CN115386128A

CN115386128A - Preparation method and application of heparinoid polymer patterned surface

Info

Publication number: CN115386128A
Application number: CN202211135315.8A
Authority: CN
Inventors: 刘小莉; 孙薇; 雷姣; 陈红
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2022-11-25
Anticipated expiration: 2042-09-19
Also published as: CN115386128B

Abstract

The invention discloses a preparation method and application of a heparinoid polymer patterned surface. The biological function of the material surface can be influenced by the surface topology. The roughness, specific geometry, etc. of the material surface all affect the protein and cellular behavior of the surface. The invention combines heparinoid polymers with different chemical compositions with the surface of a micron-sized patterned gold film. A gold film was deposited on a Polydimethylsiloxane (PDMS) template (circular array pattern) using a chloroauric acid reduction method, and a patterned gold film was introduced to the flat PDMS substrate surface by transfer printing, forming a patterned material surface. By means of visible light initiated grafting polymerization and gold-sulfur bond self-assembly method, homogeneous or heterogeneous heparin-like polymer is modified on the surface of the patterned material, and the influence of the modified surface on the adsorption of vascular cells and vascular endothelial growth factors is studied.

Description

Preparation method and application of heparinoid polymer patterned surface

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a preparation method and application of a heparinoid polymer patterned surface.

Background

The behavior of vascular cells at the surface of a material can affect the physiological and pathological reactions between the surface and the blood. The blood compatibility of the biomaterial can be optimized by adjusting the behavior of the vascular cells on the surface, improving long-term patency. At present, some macromolecules with special functions, such as heparin, heparinoid polymers, vascular Endothelial Growth Factor (VEGF) and the like, are used for modifying the surface of biological materials and play a role in regulating the biological performance of the surface. The biological material has special performance and special function, and is used in diagnosing diseases, treating diseases, repairing damaged biological tissue, strengthening or restoring tissue and organ function of biological physiological system without affecting human body tissue and blood. The surface interface of the biological material refers to the interface of the biological material when the biological material is in contact with the external environment of the material, and has important influence on the performance of the material. Due to the existence of the self-protection mechanism of the body, the material surface interface can interact with the physiological environment to different degrees when contacting with the physiological system. Whether the organism can generate adverse reactions such as inflammation and the like (namely whether the organism has biocompatibility) in the interaction process is a mark for distinguishing the surface interface of the biological material from other materials, and is also the key of whether the biological material can be safely used. Heparinoid polymers have been widely studied because of their biological functions similar to those of natural heparin and their controllable synthetic molecular weights. Studies have shown that the chemical properties of heparinoid polymers, including the degree of sulfonation and the composition of functional groups, play a unique role in the surface properties of materials. In addition to surface chemical composition, surface morphology is also an important factor affecting cell behavior, and the prior art has prepared stripe and lattice patterns of different widths and diameters (1.8 μm,2 μm,10 μm), and found that the pattern size affects the proliferation density of endothelial cells. Wherein the cell proliferation density of 2 μm stripes and 1.8 am lattice pattern can reach 4.5 times of that of a flat surface. Prior art sulfated hyaluronic acid (HyalS, 3.5 sulfate groups per disaccharide unit) was photo-fixed on the surface of micro-stripes (10, 25, 50 and 100 μm), and it was found that cells on a 100 μm stripe pattern were in a spread state, while cells on the surface of a smaller size, especially a 10 μm stripe pattern, were in a long spindle shape. The combination of the heparinoid polymer and the surface topological pattern has a regulating effect on the cell behavior. However, in the prior art, the preparation of many topological patterns requires expensive instruments and equipment or harsh preparation conditions, and may cause damage to the surface of the material during the preparation of the topological patterns. Therefore, there is a need to develop a way to build patterned material surfaces that is gentle, simple and convenient.

Disclosure of Invention

The invention discloses preparation of a heparinoid polymer patterned surface and influence thereof on vascular cell behaviors. Respectively grafting a heparinoid polymer (pSS) containing a sulfonic acid group, a heparinoid polymer (pMAG) containing a sugar group and a copolymer (pS) containing a sulfonic acid group and a sugar group on the surface of Polydimethylsiloxane (PDMS) by a visible light-initiated grafting polymerization methodco-pM). Meanwhile, a patterned gold film is deposited on a PDMS template with a circular array pattern with the diameter of 300 mu m by a chloroauric acid reduction method, the inner circle with the diameter of 300 mu m is a PDMS exposed area which is not covered by the gold film, and the outer circle is a gold film covered area. Then, transferring the patterned gold film to the surface of heparinoid PDMS, and self-assembling through a gold-sulfur bond, modifying the same heparinoid polymer grafted on PDMS in the gold film area, and constructing the topological patterned surface with the heparinoid polymer in homogeneous distribution. Successful preparation of homogeneous patterned surfaces of heparinoid polymers was demonstrated by a series of polymer and surface characterization tests. Cell experiment results show that for a flat (flat) surface, the surface (pS) only containing sulfonic acid groups, the surface (pSM) simultaneously containing the sulfonic acid groups and the glycosyl groups have the effect of promoting the adhesion and proliferation of vascular cells, and the surface (pM) only containing the glycosyl groups has the effect of inhibiting the adhesion and proliferation of the vascular cells. After the homogeneous pattern structure of the heparinoid polymer is introduced, the promoting effect of pS and pSM on vascular cells, in particular human umbilical vein vascular endothelial cells (HUVECs) is further enhanced. Among these, the overall enhancement of the homogeneously patterned pSM surface was optimal, with the highest HUVECs proliferation density, the best spreading status, and the highest HUVECs activity. Respectively modifying pSS and pMAG on the PDMS exposed area and the patterned gold film covered area by using a visible light-initiated graft polymerization method and a gold-sulfur bond self-assembly methodThe patterned surface of heterogeneous distribution of heparinoid polymers, namely, the pSS component is distributed on the PDMS area, the pMAG component is distributed on the surface of the gold membrane area (PS-M), the pSS component is distributed on the gold membrane area, and the pMAG component is distributed on the surface of the PDMS area (PM-S). Successful preparation of the heparinoid polymer heterogeneous patterned surface was demonstrated by water contact angle testing, infrared spectroscopy and energy spectral surface scanning analysis. Cell experiment results show that when sulfonic acid groups and sugar groups are heterogeneously distributed on the same surface on a heparinoid heterogeneously patterned surface, vascular cells are guided by the two chemical components and tend to grow in the regions modified by the sulfonic acid groups. Research also finds that heterogeneous patterned PS-M surfaces are more beneficial to proliferation and spreading of vascular cells than PM-S surfaces, and shows that the patterned distribution mode of chemical components of the heparinoid polymers on the material surfaces also has influence on the behavior of the vascular cells on the surfaces, and the obvious difference of VEGF adsorption amounts on the PS-M surfaces and the PM-S surfaces indicates the difference of the behavior of the vascular cells on the two heterogeneous patterned surfaces.

The invention adopts the following technical scheme:

a preparation method of a patterned surface of a heparinoid polymer comprises the following steps of modifying the heparinoid polymer on the surface of bromine-containing polydimethylsiloxane (PDMS-Br), transferring a patterned gold film to the surface of the Polydimethylsiloxane (PDMS) modified by the heparinoid polymer, and assembling a sulfhydrylation heparinoid polymer on the surface of the gold film through gold-sulfur bond self-assembly to obtain the patterned surface of the heparinoid polymer.

In the invention, the heparinoid polymer is a heparinoid polymer pSS containing sulfonic acid group, a heparinoid polymer pMAG containing sugar group or a copolymer pS-containing sulfonic acid group and sugar groupco-pM; the molecular weight of the heparinoid polymer is 0.8 × 10 ⁴ ～2× 10 ⁴ g mol ^-1 。

In the invention, when the heparinoid polymer modified on the surface of PDMS-Br and the thiolated heparinoid polymer assembled on the surface of the gold film through gold-sulfur bond self-assembly are the same heparinoid polymer, the patterned surface of the heparinoid polymer is a homogeneous patterned surface of the heparinoid polymer; when the heparinoid polymer modified on the PDMS-Br surface and the thiolated heparinoid polymer assembled on the gold film surface through gold-sulfur bond self-assembly are different heparinoid polymers, the patterned surface of the heparinoid polymer is a heparinoid polymer heterogeneous patterned surface.

In the invention, the surface of PDMS-Br is a flat surface; the patterned gold film is a circular array patterned gold film.

In the invention, firstly, the heparinoid polymer with the dithioester bond is prepared by RAFT polymerization, and then the dithioester bond at the tail end of the heparinoid polymer is reduced into sulfydryl by ethanolamine, thus obtaining the sulfhydrylation heparinoid polymer.

In the invention, 10-undecene-2-bromoisobutyrate is added into the raw materials for preparing PDMS to prepare PDMS-Br, and the raw materials for preparing PDMS and the preparation method are conventional technologies.

In the invention, PDMS-Br is immersed into a solution containing a heparinoid polymer monomer and a photoinitiator, and then the surface of PDMS-Br is modified with the heparinoid polymer through a light irradiation reaction. And (3) soaking the PDMS with the transferred patterned gold film into a thiolated heparan polymer solution for 10-25 hours, and assembling the thiolated heparan polymer on the surface of the gold film to obtain the patterned surface of the heparan polymer. Patterning gold films and their transfer are conventional techniques.

The application of the patterned surface of the heparinoid polymer in regulating the behavior of the vascular cells, or the application of the patterned surface of the heparinoid polymer in preparing a material for regulating the behavior of the vascular cells.

The patterned surface of the heparinoid polymer is applied to promotion of endothelial cell adhesion proliferation and inhibition of smooth muscle cell adhesion proliferation, or is applied to preparation of a material for promoting endothelial cell adhesion proliferation and inhibition of smooth muscle cell adhesion proliferation.

The application of the patterned surface of the heparinoid polymer disclosed by the invention in improving the survival of vascular cells or the application of the patterned surface of the heparinoid polymer disclosed by the invention in preparing a material for improving the survival of vascular cells.

The application of the patterned surface of the heparinoid polymer in improving VEGF adsorption or the application of the patterned surface of the heparinoid polymer in preparing the VEGF adsorption material is disclosed.

Different heparinoid polymers have different effects on the surface cell behavior after being uniformly distributed on the surface of the patterned gold film. The sulfonic acid group has the promotion effect on the growth of blood vessel cells, the glycosyl group has the inhibition effect on the growth of blood vessel cells, and the activity and the adhesion proliferation state of HUVECs on the patterned surface with the homogeneous distribution of the copolymer of the sulfonic acid group and the glycosyl group are optimal. Compared with a homogeneous patterned surface, the surface of the heterogeneous patterned material has a regional function, the surface can induce the adhesion of cells on the surface of the material, control the spatial distribution of the cells, influence the growth morphology, migration, proliferation, differentiation and other behaviors of the cells, and can define the mechanism of sensing a complex growth environment by the cells through the behaviors of the cells on the heterogeneous patterned surface.

Drawings

FIG. 1 is a schematic diagram of (a) preparation and (b) chemical composition distribution of patterned PDMS-Au, pS, pSM and pM surfaces of a homogeneous patterned surface of heparinoid polymer; a preparation schematic diagram of a PS-M heterogeneous patterned surface of a heparinoid polymer (c) and a chemical component distribution schematic diagram of the surfaces of PS-S, PM-M, PS-M and PM-S samples (d).

FIG. 2 shows (a) a reaction scheme for synthesizing a polymer pSS-SH; (b) an infrared spectrogram; (c) a UV spectrogram; (d) nuclear magnetic resonance spectrum.

FIG. 3 shows the polymer pS-co-pM-SH according to the reaction formula (a); (b) an infrared spectrogram; (c) ultraviolet spectra; (d) nuclear magnetic resonance spectrum.

FIG. 4 is a (a) synthesis scheme for the polymer pMAG-SH; (b) an infrared spectrum; (c) ultraviolet spectra; (d) nuclear magnetic resonance spectrum.

FIG. 5 is a graph showing the static water contact angle (a) of the PDMS-Au, pS, pSM and pM surfaces, which are Flat (Flat) and patterned (Pattern), and the change of the static water contact angle during the preparation of the PS-M and PM-S surfaces of the heparinoid heterogeneous patterned surfaces (b).

Fig. 6 is an EDS surface scan of Au, si, S elements of a flat and patterned pS surface.

FIG. 7 is an EDS profile scan of Au, si, N, S elements of a planarized and patterned pSM surface.

FIG. 8 is an EDS profile of Au, si, N elements (a) on the surface of a flattened and patterned pM and an EDS profile of Si, au, S, N elements (b) on the surface of a PS-M sample.

FIG. 9 is a photograph of fluorescent staining of dead and live cells of HUVECs after culture on flat and patterned PDMS-Au, pS, pSM and pM surfaces for (a) 4 h and (b) 48 h.

FIG. 10 shows cell activity of HUVECs after 4 h and 48 h culture on flat and patterned PDMS-Au, pS, pSM and pM surfaces (a) and HUVECs after 4 h and 48 h culture on PS-S, PM-M, PS-M and PM-S surfaces (b).

FIG. 11 is a graph of the topography and cell density and density difference (c) of HUVECs after 4 h (a) and 48 h (b) incubation on flat and patterned PDMS-Au, pS, pSM, pM, PS-M and PM-S surfaces (mean. + -. Standard deviation, n =6, · xp＜0.001）。

FIG. 12 is a graph of (a) spreading area and (b) cell aspect ratio (mean. + -. Standard deviation, n = 6;. X.) of HUVECs after 4 h and 48 h incubation on flat and patterned PDMS-Au, pS, pSM, pM, PS-M, and PM-S surfacesp＜0.05，**p＜0.01，***p＜0.001）。

FIG. 13 is a photograph of fluorescent staining of dead and live cells of HUVSMCs after culturing on the surface of flat and patterned PDMS-Au, pS, pSM, pM, PS-M and PM-S for (a) 4 h and (b) 48 h.

FIG. 14 shows cell viability of HUVSMCs after 4 h and 48 h incubation on smooth and patterned PDMS-Au, pS, pSM, pM, PS-M and PM-S surfaces.

FIG. 15 is a graph of the morphology of HUVSMCs after culturing (a) 4 h and (b) 48 h on the surface of smooth and patterned PDMS-Au, pS, pSM, pM, PS-M and PM-S and (c) the cell density and density difference (mean. + -. Standard deviation, n =6, and n is a linear variable number, all the materials arep＜0.01，***p＜0.001）。

FIG. 16 shows (a) cell spreading area and (b) cell aspect ratio of HUVSMCs after 4 h and 48 h incubation on flat and patterned PDMS-Au, pS, pSM and pM surfaces(mean ± standard deviation, n = 6;)p＜0.05，**p＜0.01，***p＜0.001）。

FIG. 17 shows (a) spread area and (b) cell aspect ratio (mean. + -. Standard deviation, n =6, and n is a linear variable number, all the materials arep＜0.01，***p＜0.001）。

Figure 18 shows VEGF adsorption (mean ± standard deviation, n = 6: ×) on planarized and patterned PDMS-Au, pS, pSM and pM surfacesp＜0.001）。

FIG. 19 shows the VEGF adsorption amounts of patterned PS-S, PM-M, PS-M and PM-S surfaces (mean. + -. Standard deviation, n =6, and n is a linear chain, all points of the Chinese characterp＜0.001）。

Detailed Description

In the prior art, the preparation of a plurality of topological patterns requires expensive instruments and equipment or harsh preparation conditions, and the surface of a material can be damaged in the preparation process of the topological patterns. In the invention, the surface of PDMS-Br is modified with a heparinoid polymer by a visible light-initiated grafting polymerization method. Meanwhile, a patterned gold film was deposited on the patterned template using a chloroauric acid reduction method. Then, the patterned gold film was transferred to the heparinized PDMS surface by transfer printing, and the gold film area was modified with a thiolated heparinoid polymer by gold-sulfur bond self-assembly. The surface of the sample prepared by the invention is a homogeneous patterned surface of the heparinoid polymer, namely, the same heparinoid polymer is modified in each area of the patterned surface, or a polymer (pSS) containing sulfonic acid groups and a polymer (pMAG) containing glycosyl groups are respectively modified on PDMS and gold film covered areas by a visible light-initiated graft polymerization method and a gold-sulfur bond self-assembly method, so that the patterned surface with heterogeneous distribution of the pSS and the pMAG is prepared. In the present invention, the heparinoid polymer is pSS, pMAG, pS-copM, chemical composition of the pattern surface see figure 1.

The raw materials adopted by the invention are all commercial products, and the specific preparation operation and test are conventional technologies. All experiments herein were performed independently, with each condition containing at least three replicates per experiment. The experimental results are expressed by mean value plus or minus standard deviation, and are compared and verified by T.TEST methodSignificant difference between test group and control group (.)p＜0.05，**p＜0.01，***p＜0.001）。

The main experimental materials and reagents are as follows.

Other reagents were purchased from chemical reagents of the national drug group, ltd, and were used after conventional purification.

Synthesis example

Synthesis of 10-undecene-2-bromoisobutyrate. 10-undecen-1-ol (2.12 g,12.5 mmol) and triethylamine (1.38 g,13.7 mmol) were weighed out and placed in a 50 mL round-bottomed flask and completely dissolved in 15 mL anhydrous dichloromethane. After stirring for 30 minutes under nitrogen and ice bath conditions, the ice bath and conventional stirring were maintained and BIBB (1.7 mL,13.7 mmol) was added dropwise to the flask. After the dropwise addition, the ice bath stirring condition is kept for 1 h, and then the reaction system is moved to the room temperature condition and stirred overnight. After the reaction is finished, removing triethylamine hydrochloride serving as a reaction byproduct by suction filtration. The reaction solution was transferred to a separatory funnel, and a saturated potassium hydrogen carbonate solution was added thereto, followed by shaking to wash the reaction solution. The reaction solution was collected, the waste liquid layer was discarded, and washing was repeated 3 times. When the washing is carried out for the last time, the mixture is collected into a dry container, anhydrous magnesium sulfate is added, and the mixture is kept stand, dried and spin-dried. The crude product was purified by silica gel column chromatography using n-hexane, ethyl acetate =20 (v/v) as a developing agent, and the purified product was spin-dried to obtain a pale yellow liquid, which was dried in a vacuum oven at normal temperature for 48 hours. ¹ H NMR（400 MHz，CD ₃ OD），δ（ppm）: 5.79-5.87（m，1H，=CH），4.89-5.02（d，2H，=CH ₂ ），4.19（t，2H，-CH ₂ O），2.06（m，2H，=CHCH ₂ ），1.93（s，6H，-CH ₃ ），1.70（m，2H，-CH ₂ CH ₂ O），1.41（m，2H，-CH ₂ CH ₂ CH ₂ O），1.35（m，10H，-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -）。

And (3) synthesizing methacrylamidoglucose. D- (+) -glucosamine hydrochloride (5 g,27.9 mmol) and potassium carbonate (3.2 g,23.1 mmol) were weighed into a 250 mL round bottom flask and dissolved in 120 mL of anhydrous methanol. Methacryloyl chloride (1.8 mL,18.6 mmol) was added dropwise with regular stirring under nitrogen and ice bath. After the dropwise addition, the ice-bath condition was kept and the stirring was continued for 1 h, and then the reaction system was moved to the room temperature condition and stirred overnight. After the reaction was completed, the solid was removed by suction filtration, and the filtrate was concentrated. The product was purified by silica gel column chromatography with methanol as developing solvent dichloromethane =1 (v/v) and the product was dried by spinning to give a white solid, which was dried in a vacuum oven at room temperature for 48 h. ¹ H NMR（D ₂ O，400 MHz），δ（ppm）: 5.69（s，1H，=CHH），5.46（s，1H，=CHH），5.21（d，0.53H，anomeric α-CH），4.70-4.74（d，0.53H，anomeric β-CH），3.40-4.00（m，6H，sugar moiety 6×CH），1.93（s，3H，-CH ₃ ）。

And (3) synthesizing a heparinoid polymer. The heparinoid polymer sodium polystyrene sulfonate (pSS) is prepared by reversible addition-fragmentation chain transfer polymerization (RAFT) method. The molar charge ratio in the polymerization is [ monomer ]] ₀ Chain transfer agent] ₀ [ initiating agent ]] ₀ 1, = 300. The monomer sodium Styrene Sulfonate (SS) (370.5 mg,1.8 mmol), the chain transfer agent CPADB (3.35 mg,0.012 mmol) and the initiator AIBN (1 mg,0.006 mmol) were dissolved in 5 mL of a solution (DMF: H) ₂ O =1, v/v). After nitrogen is introduced for 30min, the reaction flask is transferred into a glove box and reacted for 12 h under the environment of oil bath at 70 ℃. After the reaction, the reaction flask was taken out, and the stopper was opened to allow the reaction solution to contact air to terminate the polymerization. The product was transferred into a dialysis bag (molecular weight cut-off 3500 Da) for three days, and then lyophilized in a lyophilizer to finally obtain the pink flocculent product pSS.

For polymethacrylamidoglucose (pMAG), monomersN-the input of Methacrylamidoglucose (MAG) is 444.6 mg (1.8 mmol); for copolymers of SS and MAG (pS-co-pM), the input amount of the monomer SS is 185.2 mg (0.9 mmol), the input amount of the monomer MAG is 222.3 mg (0.9 mmol), and the specific preparation process is consistent with that of pSS.

The procedure for the preparation of pSS with green fluorescent label is similar to the above procedure, but 5% fluorescein O-methacrylate is added at the same time when the reagents are added, and the whole procedure is carried out in the dark.

Thiolation of heparinoid polymers. And dissolving the polymer pSS in pure water, dropwise adding ethanolamine under stirring at normal temperature, reacting for 5 hours, and reducing disulfide bonds at the tail end of the polymer into sulfydryl (-SH). After the reaction is finished, the product is transferred into a dialysis bag (molecular weight cut-off is 3500 Da) for dialysis for three days, and then the dialysis bag is placed into a freeze dryer for freeze drying, and finally the white floccule prepared is sulfhydrylation pSS (pSS-SH).

The polymer used for the thiolated pMAG (pMAG-SH) is pMAG, the thiolated pS-co-pM（pS-copM-SH) the polymer used is pS-copM, prepared in accordance with the thiolated pSS.

The preparation of PDMS and PDMS-Br surfaces, raw materials and preparation methods are conventional techniques. Component a (mixture containing prepolymer and platinum catalyst) and component B (crosslinker, polydimethylsiloxane prepolymer mixture containing vinyl and Si-H) in Sylgard 184 were mixed and stirred well in a mass ratio of 10. And after uniformly stirring, vacuumizing and defoaming the mixture in a vacuum drying oven, pouring the mixture on a patterned silicon template (a circular array with the diameter of 300 mu m), defoaming the mixture by using the vacuum drying oven again, placing the defoamed mixture in a 60 ℃ drying oven, curing the defoamed mixture for 8 hours, taking the defoamed mixture out to obtain patterned PDMS, and cutting the patterned PDMS for later use. And (3) for flat PDMS, vacuumizing and defoaming the mixture in a vacuum drying oven, pouring the mixture into a disposable culture dish with the diameter of 9 cm, defoaming the mixture in the vacuum drying oven again, placing the defoamed mixture in a 60 ℃ drying oven, curing the defoamed mixture for 8 hours, taking out the solidified mixture to obtain flat PDMS, and cutting the flat PDMS for later use.

The preparation of PDMS-Br surfaces was similar to that described above. Uniformly mixing and stirring the component A and the component B of Sylgard 184 and the synthesized component C, namely 10-undecylene-2-bromoisobutyrate, according to the mass ratio of 10.

Example construction of a homogeneous patterned surface of a heparinoid Polymer

And (3) modifying the surface of PDMS-Br with a heparinoid polymer. SS (1.2372 g,6 mmol) was dissolved in 12 mL of solvent (methanol: water =1, 2,v/v) and the photoinitiator Mn was added in the dark ₂ (CO) ₁₀ (5 mg,0.0128 mmol), adding a flat PDMS-Br sample into the reaction solution, introducing nitrogen into the reaction system for 30min, sealing the reaction bottle, and reacting for 1 h under the irradiation of visible light at 50 ℃ (I) _420nm = 0.2 mW/cm ² ). After the reaction is finished, the sample is alternately washed three times by water and ethanol, and the sample is dried by a vacuum drying oven to obtain a PDMS surface modified by pSS (PDMS-pSS).

For the preparation of a pMAG modified PDMS surface (PDMS-pMAG), the input of MAG was 1.482 g (6 mmol); for pS-coPreparation of a pM modified PDMS surface (PDMS-pSM), with a 0.6186 g (3 mmol) SS input and a 0.7410 g (3 mmol) MAG input, the rest of the preparation was identical to the above PDMS-pSS surface.

And (5) preparing a gold film. Under ice bath conditions, 1 g (0.0029 mmol) of chloroauric acid was added to 20 mL of ultrapure water, and water was added to 50 mL under ordinary stirring to give a chloroauric acid solution. Potassium bicarbonate (0.2 g,2 mmol) and glucose (0.02 g,0.112 mmol) were weighed into a centrifuge tube, 3 mL of ultrapure water, the above chloroauric acid solution and ultrasonic deaeration were added, the pH of the mixed solution was adjusted to 9.5 with NaOH solution, then deaeration in the mixed solution was performed, the mixed solution was covered with flat PDMS or patterned PDMS, and the mixture was allowed to stand at 37 ℃ for 6 hours with a 100 g weight under pressure. Taking out to obtain a smooth gold film or a patterned gold film template, storing in ultrapure water, and cutting into proper sizes when in use.

And (4) transferring the patterned gold film. The heparinoid polymer-modified PDMS samples (PDMS-pSS, PDMS-pMAG or PDMS-pSM) and the patterned gold membrane template were placed right side up in 6-well plates. The surface was hydroxylated by irradiating for 70 s with a plasma cleaner. After being taken out, the irradiated surfaces of the two samples are jointed, pressure is applied by a 100 g weight, and the samples are kept stand in an oven at 60 ℃ for 5 min. After being taken out, the two sample surfaces which are tightly attached are mutually stripped, and the patterned gold film can be successfully transferred to the surfaces of various modified PDMS samples.

And replacing the patterned gold film template with a flat gold film template, and obtaining a flat gold film modified PDMS sample by the same steps.

Self-assembly of thiolated heparan-like polymers. The thiol-modified heparinoid polymer prepared above was dissolved in ultrapure water to prepare a solution of 2 mg/mL. The modified PDMS sample with the transferred smooth gold film or the patterned gold film is soaked in the thiolated heparan-like polymer solution and soaked overnight at room temperature. And washing the gold film with ultrapure water for three times, and then drying the gold film in vacuum, thereby introducing the heparan polymer on the surface of the smooth gold film or the patterned gold film.

As shown in fig. 1 (a), the homogeneous patterning surface of heparinoid polymer is constructed by visible light-induced graft polymerization on the surface of PDMS-Br and self-assembly of gold-sulfur bonds of thiolated polymer on the surface of deposited gold film. The preparation process will now be described by way of example of homogeneously modifying the patterned surface of pSS. Firstly, modifying a heparinoid polymer pSS on a smooth PDMS-Br surface by a visible light graft polymerization method. Then, a gold film was deposited on the patterned PDMS (circular array having a diameter of 300 μm) by a chloroauric acid reduction method, and the patterned gold film was transferred to the surface of the PDMS modified with the heparan polymer. Finally, pSS-SH is assembled in the gold film covered area by gold-sulfur bond self-assembly. In this way, a homogeneous pS surface with a circular array pattern of 300 μm diameter was prepared. By the above method, a surface (PDMS-Au) without modification of the heparinoid polymer is also obtained; homogeneously modifying the surface of pMAG (pM); homogeneous modification of pS-co-the surface of pM (pSM). FIG. 1 (b) is a schematic diagram showing the distribution of chemical components on the surfaces of PDMS-Au, pS, pSM and pM of the patterned samples obtained as described above.

The construction of the heterogeneous patterned surface of the heparinoid polymer is realized by visible light-initiated graft polymerization on the surface of PDMS-Br and gold-sulfur bond self-assembly of the thiolated polymer on the surface of the deposited gold film. The heterogeneously distributed polymers chosen for this experiment were pSS and pMAG. As shown in FIG. 1 (c), for example, in the preparation of PS-M, first, a PDMS-pSS surface is prepared by grafting pSS onto a flat PDMS-Br surface by visible light-induced graft polymerization. Meanwhile, a gold film was deposited on the patterned PDMS (circular array with a diameter of 300 μm) by a chloroauric acid reduction method, and the patterned gold film was transferred to the surface of the PDMS-pSS. Finally, pMAG-SH was assembled in the gold membrane covered area by gold-sulfur bond self-assembly. In this way, PS-M samples with heterogeneously patterned distribution of heparinoid polymers were prepared. The PM-S sample was obtained by modifying pMAG in the PDMS region and pSS in the gold membrane region in a similar manner as described above. In the same manner, a surface modified with pSS in both PDMS and gold membrane areas (PS-S) and a surface modified with pMAG in both PDMS and gold membrane areas (PM-M) were obtained. FIG. 1 (d) is a schematic of the chemical composition distribution of the PS-S, PM-M, PS-M, and PM-S surfaces of the patterned samples obtained in this work.

Example two polymers and surface characterization

All synthetic monomers and polymers are analyzed by NMR ¹ H-NMR). The polymers were also characterized by fourier infrared spectroscopy (FTIR) and ultraviolet spectroscopy. The molecular weight and molecular weight distribution of the polymer were characterized using Gel Permeation Chromatography (GPC). The modification of the polymer on the surface was characterized by water contact angle, energy spectrometer (EDS) and fluorescence inverted microscope.

The invention prepares heparinoid polymers pSS, pMAG and pS-containing peptide with dithioester bond by RAFT polymerizationco-pM. Then, the disulfide bond at the end of the polymer was reduced to a mercapto group with ethanolamine. FIG. 2, FIG. 3 and FIG. 4 show thiolated heparinoid polymers pSS-SH, pS-co-synthesis steps of pM-SH and pMAG-SH, and data characterizing the thiolated products of the heparinoid polymer by means of Fourier Infrared Spectroscopy, UV Spectroscopy and Nuclear magnetic resonance spectroscopy, table 1 shows the molecular weight and molecular weight distribution of the thiolated heparinoid polymer. FIG. 2 (a) shows a synthesis step of pSS-SH, in which pSS is synthesized by RAFT polymerization and then reduced to pSS-SH by ethanolamine. FIG. 2 (b) is an FTIR spectrum of pSS-SH, 1171 cm ^-1 Counter-stretching vibration peak at S = O, 1625 cm ^-1 The peak is the stretching vibration peak of phenyl. FIG. 2 (c) is a UV spectrum of pSS polymerThe signal peak (about 310 nm) of the dithio-ester bond disappears in the spectrogram of the thiolated polymer, which indicates that the dithio-ester bond is successfully reduced to the sulfhydryl group. FIG. 2 (d) is of pSS-SH ¹ H NMR spectrum. Wherein the peaks at δ 6.7 and δ 7.6 correspond to H (positions 1 and 2) on the benzene ring of the side chain, respectively. δ 1.7 corresponds to H (positions 3 and 4)) on the methylene and methine groups in the polymer backbone. The molecular weight of the pSS-SH polymer was 15000 and the molecular weight distribution was 1.1 (Table 1). FIG. 3 (a) shows pS-co-synthesis step of pM-SH, C = O stretching vibration peak, N-H bending vibration peak and S = O reverse stretching vibration peak appear in FTIR spectrum of fig. 3 (b). Figure 3 (c) the disulfide bond signal disappears in the uv spectrum. Characteristic peaks in the NMR spectrum of FIG. 3 (d) are labeled. pS-coThe molecular weight of the-pM-SH polymer was 12000, with a molecular weight distribution of 1.2 (Table 1). FIG. 4 (a) is the synthesis procedure of pMAG-SH, FIG. 4 (b) is the FTIR spectrum of pMAG-SH, 1631 cm ^-1 At C = O tensile vibration peak, 1531 cm ^-1 The peak is the N-H bending vibration peak. FIG. 4 (c) the disulfide bond signal disappears in the UV spectrum. Characteristic peaks in the NMR spectrum of FIG. 4 (d) are labeled. The molecular weight of the pMAG-SH polymer was 10000, with a molecular weight distribution of 1.1 (Table 1). The above data indicate that pSS-SH, pS-coThe preparation of the-pM-SH and pMAG-SH polymers was successful.

The wettability of the homogeneously patterned surface of heparinoid polymers was characterized by static water contact angle test (fig. 5 a). For a flat sample surface, the water contact angle of PDMS-Au is about 98 deg.. After self-assembly modification of pSS and pMAG, the surface water contact angles dropped to 68 ° and 75 °, respectively; when pS-coAfter pM was modified to the surface, the surface water contact angle dropped to 70 °, and for patterned surfaces, the introduction of the pattern increased the water contact angle for all surfaces (9 ° -13 °). Wettability of the heterogeneous patterned surface of heparinoid polymers was characterized by static water contact angle testing. FIG. 5b shows the water contact angle change in the process of preparing the surface of a heparinoid heterogeneous patterned sample, the water contact angle of PDMS-Br is 108 degrees, the water contact angle of PDMS-pSS surface is reduced to 22 degrees, the water contact angle of the surface is increased to 90 degrees by introducing a patterned gold membrane, and the PS-M surface water contact angle finally obtained after self-assembly of pMAG-SHThe contact angle drops to 72 deg.. The water contact angle of the PDMS-pMAG surface is 62 degrees, the introduction of the patterned gold film enables the water contact angle of the surface to be increased to 95 degrees, and after the pSS-SH is self-assembled, the finally obtained water contact angle of the PM-S surface is reduced to 70 degrees.

The element distribution of the flat and patterned heparinoid polymer modified surface is characterized by EDS surface scanning analysis, and visual element distribution data is obtained. The Au element is a characteristic element of a gold film, the Si element is a characteristic element of PDMS, S is a characteristic element of pSS, and N is a characteristic element of pMAG. As shown in FIGS. 6, 7 and 8a, the flat pS, pSM and pM surfaces had uniform distribution areas and signal intensities of the respective characteristic elements. Whereas for the patterned surface, taking the patterned pS sample (fig. 6) as an example, au, si, S exhibited different elemental distributions and signal intensities in different regions. Au elements with stronger signals are uniformly distributed in the area covered by the patterned gold film outside the circular area; the Au element signal of the PDMS area which is not covered by the gold film in the circle is very weak, and the strength of the Si signal in the round area covered by the PDMS is obviously higher than that in the area outside the circle; the S element was homogeneously distributed inside and outside the circular area, indicating successful incorporation of polymer pSS in both areas of the patterned surface (circular area and outside the circular area). Whereas for the patterned pSM sample (fig. 7), both N and S elements had significant signal both inside and outside the circle; for the patterned pM sample (fig. 8 a), the N element has a clear signal both inside and outside the circle. As shown in fig. 8b, the Si and S signals are distributed across the PS-M surface, with the Si signal being stronger in the PDMS regions within the 300 μ M circle. The S signal also has obvious signals in the Au coverage area, and is the signal of the S distribution on the Au area on the sulfhydryl group carried by the self-assembly polymer. Au signals are uniformly distributed in the area outside the circle and are in a patterning distribution state, which indicates that the patterned gold film is successfully transferred. The areas where the N signal was detected to be denser were mainly distributed in the out-of-circle areas, indicating successful self-assembly of pMAG in the gold membrane covered area. The element distribution conditions of the surfaces of the various heparin polymers indicate the successful modification of the heparin polymers on the flat and patterned surfaces.

The pattern morphologies of the patterned PDMS template and the patterned gold film were characterized by bright field photography with a fluorescence inverted microscope, and a circular pattern with a diameter of about 300 μm and uniform distribution was observed on the surface of the PDMS template. A patterned gold film (gold area) was observed on the surface of the patterned gold film transferred to the sample surface using the PDMS stamp, and the area inside the circle was the bare PDMS substrate, and the diameter of the circle was also about 300 μm, consistent with the size of the stamp. pSS-SH molecules with green fluorescence are self-assembled on the surface of the flat and patterned gold film, and then a fluorescence signal is observed by a fluorescence inverted microscope. Uniformly distributed fluorescent signals are observed on the surface of the smooth gold film, obvious patterned distribution of fluorescent molecules is observed on the surface of the patterned gold film, and the fluorescent signals are concentrated in the gold film coverage area. The above shows that the transfer printing method can successfully obtain the circular array patterned gold film surface with the diameter of 300 μm, and the thiolated heparan polymer can be successfully self-assembled on the gold film.

Example experiments on dead and alive staining of three vascular cells

HUVECs were plated at 25000 cells/cm ² Density on the sample surface at 5% CO ₂ Incubated at 37 ℃ for 4 h and 48 h. After completion of incubation, the cell culture medium was aspirated, the surface of the sample was treated with a mixed staining solution (diluted with ECM) of Calcein-AM (stained viable cells appear green fluorescence) and PI (stained dead cells appear red fluorescence), and incubated in a 37 ℃ incubator for 20 min. Immediately after the waste liquid is sucked out, a dead and live staining fluorescence image of cells on the surface of the sample is shot by an inverted fluorescence microscope. At least 10 pictures of three replicates of each set of samples were taken, and the number of live and dead cells was counted using Image J and the ratio was calculated. The dead-live staining process of HUVSMCs was similar to HUVECs except that the medium used during the culture and the solvent used to dilute the staining solution was SMCM.

HUVECs were seeded on flat and patterned PDMS-Au, pS, pSM and pM sample surfaces and dead and viable cells on the sample surfaces were stained simultaneously with a mixed staining solution of Calcein-AM and PI after incubation for 4 h and 48 h. The cell viability was calculated by counting dead and viable cells and using the formula: cell activity (%) = number of living cells/(number of living cells + number of dead cells) × 100%. As shown in FIG. 9 (a), after HUVECs were cultured on the surface of each sample for 4 h, the cell activity on the surface of the flat PDMS-Au sample reached 96%, and the HUVECs activity on the surface of the pS and pSM samples approached 96% and 97%, respectively. Whereas the HUVECs activity of the pM surface containing only sugar groups was only 73%. After the surface is introduced with a pattern structure, the HUVECs activity of the patterned PDMS-Au surface is reduced compared with that of a flat surface. The HUVECs activity on the patterned pS, pSM and pMAG samples was 96%, 98% and 79%, respectively, which was comparable to the HUVECs activity of the corresponding flat surfaces (fig. 10 a). As shown in FIG. 9 (b), HUVECs activity increased on most sample surfaces after 48 h incubation of HUVECs on each sample surface. For flat surfaces, the HUVECs activity of PDMS-Au, pS and pSM were all around 97%, with data approaching or slightly increasing with 4 h. HUVECs activity of pM surface reached 93%, but was still lower than other flat surfaces. HUVECs activity was reduced by 8% and 20% for patterned PDMS-Au and patterned pM surfaces, respectively, compared to a flat surface. Whereas HUVECs activity on patterned pS surfaces was comparable to flat pS surfaces. HUVECs on the patterned pSM surface were almost fully viable with cell viability exceeding 99% (FIG. 10 a). FIG. 10b shows that the HUVECs activity of PS-M and PM-S is 90% and 81%, respectively, and the fluorescence staining of the dead cells shows that the adhesion distribution of PS-M live cells is relatively uniform, and the live cells on the surface of PM-S are basically adhered to the outside circle area of the modified pSS.

Example four vascular cell adhesion and proliferation experiments

HUVECs were plated at 25000 cells/cm ² Density species on the sample surface at 5% CO ₂ Incubate at 37 ℃ for 4 h and 48 h. After incubation was complete, the samples were soaked once in Phosphate Buffered Saline (PBS) for a short period of time. After fixing the cells with 4% paraformaldehyde for 15 min, the residual paraformaldehyde was washed three times with PBS. Cells were subjected to membrane rupture treatment by adding 0.1% Triton X-100 and left to stand for 5 min, and washed three times with PBS. Then, the sample surface was treated with 3% BSA in PBS for 40 min. The samples were washed thoroughly with Phalloidin-FITC for 40 min in the dark, washed three times with PBS, then with DAPI for 5 min in the dark, washed twice with PBS for a short time, then soaked in PBS for 10 min, and finally the PBS was blotted dry. The sample was placed on a slide with the front side down and the fluorescence image of the cells on the surface of the sample was taken with an inverted fluorescence microscope. Each set of samples was run in triplicate,at least 10 photographs were taken, and the cell density, spreading area and aspect ratio were calculated using Image J. HUVSMCs were cultured in a similar manner to HUVECs, using SMCM as the medium.

FIG. 11 (a) shows the adhesion of HUVECs to the surface of each sample after 4 hours of culture. For a flat surface, the adhesion form of HUVECs on PDMS-Au is irregular polygon, and the cell density is 86 cells/mm ² (FIG. 11 (c)). While the morphology of flat pS and pSM surface-adhered HUVECs was essentially irregular polygonal, with cells present in a larger spreading area (fig. 11 (a)). The number of HUVECs adhered to the surface of the flat pS sample was reduced to 62 cells/mm compared to the surface of the flat PDMS-Au sample ² (ii) a On the surface of the flat pSM sample, the adhesion number of HUVECs is obviously increased, and is 170 cells/mm ² (FIG. 11 (c)). On the flat pM surface, the HUVECs adhered basically in a contracted state (FIG. 11 (a)), and the cell adhesion quantity is reduced by 65% compared with that on the flat PDMS-Au surface, which is 30 cells/mm ² (FIG. 11 (c)). For the patterned surfaces, the amount of HUVECs adhesion on the patterned PDMS-Au, pSM, and pM surfaces decreased by 48%, 36%, and 23%, respectively, compared to the corresponding flat surfaces (fig. 11 (a)). While the morphology of HUVECs on the patterned pS surface spread in a healthy spindle or circle shape, the number of HUVECs adhered was also significantly higher than that on the flat pS surface, and the cell adhesion density was increased by 96% compared with that on the flat pS surface (FIG. 11 (c)). FIG. 11 (b) shows the cell growth of HUVECs after 48 h incubation on the surface of each sample. For the flat surface, HUVECs on the flat PDMS-Au, pS and pSM surfaces spread obviously in a polygonal spreading state compared with 4 h. While HUVECs on a flat pM surface remained contracted. HUVECs density of PDMS-Au and pM sample surface is 82 cells/mm respectively ² And 30 cells/mm ² Comparable to the 4 h cell density of the corresponding surface. The cell density of the flat pS and pSM surfaces at 48 h is obviously increased compared with the corresponding cell density at 4 h, and respectively reaches 158 cells/mm ² And 183 cells/mm ² (FIG. 11 (c)). For patterned surfaces, the HUVECs density of PDMS-Au and pM surfaces were comparable to the corresponding flat surfaces. The HUVECs density on the patterned pS and pSM surfaces is obviously increased compared with that on the flat surface, and respectively increased25% and 40% are added. It is noteworthy that the HUVECs density on the patterned pSM surface was maximal after 48 h incubation of the HUVECs (fig. 11 (c)), and that the spread state of the HUVECs on the surface was excellent as observed from the fluorescence map, and the HUVECs grown on the surface formed a large area of close arrangement and connection and appeared very healthy fusiform (fig. 11 (b)). After incubation of HUVECs on the surface of each sample for 4 h, the HUVECs are in a polygonal incompletely spread state on PS-M and PM-S surfaces with heterogeneous patterned distribution of heparinoid polymers. By observing the area of cell distribution, it was found that HUVECs on the PS-M surface tended to adhere at the location where the PDMS modified pSS in the circle or the border of the circle between pSS and pMAG modification, while HUVECs on the PM-S surface tended to adhere at the area where pSS was modified on the gold membrane outside the circle. The adhesion density of HUVECs on the surfaces of PS-M and PM-S is 121 cells/mm respectively ² And 128 cells/mm ² Comparable to the HUVECs adhesion density of PS-S homogeneous surfaces. After the HUVECs are incubated on the surfaces of the samples for 48 hours, HUVECs on the surfaces of PS-M and PM-S surfaces show more obvious regional distribution on the surfaces of the samples with heterogeneous patterned distribution of heparan-like polymers. On the surface of PS-M, HUVECs area adheres in a circle (PDMS area of modified pSS), and cells spread in a polygonal or spindle shape in a good state; on the surface of PM-S, HUVECs are mainly distributed outside the circle (gold membrane area of modified pSS), cells are mainly in a polygonal spreading state, and part of cells are in a slightly curled state. HUVECs density of PS-M and PM-S are respectively 140 cells/mm ² And 126 cells/mm ² . This indicates that the combination of the heparinoid polymer on the surface of the material and the surface pattern can generate a synergistic effect.

As shown in FIG. 12 (a), the average spreading area of HUVECs single cells on the surface of flat PDMS-Au, pS and pSM after 4 h culture is 900 μm ² Left and right, and flat pM surface had a small spread area of HUVECs of 335. Mu.m ² . The HUVECs spread over the patterned pS and pSM surfaces were larger than the corresponding flat surfaces, 1151 μm each ² And 1576 μm ² The cell aspect ratios were all around 2.2 (fig. 12 (b)). The cell aspect ratio is the ratio of the length stretched longitudinally and the width stretched transversely as the cells are grown, and this data can be reversedThe cells should have a tendency to stretch longitudinally. The larger the value, the more pronounced the morphology of the longitudinal tensile growth of the cells. The data thus show that HUVECs on the surface of pS and pSM have a longitudinal stretch length 2 times longer than the transverse spread length, and have a fusiform spread morphology. And the spreading areas of HUVECs on the patterned PDMS-Au and pM surfaces are smaller than those on the corresponding flat surfaces, and are 360 mu m ² And 328 μm ² FIG. 12 (a), the aspect ratio of the cells was close to 1 (FIG. 12 (b)), and many cells adhered to the surface in the form of non-spread spheres. The cell spreading areas of PS-M and PM-S on the heparin polymer heterogeneous patterned surface are 812 mu M respectively ² And 851 μm ² The cell aspect ratio was 1.5 and 1.7, respectively.

As shown in FIG. 12 (a), the spread area of HUVECs on each surface was increased as a whole as the cell culture time was extended to 48 hours. The spreading area of HUVECs on the smooth PDMS-Au surface is 4349 μm ² . The spread area of HUVECs on the flat pS and pSM surfaces was slightly lower than that on the flat PDMS-Au surface, at 3600 μm ² Left and right. Whereas HUVECs spreading was significantly inhibited at 588 μm on a flat pM surface ² The cell aspect ratio was still about 1 (FIG. 12 (b)), and was substantially in the form of incompletely spread round spheres. After the surface pattern was introduced on the surface, the ratio of the HUVECs spreading area of the patterned PDMS-Au surface to the flat PDMS-Au surface decreased by 45%. The HUVECs spreading area of the patterned pM increased 101% compared to the flat pM surface, but was still significantly lower than the cell area of the patterned PDMS-Br surface (FIG. 12 (a)). And the introduction of the pattern structure on the surface of the pS and pSM samples obviously promotes the spreading of HUVECs, and the cell spreading area is greatly increased. Wherein the spreading area of HUVECs on the surface of the patterned pS is 6809 mu m ² The cell aspect ratio was 2.1, i.e., the stretched length of the cells was about 2.1 times the spreading width, and the HUVECs spread substantially in a fusiform pattern on the surface. Whereas the spread area of HUVECs on the patterned pSM surface was the largest of all samples, reaching 10554 μm ² The cell aspect ratio was 2.3 (FIG. 12 (b)), and HUVECs spread over a large area while maintaining the overall fusiform morphology. HUVECs spreading areas of PS-M and PM-S heterogeneous patterned surfaces of heparan polymer are 5769 mu M respectively ² And 3618 μm ² Cell aspect ratios of 1.9 and 2.1, respectively, with PThe cell aspect ratio of the S-S surface is comparable. HUVECs on the PS-M surface spread out in a substantially fusiform manner, while HUVECs on the PM-S surface are in a stretched state, but slight curling reduces the spread area.

In combination with the above data, for flat surfaces, sulfonic acid groups are important groups for promoting the adhesion and proliferation of HUVECs, and sugar groups have certain inhibitory effects on the adhesion and proliferation of HUVECs. When the sulfonic acid group and the glycosyl group exist on the surface of the smooth gold film, the two groups can generate synergistic action to promote the adhesion and proliferation of HUVECs. For the patterned surface, the gold film with a circular array pattern of 300 μm diameter combined with pMAG, which inhibits HUVECs adhesion proliferation, still inhibited HUVECs adhesion and proliferation, and further enhanced HUVECs adhesion and proliferation promoting effect when combined with pSS. Thereby patterning the structure and pS-coWhen pM (the sulfonic acid group and the glycosyl group exist at the same time) is combined, the combined action of the sugar, the sulfonic acid group and the patterned structure can lead the surface to have all-round promotion effect on HUVECs, and the HUVECs have the highest HUVECs proliferation density, the best spreading state and the highest HUVECs activity. The pSS and pMAG components were introduced to the surface of the material by heterogeneous distribution, and different spatial distribution patterns were found to have different effects on the behavior of HUVECs. It is shown that the cellular behavior of heparinized material surfaces containing sulfonic acid groups and sugar groups is influenced by the spatial distribution pattern of the surface chemical components. In the present invention, heterogeneously patterned PS-M surfaces are more favorable to the proliferation and spreading of HUVECs than PM-S surfaces.

As shown in FIG. 13 (a), HUVSMCs were cultured on the surface of each sample for 4 hours, and then the cells on each surface were distributed uniformly. The HUVSMCs activity for the smooth PDMS-Au surface was 84% and comparable for the smooth pSM surface, while the HUVSMCs activity for the smooth pS sample surface was slightly higher, 89% (fig. 14). HUVSMCs activity was 73% for pM surface containing only saccharide groups, which was reduced by 11% compared to a smooth PDMS-Au surface. The HUVSMCs activity was decreased for the patterned PDMS-Au, pSM and pM surfaces compared to the corresponding flat surfaces, while the HUVSMCs activity was slightly increased for the pS surface. The HUVSMCs activity of the heparinoid polymer heterogeneous patterned surfaces PS-M and PM-S was 79% and 70%, respectively.

As shown in FIG. 13 (b), after HUVSMCs were cultured on the surface of each sample for 48 hours, the activity of HUVSMCs was decreased on most of the sample surfaces, which was different from the tendency of HUVECs on the sample surfaces. For flat surfaces, the HUVSMCs activity on PDMS-Au, pS, pSM and pM surfaces ranged from 63 to 70% (FIG. 14), which is a 14%, 21% and 8% decrease compared to the corresponding data for 4 h, respectively. Among them, the pSM flat surface with the best HUVECs activity (97%) had the lowest HUVSMCs activity, 63%. Compared with a flat surface, the activity of HUVSMCs on the patterned PDMS-Au surface is reduced to a certain degree, and the activity (72 to 75%) of the HUVSMCs on the patterned surface modified by different heparinoid polymers is slightly increased compared with that of the corresponding flat surface.

FIG. 15 (a) shows the adhesion of HUVSMCs to the surface of each sample after 4 hours of culture. For flat surfaces, HUVSMCs spread slightly over the surface of each sample in a substantially irregular polygon. HUVSMCs density of the smooth PDMS-Au sample surface is 46 cells/mm ² (ii) a In contrast, HUVSMCs density of both the sulfonic acid group-containing planarized pS and pSM was increased, at 68 cells/mm, respectively ² And 89 cells/mm ² (ii) a Whereas flat pM containing only saccharide groups had a lower HUVSMCs density of only 16 cells/mm ² (FIG. 15 (c)). For the patterned surface, HUVSMCs except for the patterned pM surface were in an atrophic state, and HUVSMCs of the remaining patterned pM surface were in a polygonal spread state tending to stripe (fig. 15 (a)). HUVSMCs densities of patterned PDMS-Au, pS, pSM and pM sample surfaces were increased by 9%, 102%, 47% and 86%, respectively, compared to the corresponding flat surfaces (fig. 15 (c)). On the heterogeneously patterned distribution surface of the heparinoid polymer, HUVSMCs on the PS-M surface are distributed more uniformly, while on the PM-S surface, HUVSMCs tend to adhere to the gold membrane area outside the circle for modifying pSS, the cell morphology is polygonal, and the HUVSMCs on the PS-M surface and the PM-S surface have the density of 62 cells/mm respectively ² And 48 cells/mm ² 。

FIG. 15 (b) shows the cell growth of HUVSMCs after 48 h incubation on the surface of each sample. For flat surfaces, HUVSMCs were observed to have a pronounced longitudinally stretched morphology on each surface. HUVSMCs density of PDMS-Au is 112 cells/mm ² . HUVSMCs density of flat pS and pSM increased to 131 cells/mm, respectively ² And 153 cells/mm ² (ii) a pM of HUVSMCs had a low density of 52 cells/mm ² (FIG. 15 (c)). For the patterned sample surface, the cells all exhibited a longitudinally extended spreading morphology, in which the HUVSMCs spread out more neatly on the pSM surface than on the other sample surfaces, and adjacent HUVSMCs tended to spread out in the same direction (fig. 15 (b)). HUVSMCs densities of patterned PDMS-Au, pS, pSM and pM surfaces increased by 2%, 12%, 25% and 23%, respectively, compared to the corresponding flat surfaces (fig. 15 (c)). For heparinoid polymer heterogeneous patterned surfaces, as shown in fig. 15 (a), HUVSMCs on PS-M surface tended to grow in the circle (PDMS area of modified PSs), tensile spreading state of cells was good; whereas HUVSMCs on the surface of PM-S tended to grow out of the circle (modifying the gold membrane area of pSS), the cells were stretched and slightly curled. The number of HUVSMCs in the pSS modified region was significantly higher than in the pMAG modified region for the same sample surface. HUVSMCs number density of the PS-M sample surface was slightly higher than the PS surface, 160 cells/mm ² HUVSMCs density of PM-S sample surface 108 cells/mm ² . In the present invention, after pSS and pMAG are introduced to the surface by heterogeneous patterning, PS-M showed a stronger HUVSMCs promotion effect than PM-S surface (HUVSMCs density of PS-M surface is about 1.5 times of PM-S surface), indicating that heterogeneous patterned surface based on heparinoid polymer has unexpected effect on HUVSMCs behavior.

As shown in FIG. 16 (a), the average spreading area of the HUVSMCs on the flat PDMS-Au surface after 4 h culture was 606 μm ² HUVSMCs spread over a flat pM surface with an area of 537 μm ² The cell aspect ratios of HUVSMCs were 1.7 and 1.2 for both surfaces, respectively (fig. 16 (b)), where HUVSMCs were substantially spherical. The HUVSMCs spread area of the flat pS and pSM surfaces was significantly increased compared to the flat PDMS-Au surface, 1991 μm each ² And 2579 μm ² The cell aspect ratios of HUVSMCs flattened against pS and pSM surfaces were 2.9 and 3.6, respectively (fig. 16 (b)), indicating that HUVSMCs on both surfaces were in a stretched state. Guiding at the surfaceAfter patterning, the spread area of HUVSMCs of the patterned PDMS-Au and pM surfaces reached 2491 μm ² And 844 μm ² HUVSMCs with aspect ratios of 3.2 and 1.5, the spreading area and cell aspect ratio increased over the flat PDMS-Au and pM surface, but the spreading and stretching increase of patterned pM HUVSMCs was not significant compared to the flat surface. Whereas the HUVSMCs spreading area of the patterned pS and pSM surfaces decreased by 54% and 16% compared to the corresponding flat surfaces (fig. 16 (a)), with aspect ratios of 1.4 and 3.2, respectively (fig. 16 (b)). Compared with other patterned surfaces, the HUVSMCs of the patterned pS surface have obviously reduced spreading area and cell aspect ratio compared with the HUVSMCs of the flat pS surface, and the introduction of the pattern structure has larger influence on the adhesion state of the HUVSMCs on the pS surface. In contrast, the spread area and the cell aspect ratio of HUVSMCs adhesion on the flat and patterned pSM surface were large, and the numerical changes before and after patterning were not significant, indicating that the introduction of the pattern structure had a small effect on the adhesion state of HUVSMCs on the pSM sample surface. HUVSMCs of the PS-M and PM-S heterogeneous patterned surfaces of the heparinoid polymer have higher spreading areas than those of the PS-M and PM-S heterogeneous patterned surfaces of the heparinoid polymer, and the spreading areas are 1151 mu M respectively ² And 2870 μm ² (FIG. 17 (a)) and cell aspect ratios of 1.5 and 1.3 (FIG. 17 (b)), respectively, the data show that HUVSMCs on the surfaces of PS-M and PM-S spread out in the aspect ratio, but the cells do not have large length-width differences and are polygonal in shape due to the spreading in both the aspect and aspect directions, thus confirming the cell state observed in the fluorescence map of the cells.

Whereas in 48 h of culture, the spread area of HUVSMCs cells on the surface of each sample was increased as a whole in comparison with 4 h, and more pronounced longitudinal extension occurred (fig. 16 (b)). For a flat surface, the HUVSMCs spreading area of PDMS-Au, pS and pSM surfaces is relatively close to 3300 mu m ² -4000 μm ² In between, the cell aspect ratios of the three flat-surfaced HUVSMCs are all 5-6, with a pronounced longitudinal elongation. Whereas the HUVSMCs spread over 1585 μm for a flat pM surface ² The cell aspect ratio was 3.7, and although HUVSMCs had a spread and stretched state compared to 4 h, the extent of spread stretching of cells was less compared to other flat surfaces. The cell aspect ratio of HUVSMCs for all surfaces after the introduction of the surface pattern was in3-6, all presenting stretching state. HUVSMCs spreading area of patterned PDMS-Au and pM surface was comparable, approximately 2600 μm ² . There was a significant increase in HUVSMCs spreading area for the patterned pS and pSM samples compared to the corresponding flat surfaces, by 81% and 109%, respectively. HUVSMCs spread area was largest for the patterned pSM surface with an aspect ratio of 6, with the HUVSMCs stretched morphology most evident in all patterned surfaces. HUVSMCs spreading area for PS-M and PM-S was 4143 μ M for heparinoid polymer hetero-patterned surface, respectively ² And 3541 μm ² The cell aspect ratios of HUVSMCs for both surfaces slightly exceeded the PS-S surface, 4.5 and 4.7 respectively (fig. 17 (b)), indicating that HUVSMCs predominate in longitudinal stretching when both surfaces are spread.

The behavior of two kinds of vascular cells on the surface is comprehensively analyzed. HUVECs and HUVSMCs planted at the same planting ratio showed different cell activities on different samples. The cellular activity of HUVECs on the surface was generally higher than that of HUVSMCs in terms of overall cellular activity. Compared with the flat and patterned PDMS-Au surface, the flat and patterned pM surface has certain inhibition effect on two kinds of vascular cells, and the flat and patterned pS and pSM surfaces have certain promotion effect on the growth of the two kinds of vascular cells. Among all samples, the surface of the patterned pSM sample was the most potent promoter of vascular cells, and the surface was prominent in promoting cell adhesion and proliferation of HUVECs and HUVSMCs. However, the patterned pSM surface can control the activity of HUVSMCs to 76% while maintaining the activity of HUVECs above 99%.

Example five VEGF adsorption experiments

Each sample was sterilized by soaking in 75% ethanol 3 times for 10 minutes each, and then soaked in sterile PBS 3 times for 10 minutes each, and air-dried in a sterile environment. Each sample was immersed vertically in 250. Mu.L of endothelial cell culture medium and placed in 5% CO ₂ And incubating for 3 hours in a cell incubator in a constant temperature environment of 37 ℃. After removal, the samples were washed twice with 250 μ L PBS. After the sample is taken out, the washing solution and the soak solution are combined into a mixed solution. And (3) testing the residual amount of the VEGF in the mixed solution by using an ELISA special kit for VEGF according to the operation of a kit instruction. Detection by microplate readerAnd measuring the OD value of the mixed solution, and calculating the VEGF adsorption quantity on the surface of the sample. The experiment was performed in triplicate.

VEGF can specifically regulate the behavior of endothelial cells and promote the growth of vascular endothelial cells. Research has shown that heparin and heparinoid polymers interact with VEGF through specific domains. Different adsorption capacities of different heparinoid polymer samples to VEGF may cause differences in adhesion and proliferation of endothelial cells on the surface. Fig. 18 and 19 show the adsorption amount of VEGF on different sample surfaces, wherein VEGF is an important factor capable of promoting the growth of vascular endothelial cells, the distribution of VEGF on the heparin-like heterogeneous patterned surface where pSS and pMAG components coexist can be influenced by the distribution space of the heparin-like polymer, and the pSS component is more favorable for adsorbing VEGF on the heparin-like heterogeneous patterned surface when the PDMS area is modified than the gold membrane area is modified, so that the growth of HUVECs is better promoted.

Researches show that the pattern on the surface of the biological material can influence protein adsorption and cell behavior on the surface, has guiding effect on the growth direction, spreading area and the like of cells, and can generate synergistic effect when some chemical components with special functions are organically combined with the patterned surface to endow the surface of the material with special functions. The method prepares the sulfhydrylation heparinoid polymer by RAFT polymerization and ethanolamine reduction, then adopts visible light to initiate induction graft polymerization, gold film deposition by chloroauric acid reduction, transfer printing and gold-sulfur bond self-assembly to prepare the homogeneous/heterogeneous modified patterned (circular array with the diameter of 300 mu m) gold film surface of the heparinoid polymer, and researches the influence of the homogeneous/heterogeneous patterned surface of the heparinoid polymer on the behavior of vascular cells. The specific conclusions are as follows:

(1) Compared with the unmodified surface, the pS and pSM flat surfaces have the function of promoting the adhesion and proliferation of vascular cells, and the pM flat surface has the function of inhibiting the adhesion and proliferation of vascular cells. After the surface is introduced with the pattern, the patterning topological structure obviously enhances the promoting effect of pS and pSM on vascular cells, particularly HUVECs. The overall enhancement of the patterned pSM surface was optimal in all samples, reaching the highest HUVECs proliferation density, the best spreading state and the highest HUVECs activity.

(2) The PS-S on the homogeneous patterned surface of the heparinoid polymer has a promoting effect on the growth of vascular cells, and the PM-M has an inhibiting effect on the growth of the vascular cells. Compared with PS-S and PM-M, the PS-M and PM-S surfaces with heterogeneous distribution of heparinoid polymers have certain guidance on the adhesion and proliferation of HUVECs and HUVSMCs. Vascular cells, guided by sulfonic acid groups and sugar groups at different areas on the surface, tend to grow in the sulfonic acid group-modified areas more favorable for cell adhesion and proliferation. In addition, PS-M and PM-S surfaces show different vascular cell behaviors, and the proliferation density and the spreading area of vascular cells on the PS-M surface are obviously higher than those on the PM-S surface, which indicates that the cellular behavior of the heparinized material surface containing sulfonic acid groups and sugar groups can be influenced by the spatial distribution mode of surface chemical components. The PS-M surface (pSS components distributed in PDMS area) is more favorable for the proliferation and spreading of vascular cells than the PM-S surface (pSS components distributed in gold area).

(3) VEGF experiments show that a certain relation exists between the adsorption quantity of VEGF on the surface of each sample and the cell density of blood vessels on the surface, the adsorption quantity of VEGF on the surface of the sample can be influenced to further influence the adhesion and proliferation behaviors of HUVECs on the surface, and the adsorption quantity of VEGF on the PS-M surface is obviously higher than that on the PM-S surface.

Claims

1. A preparation method of a patterned surface of a heparan polymer is characterized by comprising the following steps of modifying a heparan polymer on a surface of polydimethylsiloxane containing bromine, transferring a patterned gold film to the surface of polydimethylsiloxane modified by the heparan polymer, and assembling a thiolated heparan polymer on the surface of the gold film through gold-sulfur bond self-assembly to obtain the patterned surface of the heparan polymer.

2. The method of claim 1, wherein the heparinoid polymer is a heparinoid polymer containing sulfonic acid groups, a heparinoid polymer containing sugar groups or a copolymer containing sulfonic acid groups and sugar groups; the molecular weight of the heparinoid polymer is 0.8× 10 ⁴ ～2× 10 ⁴ g mol ^-1 。

3. The method of claim 1, wherein the surface of the polydimethylsiloxane polymer containing bromine is a flat surface; the patterned gold film is a circular array patterned gold film.

4. The method for preparing the patterned surface of heparinoid polymer according to claim 1, wherein the heparinoid polymer having a dithioester bond is prepared by polymerization, and then the dithioester bond at the end of the heparinoid polymer is reduced to a thiol group with ethanolamine to obtain a thiolated heparinoid polymer; the bromine-containing polydimethylsiloxane was prepared by adding 10-undecene-2-bromoisobutyrate to the starting material for preparing the polydimethylsiloxane.

5. The method for preparing the patterned surface of the heparinoid polymer according to claim 1, wherein the bromide-containing polydimethylsiloxane is immersed in a solution containing a heparinoid monomer and a photoinitiator, and then the illumination reaction is performed to modify the heparinoid polymer on the surface of the bromide-containing polydimethylsiloxane; and (3) soaking the polydimethylsiloxane transferred with the patterned gold film into the thiolated heparan polymer solution for 10-25 hours, and assembling the thiolated heparan polymer on the surface of the gold film to obtain the patterned surface of the heparan polymer.

6. The patterned surface of heparinoid polymer prepared according to the method of claim 1.

7. Use of the patterned surface of heparinoid polymers of claim 6 for modulating vascular cell behaviour or for the preparation of a material for modulating vascular cell behaviour.

8. The use of the patterned surface of heparinoid polymer of claim 6 for promoting endothelial cell adhesion proliferation and inhibiting smooth muscle cell adhesion proliferation, or for the preparation of a material for promoting endothelial cell adhesion proliferation and inhibiting smooth muscle cell adhesion proliferation.

9. Use of the heparinoid polymer patterned surface of claim 6 for increasing vascular cell survival or for the preparation of a material for increasing vascular cell survival.

10. Use of the heparinoid polymer patterned surface of claim 6 for increasing adsorption of vascular endothelial growth factor or for the preparation of an adsorption material for increasing adsorption of vascular endothelial growth factor.