Ciclosporin-loaded antioxidant antibacterial microneedle as well as preparation method and application thereof

Abstract

The invention discloses a cyclosporin-loaded antioxidant antibacterial microneedle and a preparation method and application thereof, the preparation method comprises the following steps: adding tannic acid / silver nanoparticles and a micelle solution A wrapping cyclosporin into a 3-(aminomethyl) phenylboronic acid modified hyaluronic acid solution to obtain a solution E; adding the solution E into a microneedle mold, and drying to obtain a microneedle array layer; and dropwise adding a hyaluronic acid solution above the microneedle array layer, drying, and removing the template to obtain the microneedle patch. According to the microneedle-mediated cyclosporin transdermal drug delivery mode, the drug utilization efficiency of an existing clinical oral drug delivery mode can be remarkably improved, and the drug use dosage and potential toxic and side effects are reduced. The microneedle prepared by the invention has ROS (reactive oxygen species) removal and antibacterial effects, and is combined with cyclosporine to obtain a good effect of synergistically treating atopic dermatitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an antioxidant and antibacterial microneedle loaded with cyclosporine, and a preparation method and application thereof. Background Art

[0002] Atopic dermatitis is the chronic inflammatory skin disease with the highest incidence rate in the world. Its main clinical manifestations are eczematoid skin lesions, itching and dry skin, which seriously affect the quality of life of patients. At present, the clinical drug treatment options for atopic dermatitis include emollients or moisturizers, topical glucocorticoids, topical calcineurin inhibitors and systemic immunosuppressants such as cyclosporine, azathioprine, methotrexate and mycophenolate mofetil. Among them, cyclosporine A is a calcineurin inhibitor that can exert an immunosuppressive effect by inhibiting the activation of T lymphocytes. It is widely used in patients with severe atopic dermatitis who are ineffective for conventional local treatments and is a clinical first-line drug for the treatment of atopic dermatitis. However, the cyclosporine drugs currently used in clinical practice are mostly systemically administered, such as oral 3-5 mg / kg for 6 weeks. Clinical data show that although cyclosporine takes effect quickly in the treatment of severe atopic dermatitis, it is easy to cause serious complications such as kidney damage and hypertension due to its high dose, especially long-term use, and is often accompanied by adverse symptoms such as headache, nausea and tremor. Therefore, there is an urgent need to develop a safe and efficient method for administering cyclosporine to solve the bottleneck problem of clinical drug safety.

[0003] Transdermal administration is a common method of drug delivery. Direct local application of drugs at the site of dermatitis lesions is beneficial to minimize systemic toxicity. In view of the above advantages, creams, creams, and gels have been studied. However, due to the significant increase in epidermal thickness at the site of lesions, the drug penetration rate is often low and the clinical efficacy is poor. At present, some attempts to enhance the local penetration of cyclosporine have also been reported, including microemulsion method, penetration-enhancing dosage form, and solid lipid nanoparticles, but their penetration-enhancing efficiency still needs to be improved.

[0004] Microneedle array is a small needle dot matrix integrated device with a length of less than 1 mm. Studies have shown that microneedles can effectively penetrate tissue and skin, create microchannels on the skin surface in a minimally invasive manner, and allow drugs to enter the body through the microchannels. It is a new and efficient transdermal drug delivery technology. At present, there is a lack of research on the use of soluble microneedles loaded with cyclosporine for the treatment of atopic dermatitis, and no patents have been applied for or related products have been launched at home and abroad. Therefore, the construction of a new method of cyclosporine delivery based on a microneedle drug delivery system has important research significance and clinical value, and its therapeutic effect and safety urgently need to be further explored. Summary of the invention

[0005] The purpose of the present invention is to provide a cyclosporine-loaded antioxidant and antibacterial microneedle and a preparation method and application thereof in view of the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention is to provide a method for preparing cyclosporine-loaded antioxidant and antibacterial microneedles, comprising the following steps:

[0008] Step 1: adding a thermosensitive hydrogel and a nonionic surfactant to anhydrous ethanol to dissolve to obtain a first solution; dissolving cyclosporine in anhydrous ethanol to obtain a second solution; then mixing the first solution and the second solution, reacting at 10-35° C. for 30-60 minutes, removing the ethanol at 40-45° C. using a rotary evaporator, and hydrating the complex with ionized water to obtain a micelle solution A encapsulating cyclosporine;

[0009] Step 2, mixing the tannic acid aqueous solution and the silver nitrate aqueous solution, stirring and reacting at 10-35° C. for 1-2 hours to prepare tannic acid / silver nanoparticles;

[0010] Step 3, activating hyaluronic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-acetylsuccinimide, and reacting with primary amine 3-aminophenylboronic acid to undergo an amidation reaction to prepare 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid;

[0011] Step 4, preparing an aqueous solution of hyaluronic acid modified with 3-(aminomethyl)phenylboronic acid, and adding tannic acid / silver nanoparticles and micelle solution A encapsulating cyclosporine thereto to obtain solution E;

[0012] Step 5, adding the solution E into a microneedle mold and drying to obtain a microneedle array layer;

[0013] Step six, dripping hyaluronic acid solution on the microneedle array layer, drying, removing the template, and obtaining a microneedle patch.

[0014] Furthermore, the thermosensitive hydrogel is Pluronic F127.

[0015] Furthermore, the nonionic surfactant is Pluronic F68.

[0016] Furthermore, the concentration of the tannic acid aqueous solution is 1-5wt%; the concentration of the silver nitrate aqueous solution is 1-5wt%.

[0017] Furthermore, the specific method of step three includes:

[0018] 1) Dissolve hyaluronic acid in deionized water, then add dimethyl sulfoxide and primary amine 3-aminophenylboronic acid to the hyaluronic acid solution, stir to dissolve, and record as solution C;

[0019] 2) 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-acetylsuccinimide were dissolved in dimethyl sulfoxide by stirring, and the solution was recorded as solution D;

[0020] 3) adding the solution D dropwise into the solution C, adjusting the pH of the reaction system to 4.5-5.0, and reacting at 10-35° C. for 24-36 hours; after dialysis and freeze-drying, 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid is obtained.

[0021] Furthermore, in step 4, in the solution E, the cyclosporine content is 0.1-1 μg / mL, the concentration of tannic acid / silver nanoparticles is 0.01-0.05 g / mL, and the concentration of 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid is 0.02 g / mL.

[0022] The second aspect of the present invention is to provide an antioxidant and antibacterial microneedle loaded with cyclosporine prepared by the above-mentioned preparation method.

[0023] The third aspect of the present invention is to provide the use of the above-mentioned cyclosporine-loaded antioxidant and antibacterial microneedles in the preparation of products for treating atopic dermatitis.

[0024] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0025] The microneedle-mediated cyclosporine transdermal administration method of the present invention can significantly improve the drug utilization efficiency of the existing oral administration method in clinical practice, and reduce the drug dosage and potential toxic side effects.

[0026] The microneedle prepared by the present invention has ROS scavenging and antibacterial effects, and can be used in combination with cyclosporine to achieve a good effect of synergistically treating atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a photo of the antioxidant and antibacterial microneedles prepared in the present invention.

[0028] Figure 2 This is a flat plate image of the inhibition zone of the microneedle patch of the present invention.

[0029] Figure 3 This is a diagram showing the therapeutic effect of the microneedle patch of the present invention on mice with atopic dermatitis. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0031] Example 1

[0032] This embodiment provides a method for preparing cyclosporine-loaded antioxidant and antibacterial microneedles, which specifically comprises the following steps:

[0033] Step 1: at room temperature, 26.7 mg of Pluronic F127 and 13.3 mg of Pluronic F68 are added to 1 mL of anhydrous ethanol and dissolved, and 100 μg of cyclosporine is dissolved in 1 mL of anhydrous ethanol; then the two solutions are mixed and reacted at room temperature for 30 minutes, and the ethanol is removed at 40° C. using a rotary evaporator, and a layer of complex film is observed to be formed at the bottom of the flask, and the complex is hydrated with 2 mL of deionized water to obtain a micelle solution A encapsulating cyclosporine.

[0034] Step 2: Mix 1 wt % tannic acid aqueous solution and 1 wt % silver nitrate aqueous solution, and stir at room temperature for 1 hour to prepare tannic acid / silver nanoparticles.

[0035] Step 3: 2 g of hyaluronic acid was weighed and dissolved in 100 mL of deionized water, and then 40 mL of dimethyl sulfoxide and 1.4 g of primary amine 3-aminophenylboronic acid were weighed and added to the above hyaluronic acid solution, and stirred to dissolve, which was recorded as solution C; 1.9 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1.1 g of N-acetylsuccinimide were stirred and dissolved in 60 mL of dimethyl sulfoxide, which was recorded as solution D;

[0036] At room temperature, solution D was added dropwise to solution C while stirring, and the pH of the reaction system was adjusted to 4.5 with 5M HCl solution. The reaction was carried out at room temperature for 24 hours. After dialysis and freeze-drying, 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid was obtained.

[0037] Step 4, taking 2g of 3-(aminomethyl)phenylboronic acid modified hyaluronic acid and dissolving it in 98mL of deionized water to prepare a 3-(aminomethyl)phenylboronic acid modified hyaluronic acid aqueous solution; adding 5g of tannic acid / silver nanoparticles and 2mL of cyclosporine-encapsulating micelle solution A prepared in step 1 to obtain solution E; wherein the cyclosporine content in solution E is 1μg / mL, the concentration of tannic acid / silver nanoparticles is 0.05g / mL, and the concentration of 3-(aminomethyl)phenylboronic acid modified hyaluronic acid is 0.02g / mL.

[0038] Step 5: Take 100 μl of solution E and add it into the microneedle mold. Fill the hole area of ​​the template by vacuuming or centrifuging, and dry it to obtain a microneedle array layer.

[0039] Step 6: Add 2wt% hyaluronic acid solution, 0.5mL, onto the microneedle array layer, dry, remove the template, and obtain a microneedle patch. Figure 1 shown.

[0040] Example 2

[0041] This embodiment provides a method for preparing cyclosporine-loaded antioxidant and antibacterial microneedles, which specifically comprises the following steps:

[0042] Steps 1 to 3 are the same as those in Example 1.

[0043] Step 4: dissolving 2 g of 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid in 99.8 mL of deionized water to prepare an aqueous solution of 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid; adding 1 g of tannic acid / silver nanoparticles and 0.2 mL of cyclosporine-encapsulating micelle solution A prepared in step 1 to obtain solution E; wherein the cyclosporine content in solution E is 0.1 μg / mL, the concentration of tannic acid / silver nanoparticles is 0.01 g / mL, and the concentration of 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid is 0.02 g / mL.

[0044] Steps 5 to 6 are the same as those in Example 1.

[0045] Example 3

[0046] This embodiment provides a method for preparing cyclosporine-loaded antioxidant and antibacterial microneedles, which specifically comprises the following steps:

[0047] Steps 1 to 3 are the same as those in Example 1.

[0048] Step 4, taking 2g of 3-(aminomethyl)phenylboronic acid modified hyaluronic acid and dissolving it in 98mL of deionized water to prepare an aqueous solution of 3-(aminomethyl)phenylboronic acid modified hyaluronic acid; adding 2.5g of tannic acid / silver nanoparticles and 2mL of micelle solution A encapsulating cyclosporine prepared in step 1 to obtain solution E; wherein the cyclosporine content in solution E is 1μg / mL, the concentration of tannic acid / silver nanoparticles is 0.025g / mL, and the concentration of 3-(aminomethyl)phenylboronic acid modified hyaluronic acid is 0.02g / mL.

[0049] Steps 5 to 6 are the same as those in Example 1.

[0050] Current studies believe that oxidative stress is an important pathological factor that promotes the development of atopic dermatitis. Excessive oxidative stress in the skin lesions produces a large amount of reactive oxygen species, which in turn triggers chronic inflammation, and thus forms a vicious cycle that aggravates the severity of atopic dermatitis. On the other hand, studies have shown that local skin microecological imbalance promotes the occurrence of atopic dermatitis. The diversity of the bacterial flora in the skin lesions of patients with atopic dermatitis is reduced compared with that of normal skin flora, mainly manifested in a significant increase in the abundance of pathogenic Staphylococcus aureus, while the abundance of coagulase-negative Staphylococci with antibacterial ability is significantly reduced. Staphylococcus aureus is an important clinical pathogen that causes a series of diseases such as skin abscesses and bacteremia. Staphylococcus aureus on the skin surface of patients with atopic dermatitis can release a large amount of exocrine toxins, proteases, superantigens, etc., which can aggravate the itching of the skin of patients with atopic dermatitis and cause skin infections, aggravate inflammatory reactions and cause tissue damage and lesions. Based on the above pathological mechanism, removing bacteria, reducing ROS and inhibiting inflammation are expected to alleviate the clinical symptoms of atopic dermatitis, restore the microenvironmental balance of the skin lesions and promote repair. Therefore, the following Verification Examples 1 and 2 of the present invention respectively carried out DPPH free radical scavenging experiments and antibacterial (Staphylococcus aureus) performance experiments.

[0051] Verification Example 1

[0052] The antioxidant properties of the microneedles (prepared in Example 1) were tested by DPPH free radical scavenging experiment. The specific method is as follows: (1) Preparation of DPPH test solution: Dissolve 1 mg of DPPH solid in 24 mL of anhydrous ethanol, ultrasonicate for 5 minutes, shake thoroughly, make the upper and lower parts uniform, and store it away from light. Take 1 mL of the above-prepared DPPH solution, add 0.5 mL of anhydrous ethanol to dilute it so that its absorbance is between 0.6-10. If the absorbance is too large, continue to add solvent; if the absorbance is too small, add DPPH solid or original solution. (2) Take 1.0 mL of DPPH solution and add a small amount of sample. Incubate in a 37°C oven for half an hour, then detect the absorption wavelength of the reaction solution at a wavelength of 519 nm, and calculate the absorbance according to the formula. The results show that the DPPH free radical scavenging of the microneedle patch reaches more than 70%.

[0053] Verification Example 2

[0054] The antibacterial properties of the microneedles were tested using the inhibition zone method. The specific method is: agarose plates were coated with a suspension of Staphylococcus aureus. After incubation for 12 hours, the microneedle patch (prepared in Example 1) was placed on the plate and incubated for another 12 hours to observe the inhibition zone. The results are shown in Figure 2. Figure 2 As shown: an antibacterial zone with a width of about 2 mm appeared around the microneedle, indicating that the microneedle has a significant inhibitory effect on Staphylococcus aureus.

[0055] Verification Example 3

[0056] The atopic dermatitis mouse model was used to evaluate the therapeutic effect of microneedles. The specific methods are as follows: (1) Construction of an atopic dermatitis mouse model: 0.5 nmol calcipotriol ethanol solution was evenly applied to the inner and outer sides of the mouse ears, 20 μl each time, and the drug was administered continuously for 12 days. (2) The microneedle patch was applied to the inside of the mouse ear, and the patch was replaced every two days. Photos were taken on the 1st, 3rd, 5th, and 7th days after microneedle application to observe the redness and swelling of the skin. The results are as follows Figure 3 Compared with the model group, the redness, swelling and inflammation of the ear skin of mice in the microneedle group were significantly reduced, indicating that the microneedle can effectively alleviate the symptoms of atopic dermatitis.

[0057] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing cyclosporine-loaded antioxidant and antibacterial microneedles, characterized in that: The steps include: Step 1: adding a thermosensitive hydrogel and a nonionic surfactant to anhydrous ethanol to dissolve to obtain a first solution; dissolving cyclosporine in anhydrous ethanol to obtain a second solution; then mixing the first solution and the second solution, reacting at 10-35° C. for 30-60 minutes, removing the ethanol at 40-45° C. using a rotary evaporator, and hydrating the complex with ionized water to obtain a micelle solution A encapsulating cyclosporine; Step 2, mixing the tannic acid aqueous solution and the silver nitrate aqueous solution, stirring and reacting at 10-35° C. for 1-2 hours to prepare tannic acid / silver nanoparticles; Step 3, activating hyaluronic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-acetylsuccinimide, and reacting with primary amine 3-aminophenylboronic acid to undergo an amidation reaction to prepare 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid; Step 4, preparing an aqueous solution of hyaluronic acid modified with 3-(aminomethyl)phenylboronic acid, and adding tannic acid / silver nanoparticles and micelle solution A encapsulating cyclosporine thereto to obtain solution E; Step 5, adding the solution E into a microneedle mold and drying to obtain a microneedle array layer; Step six, dripping hyaluronic acid solution on the microneedle array layer, drying, removing the template, and obtaining a microneedle patch.

2. The method for preparing the cyclosporine-loaded antioxidant and antibacterial microneedles according to claim 1, characterized in that: The thermosensitive hydrogel is Pluronic F127.

3. The method for preparing the cyclosporine-loaded antioxidant and antibacterial microneedles according to claim 1, characterized in that: The nonionic surfactant is Pluronic F68.

4. The method for preparing the cyclosporine-loaded antioxidant and antibacterial microneedles according to claim 1, characterized in that: The concentration of the tannic acid aqueous solution is 1-5wt%; the concentration of the silver nitrate aqueous solution is 1-5wt%.

5. The method for preparing the cyclosporine-loaded antioxidant and antibacterial microneedles according to claim 1, characterized in that: Step 3: Specific methods include: 1) Dissolve hyaluronic acid in deionized water, then add dimethyl sulfoxide and primary amine 3-aminophenylboronic acid to the hyaluronic acid solution, stir and dissolve, and record as solution C; 2) 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-acetylsuccinimide were dissolved in dimethyl sulfoxide by stirring, and the solution was recorded as solution D; 3) adding the solution D dropwise into the solution C, adjusting the pH of the reaction system to 4.5-5.0, and reacting at 10-35° C. for 24-36 hours; after dialysis and freeze-drying, 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid is obtained.

6. The method for preparing the cyclosporine-loaded antioxidant and antibacterial microneedles according to claim 1, characterized in that: In step 4, in the solution E, the cyclosporine content is 0.1-1 μg / mL, the concentration of tannic acid / silver nanoparticles is 0.01-0.05 g / mL, and the concentration of 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid is 0.02 g / mL.

7. An antioxidant and antibacterial microneedle loaded with cyclosporine prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the cyclosporine-loaded antioxidant and antibacterial microneedles according to claim 7 in the preparation of a product for treating atopic dermatitis.

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