Alkannin-loaded hydrogel dressing for treating specific dermatitis

Abstract

The invention discloses an alkannin-loaded hydrogel dressing for treating specific dermatitis, and belongs to the technical field of biological medicines. The hydrogel dressing comprises an alkannin liposome and a hydrogel matrix, the alkannin liposome is prepared from alkannin, lecithin and cholesterol, and the hydrogel matrix is prepared from carboxymethyl chitosan, tannic acid, CuSO4 and water. According to the invention, the alkannin is firstly prepared into the liposome and then dispersed into the hydrogel to obtain the hydrogel dressing, and the combined preparation not only enhances the penetrability and stability of the alkannin, but also can locally act on the affected part for a long time in a continuous release manner, so that the inflammatory response is remarkably reduced, and the repair of the skin barrier is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a hydrogel dressing loaded with shikonin for the treatment of atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a common chronic inflammatory skin disease, usually accompanied by significant itching, affecting people of all ages worldwide. Although its pathogenesis is not fully understood, it is currently believed to be caused by a combination of genetic factors, immune imbalance, and environmental stimuli. AD patients have impaired skin barrier function, leading to the invasion of external sensitizers and an overreaction of the immune system. With the increasing incidence of AD, especially in children and adolescents, AD significantly impacts patients' quality of life. Current commonly used medications for AD include topical corticosteroids and immunosuppressants, but long-term use of these drugs may cause side effects, thus necessitating the development of new treatment methods.

[0003] Shikonin is a natural organic compound extracted from the plant *Lithospermum erythrorhizon*, belonging to the Boraginaceae family. Shikonin has been used in traditional Chinese medicine for thousands of years, widely applied to treat skin diseases, burns, wounds, and inflammation. Studies have shown that shikonin possesses various biological activities, including anti-inflammatory, antibacterial, antioxidant, and wound-healing-promoting effects, thus attracting widespread attention in modern dermatological drug development. However, the clinical application of shikonin still faces some limitations, mainly including poor water solubility, low bioavailability, and poor stability in vivo, which restricts its widespread use in the treatment of skin diseases. Although shikonin has shown strong anti-inflammatory and skin-repairing effects in in vitro studies, its poor water solubility prevents it from effectively reaching the deep layers of the skin when applied directly, and it is easily blocked by the skin barrier. Furthermore, the poor stability of shikonin also limits its effectiveness in conventional dosage forms. Therefore, to overcome these limitations, researchers have recently explored encapsulating shikonin in liposomes to improve its bioavailability and efficacy. Liposomes can effectively encapsulate shikonin, preventing its metabolic degradation in the body, and improve drug delivery efficiency by promoting skin permeability.

[0004] Hydrogels are polymeric materials with high hydrophilicity, capable of absorbing and retaining large amounts of moisture. Due to their softness, transparency, non-irritation, and ease of application, hydrogels are widely used in skin care and drug delivery. In the treatment of atopic dermatitis (AD), hydrogel formulations offer significant advantages, particularly in topical skin treatment. A key characteristic of AD is impaired skin barrier function, leading to moisture loss and dry skin. Hydrogels possess strong water absorption and moisturizing properties, forming a hydrating protective layer on the skin surface to prevent moisture evaporation, alleviate dryness symptoms, and restore skin barrier function. Hydrogels not only provide long-lasting hydration but also serve as a drug delivery system, extending the duration of drug efficacy by controlling the drug release rate. This is particularly important for relieving itching and inflammation in AD patients, reducing the need for frequent medication use and improving treatment adherence. The moisture in hydrogels helps lower skin temperature, alleviating redness and heat caused by AD.

[0005] In addition, hydrogels have wound-healing properties and can accelerate the skin repair process. In the process of skin damage and chronic inflammation in AD patients, hydrogels help provide a suitable moist environment, promote cell regeneration, and reduce scar formation.

[0006] Therefore, this invention designs and prepares shikonin liposomes and disperses them in a hydrogel to achieve precise treatment of atopic dermatitis. Summary of the Invention

[0007] One objective of this invention is to provide a hydrogel dressing loaded with shikonin, comprising shikonin liposomes and a hydrogel matrix; The shikonin liposomes are made of shikonin, lecithin and cholesterol, with a mass ratio of shikonin, lecithin and cholesterol of 0.5~5:10~25:0.5~2. The hydrogel matrix is ​​made of carboxymethyl chitosan, tannic acid, CuSO4 and water, with a concentration of 2.5 g / 100 mL for carboxymethyl chitosan, a concentration of 1.67-3.33 g / 100 mL for tannic acid, and a concentration of 66.6 mg / 100 mL for CuSO4. The volume ratio of the shikonin liposomes to the hydrogel matrix is ​​42:100.

[0008] Furthermore, the concentration of the tannic acid is 1.67 g / 100 mL.

[0009] The shikonin liposomes of the present invention can be prepared using methods commonly used in the art, such as thin-film evaporation, reverse evaporation, solvent injection, and double emulsion. In a specific embodiment of the present invention, thin-film evaporation is used.

[0010] A second objective of this invention is to provide a method for preparing the above-mentioned hydrogel dressing loaded with shikonin, comprising the following steps: Step 1: Prepare shikonin liposomes; Step 2: Take tannic acid and add it to water to obtain a tannic acid solution. Adjust the pH of the tannic acid solution to 7.0~8.0, add carboxymethyl chitosan solution and shikonin liposomes, stir and mix, and then add CuSO4 solution to obtain the hydrogel dressing.

[0011] Furthermore, the shikonin liposomes are prepared using a thin-film dispersion method. Specifically: shikonin, soybean lecithin, and cholesterol are mixed and dissolved in anhydrous ethanol solution; the mixture is transferred to a round-bottom flask, and rotary evaporated to remove the ethanol, yielding a thin film; subsequently, hydration, ultrasonic disruption, dialysis, and extrusion are performed to obtain liposomes.

[0012] Furthermore, in step 2, NaOH solution is used to adjust the pH.

[0013] Furthermore, the concentration of the NaOH solution is 0.1 M.

[0014] The inventors' preliminary experiments revealed that during hydrogel preparation, the pH of the solution must be controlled between 7.0 and 8.0 for gel formation to occur. Furthermore, the order of solution addition is crucial: first, the carboxymethyl chitosan and tannic acid solutions must be thoroughly mixed, followed by the dropwise addition of CuSO4 solution. Mixing the carboxymethyl chitosan and CuSO4 solution first easily leads to the formation of localized blue gels with lower gel strength and incomplete mixing. To control gel strength, the concentration of tannic acid needs to be maintained between 4% and 8% (g / mL); at lower concentrations (1% to 2%), the hydrogel cannot form.

[0015] A third objective of this invention is to provide the application of the above-mentioned hydrogel dressing loaded with shikonin in the preparation of products for treating inflammatory skin diseases. Further, the inflammatory skin disease is atopic dermatitis.

[0016] This invention first prepares shikonin into liposomes, then disperses it in a hydrogel to obtain a hydrogel dressing for precise treatment of atopic dermatitis. This combined formulation not only enhances the penetration and stability of shikonin but also provides sustained local release, significantly reducing inflammation and aiding in skin barrier repair. This novel therapy holds promise as an important strategy for future atopic dermatitis treatment, particularly by offering a safer and more effective treatment option while reducing the side effects of traditional medications. Attached Figure Description

[0017] Figure 1 This is a hydrated particle size distribution diagram of shikonin liposomes.

[0018] Figure 2 This is a TEM image of shikonin liposomes.

[0019] Figure 3 This is a diagram showing shikonin liposomes and the in vitro release of shikonin.

[0020] Figure 4 The particle size results of shikonin after storage at 4 ℃ for 14 days are shown in the figure.

[0021] Figure 5 The results show the viscosity and strength of blank hydrogels composed of different tannic acids, as determined by a rheometer.

[0022] Figure 6 The infrared spectrum of the hydrogel was measured using an infrared spectrophotometer.

[0023] Figure 7 This is a diagram of the internal structure of the hydrogel as determined by scanning electron microscopy.

[0024] Figure 8 This is a frequency scan diagram of the drug-loaded hydrogel, measured using a rheometer.

[0025] Figure 9 This is a graph showing the adhesion of the hydrogel.

[0026] Figure 10 To determine the antioxidant properties of shikonin solutions of different concentrations.

[0027] Figure 11 To investigate the antioxidant capacity of shikonin liposomes and shikonin-loaded liposome hydrogels.

[0028] Figure 12 For the determination of the in vitro release rate of drug-loaded hydrogels (including in vitro dialysis method and ex vivo skin assay method).

[0029] Figure 13 Images showing the results of H&E staining and toluidine blue staining of skin after treatment in different treatment groups.

[0030] Figure 14 The graph shows the IgE concentration results after treatment in different treatment groups.

[0031] Figure 15 The spleen index is for mice.

[0032] Figure 16 TEWL results of skin after treatment in different treatment groups. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Example 1

[0034] I. Preparation and Characterization of Shikonin Liposomes Accurately weigh shikonin, soybean lecithin, and cholesterol (mass ratio 1:20:1) and add them to a centrifuge tube. Add 5 mL of anhydrous ethanol solution and sonicate for 5 min until completely dissolved. Transfer the solution to a round-bottom flask and rotary evaporate at 37 °C to remove ethanol under vacuum of 0.03–0.04 MPa to form a thin film. Vacuum dry the round-bottom flask overnight at 37 °C to remove excess ethanol. Then, add 20 mL of PBS, sonicate for 10 min, and magnetically stir at 37 °C for 2 h to completely hydrate the film. Then, sonicate for 10 min using a cell disruptor. Dialyze using a MWCO 8000 Da dialysis bag to remove unencapsulated shikonin. Finally, extrude the prepared liposomes using a liposome extruder (membrane pore size 100 nm) 100 times.

[0035] Shikonin liposomes were prepared by thin-film dispersion, and their particle size and potential were measured. The results are as follows: Figure 1 As shown in the figure. The hydrated particle size of the shikonin liposomes was 92.72 ± 1.63 nm, the PDI was 0.203 ± 0.06, and the zeta potential was -16.54 ± 1.34 mV, indicating that the prepared shikonin liposomes had good dispersibility. Next, its morphology was characterized by TEM, and the results are shown in the figure. Figure 2 As shown in the figure, the prepared liposomes have a uniform particle size, with a visible membrane layer and a particle size of approximately 90 nm, consistent with the hydrated particle size determination. The in vitro release rate of the shikonin liposomes was determined by dialysis, and the results are as follows. Figure 3 As shown in the figure, shikonin is poorly soluble in water; therefore, shikonin powder floats on the surface of the dissolution medium, and only trace amounts of shikonin are released. For shikonin liposomes, the cumulative release significantly increases over time, reaching approximately 80% after 24 hours, exhibiting a certain sustained-release effect. Finally, the storage stability of shikonin liposomes at 4 °C was evaluated by measuring their hydrated particle size, and the results are shown in the figure. Figure 4 As shown in the figure. The results showed that the particle size of shikonin liposomes remained essentially unchanged after 14 days of storage at 4 ℃, indicating good storage stability. The drug loading and encapsulation efficiency of the shikonin liposomes were determined by ultraviolet spectrophotometry, and the drug loading was 3.98 ± 0.56%, and the encapsulation efficiency was 92.34 ± 2.54%.

[0036] II. Preparation and Characterization of Hydrogels 1. Investigation of tannic acid concentration Preparation of blank hydrogel: The hydrogel was prepared under magnetic stirring. 5 mL of tannic acid solution was added to a vial, and the pH was adjusted to 7.5 using 0.1 M NaOH. Next, 5 mL of 6% (6 g / 100 mL) carboxymethyl chitosan solution was added, and the mixture was magnetically stirred for 5 min until homogeneous, forming a flowable semi-solid gel. Then, 2 mL of 0.4% (0.4 g / 100 mL) CuSO4 solution was added to the above solution, and the mixture was allowed to stand for 3 min to form a homogeneous gel.

[0037] The effect of different concentrations of tannic acid (TA) (2%, 4%, and 8%) on hydrogels was investigated. When the tannic acid concentration was 2%, no gel could form; as... Figure 5 As shown, the viscosity and gel strength of the formed hydrogel both decrease when the tannic acid concentration increases.

[0038] Hydrogels with good hygroscopic properties can effectively reduce the production of wound exudate, keeping the wound in a suitable and moderate environment, which is conducive to wound healing. Therefore, the hygroscopic properties of the prepared hydrogel were measured and the results are shown in Table 1.

[0039] Table 1. Hygroscopic properties of hydrogels prepared with different TA concentrations

[0040] In the table, W0 is the initial weight, and W1 is the weight after moisture absorption.

[0041] The results showed that the hydrogel prepared with 4% tannic acid had a stronger moisture absorption capacity than the hydrogel prepared with 8% tannic acid.

[0042] The humidification capacity of hydrogels is also an important indicator for evaluating their performance. A 35% gelatin solution was used to simulate a dry skin wound. In this experiment, when the hydrogel came into contact with gelatin, the amount of water lost by the hydrogel was the water absorbed by the gelatin. This water loss typically represents the hydrogel's ability to provide a moist environment for the dry skin wound. The results showed that the hydrogel prepared with 4% tannic acid had a stronger humidification capacity than the hydrogel prepared with 8% tannic acid.

[0043] Based on the above results, considering the viscosity, gel strength, humidification and hygroscopic properties of the hydrogel, 4% tannic acid was selected for the preparation of the hydrogel.

[0044] The mixing order is particularly important in the preparation of hydrogels. Carboxymethyl chitosan (CMCS) and tannic acid (TA) can be cross-linked via hydrogen bonds. The numerous phenolic hydroxyl groups (-OH) on the TA molecule can form a dense hydrogen bond network with the hydroxyl groups (-OH), carboxyl groups (-COOH), and unsubstituted amino groups (-NH2) on the CMCS molecule; while Cu2+ It can coordinate with the carboxyl groups on tannic acid and carboxymethyl chitosan to form very stable complexes, thereby enhancing the strength of the hydrogel. In this invention, if carboxymethyl chitosan and CuSO4 are mixed first, local blue gels are easily formed with low gel strength and uneven mixing; therefore, the order of addition must be that the carboxymethyl chitosan and tannic acid solutions are mixed evenly first, and then the CuSO4 solution is added dropwise.

[0045] The interaction between tannic acid, carboxymethyl chitosan, and CuSO4 was analyzed using Fourier transform infrared spectroscopy (FT-IR), and the results are as follows: Figure 6 As shown. The results indicate that TA and Cu 2+ As a crosslinking agent, it can form hydrogels through ionic bonds and hydrogen bonds.

[0046] 2. Preparation of drug-loaded hydrogel: The drug-loaded hydrogel was prepared under magnetic stirring. 5 mL of 4% tannic acid solution was taken, and the pH was adjusted to 7.5 with 0.1 M NaOH. Then, 5 mL of 6% carboxymethyl chitosan solution and 5 mL of liposome solution were added, and the mixture was magnetically stirred for 5 min until homogeneous, forming a flowable semi-solid gel. Next, 2 mL of 0.4% CuSO4 solution was added to the above solution, and the mixture was allowed to stand for 3 min to form a homogeneous gel.

[0047] The formation of hydrogels was investigated using the inverted bottle method, such as... Figure 7 As shown in the figure, the hydrogels prepared from tannic acid, carboxymethyl chitosan, and CuSO4 did not exhibit significant flow, indicating successful hydrogel preparation. The microstructure of the hydrogels was observed using scanning electron microscopy, and the results are as follows. Figure 7 As shown in the figure, the prepared hydrogel exhibits a loose and porous network structure, which is beneficial for the encapsulation and release of shikonin liposomes. The rheological properties of the prepared hydrogel are as follows: Figure 8 As shown, the storage modulus (G') of the hydrogel is much greater than the loss modulus (G'') over time, indicating that the prepared hydrogel is a stable solid state.

[0048] Because tannic acid in the hydrogel contains polyphenol groups, it exhibits a strong binding affinity to the thiol and amino groups of peptides and proteins on the tissue surface. Therefore, the adhesion of the prepared hydrogel to the tissue was investigated. Figure 9 As shown, the prepared hydrogel can effectively adhere to fingers and detached pig skin, demonstrating its high tissue adhesion properties.

[0049] Next, the antioxidant properties of different concentrations of shikonin solutions were investigated, and the specific procedures are as follows. The experiment was divided into three groups: a blank control group, a sample group, and a sample control group. 2 mL of anhydrous ethanol and 1 mL of 0.08 mg / mL DPPH anhydrous ethanol solution were used as the blank control group; 2 mL of shikonin or shikonin preparations (shikonin liposomes and hydrogels with equivalent shikonin content) and 1 mL of DPPH anhydrous ethanol solution were used as the sample group; and 2 mL of shikonin or shikonin preparations (shikonin liposomes and hydrogels with equivalent shikonin content) and 1 mL of anhydrous ethanol solution were used as the sample control group. All solutions were mixed thoroughly and incubated at 37°C in the dark for 30 min. Then, the absorbance of each component was measured at 517 nm using a UV spectrophotometer. The DPPH scavenging rate was calculated using the following formula:

[0050] Where A0 is the absorbance of the blank control group, A s A represents the absorbance of the sample group. c This indicates the absorbance of the sample control group.

[0051] like Figure 10 As shown in the figure. The results indicate that the DPPH scavenging rate of shikonin is concentration-dependent. Within the concentration range of 0.2–0.8 mg / mL, the DPPH scavenging rate of shikonin increases with increasing concentration. When the concentration increases to 1.0 mg / mL, the DPPH scavenging rate of shikonin reaches 70%, and there is no significant difference compared with the 0.8 mg / mL concentration, indicating that shikonin itself has strong antioxidant activity.

[0052] Since tannins and other substances also possess certain antioxidant activity, the antioxidant capacity of the aforementioned shikonin liposomes and drug-loaded hydrogels was determined, and the results are as follows: Figure 11 As shown in the figure. The results indicate that shikonin liposomes exhibit strong DPPH scavenging ability. Under the same conditions, the antioxidant capacity of the hydrogel loaded with shikonin liposomes reached 80%, which was superior to that of shikonin liposomes. This suggests that the presence of the hydrogel did not affect the antioxidant effect of shikonin, but rather enhanced its antioxidant capacity.

[0053] PBS solution containing 30% ethanol at pH 7.4 was used as the diffusion medium in the lower chamber. The in vitro release rate was investigated using a dialysis membrane and normal mouse skin as simulated physiological environments. The results are as follows: Figure 12 As shown, shikonin exhibits a slow release behavior in both models.

[0054] III. Pharmacodynamic evaluation of drug-loaded hydrogels for atopic dermatitis A mouse model of Alzheimer's disease (AD) was induced by topical application of 2,4-dinitrochlorobenzene (DNCB) to the dorsal skin. Mice were randomly assigned to four groups: a control group (Obstructive Doppler), a model group (MOD group), a positive control group (mometasone furoate cream, MOM group), a blank gel group (CTCu group), and a drug-loaded hydrogel group (CTCu@ZCS group, 0.8 mg / mL). The mice were shaved one day prior to the experiment. The MOD, MOM, CTCu, and CTCu@ZCS groups were sensitized with 100 μL of 5% DNCB (dissolved in acetone and olive oil in a 3:1 (v / v) solution). The same volume of acetone / olive oil in a 3:1 (v / v) solution was used for the control group for two days. Starting on day 4, the Control and MOD groups were treated with PBS, the MOM group with mometasone furoate cream, and the CTCu and CTCu@ZCS groups with blank hydrogel and drug-loaded hydrogel applied to their backs, respectively, for 12 days. On day 14, all mice were anesthetized with 20% urethane and sacrificed. Samples were taken to measure skin lesion index, IgE level, TEWL level, and spleen index. Simultaneously, H&E was used to assess skin lesion condition, and toluidine blue staining was used to examine mast cell count.

[0055] like Figure 13 As shown in the figure, the results indicated that the skin thickness and mast cell count in the MOD group were significantly higher than those in the Control group. Furthermore, treatment of mice with anti-inflammatory agents such as CTCu@ZCS often resulted in reduced skin thickness and a decrease in mast cell count, suggesting that these treatments have the potential to alleviate skin inflammation and restore skin barrier function.

[0056] The results are as follows Figure 14 As shown in the figure. The results indicate that IgE levels were significantly elevated in the MOD group, while serum IgE levels significantly decreased after CTCu@ZCS treatment, which can effectively regulate IgE levels and thus treat AD.

[0057] The results are as follows Figure 15 As shown, compared to the control group (Control group), the spleen index of mice treated with DNCB was significantly increased. This phenomenon indicates that splenomegaly is caused by the inflammatory response induced by atopic dermatitis. After treatment with CTCu@ZCS preparation, the spleen index of mice was significantly reduced compared to the model group (MOD group). This change indicates that CTCu@ZCS preparation has a significant alleviating effect on AD symptoms, demonstrating its good anti-inflammatory effect.

[0058] The results are as follows Figure 16As shown in the results, the TEWL value was significantly higher in the MOD group than in the normal mice (Control group), further confirming the impairment of skin barrier function. However, treatment with CTCu@ZCS significantly reduced the TEWL value, indicating that this treatment can improve skin barrier function and thus alleviate AD symptoms.

Claims

1. A hydrogel dressing loaded with shikonin, characterized in that, Including shikonin liposomes and hydrogel matrix; The shikonin liposomes are made of shikonin, lecithin and cholesterol, with a mass ratio of shikonin, lecithin and cholesterol of 0.5~5:10~25:0.5~2. The hydrogel matrix is ​​made of carboxymethyl chitosan, tannic acid, CuSO4 and water, with a concentration of 2.5 g / 100 mL for carboxymethyl chitosan, a concentration of 1.67-3.33 g / 100 mL for tannic acid, and a concentration of 66.6 mg / 100 mL for CuSO4. The volume ratio of the shikonin liposomes to the hydrogel matrix is ​​42:

100.

2. The hydrogel dressing according to claim 1, characterized in that, The concentration of the tannic acid is 1.67 g / 100 mL.

3. The method for preparing the hydrogel dressing loaded with shikonin according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Prepare shikonin liposomes; Step 2: Take tannic acid and add it to water to obtain a tannic acid solution. Adjust the pH of the tannic acid solution to 7.0~8.0, add carboxymethyl chitosan solution and shikonin liposomes, stir and mix, and then add CuSO4 solution to obtain the hydrogel dressing.

4. The preparation method according to claim 3, characterized in that, The shikonin liposomes were prepared using a thin-film dispersion method. Specifically: shikonin, soybean lecithin, and cholesterol were mixed and dissolved in anhydrous ethanol; the mixture was transferred to a round-bottom flask and rotary evaporated to remove the ethanol, yielding a thin film; subsequently, the film was hydrated, ultrasonically broken up, dialyzed, and extruded to obtain liposomes.

5. The preparation method according to claim 3, characterized in that, In step 2, NaOH solution is used to adjust the pH.

6. The preparation method according to claim 5, characterized in that, The concentration of the NaOH solution is 0.1 M.

7. The use of the hydrogel dressing loaded with shikonin according to claim 1 in the preparation of products for the treatment of inflammatory skin diseases.

8. The application according to claim 7, characterized in that, The inflammatory skin disease mentioned is atopic dermatitis.

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