Construction method of anti-protein adsorption and anti-thrombus graphene oxide coating
By constructing a graphene oxide coating on the surface of a small-diameter artificial blood vessel and grafting heparin and polyethylene glycol molecules, the problems of insufficient anti-protein adsorption and anti-thrombotic properties in existing technologies have been solved, and the stability and patency of biomaterials have been improved.
Patent Information
- Application Number
- CN202410011048.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-04
- Publication Date
- 2026-01-02
AI Technical Summary
Existing technologies for small-diameter artificial blood vessel products lack interface modification methods that simultaneously possess anti-protein adsorption and anti-thrombotic properties, leading to serious problems in neointimal formation and thrombosis, which affect the stability and patency of blood vessels.
A method for constructing graphene oxide coatings was adopted, in which heparin and polyethylene glycol were grafted onto carboxylated graphene oxide nanoparticles to adjust the charge and molecular weight, thereby modifying the surface of biomaterials and forming a coating that resists protein adsorption and thrombosis.
It significantly improved the anti-protein adsorption and anti-thrombotic properties of biomaterials, improved neointimal formation, and enhanced the patency and stability of small-diameter blood vessels.
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Figure CN121243483A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for constructing a graphene oxide coating that resists protein adsorption and thrombosis. Background Technology
[0002] When small-diameter artificial blood vessels are implanted, the ideal scenario is rapid regeneration of functionalized host cells to achieve perfect antithrombotic and growth properties. However, neointima formation after implantation remains a significant challenge for tissue regeneration. It is well known that protein adsorption, platelet adhesion, and neutrophil infiltration occur immediately upon contact between biomaterials and biological systems. These two events mediate subsequent cell adhesion, proliferation, differentiation, and regeneration, ultimately affecting the fate of the biomaterial. However, current research on cardiovascular biomaterials, particularly small-diameter vessels, rarely focuses on these processes. Modifications to the physicochemical properties and topography of the transplanted vessel interface can significantly influence the regulation of neointima formation and the long-term performance of vascular regeneration. Protein adsorption, mural thrombus formation, neutrophil adhesion, proliferation, and regression have been observed in the early neointima of small-diameter vessels and bovine pericardial valves. The protein adsorption process, as well as subsequent cell adhesion, differentiation, and neointima formation, are not influenced by a single property but by several properties of the material. By designing specific molecules or structures to promote the selective binding of desired protein molecules, or reducing the adsorption of plasma proteins and the binding of blood cells, it may be possible to regulate the formation and development of neointima. During the transplantation of xenograft acellular small-diameter vessels, the lack of endothelial cell coverage exposes the vascular basement membrane, collagen fibers, fibronectin, and other components. Upon contact with the blood system, this can trigger a series of biological reactions, leading to thrombosis or severe neointima formation and ultimately vascular occlusion. Therefore, research on the early stabilization and regenerative repair of small-diameter artificial blood vessels is crucial in the fields of medical products and biomaterials. Research into preparation technologies that enhance the anti-protein adsorption and anti-thrombotic capabilities of small-diameter blood vessels is urgently needed.
[0003] In the field of medical biomaterials, there are currently no small-diameter artificial blood vessel products that meet clinical application requirements and have been commercialized. The most common and effective method is to specifically modify the blood-contact interface to improve its blood compatibility. The most common methods for improving blood compatibility at the material interface include physical, chemical, and biological surface modifications, such as plasma treatment, corona treatment, and gamma-ray laser etching; surface oxidation, chemical grafting, and surface coatings. However, these techniques generally target antibacterial or antithrombotic effects and cannot simultaneously exert anti-protein adsorption and antithrombotic effects, nor do they significantly improve the formation of neointimal membranes. Therefore, there is a need to develop a better interface modification technology to improve the biocompatibility and stability of materials. Summary of the Invention
[0004] The purpose of this invention is to provide a method for constructing a graphene oxide coating that resists protein adsorption and thrombosis, in order to solve the problem mentioned in the background art that there are currently no small-diameter artificial blood vessel products with performance that meet the requirements for clinical application and have been commercialized, and that commonly used processing methods still have defects.
[0005] To achieve the above objectives, the present invention provides a method for constructing a graphene oxide coating with anti-protein adsorption and anti-thrombotic properties, comprising the following steps:
[0006] Step 1: Carboxylate graphene oxide to obtain carboxylated graphene oxide nanoparticles.
[0007] Step 2: Graft heparin onto carboxylated graphene oxide nanoparticles;
[0008] Step 3: The combination of graphene oxide-heparin nanoparticles and polyethylene glycol includes the following specific steps: The graphene oxide-heparin nanoparticles obtained in Step 2 are prepared into an aqueous solution, and EDC and NHS are added for reaction activation; after activation, straight-chain or four-arm aminated polyethylene glycol with a molecular weight of 4000-8000 is added, and the pH of the reaction system is adjusted to 7.2-7.6. The graphene oxide-heparin nanoparticles are redispersed under ultrasound, with a polyethylene glycol to graphene oxide mass ratio of 3-5:1. The reaction system is continuously stirred; half the mass of the previously added EDC is added, and the pH of the reaction system is controlled at 7.2-7.6; after the reaction, dialyze using a first dialysis bag. After dialysis, graphene oxide-heparin-polyethylene glycol nanoparticles are obtained.
[0009] Step 4: Combining graphene oxide-heparin-polyethylene glycol nanoparticles with biomaterials, including the following specific steps: Preparing graphene oxide-heparin-polyethylene glycol nanoparticles into an aqueous solution, adding EDC and NHS for activation; adjusting the pH to 7.2–7.6, ultrasonically dispersing the nanoparticles, placing the biomaterials in the solution for reaction; after the reaction, rinsing the biomaterials with PBS to obtain biomaterials modified with a graphene oxide-heparin-polyethylene glycol nanocoating. The coating on the biomaterials is the graphene oxide coating with anti-protein adsorption and anti-thrombotic properties.
[0010] In one specific embodiment, the biomaterial is a decellularized small-diameter blood vessel or glutaraldehyde-fixed bovine pericardium biomaterial.
[0011] In one specific implementation, step 1 includes the following specific steps:
[0012] Graphene oxide nanoparticles were prepared into an aqueous solution. Sodium hydroxide solution was added first, and the reaction was carried out under ultrasonic dispersion. Sodium hypochlorite solution was then added, and the reaction was carried out under ultrasonic dispersion. After the reaction was completed, the mixture was centrifuged, the supernatant was discarded, and the graphene oxide was washed with dilute hydrochloric acid. The mixture was centrifuged again, and the graphene oxide was washed with deionized water until the pH value was 7.0-7.6. Finally, the mixture was dialyzed using a second dialysis bag to obtain carboxylated graphene oxide.
[0013] In one specific implementation, in step 1,
[0014] The graphene oxide nanoparticles are 50-500 nm in size. The concentration of the aqueous solution of the graphene oxide nanoparticles is 0.5 mg / ml to 1 mg / ml. The final concentration of the sodium hydroxide solution is 5 mmol / ml to 10 mmol / ml. The ultrasonic dispersion reaction time after adding the sodium hydroxide solution is 1-2 hours. The final concentration of the sodium hypochlorite solution is 3 mmol / ml to 5 mmol / ml. The ultrasonic dispersion reaction time after adding the sodium hypochlorite solution is 4-5 hours. The centrifugation speed is 2000-4000 rpm. The concentration of dilute hydrochloric acid is 1%. The dilute hydrochloric acid washing and centrifugation are performed 3 times. The second dialysis bag is a 5 KD dialysis bag. The dialysis time is 24-48 hours.
[0015] In one specific implementation, step 2 includes the following specific steps:
[0016] First, prepare a heparin aqueous solution, add EDC, and add adipic acid dihydrazide to make the mass ratio of adipic acid dihydrazide to heparin 3-5:1, and adjust the pH to 7.2-7.6. Stir the reaction at room temperature, add half the mass of EDC added earlier, and control the pH of the reaction system at 7.2-7.6; dialyze using a third dialysis bag, and freeze-dry after dialysis to obtain amino-grafted heparin;
[0017] The carboxylated graphene oxide nanoparticles obtained in step 1 were prepared into an aqueous solution. EDC and NHS were added, and the pH was adjusted to 5.2–5.8. The reaction was carried out under ultrasonic dispersion in the dark to activate the carboxyl groups of the graphene oxide nanoparticles. Amino-grafted heparin was added to make the mass ratio of heparin to graphene oxide nanoparticles 5–10:1. The pH was adjusted to 7.2–7.6, and the graphene oxide-heparin nanoparticles were dispersed again under ultrasonic dispersion. The reaction was carried out at room temperature in the dark. Half the mass of EDC added previously was added, and the pH of the reaction system was controlled at 7.2–7.6. After the reaction was completed, the nanoparticles were dialyzed using a fourth dialysis bag. After dialysis, they were lyophilized for later use to obtain graphene oxide-heparin nanoparticles.
[0018] In one specific embodiment, the concentration of the heparin aqueous solution is 5 mg / ml to 10 mg / ml, the concentration of the added EDC is 5 to 10 mg / ml, and the stirring reaction time at room temperature is 24 to 36 hours; the concentration of the aqueous solution prepared from carboxylated graphene oxide nanoparticles is 0.5 mg / ml to 1 mg / ml, and the concentration of the added NHS is 3 to 8 mg / ml.
[0019] In one specific embodiment, the light-protected reaction time before activating the carboxyl groups of the graphene oxide nanoparticles is 30 min to 60 min, and the reaction time at room temperature under light-protected conditions after ultrasonic redispersion of the graphene oxide-heparin nanoparticles is 24 to 36 hours. The third dialysis bag is a 5KD dialysis bag, and the fourth dialysis bag is a 50KD dialysis bag. The dialysis time for both is 24 to 48 hours.
[0020] In one specific embodiment, the dialysis time of the dialysis bag is 24-48 hours; in steps 3 and 4, during the reaction activation, the final concentration of added EDC is 5-10 mg / ml, the final concentration of added NHS is 3-8 mg / ml, the pH is adjusted to 5.2-5.8, and the reaction is carried out under ultrasonic dispersion and in the dark for 30-60 minutes.
[0021] In one specific embodiment, in step 3, the concentration of the aqueous solution of graphene oxide-heparin nanoparticles is 0.5–1 mg / ml, and the continuous stirring reaction time is 24–48 h. The continuous stirring reaction is carried out using a magnetic stir bar with a stirring speed of 500–800 rpm. The first dialysis bag is a 50 kDa dialysis bag, and the dialysis time is 24–48 h. After dialysis, the obtained graphene oxide-heparin-polyethylene glycol nanoparticles need to be lyophilized for storage.
[0022] In one specific embodiment, in step 4, the concentration of the aqueous solution of graphene oxide-heparin-polyethylene glycol nanoparticles is 1-2 mg / ml, and the reaction time for placing the biomaterial in the solution is 12-24 hours.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] This invention improves the biocompatibility of medical cardiovascular biomaterials, enhances their anti-protein adsorption and anti-thrombotic properties, and improves the stability of biomaterials in vivo. This invention also regulates the formation of new intima in vivo, improving the patency rate of artificial small-diameter blood vessels.
[0025] This invention grafts heparin and polyethylene glycol molecules onto graphene oxide, fully utilizing the effects of both. After modifying the biomaterial, it significantly enhances its ability to resist protein adsorption and mural thrombosis, which is more conducive to achieving better patency of small-diameter artificial blood vessels in vivo.
[0026] The charge of the heparin-grafted graphene oxide-heparin nanoparticles of the present invention is controlled at around -15mV to -20mV. Polyethylene glycol of appropriate molecular weight further reduces the charge of the graphene oxide-heparin nanoparticles to around -25mV to -30mV, which is close to the surface charge of the cell membrane, thus better exerting the anti-protein adsorption effect.
[0027] In this invention, the grafting sequence of the nanoparticles is as follows: using graphene oxide as a carrier, heparin molecules are grafted first, followed by polyethylene glycol molecules. By modifying the nanoparticles onto biomaterials, the anticoagulant properties of heparin molecules can be preferentially utilized to inhibit thrombus formation, while the grafted polyethylene glycol plays an anti-protein adsorption role.
[0028] In each step of the graphene oxide reaction process, the pH is controlled at around 7.2 to 7.6, and the graphene oxide is ultrasonically dispersed to control its aggregation and improve grafting efficiency.
[0029] Figure 5 The study showed that after the nano-coating was grafted onto small-diameter blood vessels, all exposed fibers of the basement membrane were covered by nanoparticles, forming a relatively smooth interface. This indicates that the grafting efficiency of graphene-heparin-polyethylene glycol nanoparticles onto blood vessels is very high, and the coverage is complete. This alters the interfacial properties of decellularized small-diameter blood vessels, resulting in anti-protein adsorption and anti-platelet adhesion effects. Figure 6 and Figure 7 This also indicates that the coating is performing well.
[0030] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description
[0031] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:
[0032] Figure 1 This is a comparison diagram of the Zeta potentials of different groups of graphene oxide nanoparticles;
[0033] All three groups of nanoparticles were negatively charged, and there were significant differences between the groups. GC was carboxylated graphene oxide, GH was graphene oxide-heparin nanoparticles, and GHP was graphene oxide-heparin-polyethylene glycol nanoparticles. "***" represents P<0.0001.
[0034] Figure 2 This is a comparison chart of the particle size measurement results for each group of graphene oxide nanoparticles:
[0035] Among them, GC is carboxylated graphene oxide, GH is graphene oxide-heparin nanoparticles, and GHP is graphene oxide-heparin-polyethylene glycol nanoparticles.
[0036] Figure 3 Here are the infrared spectra of each group of nanoparticles:
[0037] Where Wavenumber represents wavelength; Transmittance represents transmittance. GO represents carboxylated graphene oxide; GC represents carboxylated graphene oxide nanoparticles; GH represents heparin-grafted carboxylated graphene oxide nanoparticles; GHP represents heparin and PEG-grafted graphene oxide nanoparticles.
[0038] Figure 4 A- Figure 4 Display D shows the full spectrum of elements and their content in each group of nanoparticles, as well as the elemental orbitals and their contents corresponding to each peak position. Binding energy represents the binding energy; intensity represents the spectral line intensity. GO represents carboxylated graphene oxide; GC represents carboxylated graphene oxide nanoparticles; GH represents heparin-grafted carboxylated graphene oxide nanoparticles; GHP represents heparin and PEG-grafted graphene oxide nanoparticles. C represents carbon; O represents oxygen; N represents nitrogen; and S represents sulfur.
[0039] Figure 5 The following are the scanning electron microscopy and water contact angle results of each group of nanoparticle-modified decellularized small-diameter blood vessels:
[0040] Figure 5 A and Figure 5 In B, SEM images of the three groups of blood vessels showed that the fibrous course and voids of the vascular basement membrane were visible in the DO group, while the vascular basement membrane fibers in the GC and GHP groups were completely covered by nanoparticles, with no fibers exposed. Figure 5 A shows the 5000x images in each group. Figure 5 B displays the 10000x images for each group. Figure 5C shows the hydrophilicity results of each group of blood vessels. The water contact angle of the DO group was significantly higher than that of the GC and GHP groups; n=5; where Water contact angle is the water contact angle. DO is decellularized photooxidized bovine internal artery; GC is DO blood vessel modified with carboxylated graphene oxide nanoparticles; GHP is DO blood vessel modified with graphene oxide-heparin-polyethylene glycol nanoparticles.
[0041] Figure 6 These are the platelet adhesion results of blood vessels modified with various nano-coatings:
[0042] Figure 6 A and Figure 6 In B, the antiplatelet adhesion assay results of the three groups of blood vessels showed that a large number of platelets adhered and activated on the vascular lumen surface of the DO-BIMA and GC-BIMA groups. Only a small number of platelets adhered on the vascular lumen surface of the GHP-BIMA group. Figure 6 A shows the 10000x images in each group. Figure 6 B displays the images for each group at 30000x. Figure 6 The levels of LDH enzyme in the blood vessels of each group shown in C were significantly lower in the GHP-BIMA group than in the other two groups, and the LDH level in the GC-BIMA group was also significantly lower than that in the DO group.
[0043] Wherein, LDH represents lactate dehydrogenase; DO represents decellularized photooxidized bovine internal artery; GC represents DO vessels modified with carboxylated graphene oxide nanoparticles; and GHP represents DO vessels modified with graphene oxide-heparin-polyethylene glycol nanoparticles.
[0044] Figure 7 These are the results of various groups of nano-coatings modifying blood vessels to resist the adsorption of BSA-FITC and FBG-FITC proteins:
[0045] Experimental results showed that the fluorescence intensity of the DO-BIMA group was significantly higher than that of the GC-BIMA and GHP-BIMA groups. Figure 7 A shows the fluorescence image in DO-BIMA; Figure 7 B shows the fluorescence image in GC-BIMA; Figure 7 C shows the fluorescence image in GHP-BIMA. Figure 7 D and Figure 7 E shows the quantitative analysis of three groups of green fluorescence intensities; n=6. BSA-FITC represents bovine serum albumin labeled with green fluorescence; FBG-FITC represents fibrinogen labeled with green fluorescence. Green Intensity indicates the intensity of green fluorescence. DO represents decellularized photooxidized bovine internal artery; GC represents DO vessels modified with carboxylated graphene oxide nanoparticles; GHP represents DO vessels modified with graphene oxide-heparin-polyethylene glycol nanoparticles.
[0046] Figure 8 It is a calf pericardium material modified with GHP nanoparticles and crosslinked with glutaraldehyde:
[0047] Figure 8 This image shows the SEM results of smooth and rough surfaces of bovine pericardium modified with GHP nanoparticles and cross-linked with glutaraldehyde. In the glutaraldehyde-cross-linked group alone, fibrous patterns and voids are visible. After GHP coating modification, the fibers and voids in both the smooth and rough surfaces of the pericardium are completely covered by nanoparticles, with no fibers exposed. The image shown is at 30000x. GA represents glutaraldehyde-cross-linked bovine pericardium; GHP represents glutaraldehyde-cross-linked bovine pericardium modified with graphene oxide-heparin-polyethylene glycol nanoparticles. S represents the smooth surface of the bovine pericardium, and R represents the rough surface. Detailed Implementation
[0048] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.
[0049] Example 1
[0050] Nanoparticles of graphene oxide with a size of 50nm to 200nm were used to modify decellularized bovine mammary arteries.
[0051] 1. Carboxylation of graphene oxide
[0052] Prepare a 1 mg / ml aqueous solution of graphene oxide nanoparticles (50–200 nm). First, add sodium hydroxide to a final concentration of 5 mmol / ml and react under ultrasonic dispersion for 2 hours. Then, add sodium hypochlorite to a final concentration of 5 mmol / ml and react under ultrasonic dispersion for 5 hours. After the reaction, centrifuge at 2000 rpm, discard the supernatant, add 1% dilute hydrochloric acid to wash the graphene oxide, centrifuge again, and wash a total of 3 times. Then, add deionized water to wash the graphene oxide until the pH value is 7.2. Finally, dialyze using a 5 KD dialysis bag for 48 hours. Carboxylated graphene oxide can be obtained, and the graphene oxide solution can be observed to change from pale yellow to black.
[0053] 2. Carboxylated graphene oxide nanoparticles grafted with heparin
[0054] First, prepare a 5 mg / ml aqueous solution of heparin (average molecular weight 12000). Add EDC to achieve a final EDC concentration of 5 mg / ml. Then, add adipic acid dihydrazide to achieve a mass ratio of adipic acid dihydrazide to heparin of 3:1. Adjust the pH to 7.4 and stir the reaction at room temperature for 24 hours. Monitor the pH change every 12 hours, adding half the previously added EDC mass each time, maintaining the pH of the reaction system at 7.4. Dialyze the mixture using a 5 kDa dialysis bag for 24 hours. After dialysis, freeze-dry the mixture for later use to obtain amino-grafted heparin.
[0055] The synthesized carboxylated graphene oxide nanoparticles were prepared into a 0.5 mg / ml aqueous solution. EDC / NHS was added to achieve a final EDC concentration of 5 mg / ml and a final NHS concentration of 3 mg / ml. The pH was adjusted to 5.5, and the mixture was ultrasonically dispersed and reacted in the dark for 30 min to activate the carboxyl groups of the graphene oxide nanoparticles. Amino-grafted heparin was then added to achieve a heparin-to-graphene oxide nanoparticle mass ratio of 5:1. The pH was adjusted to 7.4, and the graphene oxide-heparin nanoparticles were ultrasonically dispersed again. The reaction was carried out at room temperature in the dark for 24 hours. The pH was monitored every 12 hours, and half the previously added EDC mass was added to maintain the pH at 7.4. After the reaction, the mixture was dialyzed through a 50 kDa dialysis bag for 48 hours. After dialysis, the nanoparticles were lyophilized for later use to obtain graphene oxide-heparin nanoparticles.
[0056] 3. Combination of graphene oxide-heparin nanoparticles with polyethylene glycol
[0057] The prepared graphene oxide-heparin nanoparticles were dissolved in water at a concentration of 1 mg / ml. EDC / NHS was added to achieve a final EDC concentration of 8 mg / ml and a final NHS concentration of 5 mg / ml. The pH was adjusted to 5.5. The mixture was then ultrasonically dispersed and reacted in the dark for 60 min to activate the carboxyl groups on the graphene oxide and heparin. After activation, straight-chain or four-arm amino-modified polyethylene glycol (PEG) with a molecular weight of 4000 was added. The pH of the reaction system was adjusted to 7.5. The graphene oxide-heparin nanoparticles were redispersed ultrasonically at a PEG-to-graphene mass ratio of 4:1. The reaction system was continuously stirred on a magnetic stir bar at 600 rpm for 48 h. The pH was monitored every 12 hours, and half the amount of EDC added was added to maintain the pH at 7.5. After the reaction, the mixture was dialyzed for 48 h using a 50 kDa dialysis bag. After dialysis, the nanoparticles were lyophilized for later use to obtain graphene oxide-heparin-PEG nanoparticles.
[0058] 4. Combination of graphene oxide-polyethylene glycol-heparin nanoparticles with biomaterials
[0059] Graphene oxide-heparin-polyethylene glycol nanoparticles were prepared into a 1 mg / ml aqueous solution. EDC / NHS was added to achieve a final EDC concentration of 5 mg / ml and a final NHS concentration of 3 mg / ml. The pH was adjusted to 5.5, and the mixture was ultrasonically dispersed and reacted in the dark for 30 min to activate the carboxyl groups on the graphene oxide. The pH was then adjusted to 7.5, and the mixture was ultrasonically dispersed thoroughly. Decellularized small-diameter blood vessels were placed in the solution and reacted for 12 hours. After the reaction, the small-diameter blood vessel material was rinsed with PBS to obtain a biomaterial modified with a graphene oxide-heparin-polyethylene glycol nanocoating. Scanning electron microscopy showed that graphene oxide was successfully loaded onto the basement membrane, completely covering components such as collagen fibers.
[0060] Example 2
[0061] Glutaraldehyde-crosslinked bovine pericardium was constructed using graphene oxide nanoparticles with a size of 200nm–500nm.
[0062] 1. Carboxylation of graphene oxide
[0063] Prepare a 1 mg / ml aqueous solution of graphene oxide nanoparticles with a density of 200 nm to 500 nm. First, add sodium hydroxide to a final concentration of 8 mmol / ml and react under ultrasonic dispersion for 2 hours. Then, add sodium hypochlorite to a final concentration of 8 mmol / ml and react under ultrasonic dispersion for 5 hours. After the reaction, centrifuge at 3000 rpm, discard the supernatant, add 1% dilute hydrochloric acid to wash the graphene oxide, centrifuge again, and wash three times in total. Then, wash the graphene oxide with deionized water until the pH reaches 7.2. Finally, dialyze using a 5 KD dialysis bag for 48 hours. Carboxylated graphene oxide is obtained, and the graphene oxide solution changes from pale yellow to black.
[0064] 2. Carboxylated graphene oxide nanoparticles grafted with heparin
[0065] First, prepare a 5 mg / ml aqueous solution of heparin (average molecular weight 12000). Add EDC to achieve a final EDC concentration of 5 mg / ml. Then, add adipic acid dihydrazide to achieve a mass ratio of adipic acid dihydrazide to heparin of 3:1. Adjust the pH to 7.4 and stir the reaction at room temperature for 24 hours. Monitor the pH change every 12 hours, adding half the previously added EDC mass each time, maintaining the pH of the reaction system at 7.4. Dialyze the mixture using a 5 kDa dialysis bag for 24 hours. After dialysis, freeze-dry the mixture for later use to obtain amino-grafted heparin.
[0066] The synthesized carboxylated graphene oxide nanoparticles were prepared into a 0.5 mg / ml aqueous solution. EDC / NHS was added to achieve a final EDC concentration of 6 mg / ml and a final NHS concentration of 4 mg / ml. The pH was adjusted to 5.5, and the mixture was ultrasonically dispersed and reacted in the dark for 30 min to activate the carboxyl groups of the graphene oxide nanoparticles. Amino-grafted heparin was then added to achieve a heparin-to-graphene oxide nanoparticle mass ratio of 6:1. The pH was adjusted to 7.4, and the graphene oxide-heparin nanoparticles were ultrasonically dispersed again. The reaction was carried out at room temperature in the dark for 24 hours. The pH was monitored every 12 hours, and half the amount of EDC added was added to maintain the pH at 7.4. After the reaction, the mixture was dialyzed through a 50 kDa dialysis bag for 48 hours. After dialysis, the nanoparticles were lyophilized for later use to obtain graphene oxide-heparin nanoparticles.
[0067] 3. Combination of graphene oxide-heparin nanoparticles with polyethylene glycol
[0068] The prepared graphene oxide-heparin nanoparticles were prepared into a 1 mg / ml aqueous solution. EDC / NHS was added to achieve a final EDC concentration of 8 mg / ml and a final NHS concentration of 5 mg / ml. The pH was adjusted to 5.5, and the mixture was ultrasonically dispersed and reacted in the dark for 60 min to activate the carboxyl groups on the graphene oxide and heparin. After activation, straight-chain or four-arm amino-modified polyethylene glycol with a molecular weight of 4000 was added, and the pH of the reaction system was adjusted to 7.5. The graphene oxide-heparin nanoparticles were redispersed ultrasonically, with a polyethylene glycol to graphene oxide mass ratio of 5:1. The reaction system was continuously stirred on a magnetic stir bar at 600 rpm for 48 h. The pH change was monitored every 12 hours, and half the mass of the previously added EDC was added to maintain the pH at 7.5. After the reaction, the mixture was dialyzed for 48 h using a 50 kDa dialysis bag. After dialysis, the nanoparticles were lyophilized for later use to obtain graphene oxide-heparin-polyethylene glycol nanoparticles.
[0069] 4. Combination of graphene oxide-polyethylene glycol-heparin nanoparticles with biomaterials
[0070] Graphene oxide-polyethylene glycol-heparin nanoparticles were prepared into a 1 mg / ml aqueous solution. EDC / NHS was added to achieve a final EDC concentration of 5 mg / ml and a final NHS concentration of 3 mg / ml. The pH was adjusted to 5.5, and the mixture was ultrasonically dispersed and reacted in the dark for 30 min to activate the carboxyl groups on the graphene oxide. The pH was then adjusted to 7.5, and the mixture was ultrasonically dispersed thoroughly. Glutaraldehyde-crosslinked bovine pericardium was placed in the solution and reacted for 12 hours. After the reaction, the bovine pericardium material was washed with PBS to obtain a biomaterial modified with a graphene oxide-polyethylene glycol-heparin nanocoating. Scanning electron microscopy showed that graphene oxide was successfully loaded onto the pericardium membrane, completely covering components such as collagen fibers.
[0071] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions and substitutions can be made without departing from the inventive concept, and all such modifications and substitutions should be considered within the scope of protection of the present invention.
Claims
1. A method for constructing a graphene oxide coating with anti-protein adsorption and anti-thrombotic properties, characterized in that, Includes the following steps: Step 1: Carboxylate graphene oxide to obtain carboxylated graphene oxide nanoparticles. Step 2: Graft heparin onto carboxylated graphene oxide nanoparticles; Step 3: The combination of graphene oxide-heparin nanoparticles and polyethylene glycol includes the following specific steps: The graphene oxide-heparin nanoparticles obtained in Step 2 are prepared into an aqueous solution, and EDC and NHS are added for reaction activation; after activation, straight-chain or four-arm aminated polyethylene glycol with a molecular weight of 4000-8000 is added, and the pH of the reaction system is adjusted to 7.2-7.
6. The graphene oxide-heparin nanoparticles are redispersed under ultrasound, with a polyethylene glycol to graphene oxide mass ratio of 3-5:
1. The reaction system is continuously stirred; half the mass of the previously added EDC is added, and the pH of the reaction system is controlled at 7.2-7.6; after the reaction, dialyze using a first dialysis bag. After dialysis, graphene oxide-heparin-polyethylene glycol nanoparticles are obtained. Step 4: Combining graphene oxide-heparin-polyethylene glycol nanoparticles with biomaterials, including the following specific steps: Preparing graphene oxide-heparin-polyethylene glycol nanoparticles into an aqueous solution, adding EDC and NHS for activation; adjusting the pH to 7.2–7.6, ultrasonically dispersing the nanoparticles, placing the biomaterials in the solution for reaction; after the reaction, rinsing the biomaterials with PBS to obtain biomaterials modified with a graphene oxide-heparin-polyethylene glycol nanocoating. The coating on the biomaterials is the graphene oxide coating with anti-protein adsorption and anti-thrombotic properties.
2. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 1, characterized in that, The biomaterial is a decellularized small-diameter blood vessel or bovine pericardium biomaterial fixed with glutaraldehyde.
3. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 1, characterized in that, Step 1 includes the following specific steps: Graphene oxide nanoparticles were prepared into an aqueous solution. Sodium hydroxide solution was added first, and the reaction was carried out under ultrasonic dispersion. Sodium hypochlorite solution was then added, and the reaction was carried out under ultrasonic dispersion. After the reaction was completed, the mixture was centrifuged, the supernatant was discarded, and the graphene oxide was washed with dilute hydrochloric acid. The mixture was centrifuged again, and the graphene oxide was washed with deionized water until the pH value was 7.0-7.
6. Finally, the mixture was dialyzed using a second dialysis bag to obtain carboxylated graphene oxide.
4. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 3, characterized in that, In step 1, The graphene oxide nanoparticles are 50-500 nm in size. The concentration of the aqueous solution of the graphene oxide nanoparticles is 0.5 mg / ml to 1 mg / ml. The final concentration of the sodium hydroxide solution is 5 mmol / ml to 10 mmol / ml. The ultrasonic dispersion reaction time after adding the sodium hydroxide solution is 1-2 hours. The final concentration of the sodium hypochlorite solution is 3 mmol / ml to 5 mmol / ml. The ultrasonic dispersion reaction time after adding the sodium hypochlorite solution is 4-5 hours. The centrifugation speed is 2000-4000 rpm. The concentration of dilute hydrochloric acid is 1%. The dilute hydrochloric acid washing and centrifugation are performed 3 times. The second dialysis bag is a 5 KD dialysis bag. The dialysis time is 24-48 hours.
5. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 1, characterized in that, Step 2 includes the following specific steps: First, prepare a heparin aqueous solution, add EDC, and add adipic acid dihydrazide to make the mass ratio of adipic acid dihydrazide to heparin 3-5:1, and adjust the pH to 7.2-7.
6. Stir the reaction at room temperature, add half the mass of EDC added earlier, and control the pH of the reaction system at 7.2-7.6; dialyze using a third dialysis bag, and freeze-dry after dialysis to obtain amino-grafted heparin; The carboxylated graphene oxide nanoparticles obtained in step 1 were prepared into an aqueous solution. EDC and NHS were added, and the pH was adjusted to 5.2–5.
8. The reaction was carried out under ultrasonic dispersion in the dark to activate the carboxyl groups of the graphene oxide nanoparticles. Amino-grafted heparin was added to make the mass ratio of heparin to graphene oxide nanoparticles 5–10:
1. The pH was adjusted to 7.2–7.6, and the graphene oxide-heparin nanoparticles were dispersed again under ultrasonic dispersion. The reaction was carried out at room temperature in the dark. Half the mass of EDC added previously was added, and the pH of the reaction system was controlled at 7.2–7.
6. After the reaction was completed, the nanoparticles were dialyzed using a fourth dialysis bag. After dialysis, they were lyophilized for later use to obtain graphene oxide-heparin nanoparticles.
6. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 5, characterized in that, The concentration of the heparin aqueous solution is 5 mg / ml to 10 mg / ml, the concentration of the added EDC is 5 to 10 mg / ml, and the stirring reaction time at room temperature is 24 to 36 hours; the concentration of the aqueous solution prepared from carboxylated graphene oxide nanoparticles is 0.5 mg / ml to 1 mg / ml, and the concentration of the added NHS is 3 to 8 mg / ml.
7. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 5, characterized in that, The light-protected reaction time before activating the carboxyl groups of graphene oxide nanoparticles is 30 min to 60 min. The reaction time at room temperature and in the dark after ultrasonic redispersing of graphene oxide-heparin nanoparticles is 24 to 36 hours. The third dialysis bag is a 5KD dialysis bag, and the fourth dialysis bag is a 50KD dialysis bag. The dialysis time for both is 24 to 48 hours.
8. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 1, characterized in that, The dialysis time using the dialysis bag is 24–48 h; in steps 3 and 4, during the reaction activation, the final concentration of added EDC is 5–10 mg / ml, the final concentration of added NHS is 3–8 mg / ml, the pH is adjusted to 5.2–5.8, and the reaction is carried out under ultrasonic dispersion in the dark for 30–60 min.
9. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 1, characterized in that, In step 3, the concentration of the aqueous solution of graphene oxide-heparin nanoparticles is 0.5–1 mg / ml, and the continuous stirring reaction time is 24–48 h. The continuous stirring reaction is carried out using a magnetic stir bar with a stirring speed of 500–800 rpm. The first dialysis bag is a 50 kDa dialysis bag, and the dialysis time is 24–48 h. After dialysis, the obtained graphene oxide-heparin-polyethylene glycol nanoparticles need to be lyophilized for storage.
10. The method for constructing the anti-protein adsorption and anti-thrombotic graphene oxide coating according to claim 1, characterized in that, In step 4, the concentration of the aqueous solution prepared from graphene oxide-heparin-polyethylene glycol nanoparticles is 1-2 mg / ml, and the reaction time for placing the biomaterial in the solution is 12-24 hours.