GREEN AND BROAD-SPECTRUM PROTEIN CROSS-LINKING METHOD

Abstract

The present invention belongs to the technical field of medical biological materials, in particular to a green and broad-spectrum protein cross-linking method. In the method, a mixed solution containing protein and silver ions is irradiated by a visible light source with a compound wavelength to obtain cross-linked protein materials with uniform morphology. Compared with the traditional protein cross-linking method, the preparation method does not involve toxic chemical reagents and is environmentally friendly; the method can cross-link a variety of proteins in a broad spectrum; the preparation method can ensure the original activity of protein to the greatest extent; the preparation method also has the advantages of simple steps and easy operation; the cross-linked protein prepared by the method has good biological activity and antibacterial properties, and has great application prospect.

Claims

1. A protein cross-linking method is characterized in that the method comprises the following steps: a compound wavelength light source or a single wavelength light source with a wavelength range of 380-780 nm is used to irradiate a mixed solution containing protein and Ag.sup.+.

2. A method for preparing cross-linked protein is characterized in that the method comprises the following steps: a compound wavelength light source or a single wavelength light source with a wavelength range of 380-780 nm is used to irradiate a mixed solution containing protein and Ag.sup.+, and then the cross-linked protein is obtained after reaction and centrifugation.

3. The method, as stated in claim 2, is characterized in that the concentration ratio of Ag.sup.+ to protein in the mixed solution is 1:1-100.

4. The method, as stated in claim 3, is characterized in that the concentration ratio of Ag.sup.+ to protein in the mixed solution is 1:10.

5. The method, as stated in claim 2, is characterized in that the reaction temperature is 0-37° C.

6. The method, as stated in claim 2, is characterized in that the reaction time is 18 min-24 h.

7. The method, as stated in claim 6, is characterized in that the reaction time is 12 h.

8. The method, as stated in claim 2, is characterized in that the proteins include collagen, bovine serum albumin, human serum albumin, egg white, casein, pepsin, and papain.

9. The method, as stated in claim 8, is characterized in that the protein is collagen.

10. The cross-linked protein prepared by any of the methods stated in claim 9.

11. The application of cross-linked protein, as stated in claim 10, in the preparation of hemostatic materials, drug sustained-release carrier materials, tissue engineering scaffold materials, artificial skin, bionic teeth, artificial blood vessels, bone repair materials and corneal graft materials.

Description

DESCRIPTION OF FIGURES

[0023] FIG. 1 Ultraviolet (UV) chromatographic, energy dispersive X-ray analysis (EDX), and X-ray photoelectron spectroscopy (XPS) images of cross-linked collagen, in which FIG. 1A (a-g in the figure respectively are the cross-linked collagen a-g prepared in Embodiment 1) is the UV detection image, and FIG. 1B is the EDX detection image of the cross-linked collagen a prepared in Embodiment 1, and FIG. 1C and FIG. 1D are the XPS detection images of the cross-linked collagen a prepared in Embodiment 1;

[0024] FIG. 2 Scanning electron microscope (SEM) image, transmission electron microscope (TEM) image, infrared chromatogram (IR) image and thermogravimetric analysis (TGA) image of cross-linked collagen, in which FIG. 2a is the SEM image, FIG. 2b is local amplification of FIG. 2a, FIG. 2c is the TEM image, FIG. 2d is local amplification of FIG. 2c, FIG. 2e is the IR detection image, and FIG. 2f is the TGA detection image;

[0025] FIG. 3 TEM images of cross-linked collagen prepared at different cross-linking times, in which the cross-linking times of FIG. 3a-f are 18 min, 30 min, 1 h, 3 h, 6 h, and 10 h, respectively;

[0026] FIG. 4 UV and TGA images of collagen prepared at different cross-linking times, in which FIG. 4a is the UV image and FIG. 4b is the TGA image;

[0027] FIG. 5 SEM and TEM images of cross-linked collagen prepared with different concentrations of collagen, in which FIG. 5a-d (protein concentrations are 1 mg/ml, 3 mg/ml, 5 mg/ml and 7 mg/ml, respectively) are SEM images, and FIG. 5e-h (protein concentrations are 1 mg/ml, 3 mg/ml, 5 mg/ml and 7 mg/ml, respectively) are TEM images;

[0028] FIG. 6 SEM and TEM images of cross-linked collagen prepared with different concentrations of AgNO.sub.3, in which FIG. 6a-d (AgNO.sub.3 concentrations are 0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml and 1 mg/ml, respectively) are SEM images, and FIG. 6e-h (AgNO.sub.3 concentrations are 0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml and 1 mg/ml, respectively) are TEM images;

[0029] FIG. 7 SEM and TEM images of cross-linked collagen prepared at different pH values, in which FIG. 7a-d (pH 3, 5, 7, 9) are SEM images, and FIG. 7e-h (pH 3, 5, 7, 9) are TEM images;

[0030] FIG. 8 SEM and TEM images of other cross-linked proteins, in which FIG. 8A-F (the proteins are bovine serum albumin, casein, human serum albumin, pepsin, papain and egg white, respectively) are TEM images, and FIG. 8a-f (the proteins are bovine serum albumin, casein, human serum albumin, pepsin, papain and egg white, respectively) are SEM images.

MODE OF CARRYING OUT THE INVENTION

[0031] In order to make the technical means, creative features, achievement goals and effects realized by the present invention easy to understand, the present invention is further described below in combination with the specific implementation mode. However, the protection scope of the present invention is not limited to the following embodiments. The methods used in one or more of the following embodiments are conventional unless otherwise specified; the materials, reagents, and other items used can be obtained commercially unless otherwise specified.

[0032] The proteins described in one or more of the following embodiments include collagen (CL), bovine serum albumin (BSA), casein, human serum albumin (HSA), pepsin, papain, and egg white.

[0033] The collagen (CL) described in one or more of the following embodiments is a biopolymer, which is the main component of connective tissue of animals and the most abundant and widely distributed functional protein in mammals. It consists of three polypeptide chains with a left-handed helical structure intertwined to form a right-handed helical structure.

[0034] The collagen (CL) described in one or more of the following embodiments may be collagen prepared from a variety of natural sources or by other means, such as natural collagen, recombinant collagen, and bionic collagen.

[0035] The Ag.sup.+ solution described in one or more of the following embodiments is AgNO.sub.3 solution, and any Ag.sup.+ solution prepared with other soluble silver salts can be used for protein cross-linking.

[0036] The wavelength of visible light described in one or more of the following embodiments is 380-782 nm, and the light source is an incandescent lamp, but is not limited to the incandescent lamp. The light source can also be fluorescent lamp, flashlight, searchlight and any other light source that can emit visible light with a wavelength of 380-782 nm.

[0037] One or more of the following embodiments are carried out at room temperature, but it should be noted that the present invention realizes protein cross-linking without affecting protein stability and activity. Therefore, protein cross-linking can occur at temperatures that can maintain protein stability and activity (0-37° C.).

[0038] In one or more of the following embodiments where the reaction pH value is not specifically specified, the reaction pH value is 7.

[0039] The specific reaction parameters such as protein type, concentration, reaction time and lighting condition involved in protein cross-linking in all the following embodiments are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Protein AgNO.sub.3 Concen- Concen- Type of tration tration Reaction pH S/N Protein (mg/ml) (mg/ml) Light Time value 1 Collagen 5 0.5 Yes 24 h 7 2 Collagen 5 0.5 No 24 h 7 3 Collagen 5 0.5 Yes 0 h 7 4 Collagen 5 0 Yes 24 h 7 5 Collagen 5 0 Yes 0 h 7 6 Collagen 0 0.5 Yes 24 h 7 7 Collagen 0 0.5 Yes 0 h 7 8 Collagen 1 0.5 Yes 12 h 7 9 Collagen 3 0.5 Yes 12 h 7 10 Collagen 5 0.5 Yes 12 h 7 11 Collagen 7 0.5 Yes 12 h 7 12 Collagen 5 0.1 Yes 12 h 7 13 Collagen 5 0.2 Yes 12 h 7 14 Collagen 5 1 Yes 12 h 7 15 Collagen 5 0.5 Yes 12 h 3 16 Collagen 5 0.5 Yes 12 h 5 17 Collagen 5 0.5 Yes 12 h 7 18 Collagen 5 0.5 Yes 12 h 9 19 Collagen 5 0.5 Yes 18 min 7 20 Collagen 5 0.5 Yes 30 min 7 21 Collagen 5 0.5 Yes 1 h 7 22 Collagen 5 0.5 Yes 3 h 7 23 Collagen 5 0.5 Yes 6 h 7 24 Collagen 5 0.5 Yes 10 h 7 25 Human 5 0.5 Yes 12 h 7 serum albumin (HSA) 26 Bovine 5 0.5 Yes 12 h 7 serum albumin (BSA) 27 Egg white 5 0.5 Yes 12 h 7 28 Pepsin 5 0.5 Yes 12 h 7 29 Papain 5 0.5 Yes 12 h 7 30 Casein 5 0.5 Yes 12 h 7

[0040] The cross-linked protein described in one or more of the following embodiments refers to the product formed after cross-linking of protein and Ag.sup.+ under visible light irradiation.

[0041] The cross-linked proteins prepared in one or more of the following embodiments have good mechanical properties and can be used to prepare hemostatic materials, drug sustained-release carrier materials, tissue engineering scaffold materials, artificial skin, bionic teeth, artificial blood vessels, bone repair materials, corneal graft materials and other medical instruments.

[0042] The power of the visible light source described in one or more of the following embodiments is 48 W, and the irradiation distance is 20 cm. However, the power and irradiation distance of the visible light source in the embodiments are not the only choices. The power and irradiation distance of the visible light source can be adjusted according to different experimental operations.

[0043] UV described in one or more of the following embodiments is ultraviolet spectrum, EDX is energy dispersive X-ray analysis, XPS is X-ray photoelectron spectroscopy, SEM is scanning electron microscope, TEM is transmission electron microscope, IR is infrared chromatography, and TGA is thermogravimetric analysis.

[0044] The UV detection described in one or more of the following embodiments is as follows: take 500 ul of the prepared cross-linked protein sample, dilute it to 3 ml, and then perform UV detection; the EDX detection described is as follows: take 500 ul of the prepared cross-linked protein sample, dilute it to 3 ml, and then perform EDX detection; the XPS detection described is as follows: take an appropriate amount of cross-linked protein sample and freeze-dry it, and then perform XPS detection; the SEM detection described is as follows: take an appropriate amount of cross-linked protein reaction solution, drop it on the silicon wafer and spray gold, and then perform SEM detection; the TEM detection described is as follows: take an appropriate amount of cross-linked protein reaction solution, drop it on the copper grid to dry, and then perform TEM detection; the IR detection described is as follows: take an appropriate amount of cross-linked protein sample and freeze-dry it, and then perform IR detection; the TGA detection described is as follows: take an appropriate amount of cross-linked protein sample and freeze-dry it, and then perform TGA detection.

Embodiment 1

1.1 Preparation of Cross-Linked Collagen

[0045] Configuration of collagen solution: dissolve 10 mg collagen solid in 1 ml water, then configure it into a collagen solution with a concentration of 10 mg/ml, and dilute it with water before use.

[0046] Configuration of AgNO.sub.3 solution: dissolve 100 mg AgNO.sub.3 solid in 1 ml water, then configure it into an AgNO.sub.3 solution with a concentration of 100 mg/ml, and dilute it with water before use.

Preparation of Cross-Linked Collagen:

[0047] a. At room temperature, the collagen solution was added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the collagen solution at a distance of 20 cm. The solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solution containing 5 mg/ml collagen and 0.5 mg/ml AgNO.sub.3. The reaction solution a was prepared after reaction for 24 hours;
b. At room temperature, the collagen solution was added to the 12-well plate, the solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solution containing 5 mg/ml collagen and 0.5 mg/ml AgNO.sub.3. The reaction solution b was prepared after reaction for 24 hours;
c. At room temperature, the collagen solution was added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the collagen solution at a distance of 20 cm. The solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solution containing 5 mg/ml collagen and 0.5 mg/ml AgNO.sub.3, and then the reaction solution c was prepared;
d. At room temperature, 5 mg/ml collagen solution was added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the collagen solution at a distance of 20 cm for 24 hours, then the reaction solution d was prepared;
e. At room temperature, 5 mg/ml collagen solution was added to the 12-well plate, and then the reaction solution e was prepared;
f. At room temperature, 0.5 mg/ml AgNO.sub.3 solution was added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the AgNO.sub.3 solution at a distance of 20 cm for 24 hours, then the reaction solution f was prepared;
g. At room temperature, 0.5 mg/ml AgNO.sub.3 solution was added to the 12-well plate, and then the reaction solution g was prepared.

[0048] Purification of cross-linked protein: The above reaction solutions a-g were centrifuged at 9500 rpm, the supernatant was discarded to retain the precipitate, and the cross-linked collagens a-g were obtained after washing and centrifugation with deionized water for 3-5 times and then drying at room temperature.

1.2 Structural Detection of Cross-Linked Collagen

[0049] The cross-linked collagens a-g were detected by UV respectively; the cross-linked collagen a was detected by EDX, XPS, SEM, TEM, IR and TGA.

[0050] The UV detection results are shown in FIG. 1A (a-g in the figure are the cross-linked collagens a-g prepared above respectively), only when collagen and Ag.sup.+ exist at the same time, and under the visible light condition, the reaction system has an obvious absorption peak at the wavelength of about 450 nm, indicating the formation of silver nanoparticles; the EDX detection result is shown in FIG. 1B, the XPS detection result is shown in FIG. 1C and FIG. 1D, and the EDX and XPS detection peaks further indicate the generation of silver nanoparticles under such conditions; the SEM, TEM, IR and TGA detection results of the cross-linked collagen a prepared above are shown in FIG. 2, in which FIG. 2a is the SEM image, FIG. 2b is the local amplification of FIG. 2a, FIG. 2c is the TEM image, FIG. 2d is the local amplification of FIG. 2c, FIG. 2e is the IR detection image, and FIG. 2f is the TGA detection image; the SEM and TEM detection show that collagen fibers are formed in the reaction solution; the IR detection result shows that the tertiary structure of collagen is still intact; the TGA detection result shows that the collagen in the reaction system accounts for nearly half of the total amount of the system. The above experimental results show that only when the protein solution, silver ion solution and visible light condition exist at the same time, the protein is cross-linked, and the structure of the prepared cross-linked protein is intact.

Embodiment 2

2.1 Preparation of Cross-Linked Collagen

[0051] The collagen solutions and AgNO.sub.3 solutions were configured as shown in 1.1 above.

Preparation of Cross-Linked Collagen:

[0052] At room temperature, the collagen solution was added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the collagen solution at a distance of 20 cm. The solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solution containing 5 mg/ml collagen and 0.5 mg/ml AgNO.sub.3. The reaction solutions were prepared by reaction for 18 min, 30 min, 60 min, 3 h, 6 h and 10 h, respectively.

[0053] Purification of cross-linked protein: The above reaction solutions were centrifuged at 9500 rpm, the supernatant was discarded to retain the precipitate, and the cross-linked collagens with different cross-linking times were obtained after washing and centrifugation with deionized water for 3-5 times and then drying at room temperature.

2.2 Structural Detection of Cross-Linked Collagen

[0054] The above-mentioned cross-linked collagens or the reaction solutions were detected by UV, TEM and TGA respectively.

[0055] The TEM detection results are shown in FIG. 3, and collagen fibers with uniform morphology are generated in the reaction solutions with different cross-linking times; the UV detection results are shown in FIG. 4a, and with the extension of the cross-linking time, the absorption peak of the reaction system at the wavelength of about 450 nm increases significantly, indicating that the formation of silver nanoparticles increases gradually with the extension of cross-linking time; the TGA detection results are shown in FIG. 4b, and the thermogravimetric trend lines do not change, and the collagen in the reaction systems with different cross-linking times accounts for nearly half of the total amount of the systems, indicating that the content of collagen fibers in the system increases gradually with the increase of silver nanoparticles. The above results indicate that the cross-linked collagen can be formed when the cross-linking time is more than 18 min, and the content of collagen fibers in the reaction system increases with the extension of cross-linking time.

Embodiment 3

3.1 Preparation of Cross-Linked Collagen

[0056] The collagen solutions and AgNO.sub.3 solutions were configured as shown in 1.1 above.

Preparation of Cross-Linked Collagen:

[0057] At room temperature, the collagen solution was added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the collagen solution at a distance of 20 cm. The solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solutions containing collagen with different concentrations (1 mg/ml, 3 mg/ml, 5 mg/ml, 7 mg/ml) and 0.5 mg/ml AgNO.sub.3. The reaction solutions were prepared after reaction for 12 hours.

[0058] Purification of cross-linked protein: The above reaction solutions were centrifuged at 9500 rpm, the supernatant was discarded to retain the precipitate, and the cross-linked collagens with different collagen concentrations were obtained after washing and centrifugation with deionized water for 3-5 times and then drying at room temperature.

3.2 Structural Detection of Cross-Linked Collagen

[0059] The above-mentioned cross-linked collagens or the reaction solutions were detected by TEM and SEM respectively.

[0060] The results are shown in FIG. 5, in which FIG. 5a-d are the SEM images of different collagen concentrations of 1 mg/ml, 3 mg/ml, 5 mg/ml and 7 mg/ml respectively, and FIG. 5e-h are the TEM images of different collagen concentrations of 1 mg/ml, 3 mg/ml, 5 mg/ml and 7 mg/ml respectively. The results show that collagen fibers with uniform morphology are generated in the reaction solutions with different collagen concentrations, indicating that the collagen concentration has little effect on collagen cross-linking, and collagen fibers with uniform morphology can be formed at any concentration.

Embodiment 4

4.1 Preparation of Cross-Linked Collagen

[0061] The collagen solutions and AgNO.sub.3 solutions were configured as shown in 1.1 above.

Preparation of Cross-Linked Collagen:

[0062] At room temperature, the collagen solution was added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the collagen solution at a distance of 20 cm. The solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solutions containing 5 mg/ml collagen and AgNO.sub.3 with different concentrations (0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml and 1.0 mg/ml). The reaction solutions were prepared after 12 hours of reaction respectively.

[0063] Purification of cross-linked protein: The above reaction solutions were centrifuged at 9500 rpm, the supernatant was discarded to retain the precipitate, and the cross-linked collagens with different AgNO.sub.3 concentrations were obtained after washing and centrifugation with deionized water for 3-5 times and then drying at room temperature.

4.2 Structural Detection of Cross-Linked Collagen

[0064] The above-mentioned cross-linked collagens or the reaction solutions were detected by TEM and SEM respectively.

[0065] The results are shown in FIG. 6, in which FIG. 6a-d are the SEM images of different AgNO.sub.3 concentrations of 0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml and 1 mg/ml respectively, and FIG. 6e-h are the TEM images of different AgNO.sub.3 concentrations of 0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml respectively. The results show that collagen fibers with uniform morphology are generated in the reaction solutions with different AgNO.sub.3 concentrations, indicating that AgNO.sub.3 concentration has little effect on collagen cross-linking, and collagen fibers with uniform morphology can be formed at any concentration.

Embodiment 5

5.1 Preparation of Cross-Linked Collagen

[0066] The collagen solutions and AgNO.sub.3 solutions were configured as shown in 1.1 above.

Preparation of Cross-Linked Collagen:

[0067] The pH value of the collagen solution was adjusted to 3, 5, 7 and 9 respectively, and at room temperature, the collagen solutions with different pH values were added to the 12-well plate, then the visible light source with a power of 48 W was used to irradiate the collagen solution at a distance of 20 cm. The solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solution containing 5 mg/ml collagen and 0.5 mg/ml AgNO.sub.3. The reaction solutions were prepared after reaction for 12 hours respectively.

[0068] Purification of cross-linked protein: The above reaction solutions were centrifuged at 9500 rpm, the precipitate was retained, and the cross-linked collagens with different pH reactions were obtained after washing and centrifugation with deionized water for 3-5 times and then drying at room temperature.

5.2 Structural Detection of Cross-Linked Collagen

[0069] The above-mentioned cross-linked collagens or the reaction solutions were detected by TEM and SEM respectively.

[0070] The detection results are shown in FIG. 7, in which FIG. 7a-d are the SEM images at pH 3, 5, 7, and 9 respectively, and FIG. 7e-h are the TEM images at pH 3, 5, 7, and 9 respectively. The results show that collagen fibers with uniform morphology are generated in the reaction solutions with different pH values, indicating that cross-linked collagen can be formed in all reaction systems with different pH values.

Embodiment 6

6.1 Preparation of Cross-Linked Protein

[0071] The protein solutions and AgNO.sub.3 solutions were configured as shown in 1.1 above, where the proteins are BSA, Casein, HSA, Pepsin, Papain and Egg White respectively.

Preparation of Cross-Linked Protein:

[0072] At room temperature, the above protein solutions were added to the 12-well plate, and the visible light source with a power of 48 W was used to irradiate the protein solution at a distance of 20 cm. The solution was stirred quickly and AgNO.sub.3 solution was added slowly to obtain the final solution containing 5 mg/ml protein and 0.5 mg/ml AgNO.sub.3. The reaction solution was prepared after reaction for 12 hours respectively.

[0073] Purification of cross-linked protein: The above reaction solutions were centrifuged at 9500 rpm, the supernatant was discarded to retain the precipitate, and the cross-linked proteins with different protein types were obtained after washing and centrifugation with deionized water for 3-5 times and then drying at room temperature.

6.2 Structural Detection of Cross-Linked Protein

[0074] The above-mentioned cross-linked proteins or the reaction solutions were detected by TEM and SEM respectively.

[0075] The detection results are shown in FIG. 8, in which FIG. 8A-F are the TEM images of BSA, casein, HSA, pepsin, papain and egg white respectively, and FIG. 8a-f are the SEM images of BSA, casein, HSA, pepsin, papain and egg white respectively. The results show that the fibers with uniform morphology are generated in the reaction systems of different proteins, indicating that the method is also suitable for the cross-linking of other proteins and has broad spectrum.

[0076] In summary, the present invention obtains a cross-linked protein by cross-linking the protein solution and the silver ion solution under visible light irradiation. The method is implemented under mild conditions without additional cross-linking agent required, and it is safe and non-toxic. The prepared cross-linked protein has good biological activity and antibacterial properties. Compared with the cross-linked protein prepared by other cross-linking methods, it is more suitable for the preparation of hemostatic materials, drug sustained-release carrier materials, tissue engineering scaffold materials, artificial skin, bionic teeth, artificial blood vessels, bone repair materials and corneal graft materials.

[0077] The above descriptions are only the details of individual exemplary implementation cases of the present invention. For the technical personnel in the field, the present invention can be modified and changed in the actual application process according to the specific preparation conditions, which is not intended to limit the present invention. Anything within the spirit and principle of the present invention shall be included in the protection scope of the present invention.