CN108484956A

CN108484956A - Degradable high polymer material and the preparation method and application thereof with patterned surface

Info

Publication number: CN108484956A
Application number: CN201810336339.7A
Authority: CN
Inventors: 丁建东; 彭媛梦
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-04-16
Filing date: 2018-04-16
Publication date: 2018-09-04

Abstract

The invention belongs to technical field of biological materials, specially a kind of degradable high polymer material and the preparation method and application thereof with patterned surface.The degradable high polymer material of the present invention, using biodegradable hydrogel as matrix, surface modification is by the pattern being made of active material；So-called patterned surface refers to forming pattern array by modification in host surface.Patterning appearance structure constructed by hydrogel surface is stablized, and ingredient can be independently of base material component.There is resulting materials good biocompatibility, controllable degradability, patterning pattern to possess response bioactive sites for further modifying；The introducing of dynamic degradation makes material have the function closer to real physiological environment.The material can be applied to the fields such as stem cell culture, organizational project, biological diagnosis, bio-identification, biomimetic material design.

Description

Degradable high polymer material with patterned surface and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a degradable high polymer material and a preparation method and application thereof.

Background

The degradability of biomaterials is a very important property to consider in designing new biomedical materials. The biomaterial has the requirements on modulus, strength and the like required by tissue repair and replacement in the initial stage after being implanted into a human body; once degraded, it involves changes in many properties of the material, such as reduced modulus and strength, loss of mass, changes in chemical composition, and the like. At present, the rules of material degradation are studied, but the research on how to simulate the dynamic change of environment and influence the behavior of the size unit in the organism, such as the adhesion and differentiation of stem cells, on the degradation of biological materials is far from enough. The intensive development of relevant basic research requires first of all the establishment of a controllable material system.

The material with biocompatibility and good controllability can introduce lower complexity into the system under the influence of multi-factor regulation and control on research cell behaviors; introducing degradable components into the existing material system is the most common method for leading the degradable components to have degradability, and the common strategy is to introduce the degradable components such as hydrolysis sensitive components or polypeptide sequences recognizable by enzyme as crosslinking units at two ends of a molecular chain of the non-degradable hydrogel so that the hydrogel obtained by synthesis has degradability. By adjusting the number of the copolymerization units, the crosslinking property and the degradability of the material can be regulated and controlled within a certain range.

Patterning technology provides a reliable material means for systematically investigating the cellular responsiveness of biological materials. The structure constructed by the patterning technology can help to understand the response information of a plurality of cell levels and even smaller molecular levels, adjust the scale, distribution and composition of the constructed pattern, and can help to reveal the rule of interaction between cells and materials. A patterning technology is introduced in the establishing process of the material system, so that huge and complicated material information can be isolated, and the complicated physiological process can be explained.

The invention builds a hydrogel material platform with a degradable substrate and a patterned surface appearance by combining a patterning technology on the basis of degradable hydrogel with good biocompatibility. The material system has adjustable degradability and good dynamic response, and can adjust and control the interaction between active bodies such as cells, tissues and the like and materials.

Disclosure of Invention

The invention aims to provide a degradable high polymer material with a patterned surface, and a preparation method and application thereof.

The degradable high polymer material with the patterned surface provided by the invention takes biodegradable hydrogel as a matrix, and the surface of the biodegradable high polymer material is modified with a pattern consisting of active substances; the term "patterned surface" refers to a pattern array formed on the surface of a substrate material by modification.

In the polymer material of the invention, the biodegradable hydrogel is based on collagen, gelatin, hyaluronic acid, chitosan, chitin, alginate, fibrin or derivatives thereof which are natural sources.

In the high molecular material, the biodegradable hydrogel is formed by combining polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polypropylene oxide, polymethyl methacrylate and the like which are artificially synthesized with a small amount of degradable polyester, polyamino acid, polypeptide, polyurethane, polyesteramide, poly (n-lactide), polyanhydride, polyalkylcyanoacrylate, polyphosphazene and polyphosphoester through copolymerization, crosslinking, blending and an interpenetrating network forming mode.

In the high molecular material, the biodegradable hydrogel is formed by polymerizing and crosslinking polyethylene glycol and oligomeric ester in a manner of forming a macromonomer.

In the polymer material of the present invention, the active substance is selected from one or more of the following substances: platinum, gold, titanium, zirconium, cobalt, nickel, silver metals, hydroxyapatite, bone cement, bioglass inorganic nonmetal, collagen, cellulose protein, fibronectin biomacromolecules and special active polypeptide sequences.

In the polymer material of the present invention, the active substance is selected from polypeptide sequences of arginine-glycine-aspartic acid (RGD) and arginine-glutamic acid-aspartic acid-valine (REDV).

In the polymer material of the present invention, the morphology scale of the patterned surface may be a macro scale or a micro scale or a combination of the two: the macro scale refers to the range (for example, the magnitude of cm) observable by naked eyes, and the micro scale refers to the micrometer scale (namely, the magnitude of mum), the nanometer scale (namely, the magnitude of nm), or the combination of the two.

The high molecular material can be degraded by hydrolysis, enzymatic hydrolysis, bacterial decomposition, thermal decomposition and oxidative degradation singly or in combination. The degradation rate can be adjusted by adjusting the ratio of the degradation components and the degradation trigger.

The invention provides a preparation method of a degradable high polymer material with a patterned surface, which mainly relates to pattern transfer and comprises the following specific steps:

(1) constructing pattern arrays with different scales on the surface of a stable substrate by using mask-assisted photoetching, block copolymer micelle self-assembly or combination of the two to obtain a patterned template;

(2) copolymerizing the modified natural polymer or artificially synthesized polymer with a degradable substance to obtain a polymer macromonomer containing a degradable component and capable of crosslinking polymerization;

(3) and modifying the surface of the substrate containing the pattern array by using a coupling agent, and combining the macromolecular macromonomer with the surface of the pattern through polymerization reaction to form hydrogel consisting of degradable macromolecules, so that the pattern is transferred to the surface of the degradable hydrogel.

In the above preparation method, the modifying of the coupling agent on the surface of the pattern substrate means introducing a bifunctional coupling agent capable of interacting with the surface pattern and the macromolecular macromonomer simultaneously to promote the pattern array transfer.

One end functional group of the bifunctional coupling agent can form stable bonding of one of covalent bond, ionic bond, metallic bond and coordination bond with the pattern array on the surface of the substrate, and the other end functional group can be olefin double bond, sulfydryl and metallic ion and is used as a monomer component to participate in the hydrogel crosslinking network polymerization process.

In the above preparation method, the degradable macromolecular macromonomer is polymerized into hydrogel by thermal crosslinking, photo crosslinking or ionic crosslinking.

In the preparation method, the mask assisted photoetching mode in the step (1) can be used for constructing a micron-scale patterned template on a stable substrate, the size of the micron-scale patterned template can be selected to be 1 mu m-5000 mu m, and the micron-scale patterned template is obtained through photoetching mask plates containing graphic arrays with different sizes.

In the preparation method, the self-assembly mode of the block copolymer micelle in the step (1) can construct a nanoscale patterned array on a stable substrate, and the dimension of the nanoscale pattern template is in the range of 5-150 nm.

The block copolymer micelle self-assembly method is used for constructing the nano patterned array on the stable substrate, and the size, distribution and components of the obtained nano pattern are adjusted by loading precursors of different types of metal salts into the selective solvent through the amphiphilic block copolymer. After the micelle array is treated by plasma, the block copolymer component can be removed, and metal is reduced from a precursor to obtain the metal array.

In the above preparation method, the patterned template in step (1) may be a micrometer-nanometer scale hybrid pattern, which is obtained by obtaining a nanometer scale pattern array template through a block copolymer self-assembly method, and then limiting the obtained nanometer scale pattern array with a micrometer scale pattern array through a spin coating-lithography step.

In the above-mentioned production method, a pattern array of a plurality of active materials can be constructed on the surface of the hydrogel by further modifying the hydrogel surface pattern array with an active material.

The degradation rate of the degradable polymer material with the patterned surface provided by the invention can be adjusted by adjusting the ratio of degradation components and degradation triggers. The patterning technology can construct a macro-scale to micro-scale multi-scale structure on the surface of the material, can become a platform for precise interaction (such as targeted biological recognition, cell membrane ligand-receptor binding and the like) involved in the research of biological materials, and the influence of the degradation process of the material on the interaction also becomes a new field of research.

The degradable polymer material with the patterned surface provided by the invention has good biocompatibility, and can be applied to the fields of biological identification and diagnosis, cell culture, culture of bacteria and other microorganisms, cell screening, screening of bacteria and other microorganisms, tissue engineering and the like.

The invention has the advantages that:

(1) the degradable high molecular biological material has good biocompatibility;

(2) the preparation method is strong in operability, batch preparation and controllability;

(3) the degradable hydrogel with the patterned and modified surface can simulate the process of real in-vitro environmental stimulation or in-vivo environmental physiological change through the dynamic change of the material property caused by degradation, and explains the dynamic environmental stimulation to organs, tissues and cells in the application process of the material;

(4) the scale, the component composition and the distribution of the patterned array can be adjusted, the patterned array can play a role in researching the response action and various characteristics of multi-scale and multi-dimensional objects such as molecules, cells, tissues and organs, the identification and monitoring of a finish machining instrument and the like, and the patterned array is applied to various in-vitro and in-vivo application fields such as stem cell culture, tissue engineering, biological diagnosis, biological identification, bionic material design and the like.

Drawings

FIG. 1 illustrates the preparation and degradation of a surface patterned hydrogel. Wherein, (a) the dotted line frame part is the synthesis of the polyethylene glycol-oligoester macromonomer of example 13, and the lower part is a schematic diagram of the polymerization transfer of the macromonomer of examples 44-60 on the surface of the patterned slide; (b) the crosslinked network structure collapses in the degradation process of the hydrogel.

FIG. 2 is the scanning electron microscope characterization image of the nanopattern array on the surface of the glass slide of example 40. Wherein (a) the segmented copolymer P18225-S2VP constructs a nano gold dot distribution map formed on a glass sheet through micelle self-assembly, and the nano distance is about 30 nm; (b) the segmented copolymer P4633-S2VP is used for constructing a nano gold dot distribution diagram formed on a glass sheet through micelle self-assembly, and the nano distance is about 64 nm; (c) the block copolymer P5052-S2VP is self-assembled by micelle to form a nano-gold dot distribution diagram on a glass slide, and the nano-distance is about 90 nm.

FIG. 3 is an atomic force microscope characterization of the surface of a P4633-S2VP nanopatterned slide obtained in example 36, as described in example 40. Wherein, (a) the slide surface nano array two-dimensional AFM represents a height image; (b) a three-dimensional height image having the same field of view as the two-dimensional height map; (c) height profile of the nanogold dot column indicated by the dotted line in the two-dimensional height image.

FIG. 4 is the scanning electron microscope characterization of the surface of the nano-patterned hydrogels obtained in examples 69-70 and 66-67. Wherein, (a) a scanning electron microscope representation picture of transferring a nano array constructed by a block copolymer P4633-S2VP to the surface of hydrogel; (b) scanning electron microscope characterization images of the nano-arrays constructed from the block copolymer P18225-S2VP transferred to the hydrogel surface.

Fig. 5 is a photograph showing the cell adhesion test of example 82 on the surface of the RGD-grafted polypeptide nanopatterned hydrogel obtained in example 80, and the fluorescent staining performed for one day (left) and seven days (right). Wherein (a) the stem cells are seeded on the surface of the patterned degradable hydrogel for one day after the fluorescence staining of the cells for the image; (b) stem cell seeding, fluorescence staining of cells seven days after patterning the degradable hydrogel surface.

Detailed Description

The invention is further described below by way of example, but not limited to these examples.

EXAMPLE 1 Synthesis of Natural Polymer derivative monomer

50g of Hyaluronic Acid (HA) with the molecular weight of 50kDa is dissolved by Milli-Q to prepare a 1 wt% solution, the pH of the solution is adjusted to 8 by using 1M sodium hydroxide solution, 50% equivalent of methacrylic anhydride is added into the solution system in a dropwise manner, and the system reacts for 24 hours under the ice bath condition. After the reaction, dialyzing by using a dialysis membrane with the molecular weight cutoff range of 5-8 kDa, changing water once per hour within the first 12 hours, and properly prolonging the time for replacing the dialysis solution to 2-3 hours later, wherein the total dialysis time is 48 hours. And (4) removing water from the dialyzed product by using a freeze dryer, taking out white powdery solid MeHA after 48 hours, and sealing and storing in a refrigerator at the temperature of-20 ℃.

Example 2

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 2000) 50g (25 mmol) transfer into three-necked reaction flask. Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, cooling the system to 80 ℃, increasing the ventilation amount of argon, sequentially adding 14.4 g (100 mmol) of racemic lactide (DL-LA) and a stannous octoate catalyst with the reactant ratio of 0.1 wt% into a three-necked bottle, and heating the system to 140 ℃ for reaction. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform, slowly transferring the reaction product liquid into diethyl ether with the volume ratio of 1:10 under the stirring condition for sedimentation, and standing in a refrigerator at the temperature of-20 ℃ after all the reaction product liquid is added to ensure that the sedimentation is complete. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 3

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 2000) 50g (25 mmol) transfer into three-necked reaction flask. Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, cooling the system to 80 ℃, increasing the ventilation amount of argon, sequentially adding 20.4g (200 mmol) of trimethylene carbonate and 0.1 wt% of stannous octoate catalyst of reactant ratio into a three-necked bottle, and heating the system to 140 ℃ for reaction. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform,slowly transferring the mixture into diethyl ether with the volume ratio of 1:10 under the stirring condition for settling, and standing in a refrigerator at the temperature of-20 ℃ after all the mixture is added so as to ensure that the mixture is completely settled. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 4

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 2000) 50g (25 mmol) transfer into three-necked reaction flask. Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, cooling the system to 80 ℃, increasing the ventilation capacity of argon, sequentially adding 11.6g (100 mmol) of glycolide and a stannous octoate catalyst with the reactant ratio of 0.1 wt% into a three-necked bottle, and heating the system to 140 ℃ for reaction. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform, slowly transferring the reaction product liquid into diethyl ether with the volume ratio of 1:10 under the stirring condition for sedimentation, and standing in a refrigerator at the temperature of-20 ℃ after all the reaction product liquid is added to ensure that the sedimentation is complete. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 5

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 2000) 50g (25 mmol) transfer into three-necked reaction flask. Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, the temperature of the system is reduced to 80 ℃, the ventilation capacity of argon is increased, 11.4 g (100 mmol) of Caprolactone (CL) and stannous octoate catalyst with the reactant ratio of 0.1 wt% are sequentially added into a three-necked bottle, the temperature of the system is raised to 140 DEG CThe reaction is carried out. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform, slowly transferring the reaction product liquid into diethyl ether with the volume ratio of 1:10 under the stirring condition for sedimentation, and standing in a refrigerator at the temperature of-20 ℃ after all the reaction product liquid is added to ensure that the sedimentation is complete. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 6

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 4000) 100g (25 mmol) transfer into three-necked reaction flask. Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, the system is cooled to 80 ℃, the aeration quantity of argon is increased, and racemic lactide (lactide) is sequentially added into a three-necked bottleD,L-LA) 14.4 g (100 mmol) of stannous octoate catalyst with the reactant ratio of 0.1 wt%, and heating the system to 140 ℃ for reaction. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform, slowly transferring the reaction product liquid into diethyl ether with the volume ratio of 1:10 under the stirring condition for sedimentation, and standing in a refrigerator at the temperature of-20 ℃ after all the reaction product liquid is added to ensure that the sedimentation is complete. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 7

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 4000) 100g (25 mmol) transfer into three-necked reaction flask. Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, the system is loweredWhen the temperature is 80 ℃, the aeration quantity of argon is increased, 10.2g (100 mmol) of trimethylene carbonate (TMC) and stannous octoate catalyst with the reactant ratio of 0.1 wt% are sequentially added into a three-necked bottle, and the system is heated to 140 ℃ for reaction. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform, slowly transferring the reaction product liquid into diethyl ether with the volume ratio of 1:10 under the stirring condition for sedimentation, and standing in a refrigerator at the temperature of-20 ℃ after all the reaction product liquid is added to ensure that the sedimentation is complete. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 8

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 4000) 100g (25 mmol) transfer into three-necked reaction flask. Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, cooling the system to 80 ℃, increasing the ventilation amount of argon, sequentially adding 11.6g (100 mmol) of Glycolide (GA) and a stannous octoate catalyst with the reactant ratio of 0.1 wt% into a three-necked bottle, and heating the system to 140 ℃ for reaction. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform, slowly transferring the reaction product liquid into diethyl ether with the volume ratio of 1:10 under the stirring condition for sedimentation, and standing in a refrigerator at the temperature of-20 ℃ after all the reaction product liquid is added to ensure that the sedimentation is complete. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 9

Weighing polyethylene glycol (with dihydroxy group) ((II))M _n= 4000) 100g (25 mmol) transfer into three-necked reaction flask.Sealing the system, introducing argon for protection, and connecting the system to a water-cooled pump for heat dissipation; the temperature of the system is raised to 120 ℃, and simultaneously stirring is started, and vacuumizing is carried out for 3 h for dewatering. After the water removal is finished, cooling the system to 80 ℃, increasing the ventilation capacity of argon, sequentially adding 11.4 g (100 mmol) of Caprolactone (CL) and a stannous octoate catalyst with the reactant ratio of 0.1 wt% into a three-necked bottle, and heating the system to 140 ℃ for reaction. After the reaction is carried out for 12 hours, the reaction system is cooled to 120 ℃, the vacuum pumping is carried out for 2-3 hours, and then the system is cooled to the room temperature. Diluting the reaction product liquid with a proper amount of chloroform, slowly transferring the reaction product liquid into diethyl ether with the volume ratio of 1:10 under the stirring condition for sedimentation, and standing in a refrigerator at the temperature of-20 ℃ after all the reaction product liquid is added to ensure that the sedimentation is complete. Standing for settling for 12 h, filtering off supernatant settling solution, removing residual diethyl ether with a freeze dryer to obtain white solid powder, sealing and storing in a refrigerator at-20 deg.C for subsequent use.

Example 10 following the basic procedure set forth in examples 2-9, bishydroxy PEGs of varying molecular weights were used to synthesize block copolymers with different polyester monomers, the reaction product properties of which are set forth in table 1 below:

TABLE 1

。

Example 11

Double bonds are introduced into two ends of the molecular chain of the ring-opening polymerization product for modification. The molecular weight of each ring-opening copolymerization product in example 10 was estimated according to the number of modifications of the oligomerization unit given in example 10; quickly weighing 10 mmol of product monomer, transferring into a reaction bottle with a branch port, and pumping and exchanging air for 3 times to ensure that the internal gas environment is in an argon atmosphere; 90 mL of dichloromethane was added to the reaction flask with a long needle, and the reaction was dissolved by stirring. The system is in an ice bath condition, after the system is completely cooled, a long needle is used for taking about 7 mL of triethylamine and adding the triethylamine into a reaction bottle; a solution of 40 mmol of acryloyl chloride in 10 mL of methylene chloride was quickly prepared in a syringe and slowly added dropwise to the reaction flask using a syringe pump. The ice bath condition is kept in the whole dripping process, the dripping speed is kept at a constant speed, the dripping is carried out for 6 hours in the ice bath from the beginning to the completion, and the reaction is continued for 18 hours at room temperature after the ice bath is removed. The mixed liquid after the reaction is light yellow, and clear and transparent product liquid is obtained after salt is filtered by a sand core funnel filled with diatomite, but at the moment, a small amount of byproduct salt is still dissolved in a dichloromethane solvent, so that a proper amount of ethyl acetate is added for extraction, and the rest salt is separated out; filtering by a sand core funnel to obtain a mixed solution of a product ethyl acetate and dichloromethane, performing rotary evaporation to remove most of the solvent, settling by using ethyl acetate with the volume of 1:10, filtering out a sediment after 24 hours, dissolving by using dichloromethane, settling again, and further removing impurities. The settled product is freeze-dried to obtain milk white solid powder, which is called oligoester-PEG-oligoester DA monomer for short, and the powder is hermetically stored in a refrigerator at the temperature of-20 ℃, and the synthetic process is schematically shown in figure 1.

Example 12

Following the basic procedure given in example 11, bishydroxy PEGs of different molecular weights and block copolymers synthesized with different polyester monomers were double-bond modified, and the reaction product properties are given in Table 2 below:

TABLE 2

。

Example 13

2.25 g of oligoLA-PEG obtained in example 12 was weighed₂₀₀₀-oligoLA DA monomer, prepared into a 45 wt% solution with Milli-Q, sealed and stirred for 12 h or more away from light until completely dissolved; cleaning the slide glass with Milli-Q ultrasonic wave, air drying at room temperature, and placing into polymerization reaction glassA frame, which is fully attached; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining oligoLA-PEG with a solid content of 45%₂₀₀₀-oligoLA hydrogel.

Example 14

2.0 g of oligoLA-PEG obtained in example 12 was weighed₂₀₀₀-oligoLA DA monomer, prepared into a solution with a concentration of 20 wt% with Milli-Q, sealed and stirred for 12 h and more away from light until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining oligoLA-PEG with a solid content of 20%₂₀₀₀-oligoLA hydrogel.

Example 15

Weighing example 12 4.0 g of oligoLA-PEG₂₀₀₀-oligoLA DA monomer, prepared into a 50 wt% solution with Milli-Q, sealed and stirred away from light for 12 h or more until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining oligoLA-PEG with 50% of solid content₂₀₀₀-oligoLA hydrogel.

Example 16

2.25 g of the oligoTMC-PEG obtained in example 12 were weighed₂₀₀₀-oligoTMC DA monomer, prepared as a 45 wt% solution with Milli-Q, sealed and stirred in dark for 12 h or more until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. To give an ol having a solids content of 45%igoTMC-PEG₂₀₀₀-oligoTMC hydrogels.

Example 17

2.25 g of the oligoGA-PEG obtained in example 12 was weighed₂₀₀₀-oligoGA DA monomer, prepared into a 45 wt% solution with Milli-Q, sealed and stirred for 12 h and above in dark place until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining the oligoGA-PEG with a solid content of 45%₂₀₀₀-oligoGA hydrogels.

Example 18

2.25 g of the oligoCL-PEG obtained in example 12 was weighed₂₀₀₀-oligoCL DA monomer, prepared into a 45 wt% solution with Milli-Q, sealed and stirred in dark for 12 h or more until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finishedCarefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening the solution by using a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining the oligoCL-PEG with a solid content of 45%₂₀₀₀-oligoCL hydrogels.

Example 19

2.25 g of oligoLA-PEG obtained in example 12 was weighed₄₀₀₀-oligoLA DA monomer, prepared into a 45 wt% solution with Milli-Q, sealed and stirred for 12 h or more away from light until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining oligoLA-PEG with a solid content of 45%₄₀₀₀-oligoLA hydrogel.

Example 20

2.25 g of the oligoTMC-PEG obtained in example 12 were weighed₄₀₀₀-oligoTMC DA monomer, prepared as a 45 wt% solution with Milli-Q, sealed and stirred in dark for 12 h or more until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, D2959 light previously prepared at a concentration of 0.1 wt% was added to the macromonomer solutionA saturated solution of the initiator (7.6 mg/mL) was stirred for 0.5 h with exclusion of light to give a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining the oligoTMC-PEG with a solid content of 45%₄₀₀₀-oligoTMC hydrogels.

Example 21

2.25 g of the oligoGA-PEG obtained in example 12 was weighed₄₀₀₀-oligoGA DA monomer, prepared into a 45 wt% solution with Milli-Q, sealed and stirred for 12 h and above in dark place until completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining the oligoGA-PEG with a solid content of 45%₄₀₀₀-oligoGA hydrogels.

Example 22

2.25 g of the oligoCL-PEG obtained in example 12 was weighed₄₀₀₀-oligoCL DA monomer, prepared as a 45% strength by weight solution with Milli-Q, sealed and stirred in the darkStirring for 12 h or more until completely dissolving; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.1 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. Obtaining the oligoCL-PEG with a solid content of 45%₄₀₀₀-oligoCL hydrogels.

Example 23

Weighing 3 g of MeHA monomer obtained in example 1, preparing a solution with the concentration of 30wt% by using Milli-Q, and stirring for 12 hours or more in a sealed and dark state until the monomer is completely dissolved; cleaning the glass slide by Milli-Q ultrasonic wave in advance, airing at room temperature, placing into a polymerization reaction glass frame, and fully attaching; while the above preparation was being carried out, a saturated solution (7.6 mg/mL) of the D2959 photoinitiator prepared in advance at a concentration of 0.05 wt% was added to the macromonomer solution and stirred for 0.5 h without light to obtain a prepolymerization solution. Transferring the prepolymerization solution into a round bottom glass reaction bottle with a branch opening, pumping nitrogen for 3 times to prevent the prepolymerization solution from inhibiting polymerization, keeping the whole process in a dark place, and then placing the reaction bottle in a dark place for taking. After the preparation is finished, carefully dropping about 300 mu L of prepolymerization solution above the clean glass slide, and fastening with a cover glass for sealing; and (3) transferring all the glass reaction frames into an ultraviolet reaction device, introducing high-purity nitrogen, discharging air in the reactor for 5min, starting an ultraviolet lamp, and polymerizing for 1 h. A MeHA hydrogel with a solids content of 30% was obtained.

Example 24

After the gel-like sheets obtained in examples 13 to 23 were sufficiently swollen in the solvent, they were taken out and their surface water was wiped off with a wiping sheet, and their compression modulus was measured with a rheometer. Selecting a flat plate clamp with the model of PU 8 and the diameter of 8 mm as a clamp of the rheometer; and (3) setting the descending speed of the clamp to be 0.005 mm/s to obtain a curve of normal stress Force of the hydrogel and Gap value of the clamp, converting according to the initial thickness value of the hydrogel sample and the stress area of the hydrogel to obtain a stress-strain (sigma-e) curve, and obtaining the compression modulus of the hydrogel according to the slope of the stress-strain curve within the range of low strain e being less than or equal to 5%.

Example 25

The hydrogels obtained in examples 13 to 23 were swollen in Phosphate Buffered Saline (PBS) for 24 hours and then taken out, and the specific linear dimensions (e.g., diameters) thereof were varied by measuring with a ruler, and the linear swelling ratio (linear swelling ratio) was obtained from the ratio of the specific side dimensions of the hydrogel samples before and after swelling

Wherein D is₁Refers to the linear dimension of the hydrogel before swelling; d₂Refers to the linear dimension of the hydrogel sample after swelling;

example 26

The hydrogels obtained in examples 13-23 were fully swollen in PBS and then their surface water was wiped off with a wipe by grasping a hydrogel sample from a solvent, weighing, and recording asm ₁(ii) a Then putting the hydrogel into a vacuum drying ovenDrying to constant weightm _0。The density of the dry polymer macromonomer powder is measured by hydrometallurgy. Volume swell ratio (Q _V) From the mass swelling ratio of (Q _m) Convert to obtain

Wherein,refers to the density of the polymer macromonomer in dry state;refers to the density of the solvent.

Example 27 construction of gold micropatterns on glass substrates

Using piranha washing liquid (H)₂SO₄: H₂O₂= 3: 1), ultrasonically cleaning the slide with ultrapure water for three times, 10 minutes each time, and then blowing and drying with nitrogen. And (3) putting the glass slide into acetone for ultrasonic treatment for 830 minutes, drying the glass slide in a baking oven at 120 ℃ for 4 hours after drying the glass slide by nitrogen, and cooling the glass slide to room temperature for later use. And uniformly coating the photoresist on the cleaned slide glass in a dark room at 3500 r/min for 20 s, and then placing the cleaned slide glass in an oven at 150 ℃ for 30 minutes. And photoetching by using a photoetching machine, selecting a micron round island with the diameter range of 4-100 mu m on a mask as a template, and increasing the solubility of the part irradiated by the ultraviolet light under the mask in an organic solvent. The exposed slide glass is put into a developing solution for 35 s and then put into ultra-pure water for fixation. The fixed slide is dried by nitrogen and then is put into a 120 ℃ oven to be dried for 30 minutes. After cooling the slide to room temperature, the slide was placed in an ion sputtering apparatus and treated with a gold spray at 8mA for 100 s. And (3) placing the glass slide after the gold spraying is finished into acetone for ultrasonic treatment for 10 minutes each time. And (5) standing and storing after nitrogen blow-drying.

Example 28 construction of gold micropatterns on glass substrates

Using piranha washing liquid (H)₂SO₄: H₂O₂= 3: 1), ultrasonically cleaning the slide with ultrapure water for three times, 10 minutes each time, and then blowing and drying with nitrogen. And (3) putting the glass slide into acetone for ultrasonic treatment for 830 minutes, drying the glass slide in a baking oven at 120 ℃ for 4 hours after drying the glass slide by nitrogen, and cooling the glass slide to room temperature for later use. And uniformly coating the photoresist on the cleaned slide glass in a dark room at 3500 r/min for 20 s, and then placing the cleaned slide glass in an oven at 150 ℃ for 30 minutes. And photoetching is carried out by using a photoetching machine, stripes with the micron width distribution of 10-100 mu m and the length of 5000 mu m on the mask plate are selected as templates, and the solubility of the part irradiated by the ultraviolet light under the mask plate in the organic solvent is increased. The exposed slide glass is put into a developing solution for 35 s and then put into ultra-pure water for fixation. The fixed slide is dried by nitrogen and then is put into a 120 ℃ oven to be dried for 30 minutes. After cooling the slide to room temperature, the slide was placed in an ion sputtering apparatus and treated with a gold spray at 8mA for 100 s. And (3) placing the glass slide after the gold spraying is finished into acetone for ultrasonic treatment for 10 minutes each time. And (5) standing and storing after nitrogen blow-drying.

Example 29 gold micropattern viewing on glass surface

By using an optical microscope, the magnification of an objective lens is 10X, the magnification of an eyepiece lens is 40X, and the gold micrometer pattern on the surface of the glass is observed in a phase difference mode, so that whether the shape of the gold pattern is complete or not can be verified.

Example 30 preparation of Block copolymer micelle solution

Selecting a P4633-S2VP polymer with the molecular weight of 115500 and the ratio of the number of styrene units to the number of 2-vinylpyridine units of 759:347, dissolving the polymer in toluene according to the concentration of 4.0mg/mL to prepare 10 mL of micelle solution, and stirring the solution for 24 hours in the dark; and calculating the amount of the required gold precursor chloroauric acid when the gold loading amount is 0.40, weighing and adding the gold precursor chloroauric acid into the polymer solution, carefully wrapping two layers of the weighed solution by using an aluminum foil, and stirring for 24 hours at room temperature in a dark place.

Example 31 preparation of Block copolymer micelle solution

Selecting a P18225-S2VP polymer with the molecular weight of 44300 and the unit number ratio of styrene to 2-vinylpyridine of 320:105, dissolving the polymer in toluene according to the concentration of 7.1mg/mL to prepare 10 mL of micelle solution, and stirring the solution for 24 hours in the dark; and calculating the amount of the required gold precursor chloroauric acid when the gold loading amount is 0.61, weighing and adding the gold precursor chloroauric acid into the polymer solution, carefully wrapping two layers of the weighed solution by using an aluminum foil, and stirring for 24 hours at room temperature in a dark place.

Example 32 preparation of Block copolymer micelle solution

Selecting a P5052-S2VP polymer with the molecular weight of 217000 and the unit number ratio of styrene to 2-vinylpyridine of 1776:304, dissolving the polymer in toluene according to the concentration of 3 mg/mL to prepare 10 mL of micelle solution, and stirring for 24 hours in the dark; and calculating the amount of the gold precursor chloroauric acid required when the gold loading amount is 0.44, weighing and adding the gold precursor chloroauric acid into the polymer solution, carefully wrapping two layers of the weighed solution with an aluminum foil, and stirring for 24 hours at room temperature in a dark place.

Example 33 preparation of Block copolymer micelle solution

Selecting a P4556-S2VP polymer with the molecular weight of 257000 and the number ratio of styrene units to 2-vinylpyridine units of 1728:732, dissolving the polymer in toluene according to the concentration of 3 mg/mL to prepare 10 mL of micelle solution, and stirring the solution for 24 hours in a dark place; and calculating the amount of the required gold precursor chloroauric acid when the gold loading amount is 0.35, weighing and adding the gold precursor chloroauric acid into the polymer solution, carefully wrapping two layers of the weighed solution by using an aluminum foil, and stirring for 24 hours at room temperature in a dark place.

Example 34 according toPS-materials with different molecular weights were used in accordance with the basic procedures given in examples 30-33b-P2VP block copolymer and chloroauric acid (HAuCl)₄·3H₂O) preparing a chloroauric acid-loaded polymer micelle solution, wherein the preparation parameters are listed in the following table 3:

TABLE 3

。

Example 35 morphological characterization of micellar solution

The dispersion uniformity of the micellar solutions loaded with chloroauric acid obtained in examples 30 to 33 was characterized by Transmission Electron Microscopy (TEM). Dipping a small amount of solution by a glass dropper, dropping the solution on a copper net observed by a transmission electron microscope, and completely volatilizing at room temperature. When the accelerating voltage is set to 200 kV and the magnification is set to 2.5 ten thousand and 5 ten thousand during the observation of the transmission electron microscope, the loading condition of the chloroauric acid in the micellar solution and the distribution condition of the micelles can be observed.

Example 36 preparation of nanopattern array on glass surface

The slide glass which is pre-processed by dipping and pulling operation is treated by the piranha washing liquid and is soaked in ultrapure water. The micellar solution obtained in example 30 was transferred into a 10 mL glass cuvette. And adhering the slide on a sample rod of a pulling machine, setting the pulling speed to be 420 mu m/s, standing the coated slide at a dark and light-resistant position for more than 24 h, and treating the coated slide by using a low-temperature plasma machine after toluene is volatilized. Setting plasma processing parameters: the power is 100W, and the flow of one path of oxygen is 100 cm³And/s, washing times are 4 times, the lowest vacuum degree is 8Pa, and the treatment time is 1 h. The treated slide is stored in a dry place in a dark place.

Example 37 preparation of nanopattern array on glass surface

The slide glass which is pre-processed by dipping and pulling operation is treated by the piranha washing liquid and is soaked in ultrapure water. The micellar solution obtained in example 31 was transferred into a 10 mL glass cuvette. And adhering the slide on a sample rod of a lifting machine, setting the lifting speed to be 210 mu m/s, standing the coated slide at a dark and light-resistant position for more than 24 h, and treating the coated slide by using a low-temperature plasma machine after toluene volatilization is finished. Setting plasma processing parameters: the power is 100W, and the flow of one path of oxygen is 100 cm³And/s, washing times are 4 times, the lowest vacuum degree is 8Pa, and the treatment time is 1 h. The treated slide is stored in a dry place in a dark place.

Example 38 preparation of nanopattern array on glass surface

The slide glass which is pre-processed by dipping and pulling operation is treated by the piranha washing liquid and is soaked in ultrapure water. The micellar solution obtained in example 32 was transferred into a 10 mL glass beaker. And adhering the slide on a sample rod of a lifting machine, setting the lifting speed to be 210 mu m/s, standing the coated slide at a dark and light-resistant position for more than 24 h, and treating the coated slide by using a low-temperature plasma machine after toluene volatilization is finished. Setting plasma processing parameters: the power is 100W, and the flow of one path of oxygen is 100 cm³And/s, washing times are 4 times, the lowest vacuum degree is 8Pa, and the treatment time is 1 h. The treated slide is stored in a dry place in a dark place.

Example 39 preparation of nanopattern array on glass surface

The slide glass which is pre-processed by dipping and pulling operation is treated by the piranha washing liquid and is soaked in ultrapure water. The micellar solution obtained in example 33 was transferred into a 10 mL glass beaker. And adhering the slide on a sample rod of a lifting machine, setting the lifting speed to be 210 mu m/s, standing the coated slide at a dark and light-resistant position for more than 24 h, and treating the coated slide by using a low-temperature plasma machine after toluene volatilization is finished. Setting plasma processing parameters: the power of the power is 100W,one-way oxygen flow is 100 cm³And/s, washing times are 4 times, the lowest vacuum degree is 8Pa, and the treatment time is 1 h. The treated slide is stored in a dry place in a dark place.

Example 40 characterization of Nanogold dot arrays on glass slide surface

The resulting nanopatterned slides of examples 36-39 were characterized using two characterization methods, Atomic Force Microscopy (AFM) and field emission scanning electron microscopy (FE-SEM). The AFM characterization parameters were as follows: operating in an air environment, the probe is set in tapping mode (Softtapping), probe model RTESP, scanning ranges 500 nm '500 nm and 800 nm' 800 nm, and the remaining parameters are defaults. The SEM characterization parameter is the accelerating voltage of 1.5 kV, a secondary electron probe (SE) is used as the probe, the aperture of the diaphragm is set to be 30 mu m, the working distance is adjusted from about 3.5 mm, and the magnification range is 20k-80 k. The patterns obtained by different copolymer templates have different nano-pitches, and the scanning electron microscope shooting results of the examples 36 to 38 are shown in FIG. 2; AFM characterization of the pattern template obtained in example 36 is shown in FIG. 3, and the size of each nano-gold dot is below 10 nm according to the height image.

Example 41, example 40 characterization by scanning electron microscopy gave the following nano-spacings on the nano-patterned slides for examples 36-39 as set forth in table 4:

TABLE 4

。

Example 42 preparation of micro-nano hybrid pattern array on glass surface

Treating the nano glass slides with different nano intervals obtained in the example 41 by using low-temperature plasma for 15 minutes, and cleaning the surfaces of the glass slides; the slide was placed in acetone, ultrasonically cleaned three times for ten minutes each time, then blown dry with nitrogen, and placed in an oven at 120 ℃ overnight. Taking out the slide, cooling to room temperature, uniformly coating the photoresist on the surface of the slide in a dark room at 3500 r/min for 20 s, and drying in an oven at 100 ℃ for 20 minutes. And photoetching by using a photoetching machine, selecting square micron islands with the sizes of 35 mu m and 65 mu m on a mask plate as a template, and increasing the solubility of the part irradiated by the ultraviolet light under the mask plate in an organic solvent. And (3) putting the exposed glass slide into a developing solution for 60 s, sequentially putting the glass slide into two cups of clean ultrapure water for 60 s for fixation, drying the fixed glass slide by using nitrogen, and putting the glass slide into a 120 ℃ drying oven for drying for 30 minutes.

7.4 g of NH were weighed₄Dissolving F in 385mL of water, stirring thoroughly to dissolve, adding 9 mL of 40% HF solution, stirring for 10 minutes to obtain 0.5M HF/NH₄And F, buffer solution. And (3) placing the processed glass slide into a prepared buffer solution for etching, taking out the glass slide every 10 seconds, fully washing the glass slide with water, putting the glass slide into the buffer solution again after the water is removed completely, and circulating the same operation until the reaction time is full. The etched glass slides were cleaned in a large number of Milli-Q cells, and then ultrasonically cleaned three times for ten minutes each time, and then dried with nitrogen. And ultrasonically cleaning the glass slide for 3 times by using acetone, degumming each time for 3 minutes, ultrasonically cleaning the glass slide for 10 minutes by using ultrapure water, and blow-drying and storing the glass slide by using nitrogen.

Example 43 slide Pattern pretreatment

The gold pattern on the slide needs to be modified with a coupling agent before transferring the hydrogel surface. The structure of the selectable coupling agent of the hydrogel crosslinking system of the polyethylene glycol and the derivatives thereof is shown as follows:

① HS-PEG-SH and a macromonomer molecular chain form physical entanglement to participate in the hydrogel polymerization process;

② ACRL-PEG-SH participates in hydrogel polymerization by chemically reacting with a macromonomer and physically entangling;

③ allyl mercaptan participates in the hydrogel polymerization by reacting with the macromonomer chemically;

④ double bond end-capped cystamine molecules participate in hydrogel polymerization through chemical reaction with macromonomers

Example 44 slide surface micropattern pretreatment

The micron round island pattern glass slide obtained in example 27 was treated with low temperature plasma for 15 min, and after heating in a 120 ℃ oven for 0.5 h, the glass slide was immersed in absolute ethanol to ensure that all gold dots were reduced to gold simple substances, and after 0.5 h, the glass slide was taken out and immersed again in an absolute ethanol solution of 2 mM bifunctional small molecule Linker (N, N-bisacryloyl cystamine) at 35 ℃ for 1.5h, and was blow-dried with high purity nitrogen before uv polymerization.

Example 45 pretreatment of slide surface with micropattern

The micro stripe pattern glass slide obtained in example 28 was treated with low temperature plasma for 15 min, and after heat treatment in an oven at 120 ℃ for 0.5 h, the slide was immersed in absolute ethanol to ensure that all gold dots were reduced to gold simple substance, taken out after 0.5 h and immersed again in an absolute ethanol solution of 2 mM bifunctional small molecule Linker (N, N-bisacryloyl cystamine) at 35 ℃ for 1.5h, and blow-dried with high purity nitrogen before uv polymerization.

Example 46 slide surface micro-Pattern pretreatment

The micro-stripe pattern glass slide obtained in example 28 was treated with low temperature plasma for 15 min, and after heating in an oven at 120 ℃ for 0.5 h, the slide was immersed in absolute ethanol to ensure that all gold dots were reduced to gold simple substance, and after 0.5 h, the slide was taken out and immersed again in an absolute ethanol solution of 5 mM bifunctional small molecule Linker (example 40 ③ allyl thiol) at 35 ℃ for 1.0 h, and was blow-dried with high purity nitrogen before uv polymerization.

Example 47 slide nanopattern pretreatment

The nanopatterned slide obtained in example 36 was treated with low-temperature plasma for 15 min, and after heating in an oven at 120 ℃ for 0.5 h, the slide was immersed in absolute ethanol to ensure that all gold dots were reduced to gold simple substances, and after 0.5 h, the slide was taken out and immersed again in an absolute ethanol solution of 2 mM bifunctional small molecule Linker (N, N-bisacryloyl cystamine) at 35 ℃ for 1.5h, and was dried with high-purity nitrogen before uv polymerization.

Example 48 slide nanopattern pretreatment

The nanopatterned slide obtained in example 37 was treated with low-temperature plasma for 15 min, and after heating in an oven at 120 ℃ for 0.5 h, the slide was immersed in absolute ethanol to ensure that all gold dots were reduced to gold simple substances, and after 0.5 h, the slide was taken out and immersed again in an absolute ethanol solution of 2 mM bifunctional small molecule Linker (N, N-bisacryloyl cystamine) at 35 ℃ for 1.5h, and was dried with high-purity nitrogen before uv polymerization.

Example 49 slide nanopattern pretreatment

The nanopatterned slide obtained in example 38 was treated with low-temperature plasma for 15 min, and after heating in an oven at 120 ℃ for 0.5 h, the slide was immersed in absolute ethanol to ensure that all gold dots were reduced to gold simple substances, and after 0.5 h, the slide was taken out and immersed again in an absolute ethanol solution of 2 mM bifunctional small molecule Linker (N, N-bisacryloyl cystamine) at 35 ℃ for 1.5h, and was dried with high-purity nitrogen before uv polymerization.

Example 50 slide nanopattern pretreatment

The nanopatterned slide obtained in example 39 was treated with low-temperature plasma for 15 min, and after heating in an oven at 120 ℃ for 0.5 h, the slide was immersed in absolute ethanol to ensure that all gold dots were reduced to gold simple substances, and after 0.5 h, the slide was taken out and immersed again in an absolute ethanol solution of 2 mM bifunctional small molecule Linker (N, N-bisacryloyl cystamine) at 35 ℃ for 1.5h, and was dried with high-purity nitrogen before uv polymerization.

Example 51 slide micro-nano hybrid Pattern Pre-treatment

The micro-nano hybrid patterned glass slide obtained in the embodiment 42 is treated by low-temperature plasma for 15 min, and is heated in an oven at 120 ℃ for 0.5 h, then the glass slide is immersed in absolute ethyl alcohol to ensure that gold dots are completely reduced to a gold simple substance, the glass slide is taken out for 0.5 h and is immersed in an absolute ethyl alcohol solution of 1.5 mM bifunctional small molecule Linker (N, N-bisacryloyl cystamine) at 35 ℃ for 1.5h, and the glass slide is dried by high-purity nitrogen before ultraviolet polymerization.

Example 52 surface micropatterned hydrogel preparation

In the same manner as in example 23, the MeHA macromonomer obtained in example 1 was formulated with Milli-Q to a concentration of 30wt%, and polymerized on the surface of the patterned glass slide containing micro round islands obtained by modifying cysteamine bisacryloyl in example 44 to obtain a hyaluronic acid HA hydrogel having a pattern of micro round islands distributed on the surface.

Example 53 surface micropatterned hydrogel preparation

The procedure was as in example 13, example 12 to obtain oligoLA-PEG₂₀₀₀oligoLA DA macromonomer was formulated with Milli-Q at a concentration of 45 wt%, and polymerized on the surface of a micron-island-containing patterned glass slide obtained by modifying cysteamine bisacryloyl in example 44 to obtain a hydrogel having a micron-island pattern distributed on the surface.

Example 54 surface micropatterned hydrogel preparation

The procedure was as in example 13, example 12 to obtain oligoLA-PEG₂₀₀₀oligoLA DA macromonomer was formulated with Milli-Q at a concentration of 45 wt%, and polymerized on the surface of a micrometer-stripe-containing patterned glass slide obtained by modifying cysteamine bisacryloyl in example 45 to obtain a hydrogel having a micrometer-stripe pattern distributed on the surface.

Example 55 surface micropatterned hydrogel preparation

The procedure was as in example 13, example 12 to obtain oligoLA-PEG₂₀₀₀oligoLA DA macromonomer was formulated with Milli-Q at a concentration of 45 wt%, and polymerized on the surface of a micro-stripe-containing patterned glass slide obtained by modifying bifunctional allylthiol in example 43 to give a hydrogel with a micro-stripe pattern distributed on the surface.

Example 56 surface nanopatterned hydrogel preparation

The procedure was as in example 13, example 12 to obtain oligoLA-PEG₂₀₀₀The oligoLA macromonomer was polymerized on the surface of the nanopatterned slide having a nano-pitch of about 64 nm obtained in example 47 to obtain a hydrogel with a nanopatterned surface.

Example 57 surface nanopatterned hydrogel preparation

The procedure was as in example 13, example 12 to obtain oligoLA-PEG₂₀₀₀The oligoLA macromonomer was polymerized on the surface of the nanopatterned slide having a nano-pitch of about 31 nm obtained in example 48 to obtain a hydrogel with a nanopatterned surface.

Example 58 surface nanopatterned hydrogel preparation

The procedure was as in example 13, example 12 to obtain oligoLA-PEG₂₀₀₀The oligoLA macromonomer was polymerized on the surface of the nanopatterned slide having a nano-pitch of about 90 nm obtained in example 49 to obtain a hydrogel with a nanopatterned surface.

Example 59 surface nanopatterned hydrogel preparation

The procedure is as in example 13oligoLA-PEG from example 11₂₀₀₀oligoLA macromonomers were polymerized on the surface of the nanopatterned slides obtained in example 50 with a nano-pitch of about 130 nm. Obtaining the hydrogel with the nano-patterned surface.

Example 60 surface micro-nano patterned hydrogel preparation

The procedure was as in example 13, example 12 to obtain oligoLA-PEG₂₀₀₀oligoLA macromonomers were polymerized on the surface of the micro-nano patterned slide obtained in example 51. Obtaining the hydrogel with the micro-nano patterned surface.

Example 61 Observation of hydrogel surface micropattern by optical microscope

The MeHA hydrogel obtained in example 52, on which the circular islands of different sizes are distributed on the surface, is swollen in Milli-Q after polymerization is completed, and after swelling is completed, is taken out and cut into a size smaller than the size of a glass slide, is placed upside down on the glass slide with the patterned side facing downward, and is observed in a phase difference mode by using a 10X objective lens, and it can be confirmed that all the circular islands of different sizes are successfully transferred to the hydrogel surface by changing the observation field.

Example 62 optical microscopy of hydrogel surface micropatterns

EXAMPLE 53 obtained oligoLA-PEG with different micron-sized circular islands distributed on the surface₂₀₀₀Swelling in Milli-Q after completing polymerization of oligoLA hydrogel, taking out and cutting into a size smaller than the size of the glass slide after swelling is completed, placing the glass slide upside down with the patterned side facing downwards, observing with a 10X objective lens in a phase difference mode, and confirming that all the micron circular islands are successfully transferred to the surface of the hydrogel by changing the observation field。

Example 63 optical microscopy of hydrogel surface micropatterns

Example 54 obtained oligoLA-PEG with stripes of different widths distributed on the surface₂₀₀₀After the polymerization of the oligoLA hydrogel is completed, the hydrogel is swelled in Milli-Q, and after the swelling is completed, the hydrogel is taken out, cut into a size smaller than the size of a glass slide, placed upside down on the glass slide with the patterned side facing downwards, and observed in a phase contrast mode by using a 10X objective lens, and it can be confirmed that the micro-stripes of all widths are successfully transferred to the surface of the hydrogel by changing the observation field.

Example 64 optical microscopy of hydrogel surface micropatterns

Example 55 obtained oligoLA-PEG with stripes of different widths distributed on the surface₂₀₀₀After the polymerization of the oligoLA hydrogel is completed, the hydrogel is swelled in Milli-Q, and after the swelling is completed, the hydrogel is taken out, cut into a size smaller than the size of a glass slide, placed upside down on the glass slide with the patterned side facing downwards, and observed in a phase contrast mode by using a 10X objective lens, and it can be confirmed that the micro-stripes of all widths are successfully transferred to the surface of the hydrogel by changing the observation field. Small molecule allyl thiols can also achieve complete transfer of larger size patterns.

Example 65 microscopic observation of micro-nano patterns on hydrogel surface

Example 60 hydrogel with square round islands of different sizes distributed on the surface and nano-pattern array contained in the round islands was obtained₂₀₀₀Swelling in Milli-Q after completion of the oligoLA hydrogel polymerization, until swelling is completeAnd then taking out the hydrogel, cutting the hydrogel into a size smaller than the size of the glass slide, placing the hydrogel on the glass slide in an inverted mode, enabling the side with the pattern to face downwards, observing the hydrogel in a phase difference mode by using a 10X objective lens, and confirming that the square micron islands of all sizes are successfully transferred to the surface of the hydrogel by changing an observation visual field.

Example 66

In order to observe the distribution condition of the nano gold dots on the surface of the hydrogel, the surface of the dehydrated sample is observed by adopting a scanning electron microscope. The nano-patterned hydrogel obtained in example 51 was swollen in Milli-Q, and was replaced with Milli-Q three times, each time for 20 min, to prepare 30%, 50%, 70%, 90%, 95%, and 100% ethanol solutions by volume, and the hydrogel swelling solvent was gradually replaced, with each gradient treatment time of 0.5 to 1 h, and after the 100% absolute ethanol treatment was completed, all the ethanol was aspirated, and the sample was evaporated overnight at room temperature.

Example 67

In order to observe the distribution condition of the nano gold dots on the surface of the hydrogel, the surface of the dehydrated sample is observed by adopting a scanning electron microscope. The nano-patterned hydrogel obtained in example 52 was swollen in Milli-Q, and was replaced with Milli-Q three times, each time for 20 min, to prepare 30%, 50%, 70%, 90%, 95%, and 100% ethanol solutions by volume, and the hydrogel swelling solvent was gradually replaced, with each gradient treatment time of 0.5 to 1 h, and after the 100% absolute ethanol treatment was completed, all the ethanol was aspirated, and the sample was evaporated overnight at room temperature.

Example 68

In order to observe the distribution condition of the nano gold dots in the micro islands on the surface of the hydrogel, the surface of the dehydrated sample is observed by adopting a scanning electron microscope. The micro-nano hybrid pattern hydrogel obtained in example 60 was swelled in Milli-Q, and the micro-nano hybrid pattern hydrogel was displaced three times with Milli-Q for 20 min each time, and an ethanol solution with a volume ratio of 30%, 50%, 70%, 90%, 95%, and 100% was prepared, and the hydrogel swelling solvent was gradually replaced, with each gradient treatment time of 0.5 to 1 h, and after the 100% absolute ethanol treatment was completed, all the ethanol was aspirated, and the sample was volatilized overnight at room temperature.

Example 69 scanning Electron microscopy of hydrogel surface nanopatterns

The bottom of the hydrogel obtained in example 66 was fixed with a conductive adhesive, and the periphery of the hydrogel was connected to the bottom with a conductive adhesive, thereby improving the conductivity of the sample. The characterization parameters are set as: the accelerating voltage is less than or equal to 1kV, the aperture of the diaphragm is 20 mu m, the probe selects secondary electrons SE or InLens (for observing more prominent surface topography), the working distance is about 2.5 mm, the secondary electrons SE or InLens are properly adjusted according to the voltage and the aperture of the diaphragm, and the result is shown in fig. 4. The nano-array pattern on the glass slide with the initial nano-spacing of about 64 nm was successfully transferred to the hydrogel surface.

Example 70 scanning Electron microscopy of hydrogel surface nanopatterns

The bottom of the hydrogel obtained in example 67 was fixed with a conductive adhesive, and the periphery of the hydrogel was connected to the bottom with a conductive adhesive, thereby improving the conductivity of the sample. The characterization parameters are set as: the accelerating voltage is less than or equal to 1kV, the aperture of the diaphragm is 20 mu m, the probe selects secondary electrons SE or InLens (for observing more prominent surface morphology), the working distance is about 2.5 mm, and the secondary electron SE or InLens is properly adjusted according to the voltage and the aperture of the diaphragm. The results are shown on the right of FIG. 4. The nano-array pattern with initial nano-spacing of about 30 nm on the slide was successfully transferred to the hydrogel surface.

Example 71 scanning Electron microscopy of hydrogel surface micro-nano patterns

The bottom of the hydrogel obtained in example 60 was fixed with a conductive adhesive, and the periphery of the hydrogel was connected to the bottom with a conductive adhesive, thereby improving the conductivity of the sample. The characterization parameters are set as: the accelerating voltage is less than or equal to 1kV, the aperture of the diaphragm is 20 mu m, the probe selects secondary electrons SE or InLens (for observing more prominent surface morphology), the working distance is about 2.5 mm, and the secondary electron SE or InLens is properly adjusted according to the voltage and the aperture of the diaphragm. The sharper edge of the micron island can be observed under the 8k magnification of a scanning electron microscope, and the etching process of the hydrofluoric acid is more complete; at the same time, under the magnification of 20k-80k, it can be observed that nano pattern arrays with different nano intervals are distributed in the micrometer round island, and the micro/nano hybrid pattern is in oligoLA-PEG₂₀₀₀Successful construction of the oligoLA hydrogel surface.

Example 72 hydrogel degradation without Pattern

Example 23 MeHA hydrogel with a solids content of 30% by weight was obtained, and was subjected to 5' -10 at 37 ℃⁴The degradation was performed by the U/mL phage H4489A hyaluronidase at sampling time points of 0 d, 1 d, 4 d, and 7 d, respectively. After the degraded sample is taken out at each stage, the enzyme solution is washed away by PBS buffer solution, and the degraded sample is soaked in the PBS buffer solution after accelerated replacement by a shaking table.

The modulus and swelling ratio of the hydrogel samples obtained in the above steps at different degradation stages were measured in the same manner as in examples 24 to 26, and the results of decreasing modulus with the progress of degradation and increasing volume swelling ratio were obtained, indicating that the obtained MeHA hydrogel samples were degraded under the selected degradation conditions.

Example 73 hydrogel degradation without Pattern

The oligoLA-PEG-oligoLA hydrogels with different solid contents obtained in examples 13-15 were degraded at 37 ℃ under 2 mol/L hydrochloric acid, and mixed with Milli-Q at a volume ratio of 1:1 with hydrochloric acid having a hydrogen ion concentration of 12 mol/L to obtain a strong acid aqueous solution, while pure PBS solution was set as a weak degradation condition. Setting the total degradation time of 12 h, and respectively setting the degradation sampling time points to be 0 h, 2h, 4 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h and 12 h, wherein 0 h represents the degradation point under the condition of no external acid. After the degradation sample is taken out at each stage, washing off acid liquor by using PBS buffer solution, accelerating replacement by using a shaking table for 0.5 hour each time for 5 times in total until the pH value of the solution is 7.4, and then soaking in the PBS buffer solution.

The modulus and the swelling ratio of the hydrogel samples obtained in the above steps at different degradation stages are measured by the same operations as in examples 24 to 26, and the results that the modulus decreases with the progress of degradation and the volume swelling ratio increases can be obtained, which shows that the hydrogel samples can be degraded, and the hydrogel with low degradation rate and solid content is faster than the hydrogel with high solid content.

Example 74 hydrogel degradation without Pattern

Example 13 obtained 45 wt% oligoLA-PEG-oligoLA hydrogel, degraded at 37 ℃ in PBS, taken out samples at 0, 4, 7, and 14 days, and measured for modulus and swelling ratio in the same manner as examples 24-26, the results of decreasing modulus with increasing degradation time and increasing volume swelling ratio were obtained, indicating that hydrogel samples can be degraded at a slower rate in biomimetic conditions in PBS buffer.

Example 75 hydrogel degradation without Pattern

Example 16A 45 wt% oligoTMC-PEG-oligoTMC hydrogel was obtained, at 37 ℃ and 5' 10 ″⁴Degradation is carried out under the condition of U/mL lipase, samples are taken out at 0 day, 4 days, 7 days and 14 days, and the modulus and the swelling ratio are measured by the same operation as in examples 24 to 26, so that the modulus drop with the decrease of the lipase can be obtainedThe result that the degradation time is prolonged and decreased and the volume swelling ratio is increased shows that the hydrogel sample can be degraded at a slower speed under the action of lipase, and the hydrogel structure can be disintegrated.

Example 76 hydrogel degradation without Pattern

Example 17 obtained 45 wt% oligoGA-PEG-oligoGA hydrogel, degraded at 37 ℃ under 2 mol/L hydrochloric acid, mixed with Milli-Q at a volume ratio of 1:1 with hydrochloric acid having a hydrogen ion concentration of 12 mol/L to obtain a strong acid aqueous solution, while setting pure PBS solution as weak degradation condition. Setting the total degradation time of 12 h, and respectively setting the degradation sampling time points to be 0 h, 2h, 4 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h and 12 h, wherein 0 h represents the degradation point under the condition of no external acid. After the degradation sample is taken out at each stage, washing off acid liquor by using PBS buffer solution, accelerating replacement by using a shaking table for 0.5 hour each time for 5 times in total until the pH value of the solution is 7.4, and then soaking in the PBS buffer solution.

The hydrogel samples obtained in the above steps and different degradation stages are subjected to modulus and swelling ratio measurement by the same operations as in examples 24 to 26, so that the results of decreasing modulus with the progress of degradation and increasing volume swelling ratio with a change rate higher than that of oligoLA-PEG-oligoLA with the same solid content can be obtained, the hydrogel samples can be degraded, and the degradation rate is faster than that of the hydrogel containing oligoLA segments.

Example 77 hydrogel degradation without Pattern

Example 18A 45 wt% oligoCL-PEG-oligoCL hydrogel was obtained and degraded at 37 ℃ with 2 mol/L hydrochloric acid, and mixed with Milli-Q at a hydrogen ion concentration of 12 mol/L in a volume ratio of 1:1 to give a strong acid aqueous solution, while setting pure PBS solution as weak degradation conditions. Setting the total degradation time of 12 h, and respectively setting the degradation sampling time points to be 0 h, 2h, 4 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h and 12 h, wherein 0 h represents the degradation point under the condition of no external acid. After the degradation sample is taken out at each stage, washing off acid liquor by using PBS buffer solution, accelerating replacement by using a shaking table for 0.5 hour each time for 5 times in total until the pH value of the solution is 7.4, and then soaking in the PBS buffer solution.

The hydrogel samples obtained in the above steps and different degradation stages are subjected to modulus and swelling ratio measurement by the same operations as in examples 24 to 26, so that the results that the modulus decreases with the progress of degradation and the volume swelling ratio increases, and the change rate is lower than that of oligoLA-PEG-oligoLA with the same solid content can be obtained.

Example 78

Selecting 45 wt% oligoLA-PEG from the results of example 73_2000-The degradation performance (modulus, swelling ratio) of the hydrogel was measured at 3 time points in the change curve of the oligoLA modulus under cell culture conditions (37 ℃, 5% CO2, cell culture medium), and the results showed that the modulus was further decreased and the volume swelling ratio was also changed, the hydrogel was degraded again under the cell culture conditions, and the degradation starting point was different from the environment, so that different degradation rates could be obtained.

Example 79 collagen modification of micropattern

The micropatterned oligoLA-PEG-oligoLA hydrogel from example 52 was allowed to wick out and carefully wipe away moisture on its surface and periphery after swelling in PBS. 1mg/mL of collagen is prepared by Milli-Q to modify the micron patterning morphology, and a collagen aqueous solution is carefully dripped to the surface of the hydrogel by using a gun head, so that the surface of the hydrogel is completely covered with the solution; the mixture was placed in a refrigerator at 4 ℃ overnight for 12 hours. The grafted sample needs to be washed with PBS 3 times in time, each time for 0.5 h. The surface of the obtained hydrogel is distributed with collagen-modified micron round islands, and the hydrogel can be used as a bracket for promoting nerve regeneration to be applied to tissue engineering.

Example 80 grafting of RGD Polypeptides

The hydrogel obtained in example 56 was swollen with PBS, and then peeled off and carefully wiped off to remove water on the surface and around the hydrogel. The surface of the polypeptide has a nanoscale patterned morphology, and a cyclic pentapeptide c (RGDFK) -SH is selected as a biomacromolecule cell adhesion related ligand to modify the patterned morphology, so as to prepare a 25 mu M RGD polypeptide solution; dripping RGD polypeptide aqueous solution on the surface of the hydrogel by using a gun head to ensure that the surface of the hydrogel is completely covered with the solution; putting into a refrigerator at 4 ℃ for overnight connection for 8 h. The grafted sample needs to be washed with PBS 3 times in time, each time for 0.5 h.

Example 81 grafting of REDV Polypeptides

The hydrogel obtained in example 56 was swollen with PBS, and then peeled off and carefully wiped off to remove water on the surface and around the hydrogel. The surface of the polypeptide has a nanoscale patterned morphology, and the patterned morphology is modified by selecting cyclic pentapeptide c (RGDFK) -SH as a biological macromolecular cell adhesion related ligand to prepare a 25 mu M REDV polypeptide solution; dripping REDV polypeptide aqueous solution on the surface of the hydrogel by using a gun head to ensure that the surface of the hydrogel is completely covered with the solution; putting into a refrigerator at 4 ℃ for overnight connection for 8 h. The grafted sample needs to be washed with PBS 3 times in time, each time for 0.5 h.

Example 82 Observation of cell adhesion contrast on the surface of nanopatterned hydrogels

Bone marrow mesenchymal stem cells (rmscs) isolated and cultured from rats were seeded on the olioLA-PEG-oligoLA hydrogel grafted with the cell adhesion polypeptide RGD obtained in example 80, cultured in a culture medium for seven days, and at the first and seventh days, samples were taken and cells were fixed with a tissue fixative for subsequent staining. After immunofluorescent staining of cytoskeleton F-actin (red), Vinculin (green) and cell nucleus (blue) of the cells, a photograph taken by a microscope is shown in FIG. 5, and it can be observed whether a microscopically visible cell adhesion contrast area appears on the hydrogel surface with or without a nanopattern (the area separated by the dotted line in the figure). The selective adhesion of cells to different regions of the hydrogel substrate can be applied to stem cell culture.

Example 83 application of nanopatterned hydrogels for cell identification and screening

Endothelial cells and smooth muscle cells were simultaneously seeded on the surface of the olioLA-PEG-oligoLA hydrogel grafted with REDV polypeptide and RGD polypeptide obtained in examples 80 to 81, and cultured in a medium for 1 day for observation. The surfaces of the hydrogel modified with the REDV nano-array have better selective adhesion to endothelial cells. The incubation was continued for 7 days, and the degradation of the hydrogel substrate did not significantly affect the selectivity described above. The method constructs the endothelial cell selective functional surface, and can be applied to related cell culture and screening.

Claims

1. A degradable high molecular material with a patterned surface is characterized in that biodegradable hydrogel is used as a matrix, and the surface of the biodegradable hydrogel is modified with a pattern consisting of active substances; the term "patterned surface" refers to a pattern array formed on the surface of a substrate material by modification.

2. The degradable polymeric material with a patterned surface of claim 1, wherein the biodegradable hydrogel is a hydrogel based on collagen, gelatin, hyaluronic acid, chitosan, chitin, alginate, fibrin or their derivatives from natural sources; or

The biodegradable hydrogel is formed by combining polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polypropylene oxide or polymethyl methacrylate which are artificially synthesized with a small amount of degradable polyester, polyamino acid, polypeptide, polyurethane, polyesteramide, poly (n-lactide), polyanhydride, polyalkylcyanoacrylate, polyphosphazene or polyphosphoester in a way of copolymerization, crosslinking, blending to form an interpenetrating network; or

The biodegradable hydrogel is formed by polymerizing and crosslinking polyethylene glycol and oligomeric ester in a manner of forming a macromonomer.

3. The degradable high molecular material with the patterned surface of claim 1 or 2, wherein the active substance is selected from one or more of the following substances: platinum, gold, titanium, zirconium, cobalt, nickel, silver metals, hydroxyapatite, bone cement, bioglass inorganic nonmetal, collagen, cellulose protein, fibronectin biomacromolecules and special active polypeptide sequences; or

The active substance is selected from polypeptide sequences of arginine-glycine-aspartic acid, arginine-glutamic acid-aspartic acid-valine.

4. The degradable polymeric material with the patterned surface of claim 3, wherein the patterned surface has a morphology and a scale of macro scale or micro scale or a combination of both, and the macro scale is a range observable by naked eyes and is in the order of-cm; microscale refers to microscale, nanoscale, or a combination thereof.

5. The degradable polymeric material with a patterned surface of claim 1, 2 or 4, wherein the degradable polymeric material is degradable by hydrolysis, enzymatic hydrolysis, bacterial decomposition, thermal decomposition, oxidative degradation, alone or in combination of more; the degradation rate is adjusted by adjusting the ratio of the degradation components and the degradation trigger.

6. The method for preparing the degradable polymer material with the patterned surface according to any one of claims 1 to 5, which mainly relates to pattern transfer, and is characterized by comprising the following steps:

(3) modifying the surface of a substrate containing a pattern array by using a coupling agent, and combining the macromolecular macromonomer with the surface of the pattern through polymerization reaction to form hydrogel consisting of degradable macromolecules so as to transfer the pattern to the surface of the degradable hydrogel;

wherein, the surface of the pattern substrate is modified by a coupling agent, which means that a bifunctional coupling agent capable of simultaneously interacting with the surface pattern and the macromolecular macromonomer is introduced to promote pattern array transfer.

7. The method according to claim 6, wherein the bifunctional coupling agent has a functional group at one end capable of forming a stable bond with one of a covalent bond, an ionic bond, a metallic bond and a coordination bond with the patterned array on the substrate surface, and a functional group at the other end capable of participating in the hydrogel crosslinking network polymerization process as a monomer component.

8. The method according to claim 6 or 7, wherein the degradable polymeric macromonomer is polymerized into the hydrogel by thermal crosslinking, photo-crosslinking, or ionic crosslinking.

9. The preparation method according to claim 6 or 7, characterized in that the mask assisted lithography mode in the step (1) is to construct a micron-scale patterned template on a stable substrate, wherein the micron-scale patterned template is 1-5000 μm in size and is obtained by a lithography mask plate containing graphic arrays with different sizes;

the self-assembly mode of the block copolymer micelle in the step (1) is to construct a nanoscale patterned array on a stable substrate, and the dimension of the nanoscale pattern template is 5-150 nm.

10. The preparation method of claim 9, wherein the block copolymer micelle self-assembly method is used for constructing a nano-patterned array on a stable substrate, and the size, distribution and components of the obtained nano-patterns are adjusted by loading precursors of different types of metal salts on the amphiphilic block copolymer in a selective solvent; and after the micelle array is subjected to plasma treatment, removing the components of the block copolymer, and reducing the metal from the precursor to obtain the metal array.

11. The method according to claim 6, 7 or 9, wherein the patterned template in step (1) is a micro-nano hybrid pattern, and the nano pattern array template is obtained by a self-assembly method of a block copolymer, and the obtained nano pattern array is defined by a micro pattern array through a spin-lithography step.

12. The method according to claim 11, wherein the hydrogel surface pattern array is further modified with an active substance to construct a pattern array of a plurality of active substances on the hydrogel surface.

13. Use of the degradable polymeric material of any one of claims 1 to 5 for biological identification and diagnosis, cell culture, culture of bacteria and other microorganisms, cell screening, screening of bacteria and other microorganisms, or tissue engineering.