Silver nano-chlorogenic acid composite hydrogel as well as preparation and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to silver nano-chlorogenic acid composite hydrogel as well as preparation and application thereof. According to the invention, the CGA and the silver nitrate are used for green synthesis of the nano-particles CGA-AgNPs, and the nano-particles have peroxide nano-enzyme activity and antibacterial activity; and the CGA-AgNPs and chitosan (CS) are cross-linked to form the silver nano-chlorogenic acid composite hydrogel CGA-AgNPs-CS, so that the uniform distribution of nano-particles in the hydrogel can be promoted. According to the invention, multi-functionalization is taken as a target, a novel hydrogel composition system is designed from the source, the provided preparation method is green, simple, rapid and environment-friendly, the obtained composite hydrogel has multiple functions, and a good foundation is laid for expansion and application of the composite hydrogel in the biomedical fields of wound repair, wearable equipment, nano antibacterial drugs and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a silver nano-chlorogenic acid composite hydrogel and its preparation and application. Background Art

[0002] Hydrogels have a three-dimensional hydrophilic polymer network structure and good biocompatibility. They have a porous structure, high water content, and controllable shape and volume, which are very similar to soft tissues such as skin. Therefore, hydrogels have important application prospects in biomedical fields such as wearable biodevices, biological tissue structure mimicry, drug delivery, skin wound treatment, regenerative medicine, and tissue engineering.

[0003] However, biomedical applications usually require hydrogels to have multiple functions, such as high requirements for multiple properties under dry and wet conditions and in vivo conditions, such as antibacterial, anti-inflammatory, antioxidant, adhesion, elasticity, conductivity, and enzyme-like activity. Hydrogels usually have weak mechanical properties and cannot adapt to the diverse requirements of biomedical applications. Therefore, the multifunctionality of gels and their optimization are still the main challenges facing current practical applications. The incorporation of functional nanomaterials such as silver nanoparticles (AgNPs) into hydrogels can introduce various new material properties, and hydrogels can also increase the retention effect of nanomaterials in the body. Generally, due to the weak interaction between nanomaterials and polymer chains in hydrogels, their uniform distribution in hydrogels is relatively difficult, which is still one of the main problems that need to be solved for the multifunctionalization of nanocomposite gels. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a chlorogenic acid (CGA)-silver nanoparticles (AgNPs)-chitosan (CS) composite hydrogel with important physiological activities such as antibacterial and antioxidant properties.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for preparing a silver nano-chlorogenic acid composite hydrogel, comprising the following steps:

[0007] (1) Mixing chlorogenic acid solution and silver nitrate solution to form a mixed solution, stirring and heating the solution until the color of the solution changes to brown-yellow, thereby obtaining a CGA-AgNPs solution;

[0008] (2) adding chitosan to the CGA-AgNPs solution obtained in step (1) and stirring, then adding an acidic solution to adjust the pH, reacting to form the silver nano-chlorogenic acid composite hydrogel.

[0009] The present invention is guided by the goal of multifunctionalization and designs a novel hydrogel component system from the source. Chlorogenic acid CGA is a polyphenolic acid that is widely present in various plants and has important physiological activities such as antibacterial and anti-inflammatory. The present invention uses CGA and silver nitrate to synthesize green nanoparticles CGA-AgNPs (AgNPs are coated with CGA), which have peroxidase-like activity and broad-spectrum antibacterial properties. During the preparation of AgNPs, CGA is both a reducing agent and a stabilizer. CGA-AgNPs are cross-linked with chitosan to form a silver nano-chlorogenic acid composite hydrogel CGA-AgNPs-CS. Chitosan is the product of the natural polysaccharide chitin removing some acetyl groups. It is the only alkaline polysaccharide among natural polysaccharides, contains many hydroxyl groups and amino groups, has good biocompatibility, and is suitable as a medical hydrogel. The unique catechol group structure of CGA can give the hydrogel multifunctionality and promote the uniform distribution of nanoparticles.

[0010] The preparation method provided by the present invention is green, simple, rapid and environmentally friendly, and the obtained composite hydrogel has excellent multifunctionality, including antibacterial, antioxidant, nanoenzyme activity, conductivity and adhesion.

[0011] Preferably, in step (1), the molar ratio of chlorogenic acid to silver nitrate in the mixed solution is chlorogenic acid:silver nitrate=(1-5):1.

[0012] More preferably, in step (1), the molar ratio of chlorogenic acid to silver nitrate in the mixed solution is chlorogenic acid:silver nitrate=4:1.

[0013] Preferably, in step (1), the heating temperature is 30-100° C., and the reaction time is more than 10 minutes.

[0014] More preferably, in step (1), the heating temperature is 80-90°C.

[0015] Preferably, in step (1), the reaction is carried out under alkaline conditions, and the reaction pH is 8-9.

[0016] The pH and temperature during the synthesis of the nanoparticles are the conditions for CGA to reduce the silver salt precursor. Within the scope of the present invention, a silver nanosol with better stability and no agglomeration can be obtained.

[0017] The synthesis of AgNPs is related to the concentration of Ag salt, CGA concentration, acidity and alkalinity of the system, etc. Generally speaking, CGA can synthesize AgNPs under weakly acidic, near-neutral and alkaline conditions, but the reaction speed is faster under alkaline conditions, and the prepared nanosilver sol is less aggregated.

[0018] Preferably, in step (2), the ratio of the volume (mL) of the CGA-AgNPs solution to the mass (g) of chitosan is CGA-AgNPs:chitosan=1:(0.05-0.09).

[0019] Preferably, in step (2), the acidic solution comprises HCl.

[0020] Preferably, in step (2), the pH is adjusted to 4-5.

[0021] In a second aspect, the present invention provides a silver nano-chlorogenic acid composite hydrogel prepared by the preparation method.

[0022] In a third aspect, the present invention provides the use of the silver nano-chlorogenic acid composite hydrogel in the preparation of antibacterial, antioxidant or wound repair products.

[0023] The composite hydrogel prepared by the present invention has multiple functions, laying a good foundation for its expanded application in biomedical fields such as wound repair, wearable devices, and nano-antibacterial drugs.

[0024] The beneficial effects of the present invention are:

[0025] This invention utilizes CGA and silver nitrate to synthesize green nanoparticles (CGA-AgNPs), which possess multifunctional antibacterial nanozyme activity. CGA-AgNPs are cross-linked with chitosan to form a silver nanochlorogenic acid composite hydrogel (CGA-AgNPs-CS), which promotes uniform distribution of the nanoparticles within the hydrogel. This invention, guided by multifunctionalization, designs a novel hydrogel component system from the ground up. The preparation method provided is green, simple, rapid, and environmentally friendly. The resulting composite hydrogel possesses multiple functions, laying a solid foundation for its expanded applications in biomedical fields such as wound repair, wearable devices, and nano-antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Transmission electron microscopy image of CGA-AgNPs.

[0027] Figure 2 The numbers of nanoparticles contained in the CGA-AgNPs solutions prepared in Comparative Example 1 (A) and Comparative Example 2 (B) and the appearance observation results of the nanoparticles are shown.

[0028] Figure 3 The results of peroxidase nanozyme activity test of CGA-AgNPs at different temperatures.

[0029] Figure 4 The results of peroxidase nanozyme activity test of CGA-AgNPs in different buffer solutions.

[0030] Figure 5 CGA-AgNPs-CS composite hydrogel and its scanning electron microscopy image.

[0031] Figure 6 Antibacterial activity of chlorogenic acid-silver nanocomposite hydrogel.

[0032] Figure 7 This is the result diagram of the effect of chitosan dosage on gel formation. DETAILED DESCRIPTION

[0033] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0034] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0035] All reagents used in the present invention were analytically pure, and the CGA solution and silver nitrate solution were prepared by weighing and dilution. In the reaction system, the concentration of the chlorogenic acid solution was 0.1 mM-1.8 mM; the concentration of the silver nitrate solution was 0.2 mM.

[0036] Example 1:

[0037] An embodiment of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel comprises the following steps:

[0038] (1) Mixing chlorogenic acid solution and silver nitrate solution to form a mixed solution, wherein the molar ratio of chlorogenic acid to silver nitrate in the mixed solution is chlorogenic acid:silver nitrate = 4:1; stirring and heating at 80°C, adjusting the pH to 8-9; reacting for 30 minutes until the color of the solution changes to brown (indicating the formation of AgNPs), thereby obtaining a CGA-AgNPs solution;

[0039] (2) 0.1304 g of chitosan (CS) was added to 2 mL of CGA-AgNPs solution in a 50°C water bath and stirred for more than 5 min. An appropriate amount of hydrochloric acid was then added dropwise to adjust the pH to 4-5. The gelation reaction was performed for 5 min to form the silver nano-chlorogenic acid composite hydrogel.

[0040] Example 2:

[0041] An embodiment of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel described in this embodiment is different from that of Example 1 only in that: in the step (1), the molar ratio of chlorogenic acid and silver nitrate in the mixed solution is chlorogenic acid: silver nitrate 1:1; the remaining steps and preparation conditions are the same as those of Example 1.

[0042] Example 3:

[0043] An embodiment of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel described in this embodiment is different from that of Example 1 only in that: in the step (1), the molar ratio of chlorogenic acid and silver nitrate in the mixed solution is chlorogenic acid: silver nitrate = 5:1; the remaining steps and preparation conditions are the same as those of Example 1.

[0044] Example 4:

[0045] An embodiment of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel described in this embodiment is different from that of Example 1 only in that: in step (2), 0.1 g of chitosan (CS) is added to 2 mL of CGA-AgNPs solution in a 50°C water bath and stirred; the remaining steps and preparation conditions are the same as those in Example 1.

[0046] Example 5:

[0047] An embodiment of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel described in this embodiment is different from that of Example 1 only in that: in step (2), 0.12 g of chitosan (CS) is added to 2 mL of CGA-AgNPs solution in a 50°C water bath and stirred; the remaining steps and preparation conditions are the same as those in Example 1.

[0048] Example 6:

[0049] An embodiment of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel described in this embodiment is different from that of Example 1 only in that: in step (2), 0.15 g of chitosan (CS) is added to 2 mL of CGA-AgNPs solution in a 50°C water bath and stirred; the remaining steps and preparation conditions are the same as those in Example 1.

[0050] Example 7:

[0051] An embodiment of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel described in this embodiment is different from that of Example 1 only in that: in step (2), 0.18 g of chitosan (CS) is added to 2 mL of CGA-AgNPs solution in a 50°C water bath and stirred; the remaining steps and preparation conditions are the same as those in Example 1.

[0052] Comparative Example 1:

[0053] A comparative example of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel in this comparative example is different from that in Example 1 only in that: in the step (1), the molar ratio of chlorogenic acid and silver nitrate in the mixed solution is chlorogenic acid:silver nitrate = 38:1; the remaining steps and preparation conditions are the same as those in Example 1.

[0054] Comparative Example 2:

[0055] A comparative example of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel in this comparative example is different from that in Example 1 only: in the step (1), the molar ratio of chlorogenic acid and silver nitrate in the mixed solution is chlorogenic acid: silver nitrate = CGA: AgNO3 = 1:15; the remaining steps and preparation conditions are the same as those in Example 1.

[0056] Comparative Example 3:

[0057] A comparative example of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel in this comparative example differs from that in Example 1 only in that: in step (2), 0.02 g of chitosan (CS) is added to 2 mL of CGA-AgNPs solution in a 50°C water bath and stirred; the remaining steps and preparation conditions are the same as in Example 1.

[0058] Comparative Example 4:

[0059] A comparative example of the silver nano-chlorogenic acid composite hydrogel of the present invention; the preparation method of the silver nano-chlorogenic acid composite hydrogel in this comparative example differs from that in Example 1 only in that: in step (2), 0.08 g of chitosan (CS) is added to 2 mL of CGA-AgNPs solution in a 50°C water bath and stirred; the remaining steps and preparation conditions are the same as in Example 1.

[0060] Experimental Example 1: Characterization of Nanoparticles CGA-AgNPs

[0061] (1) The number of nanoparticles contained in the CGA-AgNPs solutions prepared in Example 1, Comparative Example 1 and Comparative Example 2 and the appearance of the nanoparticles were observed using a transmission electron microscope. The observation results of Example 1 are as follows: Figure 1 As shown, the nanoparticles CGA-AgNPs are spherical and have an obvious thin layer of coating on the outer layer, indicating that CGA has a good stabilizing effect. The average particle size of the CGA-AgNPs prepared by the present invention is less than 100nm.

[0062] Compared with Example 1, Comparative Example 1 only produced a small amount of spherical nanoparticles ( Figure 2 A); Comparative Example 2 generates more nanoparticles, but more agglomerates ( Figure 2 B); are not conducive to the subsequent gel formation.

[0063] Considering the factors such as nanoparticle morphology, quantity, activity and cytotoxicity in subsequent applications, the reaction concentration ratio CGA:AgNO3=(1-5):1 was selected as the appropriate range.

[0064] ⑵Test the peroxidase activity of nanoparticles CGA-AgNPs.

[0065] Test method: In the presence of hydrogen peroxide, the sample is catalyzed to oxidize 3,3',5,5'-tetramethylbenzyl diamine (TMB) to produce a blue substance.

[0066] ①Peroxidase activity at different temperatures

[0067] Take 3.65 mL of HAc-NaAc buffer solution with a pH of 4, add 150 μL of TMB, 100 μL of chlorogenic acid-silver nanoparticles CGA-AgNPs (Example 1), and 100 μL of H2O2 in sequence, and react in a water bath at different temperatures (30°C-80°C) for 30 minutes. After the reaction is completed, the absorbance at 652 nm is measured (CGA-AgNPs catalyzes the oxidation of TMB in the presence of hydrogen peroxide to generate a blue substance with a characteristic absorption peak at 652 nm).

[0068] The results are as follows Figure 3 As shown, the nanoparticles CGA-AgNPs prepared by the present invention have peroxidase nanozyme activity, and their antibacterial activity provides an advantage in resisting bacterial resistance.

[0069] ②Peroxidase activity in different buffer solutions

[0070] Take 3.65 mL of HAc-NaAc buffer solution with different pH values, add 150 μL of TMB, 100 μL of chlorogenic acid-silver nanoparticles CGA-AgNPs (Example 1), and 100 μL of H2O2 in sequence, and react in a 50°C water bath for 30 min. After the reaction, measure the absorbance at 652 nm.

[0071] The results are as follows Figure 4 As shown, it can be seen that the buffer system with better peroxidase activity is pH=4.

[0072] Experimental Example 2: Characterization of Silver Nanochlorogenic Acid Composite Hydrogel

[0073] (1) Appearance observation and microscopic observation of the silver nano-chlorogenic acid composite hydrogel sample prepared in Example 1.

[0074] like Figure 5 As shown, the silver nano-chlorogenic acid composite hydrogel has a light yellow appearance and is a semi-solid substance.

[0075] Scanning electron microscopy observation results show that it has a three-dimensional network structure. Scanning electron microscopy results show that it has a honeycomb three-dimensional network structure, showing the typical characteristics of a gel. Silver nanoparticles can be seen distributed relatively evenly in the three-dimensional network structure.

[0076] ⑵Antibacterial activity test

[0077] The plate culture method was used to determine the antibacterial activity of the composite hydrogel against Escherichia coli and Staphylococcus aureus. E. coli and S. aureus are representative Gram-negative and Gram-positive bacteria that are common in nature.

[0078] Methods: The composite hydrogel was freeze-dried and dissolved in deionized water to prepare sample solutions of different concentrations, namely 1 mg / mL, 5 mg / mL, 15 mg / mL, and 20 mg / mL. 3 The bacterial strains with different CFU / mL concentrations were reacted with CGA-AgNPs hydrogel samples with different concentrations for 20 min. 500 mL of the mixture was evenly spread on the adapted culture medium plate. The blank group was directly coated with the bacterial strain for comparison.

[0079] After coating, place the culture plate at room temperature for 10 to 30 minutes until the solid culture medium completely absorbs the bacterial solution and there is no liquid on the surface. Then, cover the plate and incubate the plate upside down at 37°C for 16 hours. The antibacterial effect is evaluated based on the number of antibacterial inhibitions.

[0080] Table 1

[0081]

[0082]

[0083] The results are as follows Figure 6 As shown in Table 1, the silver nano-chlorogenic acid composite hydrogel prepared by the present invention has an excellent, broad-spectrum antibacterial effect, especially against Staphylococcus aureus. The antibacterial effect gradually increases with the increase of its concentration.

[0084] Test Example 3:

[0085] The silver nano-chlorogenic acid composite hydrogel samples obtained in Examples 4-7 and Comparative Examples 3-4 were inverted for a period of time to observe the formation of the gel.

[0086] The gel formation of each group of samples is as follows Figure 7 As shown in Figure 4, the ratio of the volume (mL) of the CGA-AgNPs solution to the mass (g) of chitosan in Examples 4-7 was 1: (0.05-0.09) and was able to form stable gels. However, the insufficient amount of chitosan in Comparative Examples 1 and 2 resulted in failure to form stable gels.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing silver nano-chlorogenic acid composite hydrogel, characterized in that: The steps include: (1) Mixing chlorogenic acid solution and silver nitrate solution to form a mixed solution, stirring and heating the solution until the color of the solution changes to brown-yellow, thereby obtaining a CGA-AgNPs solution; (2) adding chitosan to the CGA-AgNPs solution obtained in step (1) and stirring, then adding an acidic solution to adjust the pH, reacting to form the silver nano-chlorogenic acid composite hydrogel.

2. The method for preparing the silver nano-chlorogenic acid composite hydrogel according to claim 1, wherein: In the step (1), the molar ratio of chlorogenic acid to silver nitrate in the mixed solution is chlorogenic acid:silver nitrate=(1-5):

1.

3. The method for preparing the silver nano-chlorogenic acid composite hydrogel according to claim 1, wherein: In the step (1), the heating temperature is 30-100° C. and the reaction time is more than 10 minutes.

4. The method for preparing the silver nano-chlorogenic acid composite hydrogel according to claim 1, wherein: In the step (1), the reaction is carried out under alkaline conditions, and the reaction pH is 8-9.

5. The method for preparing the silver nano-chlorogenic acid composite hydrogel according to claim 1, wherein: In the step (2), the ratio of the volume (mL) of the CGA-AgNPs solution to the mass (g) of chitosan is CGA-AgNPs:chitosan=1:(0.05-0.09).

6. The method for preparing the silver nano-chlorogenic acid composite hydrogel according to claim 1, wherein: In the step (2), the acidic solution includes HCl.

7. The method for preparing the silver nano-chlorogenic acid composite hydrogel according to claim 1, wherein: In the step (2), the pH is adjusted to 4-5.

8. The silver nano-chlorogenic acid composite hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the silver nano-chlorogenic acid composite hydrogel according to claim 8 in the preparation of antibacterial, antioxidant or wound repair products.

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