Cinnamaldehyde nanometer calcium carbonate / chitosan hydrogel and preparation method and application thereof

Abstract

The application discloses a cinnamaldehyde-nano calcium carbonate / chitosan hydrogel, which comprises a hydrogel matrix formed by cross-linking of nano calcium carbonate and chitosan, and cinnamaldehyde loaded on the hydrogel matrix. The hydrogel system is prepared by using the mutual adsorption of cinnamaldehyde and nano calcium carbonate, and has the functions of loading traditional Chinese medicine components and intelligently controlling the effective release of the traditional Chinese medicine components. The hydrogel has excellent antibacterial activity and the function of accelerating wound healing, and the effect is obviously better than that of common calcium carbonate / chitosan hydrogel. The application provides a new direction for the application of the cinnamaldehyde-nano calcium carbonate / chitosan nano system, and provides a new strategy for designing advanced functional materials modified by traditional Chinese medicine components.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel, its preparation method, and its application. Background Technology

[0002] Hydrogel dressings are a well-established field of medical research. Their main functions 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. If traditional Chinese medicine ingredients could be combined with hydrogels to create herbal-loaded hydrogels, the good biocompatibility of the hydrogel could be utilized to promote wound cell growth, while the loaded herbal ingredients could provide antibacterial, anti-inflammatory, and antioxidant effects, accelerating wound healing and reducing the likelihood of drug resistance.

[0003] Cinnamaldehyde (CA) is a hydrophobic aromatic aldehyde extracted from cinnamon oil. It is an ideal natural dressing component with antibacterial and antioxidant capabilities and is the main active ingredient of cinnamon, a food and medicine homology substance. Due to its edibility, biodegradability, antibacterial, and anticancer activities, it has been widely used in the food and biomedical fields. However, the strong hydrophobicity of cinnamaldehyde makes it difficult to integrate with hydrophilic matrices, and its volatility significantly reduces its bioavailability. To incorporate this oily active ingredient into the hydrogel matrix, the paper "Construction of Tannic Acid and Cinnamaldehyde Nanofunctionalized System and Its Antibacterial and Antioxidant Properties" (Sun Xinyu, 2022) showed that combining cinnamaldehyde and tannic acid through ultrasonic-assisted emulsification to form a composite hydrogel exhibits good antibacterial and antioxidant properties. However, it cannot intelligently control the release of the active pharmaceutical ingredient. Therefore, there is an urgent need to prepare a hydrogel system loaded with cinnamaldehyde and capable of intelligent release. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned defects and deficiencies in the prior art and provide a cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel. This invention combines nano-calcium carbonate with chitosan, and then utilizes the high oil absorption value of nano-calcium carbonate to load cinnamaldehyde, thus constructing an "oil-in-water" cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

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

[0006] A third objective of this invention is to provide the application of the above-mentioned hydrogel in antibacterial and / or wound healing promotion.

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

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] The present invention provides a cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel, comprising a hydrogel matrix formed by crosslinking nano-calcium carbonate and chitosan, and cinnamaldehyde loaded on the hydrogel matrix.

[0010] Chitosan has better solubility, making it more suitable for preparing hydrogels. During the formation of hydrogels, chitosan, as the main polymer matrix, forms a stable three-dimensional network structure through 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 activities.

[0011] Nano-calcium carbonate is commonly used as a drug carrier in targeted, controlled-release drug therapy for cancer. The main mechanism is that the environment surrounding cancer cells is a low pH. When drug-loaded nano-calcium carbonate is placed in this acidic environment, it dissolves and releases the effective drug loaded within it. Simultaneously, nano-calcium carbonate can inhibit the regeneration of inflammatory cells and reduce the probability of wound infection. Since the wound environment is acidic, using nano-calcium carbonate to prepare hydrogels can also release the effective drug loaded within it.

[0012] Cinnamaldehyde is a hydrophobic aromatic aldehyde, an ideal natural dressing ingredient with antibacterial and antioxidant properties extracted from cinnamon oil.

[0013] Therefore, by combining nano-calcium carbonate with chitosan, and then utilizing the high oil absorption value of nano-calcium carbonate to load cinnamaldehyde, a water-in-oil cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel was constructed, 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 has significant research value.

[0014] This invention provides a method for preparing the above-mentioned hydrogel, the method being as follows:

[0015] S1. Chitosan is mixed with sodium carbonate, calcium hydroxide and urea, then subjected to ultra-low temperature freeze-thaw, soaked and freeze-dried to obtain nano-calcium carbonate / chitosan hydrogel.

[0016] S2. Add cinnamaldehyde aqueous solution to the surface of nano-calcium carbonate / chitosan hydrogel to obtain cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0017] This invention utilizes the natural antibacterial agent cinnamaldehyde as the active component to construct a cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel system. First, chitosan is cross-linked with nano-calcium carbonate using its self-polymerization properties to form a nano-calcium carbonate / chitosan hydrogel. Then, cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel is prepared by utilizing the high oil absorption value of nano-calcium carbonate and the adsorption effect between it and cinnamaldehyde. Regarding the specific preparation principle: Since chitosan itself is poorly soluble in pure water, the pH of the solution can be adjusted to an alkaline environment (approximately pH 12-13) by adding alkaline substances (sodium carbonate and calcium hydroxide), causing the amino (-NH2) portions of the chitosan molecules to deprotonate and form soluble chitosan salts. This step creates conditions for subsequent cross-linking and mineralization reactions. The added calcium hydroxide (Ca(OH)2) and sodium carbonate (Na2CO3) dissociate in water to release Ca... 2+ and CO3 2- Calcium carbonate (CaCO3) nanoparticles are generated through ionic reactions. These particles can act as physical cross-linking points embedded in the chitosan network, enhancing the mechanical properties of the hydrogel and enabling intelligent control of the effective release of traditional Chinese medicine components. Simultaneously, Ca... 2+ Ionic crosslinking with the amino and hydroxyl groups of chitosan promotes the formation of a three-dimensional network. Urea molecules disrupt the hydrogen bonds between chitosan molecules, promoting the detangling of molecular chains and improving solution homogeneity; simultaneously, urea acts as a low-temperature stabilizer, inhibiting excessive ice crystal growth during freeze-thaw cycles and reducing mechanical damage to the gel structure. Through repeated freeze-thaw cycles inducing physical crosslinking, rapid freezing at -80℃ causes water in the solution to form tiny ice crystals, forcing chitosan molecular chains 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 crosslinked network. Multiple freeze-thaw cycles (6 in total) significantly improve the crosslinking density and gel strength.

[0018] Furthermore, 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.

[0019] Preferably, in step S1, the mass ratio of chitosan, sodium carbonate, calcium hydroxide, and urea is 2:10.6:7:4.

[0020] Furthermore, the method of ultra-low temperature freeze-thaw in step S1 is to freeze the food in an ultra-low temperature freezer at -70 to -80°C and then thaw it at room temperature, repeating the freeze-thaw cycle 4 to 8 times.

[0021] Furthermore, the soaking conditions in step S1 are as follows: soaking in pure water until the pH value of the soaking solution is 7.3 to 7.5.

[0022] Preferably, the soaking conditions in step S1 are: soaking in pure water until the pH value of the soaking solution is 7.4.

[0023] Furthermore, in step S2, the method of adding cinnamaldehyde aqueous solution to the surface of nano-calcium carbonate / chitosan hydrogel is to take 100 μL of cinnamaldehyde homogenate and add it dropwise onto the surface of a cylindrical hydrogel with a diameter of 10 mm and a height of 5 mm.

[0024] Furthermore, the preparation method of the cinnamaldehyde aqueous solution in step S2 is ultrasonic dissolution.

[0025] This invention tested the antibacterial effect of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel in vitro, finding that the hydrogel exhibits excellent antibacterial activity against Staphylococcus aureus and Escherichia coli. By constructing a mouse wound model, the wound-healing effect of the cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel was tested, showing that the hydrogel can rapidly gel in situ at the wound site, covering irregular wounds, effectively killing bacteria, cleaning the wound microenvironment, and accelerating wound healing. This invention provides a new direction for the application of cinnamaldehyde@nano-calcium carbonate / chitosan nanosystems and also offers a new strategy for designing advanced functional materials modified with traditional Chinese medicine components.

[0026] Therefore, the present invention provides the application of the above-mentioned hydrogel in antibacterial and / or wound healing promotion.

[0027] The present invention also provides the use of the above-described hydrogel in the preparation of antibacterial and / or wound-healing products.

[0028] Furthermore, the antibacterial effect is to inhibit Staphylococcus aureus or Escherichia coli.

[0029] The present invention also provides a product comprising the above-mentioned cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention provides a cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel, comprising a hydrogel matrix formed by crosslinking nano-calcium carbonate and chitosan, and cinnamaldehyde loaded onto the hydrogel matrix. This invention utilizes the mutual adsorption between cinnamaldehyde and nano-calcium carbonate to prepare a hydrogel system loaded with traditional Chinese medicine components and intelligently controlling the effective release of these components. This hydrogel exhibits excellent antibacterial activity and wound-healing acceleration, with significantly better effects than ordinary calcium carbonate / chitosan hydrogels. This invention provides a new direction for the application of cinnamaldehyde@nano-calcium carbonate / chitosan nanosystems and a new strategy for designing advanced functional materials modified with traditional Chinese medicine components. Attached Figure Description

[0032] Figure 1 Electron micrograph of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0033] Figure 2 This is an enlarged view of the internal spatial network structure of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel.

[0034] Figure 3 Representative images of E. coli culture dishes after different grouping and treatment methods.

[0035] Figure 4 Representative images of Staphylococcus aureus culture dishes after different grouping and treatment methods.

[0036] Figure 5 Images of wound healing at different time points for different treatment groups. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] Example 1: Preparation of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel

[0040] Cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel was prepared using a combination of ion crosslinking and ultrasound-assisted methods. The specific steps included are as follows:

[0041] Dissolve 2g of chitosan in 100mL of pure water, then add 10.6g of sodium carbonate and 7.4g of calcium hydroxide, mix well, add 4g of urea, mix again, and place in a -80℃ ultra-low temperature freezer overnight. The next day, remove and place at room temperature until completely thawed, then place back in the -80℃ ultra-low temperature freezer. Repeat this freeze-thaw cycle three times. Pour the thawed mixture into 24-well and 6-well plates, place in a -80℃ ultra-low temperature freezer, and repeat the freeze-thaw cycle three times. Remove the thawed material and immerse it in pure water. When the pH of the immersion solution reaches 7.4, remove the material from the pure water, place it in a -80℃ ultra-low temperature freezer overnight, and then freeze-dry using a freeze dryer to prepare nano-calcium carbonate / chitosan hydrogel. Using an ultrasonic homogenizer, cinnamaldehyde was mixed with pure water to prepare cinnamaldehyde homogenates with concentrations of 0, 20, 60, 80, and 100 mg / mL. 100 μL of each homogenate was then dropped onto the surface of a hydrogel (a cylindrical structure 10 mm in diameter and 5 mm in height). After complete absorption, cinnamaldehyde-free nano-calcium carbonate / chitosan hydrogels (CaCO3 / CS) and cinnamaldehyde-containing nano-calcium carbonate / chitosan hydrogels (CA) with concentrations of 20, 60, 80, and 100 mg / mL were prepared. 20 @CaCO3 / CS, CA60 @CaCO3 / CS, CA 80 @CaCO3 / CS, CA 100 @CaCO3 / CS).

[0042] To simplify and greenen the construction strategy of cinnamaldehyde@nano-calcium carbonate / chitosan nanosystem, a nano-calcium carbonate / chitosan hydrogel was first prepared via ion crosslinking. Then, cinnamaldehyde was dropwise added to the nano-calcium carbonate / chitosan hydrogel, allowing it to adsorb onto the hydrogel, thus preparing the cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel. This strategy achieves the fusion of hydrophilic and hydrophobic molecules, leveraging the mutual adsorption between cinnamaldehyde and nano-calcium carbonate. The electron micrograph of the constructed cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel is shown below. Figure 1 As shown, the hydrogel has a spatial network structure inside. Further magnification reveals that the hydrogel's organic framework is wrapped with countless calcium carbonate nanoparticles with a diameter of about 50 nm. Figure 2 This demonstrates that the nano-calcium carbonate in the hydrogel is uniformly dispersed in the organic matter, providing a solid material performance foundation for drug loading and treatment. Therefore, this invention provides a new method for constructing multifunctional nanosystems loaded with cinnamaldehyde.

[0043] Example 2: Antibacterial performance test of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel

[0044] The antibacterial (Staphylococcus aureus, Escherichia coli) activity of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel was evaluated using the plate coating method and the inhibition zone method to directly reflect whether the antibacterial material can inhibit bacterial growth.

[0045] (1) Plate coating method: Six experimental groups were established and placed in 24-well culture plates. The groups were: a blank group containing 1 mL of PBS, a group containing 1 nano-calcium carbonate / chitosan hydrogel with different concentrations of cinnamaldehyde, and a group containing CaCO3. 3 / CS group, CA 20 @CaCO3 / CS group, CA 60 @CaCO3 / CS group, CA 80 @CaCO3 / CS group, CA 100 @CaCO3 / CS group. Add 10 μL of bacteria to each group to achieve a bacterial concentration of 10. 6CFU / mL bacterial suspension. After incubating the 24-well plates at 37°C 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 nutrient agar plates and incubated at 37°C for 24 hours. The colony count on the nutrient agar plates was then performed. All groups were replicated in six places. 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 control group.

[0046] (2) Inhibition zone method: Pour approximately 25 mL of sterilized agar medium into a petri dish while it is still hot. Take 200 μL of bacterial concentration of 10... 6 A bacterial suspension of CFU / mL was dropped onto an agar plate and spread evenly with a spreader. Then, a hydrogel with a different drug loading concentration was placed in the center of the agar plate; this constitutes the CaCO3 / CS group. 20 @CaCO3 / CS group, CA 60 @CaCO3 / CS group, CA 80 @CaCO3 / CS group, CA 100 The CaCO3 / CS group served as a blank control group without hydrogel. All groups were divided into six replicates, and the inhibition zone diameter was measured after incubation at 37°C for 24 hours.

[0047] The results are as follows Figure 3 As shown, the plate coating method results indicated that, compared to the control group, the number of *E. coli* colonies in the CaCO3 / CS group was significantly reduced, with an inhibition rate of 56% (p < 0.01). In the cinnamaldehyde-containing hydrogel group, colonies were almost completely eliminated on the plate, with an inhibition rate approaching 100% (p < 0.01). The inhibition zone method results showed that, compared to the control group, the CaCO3 / CS group exhibited a smaller inhibition zone. With increasing cinnamaldehyde concentration, the inhibition zone gradually increased in size, reaching a maximum of 100%, indicating a gradient effect of cinnamaldehyde's antibacterial effect. The cinnamaldehyde-free hydrogel showed a certain antibacterial effect; the mechanism may be that chitosan can penetrate the bacterial cell wall and bind to its DNA, inhibiting messenger RNA synthesis and DNA transcription, leading to bacterial death. Furthermore, chitosan can form a film on the bacterial surface, preventing nutrient uptake, which also helps chitosan kill bacteria. Cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel exhibits better antibacterial effects. Its potential antibacterial mechanism may be due to the active lipophilic component cinnamaldehyde's ability to penetrate the phospholipid bilayer of the cell membrane, disrupting bacterial cell membrane fluidity and leading to bacterial death. Similar effects were observed in antibacterial experiments against Staphylococcus aureus, with results as follows... Figure 4 As shown, the prepared cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel exhibits excellent antibacterial activity against Staphylococcus aureus and Escherichia coli.

[0048] Example 3: Test of the wound healing properties of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel

[0049] Thirty-six SPF-grade male Kunming mice (weighing 20-25g, 4 weeks old) were purchased and housed in a well-ventilated environment with suitable temperature. The mice were placed in the animal room for one week before the experiment to acclimatize, with free access to water and food during this period, and stress factors were eliminated.

[0050] (1) Animal model construction: To ensure the successful completion of the wound model, 24 hours before modeling, the hair on an appropriate area of ​​the mouse's back was removed with a shaver, and an appropriate amount of depilatory cream was evenly applied to the mouse's back. After three minutes, the depilatory cream was gently scraped off and the area was wiped clean with a cotton ball. Isoflurane was used for gas anesthesia, and the modeling experiment was carried out after complete anesthesia. Full-thickness skin tissue was removed from the mouse's back using sterilized scissors and tweezers to create 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 biting. After the mouse woke up, it was put back into the cage for observation and later use.

[0051] (2) Grouping and administration: Before administration, the experimental mice were randomly divided into 6 groups of 6 mice each. The untreated group served as the blank control. The group receiving gradient concentrations of cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel as the CaCO3 / CS group received the treatment. 20 @CaCO3 / CS group, CA 60 @CaCO3 / CS group, CA 80 @CaCO3 / CS group, CA 100 @CaCO3 / CS group. Administer medication every other day, take photos, and measure the wound width. Continue for 12 consecutive days.

[0052] The results are as follows Figure 5 As shown, the images of the mice's wounds and the trend of wound area changes at different treatment times are displayed. It can be seen that the wound healing was slowest in the blank group, followed by the CaCO3 / CS group. In contrast, the wound healing speed accelerated with increasing cinnamaldehyde concentration, 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, scavenge free radicals at the wound site, and inhibit the proliferation of inflammatory cells, thereby achieving the purpose of cleaning the microenvironment of the wound site. Simultaneously, cinnamaldehyde can also balance and restore the normal immune function of the mouse's tissues, promote vascular network reconstruction, hair follicle regeneration, and repair of damaged tissues, thereby accelerating healing. This invention provides a new direction for the application of the cinnamaldehyde@nano-calcium carbonate / chitosan nanosystem and a new strategy for designing advanced functional materials modified with food functional factors.

Claims

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

2. The preparation method according to claim 1, 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.

3. The preparation method according to claim 1, characterized in that, In step S1, the cryogenic freeze-thaw process involves freezing the food at -70 to -80°C and then thawing it at room temperature, repeating this process 4 to 8 times.

4. The preparation method according to claim 1, characterized in that, In step S1, soaking involves immersing the food in pure water until the pH of the soaking solution reaches 7.3 to 7.

5.

5. The preparation method according to claim 1, characterized in that, The cinnamaldehyde aqueous solution in step S2 is prepared by ultrasonic dissolution.

6. A cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel prepared by any one of the preparation methods described in claims 1 to 5.

7. The use of the cinnamaldehyde@nano-calcium carbonate / chitosan hydrogel of claim 6 in the preparation of products that inhibit bacteria and / or promote wound healing.

8. The application according to claim 7, characterized in that, The antibacterial effect refers to the inhibition of Staphylococcus aureus or Escherichia coli.

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