Microneedles capable of loading poorly water-soluble antibiotics, preparation method thereof, and applications thereof

Abstract

The present invention belongs to the field of microneedle patch preparations for transdermal drug delivery, specifically relating to a microneedle capable of loading a poorly water-soluble antibiotic, its preparation method, and its application. The microneedles of the present invention utilize gelatin methacrylate (GelMA) as a substrate. The poorly soluble antibiotic is encapsulated with bovine serum albumin and evenly distributed within the microneedles. The microneedles are obtained by preparing a GelMA solution, preparing BSA-encapsulated antibiotic nanoparticles, mixing the solutions, forming a microneedle mold, cross-linking and curing, and demolding and drying. The resulting microneedles exhibit excellent mechanical properties and biocompatibility. The microneedles of the present invention can locally deliver antibiotics and effectively treat localized skin infections. They offer advantages such as high drug stability, controlled release, and painless delivery. They can effectively penetrate the skin barrier, enhance drug bioavailability, mitigate the effects of the gastrointestinal environment on drug efficacy, and mitigate the "first-pass effect" in the liver, thereby improving patient compliance.

Description

Technical Field

[0001] The present invention belongs to the field of microneedle patch preparations for transdermal drug delivery, and particularly relates to a microneedle capable of being loaded with poorly water-soluble antibiotics, a preparation method thereof, and an application thereof. Background Art

[0002] Transdermal antibiotic delivery is a novel approach for treating skin and soft tissue infections (SSTIs). Compared to oral and parenteral administration, transdermal antibiotics bypass the first-pass liver effect and avoid gastrointestinal damage. This approach reduces blood concentration fluctuations, minimizes toxic side effects, and offers increased convenience, leading to high patient compliance.

[0003] The skin is the largest organ in the human body, and its stratum corneum is the main barrier to transdermal drug delivery. Microneedles are a collection of needle arrays with a length of 150-2000μm, which can directly penetrate the stratum corneum and effectively deliver antibiotics to the subcutaneous tissue without damaging the dermis or stimulating nerve endings, thus achieving painless or minimally painful drug delivery and reducing patient suffering. According to the drug release mechanism, microneedles are divided into solid microneedles, hollow microneedles, coated microneedles, soluble microneedles and hydrogel microneedles. Among them, soluble microneedles are safer, have a large drug loading capacity, can penetrate the stratum corneum for precise delivery, and greatly improve transdermal efficiency. However, since the soluble microneedle matrix is mostly water-soluble polymers, the microneedles obtained by the existing preparation method are difficult to load antibiotics that are poorly soluble in water. Summary of the Invention

[0004] To address the above technical problems, the present invention has prepared a new type of soluble microneedle and its preparation method, which allows poorly water-soluble antibiotics to be sealed in the cavity of the microneedle substrate, ensuring drug activity and enabling precise and efficient transdermal delivery, thereby achieving effective treatment of SSTI.

[0005] In a first aspect, the technical solution of the present invention provides a hydrogel microneedle loaded with a poorly water-soluble antibiotic, which is a hydrogel microneedle patch that can penetrate the skin and release the antibiotic. The hydrogel microneedle is formed by adding GelMA prepolymer into a microneedle mold.

[0006] The method for preparing the poorly soluble antibiotic-loaded microneedles according to a specific embodiment of the present invention comprises the following steps:

[0007] (1) Add the antibiotic solution to the bovine serum albumin solution and mix thoroughly by ultrasonication;

[0008] (2) rotary evaporating the mixed solution obtained in step (1) to remove the solvent and obtain a bovine serum albumin / antibiotic coating;

[0009] (3) dissolving the inclusions obtained in step (2) in water, centrifuging, and rotary evaporating to obtain bovine serum albumin / antibiotic nanoparticles;

[0010] (4) adding the bovine serum albumin / antibiotic nanoparticles and photoinitiator obtained in step (3) to the methacrylated gelatin solution, and ultrasonically mixing to obtain a prepolymer solution;

[0011] (5) adding the prepolymer solution obtained in step (4) into a microneedle mold to prepare microneedles;

[0012] (6) Irradiating the microneedles obtained in step (5) under ultraviolet light to obtain microneedles loaded with poorly soluble antibiotics.

[0013] Poorly soluble antibiotics include:

[0014] Penicillins: such as penicillin G, have low solubility in water and usually need to be made into sodium or potassium salts to increase their water solubility for clinical application.

[0015] Cephalosporins: Some cephalosporins have relatively poor solubility, such as cefazolin, which has limited solubility in water.

[0016] Macrolides: Erythromycin is a typical poorly soluble antibiotic with low solubility in water. It is generally formulated as enteric-coated tablets or esters to improve absorption and application. Azithromycin faces a similar situation, being poorly soluble in water and often available in granules or capsules.

[0017] Tetracyclines: Tetracycline is not highly soluble in water, and doxycycline is also somewhat insoluble. These drugs are usually made into hydrochlorides to increase their absorption and utilization in the body.

[0018] Aminoglycosides: gentamicin, tobramycin and other aminoglycoside antibiotics.

[0019] Among them, Equisetin is a secondary metabolite produced by deep-sea fungi (Fusarium sp. 152) and has an antibacterial effect on methicillin-resistant Staphylococcus aureus (MRSA).

[0020] According to the microneedle preparation method of a specific embodiment of the present invention, in step (1), the mass ratio of the antibiotic to the bovine serum albumin is 1:5-20; and / or,

[0021] adding an antibiotic to an organic solvent to obtain an antibiotic solution, and mixing methanol and chloroform in a volume ratio of 1:1 to obtain an organic solvent; and / or,

[0022] Add bovine serum albumin solution into water to obtain bovine serum albumin solution.

[0023] According to the method for preparing microneedles according to a specific embodiment of the present invention, in step (2), rotary evaporation is performed at 55-65° C. and 400-600 rpm.

[0024] According to the method for preparing microneedles in a specific embodiment of the present invention, in step (4), the concentration of methacrylated gelatin in the prepolymer solution is 150-250 mg / mL; and / or,

[0025] The concentration of the photoinitiator is 0.5-1 mg / mL; and / or,

[0026] The concentration of BSA / antibiotic nanoparticles was 30-40 mg / mL.

[0027] The present invention uses methacrylated gelatin (GelMA) as the substrate of the microneedle. Methacrylated gelatin is produced by reacting gelatin molecules with a concentration of 95% with methacrylic anhydride. The amino groups on the lysine residues on the gelatin molecules undergo a grafting reaction with methacrylic anhydride, and the grafting rate is 95%.

[0028] Preferably, in step (4), the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0029] According to the microneedle preparation method of the specific embodiment of the present invention, in step (5), the prepolymer solution is added to the microneedle mold twice, and the volume of the prepolymer solution added each time is equal. After each addition of the prepolymer solution, it is dried for 0.5-2 hours.

[0030] Preferably, the sample is added to the PDMS mold twice, with a single sample addition of 0.5 mL. After each addition, the sample is placed in a forced air drying oven for concentration for 1 hour to ensure the uniformity and strength of the microneedle substrate, thereby obtaining microneedles with good mechanical properties.

[0031] According to the method for preparing microneedles according to a specific embodiment of the present invention, in step (5), the prepolymer solution is added to the mold and centrifuged at 37° C. and 3500 rpm, and then dried.

[0032] According to the method for preparing microneedles according to a specific embodiment of the present invention, in step (6), the microneedles are exposed to 1W, 405nm ultraviolet light and then dried in the dark.

[0033] GelMA molecules contain methacryloyl groups, whose carbon-carbon double bonds are activated by free radicals generated by light, leading to a chain polymerization reaction. The double bonds on different GelMA molecules are interconnected through polymerization, forming a three-dimensional network structure, which solidifies the material and produces hydrogel microneedles. A single drug-loaded microneedle can withstand a force of at least 0.1 N without deformation.

[0034] The present invention also provides a poorly soluble antibiotic-loaded microneedle prepared by the above preparation method.

[0035] Furthermore, loaded microneedles were prepared using a PDMS microneedle mold. Residual air in the solution was removed by centrifugation at 3500 rpm. The resulting microneedle structure was a regular square pyramid with a height of 750 μm, a base side length of 350 μm × 350 μm, and a spacing of 720 μm. The number of microneedles was 15 × 15, with an overall size of 14.5 mm × 14.5 mm.

[0036] Beneficial effects of the present invention:

[0037] The microneedles of the present invention include methacrylated gelatin (GelMA) as a substrate, in which poorly soluble antibiotics are wrapped with bovine serum albumin and evenly distributed in the microneedles. The microneedles are obtained by preparing a GelMA solution, preparing BSA-wrapped antibiotic nanoparticles, mixing the solution, forming a microneedle mold, cross-linking and curing, and demolding and drying.

[0038] The release behavior of the microneedles of the present invention reached 43.6% within the first 1.5 hours, then gradually slowed down to reach 69% at 11 hours. After that, the drug release leveled off, reaching a cumulative drug release rate of 77.4% at 71 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 This is a transmission electron microscopy image of nanoparticles formed after Equisetin was encapsulated by bovine serum albumin.

[0041] Figure 2 Dynamic light scattering results of BSA / EQ nanoparticles

[0042] Figure 3 The morphology of microneedles loaded with BSA / EQ nanoparticles under an electron microscope; the scale bar in (A) is 100 μm, and the scale bar in (B) is 200 μm.

[0043] Figure 4 This is the morphology of microneedles loaded with BSA / EQ nanoparticles under white light.

[0044] Figure 5The antibacterial effects of Equisetin, BSA / EQ, microneedle material (GelMA), microneedle material containing BSA / EQ nanoparticles and blank control on MRSA.

[0045] Figure 6 The release efficiency of Equisetin from drug-loaded microneedles was calculated using a Franz diffusion cell experiment using mouse skin as the medium.

[0046] Figure 7 The displacement change of a single microneedle after being subjected to force.

[0047] Figure 8 The figure shows the survival rate of L929 cells at BSA / EQ concentrations of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, and 40 mg / mL in the microneedle material containing BSA / EQ nanoparticles. The leftmost bar shows the survival rate of the control group (containing only culture medium). DETAILED DESCRIPTION

[0048] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0049] The present invention provides a method for preparing microneedles loaded with the poorly soluble antibiotic yinquesetin, comprising the following steps:

[0050] (1) Add the Yinkuisetin solution to the bovine serum albumin solution and mix thoroughly by ultrasonication;

[0051] (2) rotary evaporating the mixed solution obtained in step (1) to remove the solvent and obtain a bovine serum albumin / Equisetin (BSA / EQ) coating;

[0052] (3) dissolving the inclusions obtained in step (2) in water, centrifuging, and rotary evaporating to obtain bovine serum albumin / antibiotic nanoparticles;

[0053] (4) adding the bovine serum albumin / antibiotic nanoparticles and the photoinitiator obtained in step (3) to the methacrylated gelatin solution and ultrasonically mixing to obtain a prepolymer solution; the concentration of the methacrylated gelatin is 150-250 mg / mL, the concentration of the bovine serum albumin / antibiotic nanoparticles is 30-40 mg / mL, and the concentration of the photoinitiator is 0.5-1 mg / mL;

[0054] (5) adding the prepolymer solution obtained in step (4) into a microneedle mold to prepare microneedles;

[0055] (6) The microneedles obtained in step (5) are irradiated with a 405 nm ultraviolet lamp for 0.5-5 minutes. After solidification, microneedles loaded with poorly soluble antibiotics are obtained.

[0056] In step (1), the mass ratio of the antibiotic quinolones to bovine serum albumin is 1:5-20, preferably, the mass ratio of quinolones to bovine serum albumin is 1:8-12, or the mass ratio of quinolones to bovine serum albumin is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15.

[0057] Wherein, inquinoline is dissolved in a solvent (methanol and chloroform in a volume ratio of 1:1) to obtain an inquinoline solution.

[0058] Bovine serum albumin is added to water to obtain a bovine serum albumin solution.

[0059] In step (3), the EQ particles remaining in the package can be removed by performing a rotary evaporation with water.

[0060] The encapsulation efficiency of EQ in BSA / EQ nanoparticles was 94%. The minimum inhibitory concentration of Yinkuisetin against methicillin-resistant Staphylococcus aureus (MRSA) was 1 μg / mL, and the inhibitory concentration of BSA / EQ nanoparticles against MRSA was 4 μg / mL.

[0061] In step (4), the concentration of methacrylated gelatin in the prepolymer solution is 150-250 mg / mL; preferably, the concentration of methacrylated gelatin is 190-210 mg / mL, and more preferably, the concentration of methacrylated gelatin is 200 mg / mL.

[0062] The concentration of the photoinitiator is 0.5-1 mg / mL. Among them, the photoinitiator can be selected from lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP). Preferably, the concentration of the photoinitiator is 0.5-0.7 mg / mL.

[0063] More preferably, the prepolymer solution contains GelMA at a concentration of 200 mg / mL, LAP at a concentration of 0.625 mg / mL, and BSA / EQ nanoparticles at a concentration of 33 mg / mL;

[0064] The microneedles of this invention use methacrylated gelatin (GelMA) as the substrate, in which the antibiotic Equisetin is encapsulated by bovine serum albumin and evenly distributed throughout the microneedles. The microneedles are obtained by preparing a GelMA solution, preparing BSA-encapsulated Equisetin nanoparticles, preparing a prepolymer solution, forming a microneedle mold, crosslinking and curing, and then demolding and drying. The Equisetin-loaded microneedles, encapsulated by bovine serum albumin, consist of methacrylated gelatin (GelMA) forming the microneedle body. Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) triggers the crosslinking and curing of the methacrylated gelatin to form a hydrogel needle body. After the microneedles penetrate the skin, they slowly release Equisetin.

[0065] Example 1 Preparation of microneedles loaded with antibiotic (Equisetin)

[0066] The method for preparing microneedles loaded with antibiotics (Equisetin) in this embodiment includes the following steps:

[0067] (1) Add 20 mg of Equisetin to 700 μL of a 1:1 methanol to chloroform solution and mix thoroughly by ultrasonication at 25°C for 5 min.

[0068] (2) Add 200 mg of bovine serum albumin to 4 mL of deionized water and mix thoroughly by ultrasonication at 25 °C for 5 min.

[0069] (3) Add solution (1) to solution (2) and mix thoroughly by ultrasonication at 25°C for 10 min;

[0070] (4) The solution of step (3) was subjected to rotary evaporation at 60°C and 500 rpm to remove water, methanol, and chloroform to obtain solid BSA and inclusion BSA / EQ;

[0071] (5) The BSA / EQ nanoparticles were redissolved in ultrapure water, placed in a centrifuge, and centrifuged at 11,000 rpm for 10 minutes. The rotary evaporation was repeated to obtain the BSA / EQ nanoparticles.

[0072] (6) Add 0.4 g of methacrylated gelatin to a test tube containing 1.95 mL of deionized water;

[0073] (7) Weigh 66 mg of the inclusion particles prepared in step (5) and add them to the solution in step (6);

[0074] (8) Weigh 1.25 mg of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and add it to the solution prepared in (7). Mix by ultrasonication at 35 °C for 10 min and store in the dark.

[0075] (9) Take 0.5 mL of the solution (8) and add it to the polydimethylsiloxane (PDMS) microneedle mold, centrifuge at 37°C and 3500 rpm for 5 min, and then absorb the bubbles on the mold surface;

[0076] (10) Take 0.5 mL of solution (8) again and add it to the mold of (9), centrifuge at 37°C and 3500 rpm for 5 min, and then place it in a 37°C forced air drying oven for concentration and drying for 1 h;

[0077] (11) Take out the PDMS and expose it to 1W, 405nm ultraviolet light for 1 minute, dry it in the dark for 24 hours, and then demold it.

[0078] Example 2

[0079] The BSA / EQ nanoparticles obtained in step (5) of Example 1 were selected and the morphology of the BSA-EQ nanoparticles was observed under a transmission electron microscope at 30,000 times magnification. The nanoparticles were spherical in shape with a diameter between 100 and 500 nm (e.g. Figure 1 shown).

[0080] Dynamic light scattering further confirmed that the dynamic light scattering was carried out in three parallel experiments and the average value was taken. To ensure the accuracy of the measurement data, the 5% with the highest light intensity and the 1% with the lowest light intensity were set to be eliminated. The average diameter of BSA-EQ nanoparticles is about 176.98 nm (such as Figure 2 shown).

[0081] The microneedles obtained in Example 1 were carefully observed in the field emission electron microscope scanning image. The microneedles had no obvious collapse, breakage, or deformation, and the needle tips were sharp. The needle base width was about 220 μm, the needle height was about 567 μm, and the needle spacing was about 520 μm (as shown in FIG. Figure 3 shown).

[0082] The microneedles are distributed in a 15 × 15 array under white light, with no obvious collapse, breakage, or distortion. The overall size of the microneedles is approximately 12 mm × 12 mm (e.g. Figure 4 shown).

[0083] Equisetin, BSA / EQ, microneedle material (GelMA), and microneedle material containing BSA / EQ nanoparticles were prepared into tablets, and blank controls were added and placed on agar medium coated with MRSA to observe their respective antibacterial effects. The inhibition zone areas from large to small are Equisetin, BSA / EQ, and microneedle material containing BSA / EQ nanoparticles, among which GelMA and blank control have an inhibition zone of 0. This proves that BSA / EQ and microneedle materials containing BSA / EQ nanoparticles have antibacterial ability (such as Figure 5 shown).

[0084] The release rate of Equisetin from the microneedles was calculated using a Franz diffusion cell. Throughout the experiment, the Franz diffusion cell was placed under a water bath heating device and maintained at a constant temperature of 37°C. The diffusion phase was mouse skin and the receiving cell was filled with phosphate buffer.

[0085] The results are as follows Figure 6 As shown in Figure 3, the release rate reached 43.6% in the first 1.5 hours, and then gradually slowed down to reach 69% at 11 hours. After that, the drug release tended to be stable, and the cumulative drug release rate reached 77.4% at 71 hours.

[0086] The displacement change of a single microneedle after being subjected to force is as follows Figure 7 The calculation formula is the total force divided by the total number of microneedles. When subjected to a force of 3 N, the microneedles displaced more than 0.6 mm without breaking, demonstrating their ability to penetrate the skin's surface.

[0087] Biocompatibility of microneedle materials containing BSA / EQ nanoparticles Figure 8 When the BSA / EQ concentration was within 20 mg / mL, the material had no significant inhibitory effect on cells, which was far lower than the actual usage concentration (0.02 mg / mL).

[0088] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing microneedles loaded with poorly soluble antibiotics, characterized in that: The preparation method comprises the following steps: (1) Add the antibiotic solution to the bovine serum albumin solution and mix thoroughly by ultrasonication; (2) rotary evaporating the mixed solution obtained in step (1) to remove the solvent and obtain a bovine serum albumin / antibiotic coating; (3) dissolving the inclusions obtained in step (2) in water, centrifuging, and rotary evaporating to obtain bovine serum albumin / antibiotic nanoparticles; (4) adding the bovine serum albumin / antibiotic nanoparticles and photoinitiator obtained in step (3) to the methacrylated gelatin solution and ultrasonically mixing to obtain a prepolymer solution; the concentration of the bovine serum albumin / antibiotic nanoparticles is 30-40 mg / mL; (5) adding the prepolymer solution obtained in step (4) into a microneedle mold to prepare microneedles; (6) irradiating the microneedles obtained in step (5) under ultraviolet light to obtain microneedles loaded with poorly soluble antibiotics; In step (1), the antibiotic is yinkusetin, and the mass ratio of the antibiotic to bovine serum albumin is 1:5-20; adding an antibiotic to an organic solvent to obtain an antibiotic solution, and mixing methanol and chloroform in a volume ratio of 1:1 to obtain an organic solvent; and / or, Dissolve bovine serum albumin in water to obtain a bovine serum albumin solution.

2. The method for preparing a microneedle according to claim 1, wherein: In step (2), rotary evaporation is performed at 55-65°C and 400-600 rpm.

3. The method for preparing a microneedle according to claim 1, wherein: In step (4), the concentration of methacrylated gelatin in the prepolymer solution is 150-250 mg / mL; and / or, The concentration of the photoinitiator was 0.5-1 mg / mL.

4. The method for preparing a microneedle according to claim 1, wherein: In step (4), the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

5. The method for preparing a microneedle according to claim 1, wherein: In step (5), the prepolymer solution is added to the microneedle mold twice, and the volume of the prepolymer solution added each time is equal. After each addition of the prepolymer solution, it is dried for 0.5-2 hours.

6. The method for preparing a microneedle according to claim 1 or 5, characterized in that: In step (5), the prepolymer solution is added to the mold and centrifuged at 37°C and 3500 rpm, and then dried.

7. The method for preparing a microneedle according to claim 1 or 6, characterized in that: In step (6), the microneedles are exposed to 1W, 405nm ultraviolet light and then dried in the dark.

8. The poorly soluble antibiotic-loaded microneedle prepared according to the preparation method according to any one of claims 1 to 7.

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