Cinnamaldehyde-coated nano calcium carbonate / chitosan hydrogel as well as preparation method and application thereof

Abstract

The invention discloses cinnamyl aldehyde at nano calcium carbonate / chitosan hydrogel. The hydrogel comprises a hydrogel matrix formed by cross-linking nano calcium carbonate and chitosan, and cinnamyl aldehyde loaded on the hydrogel matrix. The hydrogel system which is loaded with traditional Chinese medicine components and intelligently controls effective release of the traditional Chinese medicine components is prepared by utilizing the mutual adsorption effect of cinnamyl aldehyde and nano calcium carbonate. The hydrogel has excellent antibacterial activity and a function of accelerating wound healing, and the effect of the hydrogel is obviously superior to that of common calcium carbonate / chitosan hydrogel. A new direction is provided for application of the cinnamyl aldehyde and nano calcium carbonate / chitosan nano system, and a new strategy is provided for designing advanced functional materials modified by traditional Chinese medicine ingredients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a cinnamaldehyde@nano calcium carbonate / chitosan hydrogel and a preparation method and application thereof. Background Art

[0002] Hydrogel dressings are a well-established area of ​​medical research. Their primary benefits include promoting wound healing, inhibiting bacterial growth, reducing scarring, reducing exudation, and promoting blood circulation. However, antibiotic-loaded hydrogels can easily lead to drug resistance in patients. Combining traditional Chinese medicine (TCM) components with hydrogels to create TCM-loaded hydrogels could leverage the hydrogel's excellent biocompatibility to promote wound cell growth while also accelerating wound healing through the antibacterial, anti-inflammatory, and antioxidant properties of the loaded TCM ingredients, all without compromising drug resistance.

[0003] Cinnamaldehyde (CA), a hydrophobic aromatic aldehyde extracted from cinnamon oil, is an ideal natural dressing ingredient with antibacterial and antioxidant properties. It is the main active ingredient in cinnamon, a medicinal and edible substance. Due to its edible, biodegradable, antibacterial, and anticancer properties, it has been widely used in the food and biomedical fields. However, its strong hydrophobicity makes it difficult to integrate with hydrophilic matrices, and its volatility significantly reduces its bioavailability. To incorporate this oily active ingredient into a hydrogel matrix, the study "Construction of a Nanofunctionalized System of Tannic Acid and Cinnamaldehyde and Its Antibacterial and Antioxidant Properties" (Sun Xinyu, 2022) demonstrated that a composite hydrogel formed by combining cinnamaldehyde with tannic acid via ultrasound-assisted emulsification exhibits excellent antibacterial and antioxidant properties. However, this method lacks the ability to intelligently control the release of the active ingredient. Therefore, a hydrogel system that can load cinnamaldehyde and intelligently release the active ingredient is urgently needed. Summary of the Invention

[0004] The present invention aims to overcome the aforementioned drawbacks and deficiencies in the prior art by providing a cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel. This invention combines nano-calcium carbonate with chitosan, then leverages the high oil absorption of the nano-calcium carbonate to load cinnamaldehyde, creating an "oil-in-water" cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0005] The second object of the present invention is to provide a method for preparing the cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0006] The third object of the present invention is to provide the use of the hydrogel in inhibiting bacteria and / or promoting wound healing.

[0007] The fourth object of the present invention is to provide a product comprising the above-mentioned cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions: The invention provides a cinnamaldehyde@nano calcium carbonate / chitosan hydrogel, which comprises a hydrogel matrix formed by cross-linking nano calcium carbonate and chitosan, and cinnamaldehyde loaded on the hydrogel matrix.

[0009] Chitosan has better solubility, making it more suitable for preparing hydrogels. During the hydrogel formation process, chitosan serves as the main polymer matrix, forming a stable three-dimensional network structure through its intermolecular interactions (such as hydrogen bonds and electrostatic interactions) and interactions with other components (such as nano-calcium carbonate), thereby endowing the hydrogel with specific mechanical properties and biological activity.

[0010] Nano-calcium carbonate is commonly used as a drug carrier in targeted sustained-release drug treatments for cancer. The main mechanism is that the pH environment around cancer cells is relatively low. When the drug-loaded nano-calcium carbonate is exposed to this acidic environment, it dissolves and releases the effective drug loaded in the nano-calcium carbonate. At the same time, nano-calcium carbonate can also inhibit the regeneration of inflammatory cells and reduce the probability of wound infection. Because the wound environment is acidic, the effective drug loaded in the nano-calcium carbonate can also be released when nano-calcium carbonate is used to prepare hydrogels.

[0011] Cinnamaldehyde is a hydrophobic aromatic aldehyde extracted from cinnamon oil and is an ideal natural dressing ingredient with antibacterial and antioxidant properties.

[0012] Therefore, nano-calcium carbonate was combined with chitosan, and then cinnamaldehyde was loaded onto the nano-calcium carbonate's high oil absorption, resulting in an "oil-in-water" cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel, achieving a fusion of hydrophilic and hydrophobic molecules. This invention provides a highly competitive strategy for the development of multifunctional nanosystems loaded with cinnamaldehyde, and its practical application is of great research significance.

[0013] The present invention provides a method for preparing the above-mentioned hydrogel, and the preparation method is as follows: S1. Chitosan was mixed with sodium carbonate, calcium hydroxide, and urea, and then subjected to ultra-low temperature freeze-thaw, soaking, and freeze-drying to obtain nano-calcium carbonate / chitosan hydrogel; S2. Add the cinnamaldehyde aqueous solution dropwise onto the surface of the nano-calcium carbonate / chitosan hydrogel to obtain cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0014] The application constructs a cinnamaldehyde@nano calcium carbonate / chitosan hydrogel system with natural antibacterial agent cinnamaldehyde as an active component. First, crosslinking is performed between chitosan and nano calcium carbonate by virtue of the self-polymerization characteristics of chitosan, to become a nano calcium carbonate / chitosan hydrogel. Then, the cinnamaldehyde@nano calcium carbonate / chitosan hydrogel is prepared by virtue of the high oil absorption value of nano calcium carbonate and the adsorption effect between cinnamaldehyde. The specific preparation principle is as follows: chitosan itself is difficult to dissolve in pure water, but by adding alkaline substances (calcium carbonate and calcium hydroxide), the pH of the solution can be adjusted to an alkaline environment (about pH 12-13), so that the amino groups (-NH2) in the chitosan molecules are partially deprotonated to form soluble chitosan salt. This step creates conditions for subsequent crosslinking and mineralization reactions. The added calcium hydroxide (Ca(OH)2) and sodium carbonate (Na2CO3) dissociate into Ca 2+ and CO3 2- in water, and nano calcium carbonate (CaCO3) particles are generated through ionic reactions. These particles can be embedded in the chitosan network as physical crosslinking points, enhancing the mechanical properties of the hydrogel and enabling intelligent control of the effective release of traditional Chinese medicine ingredients. Meanwhile, Ca 2+ forms ionic crosslinking with the amino and hydroxyl groups of chitosan, promoting the formation of a three-dimensional network. Urea molecules promote the disentanglement of molecular chains by breaking the hydrogen bonds between chitosan molecules, improving the uniformity of the solution; at the same time, urea is a low-temperature stabilizer: it inhibits the excessive growth of ice crystals during freeze-thaw processes, reducing mechanical damage to the gel structure. Through repeated freeze-thaw-induced physical crosslinking, rapid freezing at -80℃ causes the water in the solution to form small ice crystals, and chitosan molecular chains are forced to aggregate and form hydrogen bonds and hydrophobic interactions; after the ice crystals melt, the chitosan chains rearrange due to thermodynamic relaxation, forming a more stable physical crosslinking network. Multiple freeze-thaw cycles (a total of 6 times) can significantly improve the crosslinking density and gel strength.

[0015] Further, the mass ratio of chitosan, sodium carbonate, calcium hydroxide, and urea in step S1 is 1:5-5.5:3.5-4:1.8-2.2.

[0016] Preferably, the mass ratio of chitosan, sodium carbonate, calcium hydroxide, and urea in step S1 is 2:10.6:7:4.

[0017] Further, the method of ultra-low temperature freezing and thawing in step S1 is to place it in an ultra-low temperature refrigerator at -70 to -80℃ and then thaw it at room temperature, with 4-8 repeated freeze-thaw cycles.

[0018] Further, the soaking conditions in step S1 are to soak in pure water until the pH value of the soaking solution is 7.3-7.5.

[0019] Preferably, the soaking conditions in step S1 are to soak in pure water until the pH value of the soaking solution is 7.4.

[0020] Further, the method for dropping the cinnamyl aldehyde aqueous solution on the surface of the nano calcium carbonate / chitosan hydrogel in step S2 is to drop 100 muL of cinnamyl aldehyde homogenate on the surface of a cylindrical hydrogel with a diameter of 10 mm and a height of 5 mm.

[0021] Further, the preparation method of the cinnamyl aldehyde aqueous solution in step S2 is ultrasonic dissolution.

[0022] The application tests the antibacterial effect of the cinnamyl aldehyde@nano calcium carbonate / chitosan hydrogel through in vitro experiments, and finds that the hydrogel has excellent antibacterial activity on Staphylococcus aureus and Escherichia coli; the application tests the effect of promoting wound healing of the cinnamyl aldehyde@nano calcium carbonate / chitosan hydrogel by constructing a mouse wound model, and finds that the hydrogel can realize in-situ rapid gelation at the wound site, cover irregular wounds, effectively kill bacteria, clean the wound microenvironment, and accelerate wound healing. The application provides a new direction for the application of the cinnamyl aldehyde@nano calcium carbonate / chitosan nanosystem, and also provides a new strategy for designing advanced functional materials modified by traditional Chinese medicine ingredients.

[0023] Therefore, the application provides the application of the above-mentioned hydrogel in bacteriostasis and / or promotion of wound healing.

[0024] The application also provides the application of the above-mentioned hydrogel in the preparation of products for bacteriostasis and / or promotion of wound healing.

[0025] Further, the bacteriostasis is for inhibiting Staphylococcus aureus or Escherichia coli.

[0026] The application also provides a product, which comprises the above-mentioned cinnamyl aldehyde@nano calcium carbonate / chitosan hydrogel.

[0027] Compared with the prior art, the application has the following beneficial effects: The application provides a cinnamyl aldehyde@nano calcium carbonate / chitosan hydrogel, which comprises a hydrogel matrix formed by cross-linking nano calcium carbonate and chitosan, and cinnamyl aldehyde loaded on the hydrogel matrix. The application utilizes the mutual adsorption of cinnamyl aldehyde and nano calcium carbonate to prepare a hydrogel system loaded with traditional Chinese medicine ingredients and capable of intelligently controlling the effective release of the traditional Chinese medicine ingredients. The hydrogel has excellent antibacterial activity and the function of accelerating wound healing, and the effect is obviously better than that of ordinary calcium carbonate / chitosan hydrogel. The application provides a new direction for the application of the cinnamyl aldehyde@nano calcium carbonate / chitosan nanosystem, and also provides a new strategy for designing advanced functional materials modified by traditional Chinese medicine ingredients. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is an electron microscope graph of the cinnamyl aldehyde@nano calcium carbonate / chitosan hydrogel.

[0029] Figure 2 This is an enlarged image of the spatial network structure inside the cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0030] Figure 3 Representative images of E. coli culture plates after different group treatments.

[0031] Figure 4 Representative images of Staphylococcus aureus culture dishes after different group treatments.

[0032] Figure 5 These are pictures of wound healing at different time points in different treatment groups. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0035] Example 1 Preparation of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel Cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel is prepared by combining ionic crosslinking and ultrasound-assisted methods. The specific steps include: Take 2g of chitosan, dissolve it in 100mL of pure water, add 10.6g of sodium carbonate and 7.4g of calcium hydroxide respectively, mix well, add 4g of urea, mix well and place in a -80℃ ultra-low temperature refrigerator overnight. Take it out the next day and place it at room temperature. After it is completely thawed, place it in a -80℃ ultra-low temperature refrigerator again. After repeated freezing and thawing 3 times, pour the thawed mixture into 24-well plates and 6-well plates, place them in a -80℃ ultra-low temperature refrigerator and repeat freezing and thawing 3 times. Take out the thawed material and soak it in pure water. When the pH value of the soaking liquid is 7.4, take the material out of the pure water, place it in a -80℃ ultra-low temperature refrigerator overnight, and then use a freeze dryer for freeze drying to prepare nano calcium carbonate / chitosan hydrogel. Using an ultrasonic homogenizer, cinnamaldehyde was mixed with pure water to form cinnamaldehyde homogenates with concentrations of 0, 20, 60, 80, and 100 mg / mL. 100 μL of the homogenate was dripped onto the surface of a hydrogel (cylindrical with a diameter of 10 mm and a height of 5 mm) and waited for it to be completely absorbed. Nano-calcium carbonate / chitosan hydrogels (CaCO3 / CS) without cinnamaldehyde and nano-calcium carbonate / chitosan hydrogels (CACO3 / CS) with concentrations of 20, 60, 80, and 100 mg / mL of cinnamaldehyde were prepared. 20 @CaCO3 / CS,CA 60 @CaCO3 / CS,CA 80@CaCO3 / CS,CA 100 @CaCO3 / CS).

[0036] In order to simplify and green the construction strategy of cinnamaldehyde@nano calcium carbonate / chitosan nanosystem, nano calcium carbonate / chitosan hydrogel was first prepared by ionic crosslinking, and then cinnamaldehyde was added to the nano calcium carbonate / chitosan hydrogel to make it adsorbed on the nano calcium carbonate / chitosan hydrogel, thus preparing cinnamaldehyde@nano calcium carbonate / chitosan hydrogel, achieving the fusion of hydrophilic and hydrophobic molecules. This strategy takes advantage of the mutual adsorption between cinnamaldehyde and nano calcium carbonate. The constructed cinnamaldehyde@nano calcium carbonate / chitosan hydrogel electron microscope image is shown below. Figure 1 As shown, the interior of the hydrogel is a spatial network structure. Further magnification reveals that the hydrogel organic skeleton is wrapped with countless calcium carbonate nanoparticles with a diameter of about 50nm ( Figure 2 ), demonstrating that the nano-calcium carbonate in the hydrogel is uniformly dispersed within the organic matter, laying a solid foundation for excellent material performance for both drug delivery and therapeutic applications. Therefore, this invention provides a new method for constructing multifunctional nanostructured systems loaded with cinnamaldehyde.

[0037] Example 2 Antibacterial performance test of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel The plate coating method and inhibition zone method were used to evaluate the antibacterial (Staphylococcus aureus, Escherichia coli) activity of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel to directly reflect whether the antibacterial material can inhibit the growth of bacteria.

[0038] (1) Plate coating method: Six experimental groups were established and placed in 24-well culture plates, including a blank group containing 1 mL PBS, a group containing 1 nano-calcium carbonate / chitosan hydrogel with different cinnamaldehyde concentrations, namely, CaCO 3 / CS Group, CA 20 @CaCO3 / CS group, CA 60 @CaCO3 / CS group, CA 80 @CaCO3 / CS group, CA 100 @CaCO3 / CS group. Each group was added with 10 μL of bacteria at a concentration of 10 6 CFU / mL of bacterial suspension. After incubating the 24-well plate in a 37°C incubator for 2 hours, 1 mL of PBS was added to each well to resuspend the bacteria. 200 μL of the bacterial suspension was spread onto a nutrient agar plate. After incubation in a 37°C incubator for 24 hours, the number of colonies on the nutrient agar plate was counted. Six replicates were set up for all groups. The inhibition rate R was calculated as R = (Qc - Qh) / Qc × 100%. Where: Qh represents the number of bacteria in the calcium carbonate / chitosan hydrogel group; Qc represents the number of bacteria in the blank group.

[0039] (2) Bacteriostatic circle method: about 25 mL of sterilized agar medium was poured into a petri dish while hot. 200 μL of bacterial suspension with a concentration of 10 6 CFU / mL was dropped on the agar plate, and the bacterial suspension was evenly coated with a coating rod. Then, one particle of hydrogel with different drug loading concentrations was placed in the center of the agar plate, namely the CaCO3 / CS group, CA 20 @CaCO3 / CS group, CA 60 @CaCO3 / CS group, CA 80 @CaCO3 / CS group, CA 100 @CaCO3 / CS group, CA

[0040] The results are shown in Figure 3 Table 2. The plate coating method results showed that, compared with the blank group, the number of E. coli colonies in the CaCO3 / CS group was significantly reduced, and the bacteriostatic rate was 56% (p<0.01). The plate colonies of the hydrogel group containing cinnamaldehyde were almost eliminated, and the bacteriostatic rate was close to 100% (p<0.01). The bacteriostatic circle method results showed that, compared with the blank group, the CaCO3 / CS group showed a smaller bacteriostatic circle. With the increase of cinnamaldehyde concentration, the bacteriostatic circle gradually increased to a maximum of 100%, indicating that the bacteriostatic effect of cinnamaldehyde showed a gradient effect. The hydrogel without cinnamaldehyde showed a certain bacteriostatic effect, and the mechanism may be that chitosan can penetrate the bacterial cell wall and combine with DNA, inhibit the synthesis of messenger RNA and the transcription of DNA, and cause the death of bacteria. In addition, chitosan can form a film on the surface of bacteria, preventing the intake of nutrients, which also helps chitosan to kill bacteria. The antibacterial effect of cinnamaldehyde@nano calcium carbonate / chitosan hydrogel is better, and the potential antibacterial mechanism may be that the active lipophilic component cinnamaldehyde can penetrate the double phospholipid layer of the cell membrane, disturb the fluidity of the bacterial cell membrane, and cause the death of bacteria. Similar effects were observed in the antibacterial experiment of Staphylococcus aureus, and the results are shown in Figure 4 Table 3. It is shown that the prepared cinnamaldehyde@nano calcium carbonate / chitosan hydrogel has excellent antibacterial activity on Staphylococcus aureus and Escherichia coli.

[0041] Example 3 Test of the Wound Healing Promotion Performance of Cinnamaldehyde@Nano Calcium Carbonate / Chitosan Hydrogel Thirty-six SPF Kunming male mice (weight 20-25 g, 4 weeks old) were purchased, and the feeding environment was fresh and circulating air with suitable temperature. The experimental mice were first placed in the animal room for one week before the experiment started, and the water and food were freely supplied during the feeding period to exclude the interference of stress factors.

[0042] (1) Animal model construction: To ensure the successful completion of the wound model, 24 hours before modeling, the back of the mouse was shaved with a shaver, and an appropriate amount of depilatory cream was evenly applied to the back of the mouse. After three minutes, the depilatory cream was gently scraped off and wiped clean with a cotton ball. Isoflurane was selected for gas anesthesia, and the modeling experiment was performed after complete anesthesia. The full-thickness skin tissue was removed from the back of the mouse with sterile scissors and forceps, resulting in a circular full-thickness excision wound with a diameter of 9 mm ± 1 mm. The mouse wound was covered with a dressing to prevent excessive bleeding and mutual tearing. After the mouse woke up, it was put back into the mouse cage for observation.

[0043] (2) Group administration: Before administration, the experimental mice were randomly divided into 6 groups, 6 mice per group. The untreated group was used as a blank control, and the gradient concentration of cinnamaldehyde@nano calcium carbonate / chitosan hydrogel was applied to the wound as the CaCO3 / CS group, CA 20 @CaCO3 / CS group, CA 60 @CaCO3 / CS group, CA 80 @CaCO3 / CS group, CA 100 @CaCO3 / CS group. Every other day, take a photo and measure the wound width. For 12 consecutive days.

[0044] The results are shown in Figure 5 The wound photos and wound area change trends of mice at different treatment times are shown. It can be seen that the wound healing of the blank group is the slowest, followed by the CaCO3 / CS group. In contrast, as the concentration of cinnamaldehyde increases, the wound healing rate increases, indicating its superior wound healing performance. The potential mechanism may be that cinnamaldehyde@nano calcium carbonate / chitosan hydrogel can release cinnamaldehyde at the wound site, which can kill bacteria around the wound, remove free radicals at the wound site, inhibit the proliferation of inflammatory cells, and thus achieve the purpose of cleaning the microenvironment of the wound site. At the same time, cinnamaldehyde can also balance and restore the normal immune function of the mouse body tissue, promote the reconstruction of blood vessel network, hair follicle regeneration, and repair of damaged tissue, thereby achieving the purpose of accelerating healing. The present application provides a new direction for the application of cinnamaldehyde@nano calcium carbonate / chitosan nanosystem and a new strategy for designing advanced functional materials modified by food functional factors.

Claims

1. A cinnamaldehyde@nano calcium carbonate / chitosan hydrogel, characterized in that: The invention comprises a hydrogel matrix formed by cross-linking nano calcium carbonate and chitosan, and cinnamaldehyde loaded on the hydrogel matrix.

2. The method for preparing the cinnamaldehyde@nano calcium carbonate / chitosan hydrogel according to claim 1, characterized in that: The preparation method is as follows: S1. Chitosan was mixed with sodium carbonate, calcium hydroxide, and urea, and then subjected to ultra-low temperature freeze-thaw, soaking, and freeze-drying to obtain nano-calcium carbonate / chitosan hydrogel; S2. Add the cinnamaldehyde aqueous solution dropwise onto the surface of the nano-calcium carbonate / chitosan hydrogel to obtain cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

3. The preparation method according to claim 2, characterized in that: In step S1, the mass ratio of chitosan, sodium carbonate, calcium hydroxide and urea is 1:5-5.5:3.5-4:1.8-2.

2.

4. The preparation method according to claim 2, characterized in that In step S1, the ultralow temperature freeze-thaw is to place the sample at an ultralow temperature of -70 to -80°C and then thaw it at room temperature, and repeat the freeze-thaw process 4 to 8 times.

5. The preparation method according to claim 2, characterized in that: In step S1, the immersion is performed in pure water until the pH value of the immersion liquid reaches 7.3 to 7.

5.

6. The preparation method according to claim 2, characterized in that: The cinnamaldehyde aqueous solution in step S2 is prepared by ultrasonic dissolution method.

7. Use of the cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel according to claim 1 in inhibiting bacteria and / or promoting wound healing.

8. Use of the cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel according to claim 1 in the preparation of products for inhibiting bacteria and / or promoting wound healing.

9. The use according to any one of claims 7 or 8, characterized in that: The antibacterial activity is the inhibition of Staphylococcus aureus or Escherichia coli.

10. A product, characterized in that The product comprises the cinnamaldehyde@nano calcium carbonate / chitosan hydrogel according to claim 1.

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