A kind of preparation method of surface for the imitating cell outer-layer membrane structure coating of Phosphorylcholine
Technical field
The invention belongs to material surface science and biological medical polymer material technical field, and in particular to a kind of surface is
The preparation method of the imitating cell outer-layer membrane structure coating of Phosphorylcholine.
Background technology
Chitosan has many advantages, such as degradability, antibiotic property, nontoxic, non-stimulated, pH responses (Carbohydrate
Polymers 2010,79:724-730), it has been widely used in the fields such as biomedicine.It is more and more research shows that:
Chitosan and its derivative material can be used for blood purification.Amino in chitosan molecule contributes to toxin a variety of in blood
Absorption, can be used for blood Absorbent (Chemical Journal of Chinese Universities 2002,23:75-77;Journal of
Microencapsulation 1993,10:475-486).Chitosan film has high dialysance, selectivity and intensity, can be with
As haemodialysis material (Journal of Applied Polymer Science 1992,46:255-261;263-269).
Although chitosan and its derivative has many advantages as blood purification material, there is also protein absorption, and blood is small
Plate sticks, the problems such as eventually leading to blood coagulation, form thrombus, so the blood compatibility for improving chitosan and its derivative material is compeled
In the eyebrows and eyelashes (Applied Surface Science 2005,241:485-492;Biomaterials 2002,23:2561-
2568;Biomaterials 2003,24:3213-3220).
Phosphorylcholine (phosphorylcholine, PC) is the terminal hydrophyllic group for forming cell membrane basic unit lecithin, is
Outer layer functional group in extracellular tunic simultaneous with positive and negative xenogenesis charge, has the ability and hydrophily of stronger combination water
Can, the surface of this structure and composition will not only be adsorbed with physiological environment interaction and depositing proteins, will not cause
Platelet activation leads to the adverse reactions such as blood coagulation, has good biocompatibility.In recent years research shows that, using phosphinylidyne
Choline group and its polymer have imitating cell outer-layer membrane structure in material surface structure, can significantly improve the blood phase of material
Capacitive.
In recent years, using approach (the Carbohydrate Polymers 2007,70 of grafting Phosphorylcholine small molecule:82-
88;Biomacromolecules 2007,8:3169-3176;Biomacromolecules 2006,7:3151-3156;
Journal of Applied Polymer Science 2003,88:489-493;Polymer International
2003,52:81-85;Journal of biomaterials science,Polymer edition 2002,13:501-
510;Colloids and Surfaces B:Biointerfaces 2009,71:268-274) modification of chitosan so that shell gathers
The blood compatibility of sugar significantly improves.But these modes are not often high in the density of the Phosphorylcholine group of material surface, limit
It has made it and has been modified the application in field and further improving for blood compatibility in bio-medical material.
For this purpose, by the methacrylic acid containing Phosphorylcholine group-methylacryoyloxyethyl Phosphorylcholine binary copolymerization
Object (PMA) polyanion carries out layer upon layer electrostatic self assembly with chitosan (polycation), obtains with imitating cell outer-layer film knot
Coating surface (the Colloids and Surfaces B of structure:Biointerfaces 2011,85:48-55).Protein adsorbs
With platelet adhesion reaction the experimental results showed that:The blood compatibility of modified rear surface is obviously improved.In view of this modification side
The all the advantages of method will provide technical support to promote the blood compatibility of bio-medical material.However, with physical absorption side
Formula is incorporated in the polymer with simulated cellulosa membrane structure coating of transplanting device surface, inevitably occurs in complex environment in vivo molten
It solves, come off.For this purpose, Lewis and Xu build equality (Biomaterials 2001,22:99-111;Biomaterials 2004,
25:3099-3108;European Polymer Journal 2004,40:291-298) respectively to containing trimethoxy silicon substrate
The polymer coating of group and Phosphorylcholine group is studied.The result shows that trimethoxy on polymer molecular chain in coating
Silicon group meets water and can hydrolyze, be crosslinked, and also covalent bond can be formed with the active group of substrate surface, so as to make Phosphorylcholine class
The performance of polymer coating is significantly improved.It can be seen that between polymer crosslinking and its with substrate surface functional group
Reaction is the key factor for improving Phosphorylcholine Type of Collective object coating performance.
Crosslinkable groups are introduced into phosphoryl choline polymer can form metastable imitating cell outer-layer membrane structure coating,
But the phosphoryl choline polymer synthesis of this structure stable coatings is complicated, using it is relatively difficult, stability is undesirable.In addition, by
The limitation of Phosphorylcholine content and coating be dry in polymer, is migrated in the Phosphorylcholine group orientation of surface during storage, causes
Make coating surface Phosphorylcholine groups density relatively low, be not easy effective simulation cell outer-layer membrane structure, blood compatibility need
It improves.Therefore, building the high imitating cell outer-layer membrane structure coating of stable structure, Phosphorylcholine groups density has important research
Meaning and application prospect.
Invention content
The technical problems to be solved by the invention are in view of the above shortcomings of the prior art, to provide a kind of surface as phosphinylidyne
The preparation method of the imitating cell outer-layer membrane structure coating of choline.By the vinyl monomer containing amphoteric ion Phosphorylcholine group,
Containing amido vinyl monomer by simple solution free radical polymerization, the phosphoryl choline polymer containing amino is synthesized, and will
It is dissolved in polar solvent with glutaraldehyde and is uniformly mixed, and coated in chitosan film surface, dries, and is then coated with the phosphinylidyne containing amino
Choline polymer solution.The phosphoryl choline polymer containing amino is coated to the Phosphorylcholine polymerization containing amino by coating
Object and glutaraldehyde coating surface, then amino and chitosan film surface amino groups and penta 2 in phosphoryl choline polymer are controlled by temperature
Aldehyde reaction makes coat inside be anchored base material while crosslinking, makes remaining phosphinylidyne courage of the aldehyde radical grafting containing amino of coating surface
Alkali polymer so that the thickness reduction of coating significantly improves the density of coating surface Phosphorylcholine, you can obtains surface phosphinylidyne courage
The high imitating cell outer-layer membrane structure coating of alkali density.
In order to solve the above technical problems, the technical solution adopted by the present invention is:
A kind of preparation method of surface for the imitating cell outer-layer membrane structure coating of Phosphorylcholine, includes the following steps:
Step 1:Vinyl monomer containing amphoteric ion Phosphorylcholine group, the vinyl monomer containing amino are led to
Solution free radical polymerization is crossed, synthesizes the phosphoryl choline polymer containing amino;
It is uniformly mixed, coats Step 2: the phosphoryl choline polymer containing amino is dissolved in glutaraldehyde in polar solvent
Modified material surface is being needed, is being dried;
Step 3: by the property material surface after being dried in the phosphoryl choline polymer solution coating step two containing amino;
Step 4: the film handled in step 3 is placed in distilled water, handled under the conditions of 80 DEG C~95 DEG C 2h~
12h;It is washed with solvent, obtains the high imitating cell outer-layer membrane structure coating of surface Phosphorylcholine density.
The vinyl monomer containing amphoteric ion Phosphorylcholine group is 2- methylacryoyloxyethyl Phosphorylcholines
Monomer, the vinyl monomer containing amino are 2- aminoethyl methacrylate hydrochloric acid salt monomers.
In step 1, the vinyl monomer containing amphoteric ion Phosphorylcholine group and the vinyl list containing amino
Body molar ratio is about (90:10)~(70:30).
In step 2, the polar solvent is methanol or ethyl alcohol.
In step 2, the phosphoryl choline polymer containing amino is dissolved in glutaraldehyde in polar solvent, the phosphorus containing amino
The concentration of phatidylcholine polymer is about 0.5~5mg/mL.
In step 2,10 microlitres/cm of volume is coated2/ face.
In two, the molar ratio of aldehyde radical is about 100 in the amino and glutaraldehyde in the phosphoryl choline polymer containing amino: (5
~20).
In step 2, it is chitosan material to need modified material.
In step 3, a concentration of 1~5mg/mL of the phosphoryl choline polymer solution containing amino of coating, solvent is first
Alcohol, 5~50 microlitres/cm of coating volume2/ face.
In step 4, solvent washing is successively with methanol, distillation water washing.
Compared with the prior art, the present invention has the following advantages:
The amino-containing phosphoryl choline polymer of the present invention is by the vinyl monomer containing Phosphorylcholine and contains ammonia
The vinyl monomer of base uses the binary randomcopolymer of radical polymerization synthesis, which mixes with glutaraldehyde
It is even, it coated in chitosan film surface, dries, is then coated with the phosphoryl choline polymer solution containing amino.It will be contained by coating
The phosphoryl choline polymer for having amino is coated to the phosphoryl choline polymer containing amino and glutaraldehyde coating surface, then passes through temperature
Amino and chitosan film surface amino groups are reacted with glutaraldehyde in degree control phosphoryl choline polymer, while being crosslinked coat inside
Base material is anchored, makes remaining phosphoryl choline polymer of the aldehyde radical grafting containing amino of coating surface so that the thickness of coating reduces
Significantly improve the density of coating surface Phosphorylcholine, you can obtain the high imitating cell outer-layer membrane structure of surface Phosphorylcholine density and apply
Layer.Stabilization, the preparation method of the high imitating cell outer-layer membrane structure coating of Phosphorylcholine groups density of the present invention are simple, condition temperature
With provide a kind of new approach to obtain the coating surface of imitating cell outer-layer membrane structure that has excellent performance;The present invention utilizes painting
The remaining aldehyde radical of coating surface is covered, is grafted the phosphoryl choline polymer containing amino, improves coating surface Phosphorylcholine group
Density significantly improves the performance of coating.The imitating cell outer-layer membrane structure coating that stabilization, the Phosphorylcholine groups density of preparation are high will
It is material implanted in blood purification, organizational project, before the fields such as medicament slow release and biosensor have wide application
Scape.
Description of the drawings
Fig. 1 is the dynamic contact angle of chitosan film of the present invention (CS) and Chitosan film (CS-PMH20GA-PMH10).
Fig. 2 is fine for the surface-element of chitosan film of the present invention (CS) and Chitosan film (CS-PMH20GA-PMH10)
Energy spectrum diagram.
Fig. 3 is the AFM figures of chitosan film of the present invention (CS) and Chitosan film (CS-PMH20GA-PMH10).
Fig. 4 is the fluorescin absorption of chitosan film of the present invention (CS) and Chitosan film (CS-PMH20GA-PMH10)
Amount.
Specific embodiment
With reference to the accompanying drawings and examples, technical scheme of the present invention is described in further detail.
Embodiment 1
Weigh 16mmol 2- methylacryoyloxyethyls Phosphorylcholines and 4mmol 2- aminoethyl methacrylate hydrochloric acid
Salt, using 0.1mmol potassium peroxydisulfates as initiator, under nitrogen protection, 70 DEG C of polymerisations for 24 hours, are dialysed, then after reaction
It is freeze-dried at -50 DEG C, obtains amino-containing phosphoryl choline polymer PMH20 (amino in 20 representation polymer building-up processes
Vinyl monomer molar ratio for 20%).The samples such as PMH10, PMH15 and PMH25 can similarly be synthesized.
With 400MHz Nuclear Magnetic Resonance with D2O is the proton magnetic of solvent test polymer.Appearance is had no at 5~7ppm,
Show gained copolymer in there is no residual monomer, and successfully synthesize the polymer, using at 3.28ppm as-N+(CH3)3Feature
Peak calculates polymer composition at 0.9~2.2ppm for methylene on main chain and the peak of pendant methyl, it is known that the polymer form with
Rate of charge is basically identical.
Phosphoryl choline polymer containing amino and glutaraldehyde are dissolved in methanol by amino and aldehyde radical molar ratio for 100: 10
In solvent after mixing, make a concentration of 1.0mg/mL of the phosphoryl choline polymer containing amino, coat 10 microlitres/cm2/ face
It is dried on chitosan material (CS) surface, then through 90 degree of processing 6h in distilled water, later successively with a large amount of methanol, distilled water
Washing, you can contrast sample (CS-PMH20/GA), then by the methanol solution of 1mg/mL PMH10 coat 10 microlitres of volume/
cm2/ face, dries, after through in distilled water 90 degree processing 6h, later successively with a large amount of methanol, distill water washing, you can obtain
The coating (CS-PMH20/GA-PMH10) of imitating cell outer-layer membrane structure.
It is 100: 10 by amino and aldehyde radical molar ratio that the present embodiment is contained the phosphoryl choline polymer of amino with glutaraldehyde
Being dissolved in methanol solvate after mixing, makes a concentration of 1.0mg/mL of the phosphoryl choline polymer containing amino, and coating 10 is micro-
Liter/cm2/ face is dried on chitosan material surface, then by 10 microlitres/cm of the methanol solution of 1mg/mL PMH10 coating volume2/
Face is dried, after through in distilled water 90 degree processing 6h, later successively with a large amount of methanol, distill water washing, you can obtain phosphinylidyne
The coating surface (CS-PMH20GA-PMH10) of the high imitating cell outer-layer membrane structure of content of choline.
As shown in Figure 1, chitosan material of the present embodiment through coating treatment and the chitosan material handled through control coatings
It compares, the surface advancing angle of the chitosan through coating treatment is relatively low, this is because the phosphoryl choline polymer containing amino and penta
The remaining aldehyde radical of dialdehyde coating surface is grafted the phosphoryl choline polymer containing amino by schiff base reaction and further improves painting
The density of layer surface Phosphorylcholine group so that coating surface hydrophily is higher, and advancing angle is relatively low.
As shown in Figure 2 and Table 1, chitosan material of the present embodiment through coating treatment and the chitosan handled through control coatings
Material is compared, N and P characteristic absorption peaks are larger on the chitosan film surface Phosphorylcholine group of modified processing, surface phosphinylidyne courage
Base groups density is higher.
The surface-element content of 1. chitosan film of table (CS) and Chitosan film (CS-PMH20GA-PMH10)
As shown in figure 3, chitosan material of the present embodiment through coating treatment and the chitosan material handled through control coatings
It compares, the surface topography of the chitosan through coating treatment is dramatically different.
As shown in figure 4, chitosan material of the present embodiment through coating treatment and the chitosan material handled through control coatings
It compares, processed chitosan fluorescin adsorbance has apparent reduction, and blood compatibility significantly improves.
Embodiment 2
Weigh 17mmol 2- methylacryoyloxyethyls Phosphorylcholines and 3mmol 2- aminoethyl methacrylate hydrochloric acid
Salt, using 0.1mmol potassium peroxydisulfates as initiator, under nitrogen protection, 70 DEG C of polymerisations for 24 hours, are dialysed, then after reaction
It is freeze-dried at -50 DEG C, obtains amino-containing phosphoryl choline polymer.
It is molten for 100: 5 by amino and aldehyde radical molar ratio with glutaraldehyde that the present embodiment is contained into the phosphoryl choline polymer of amino
In alcohol solvent after mixing, make a concentration of 0.5mg/mL of the phosphoryl choline polymer containing amino, 10 microlitres of coating/
cm2/ face is dried on chitosan material surface, then by 5 microlitres/cm of the methanol solution of 2mg/mL PMH20 coating volume2/ face, dries in the air
It is dry, after through 80 degree processing 12h in distilled water, later successively with a large amount of methanol, distill water washing, you can obtain Phosphorylcholine
The coating surface of the high imitating cell outer-layer membrane structure of content.
Embodiment 3
Weigh 12mmol 2- methylacryoyloxyethyls Phosphorylcholines and 8mmol 2- aminoethyl methacrylate hydrochloric acid
Salt, using 0.1mmol potassium peroxydisulfates as initiator, under nitrogen protection, 70 DEG C of polymerisations for 24 hours, are dialysed, then after reaction
It is freeze-dried at -50 DEG C, obtains amino-containing phosphoryl choline polymer.
It is 100: 15 by amino and aldehyde radical molar ratio that the present embodiment is contained the phosphoryl choline polymer of amino with glutaraldehyde
Being dissolved in methanol solvate after mixing, makes a concentration of 2mg/mL of the phosphoryl choline polymer containing amino, and 10 microlitres of coating/
cm2/ face is dried on chitosan material surface, then by 20 microlitres/cm of the methanol solution of 3mg/mL PMH15 coating volume2/ face,
Dry, after through in distilled water 85 degree processing 11h, later successively with a large amount of methanol, distill water washing, you can obtain phosphinylidyne courage
The coating surface of the high imitating cell outer-layer membrane structure of alkali content.
Embodiment 4
Weigh 15mmol 2- methylacryoyloxyethyls Phosphorylcholines and 5mmol 2- aminoethyl methacrylate hydrochloric acid
Salt, using 0.1mmol potassium peroxydisulfates as initiator, under nitrogen protection, 70 DEG C of polymerisations for 24 hours, are dialysed, then after reaction
It is freeze-dried at -50 DEG C, obtains amino-containing phosphoryl choline polymer.
It is 100: 20 by amino and aldehyde radical molar ratio that the present embodiment is contained the phosphoryl choline polymer of amino with glutaraldehyde
Being dissolved in alcohol solvent after mixing, makes a concentration of 3mg/mL of the phosphoryl choline polymer containing amino, and 10 microlitres of coating/
cm2/ face is dried on chitosan material surface, then by 30 microlitres/cm of the methanol solution of 4mg/mL PMH10 coating volume2/ face,
Dry, after through in distilled water 95 degree processing 2h, later successively with a large amount of methanol, distill water washing, you can obtain phosphinylidyne courage
The coating surface of the high imitating cell outer-layer membrane structure of alkali content.
Embodiment 5
Weigh 14mmol 2- methylacryoyloxyethyls Phosphorylcholines and 6mmol 2- aminoethyl methacrylate hydrochloric acid
Salt, using 0.1mmol potassium peroxydisulfates as initiator, under nitrogen protection, 70 DEG C of polymerisations for 24 hours, are dialysed, then after reaction
It is freeze-dried at -50 DEG C, obtains amino-containing phosphoryl choline polymer.
It is 100: 12 by amino and aldehyde radical molar ratio that the present embodiment is contained the phosphoryl choline polymer of amino with glutaraldehyde
Being dissolved in methanol solvate after mixing, makes a concentration of 4mg/mL of the phosphoryl choline polymer containing amino, and 10 microlitres of coating/
cm2/ face is dried on chitosan material surface, then by 40 microlitres/cm of the methanol solution of 5mg/mL PMH15 coating volume2/ face,
Dry, after through in distilled water 83 degree processing 11h, later successively with a large amount of methanol, distill water washing, you can obtain phosphinylidyne courage
The coating surface of the high imitating cell outer-layer membrane structure of alkali content.
Embodiment 6
Weigh 13mmol 2- methylacryoyloxyethyls Phosphorylcholines and 7mmol 2- aminoethyl methacrylate hydrochloric acid
Salt, using 0.1mmol potassium peroxydisulfates as initiator, under nitrogen protection, 70 DEG C of polymerisations for 24 hours, are dialysed, then after reaction
It is freeze-dried at -50 DEG C, obtains amino-containing phosphoryl choline polymer.
It is molten for 100: 7 by amino and aldehyde radical molar ratio with glutaraldehyde that the present embodiment is contained into the phosphoryl choline polymer of amino
In alcohol solvent after mixing, make a concentration of 1.5mg/mL of the phosphoryl choline polymer containing amino, 10 microlitres of coating/
cm2/ face is dried on chitosan material surface, then by 50 microlitres/cm of the methanol solution of 1.5mg/mL PMH20 coating volume2/
Face is dried, after through in distilled water 87 degree processing 8h, later successively with a large amount of methanol, distill water washing, you can obtain phosphinylidyne
The coating surface of the high imitating cell outer-layer membrane structure of content of choline.
Embodiment 7
Weigh 16mmol 2- methylacryoyloxyethyls Phosphorylcholines and 4mmol 2- aminoethyl methacrylate hydrochloric acid
Salt, using 0.1mmol potassium peroxydisulfates as initiator, under nitrogen protection, 70 DEG C of polymerisations for 24 hours, are dialysed, then after reaction
It is freeze-dried at -50 DEG C, obtains amino-containing phosphoryl choline polymer.
It is 100: 17 by amino and aldehyde radical molar ratio that the present embodiment is contained the phosphoryl choline polymer of amino with glutaraldehyde
Being dissolved in methanol solvate after mixing, makes a concentration of 2.5mg/mL of the phosphoryl choline polymer containing amino, and coating 10 is micro-
Liter/cm2/ face is dried on chitosan material surface, then by the methanol solution of 2.5mg/mL PMH10 coat 25 microlitres of volume/
cm2/ face, dries, after through in distilled water 92 degree processing 3h, later successively with a large amount of methanol, distill water washing, you can obtain
The coating surface of the high imitating cell outer-layer membrane structure of Phosphorylcholine content.
The above is only presently preferred embodiments of the present invention, and any restrictions are not done to the present invention, every according to invention skill
Any simple modification, change and the equivalent structure that art essence makees above example change, and still fall within the technology of the present invention
In the protection domain of scheme.