at 50 ℃ for 2 hours, and after removal of the sample, the surface rinse residual solution was dried.
Examples
And selecting a stainless steel sheet as a sample and performing cleaning and etching treatment. The samples were then pretreated as in example 1. The hydroformylation of phosphorylcholine was prepared according to the method of example 2, and then the aldehyde phosphorylcholine was prepared as a 200ml of 4 mg/ml aqueous solution, and added with 8mg sodium borohydride to be uniformly mixed. The pretreated stainless steel sheet material was then immersed in the aldehyde phosphorylcholine solution described above, cured at 50 ℃ for 2 hours, and after removal of the sample, the surface residual solution was rinsed and dried.
Examples
As in example 1, the sample was replaced with TPU sheet only.
Comparative example 1
Hollow dialysis fibers that have not been treated with a coating.
Comparative example 2
Preparation of phosphorylcholine Polymer 2.95 g of 2-methacryloyloxyethyl phosphorylcholine, 5.69 g of n-butyl methacrylate and 40 mg of azobisisobutyronitrile were weighed out and dissolved in 50 mL of absolute ethanol and reacted at 60℃under nitrogen atmosphere for 16 h. And (3) adopting diethyl ether precipitation for post-treatment, and drying to obtain the phosphorylcholine polymer.
The phosphorylcholine polymer coating is prepared by dissolving 1g phosphorylcholine polymer in 100 mL absolute ethyl alcohol to prepare a coating liquid, immersing the hollow dialysis fiber after the cleaning treatment in the coating liquid for 20min, taking out the hollow dialysis fiber, and drying for 2 hours at 50 ℃ to obtain a phosphorylcholine polymer coating sample.
Comparative example 3
The surface which is not coated with the coating has the PDMS material with the microstructure.
Comparative example 4
A stainless steel sheet material that has not been subjected to a coating treatment.
Test example 1 characterization of surface topography
The morphology of the surface coating of the sample was observed by scanning electron microscopy.
SEM test charts of example 1, comparative example 1 and comparative example 2 are shown in fig. 1. It has been found that hollow dialysis fiber samples with a monolayer coating material result in a significant maintenance of the dense network/microporous substrate microstructure compared to the hollow dialysis fiber samples with a polymer layer. Specifically, the maintenance rate of the microstructure of the hollow dialysis fiber material can reach more than 80% after the ultrathin monolayer treatment. And the variation is not more than 5% from front to back according to the number or area of micropores per unit area.
SEM test charts of example 2 and comparative example 3 are shown in fig. 2. The surface of the test sample is provided with a cylindrical groove structure formed by an inverted film method. It was also found that for PDMS film materials with circular micro-nanostructures on the surface, the cylindrical groove microstructure of the surface could be significantly maintained by monolayer treatment according to the invention, the coating did not cause stacking in the topology, and the thickness was not changed by more than 1% compared to the substrate itself.
Test example 2 surface phosphorus element content detection
And detecting the phosphorus element content on the surface of the material by adopting an X-ray photoelectron spectroscopy. The test data are set forth in table 1.
TABLE 1 testing of the phosphorus content of the coating surface
| Example 3 | Comparative example 4 |
| Phosphorus element content/% | 0.3 | 2.2 |
Test example 3 coating thickness test
The morphology of the coating on the surface of the sample was observed by a scanning electron microscope and the thickness of the coating was measured. The test data are set forth in table 2. In particular, the coating of the present invention, because it forms a monolayer by chemical bonding of the ends of a single molecular structure to the substrate surface, can be ultra-thin and can be at least 10 nm a or even 5nm a or less thick as calculated theoretically.
TABLE 2 coating thickness test case
| Example 2 | Example 3 | Comparative example 2 |
| Coating thickness/nm | Not detected | Not detected | 736 |
Test example 4 protein adsorption test
Protein adsorption testing of the sample surface was performed by fibrinogen (Fg) protein detection kit. The test data are shown in table 3 below. Specifically, the coating of the present invention was subjected to calculation of reduction in protein adsorption by using the bare substrate corresponding to each of the coating as a control sample. The monolayer structures of the present invention were found to reduce protein adsorption and, although ultra-thin, substantially achieve the same level of protein adsorption resistance as the polymer coating.
TABLE 3 protein adsorption conditions
| Example 1 | Comparative example 1 | Comparative example 2 | Example 3 | Comparative example 4 |
| Protein adsorption/OD value | 0.46 | 0.78 | 0.41 | 0.44 | 0.81 |
| Protein adsorption reduction/% | 41% | / | 47% | 46% | / |