Microneedle capable of loading antibiotics insoluble in water as well as preparation method and application of microneedle

Abstract

The invention belongs to the field of transdermal drug delivery microneedle patch preparations, and particularly relates to a microneedle capable of loading antibiotics insoluble in water as well as a preparation method and application of the microneedle. According to the microneedle, methylacryloylated gelatin (GelMA) serves as a base material, insoluble antibiotics are wrapped by bovine serum albumin and evenly distributed in the microneedle, and the microneedle is obtained through the steps of preparing a GelMA solution, preparing antibiotic nano-particles wrapped by the BSA, mixing the solution, forming a microneedle mold, conducting cross-linking solidification, conducting demolding and drying and the like. And the microneedle with good mechanical property and biocompatibility is obtained. The microneedle can locally deliver antibiotics and effectively treat local skin infection. The invention has the advantages of high drug stability, controllable release, painless delivery and the like, and can effectively break through the skin barrier, improve the bioavailability of the drug, avoid the influence of the gastrointestinal environment on the drug effect and the'first pass effect 'of the liver, and improve the compliance of patients.

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 loading water-insoluble antibiotics, a preparation method thereof, and an application thereof. Background Art

[0002] Transdermal administration of antibiotics is a new route for antibiotic treatment, especially suitable for skin and soft tissue infections (SSTIs). Compared with oral and injection administrations, transdermal administration of antibiotics bypasses the first-pass effect of the liver and avoids the destruction of drugs by the gastrointestinal tract. By reducing the fluctuation of blood drug concentration, reducing toxic and side reactions, and being convenient for medication, the compliance of patients is high.

[0003] As the largest organ of the human body, the stratum corneum of the skin is the main barrier for transdermal drug delivery. Microneedles are a collection of needle-like arrays with a length of 150 - 2000 μm, which can directly penetrate the stratum corneum, effectively deliver antibiotics to the subcutaneous layer, and at the same time do not damage the dermis layer and do not stimulate nerve endings, realizing painless or minimally painful drug delivery and reducing the pain of patients. 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 have high safety, a large drug loading capacity, can penetrate the stratum corneum to achieve precise delivery, and greatly improve the transdermal efficiency. However, due to the fact that the matrix of soluble microneedles is mostly water-soluble polymers, it is difficult for the microneedles obtained by existing preparation methods to load water-insoluble antibiotics. Summary of the Invention

[0004] To solve the above technical problems, the present invention prepares a novel soluble microneedle and a preparation method thereof, so that water-insoluble antibiotics are sealed in the cavity of the microneedle substrate, ensuring the drug activity, and can penetrate the skin precisely and efficiently, realizing the effective treatment of SSTIs.

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

[0006] According to the preparation method of the microneedle loaded with poorly soluble antibiotics according to the specific embodiment of the present invention, the method comprises the following steps: (1) Add an antibiotic solution to a bovine serum albumin solution and mix well by ultrasonic treatment; (2) Perform rotary evaporation on the mixture obtained in step (1) to remove the solvent, and obtain a bovine serum albumin / antibiotic inclusion; (3) Dissolve the inclusion obtained in step (2) in water, centrifuge, and perform rotary evaporation to obtain bovine serum albumin / antibiotic nanoparticles; (4) Add the bovine serum albumin / antibiotic nanoparticles obtained in step (3) and the photoinitiator into the methacrylated gelatin solution, and mix well by ultrasonic treatment to obtain a prepolymer solution; (5) Add the prepolymer solution obtained in step (4) into a microneedle mold to prepare microneedles; (6) Irradiate the microneedles obtained in step (5) under ultraviolet light to obtain microneedles loaded with poorly soluble antibiotics.

[0007] Poorly soluble antibiotics include: Penicillins: Such as penicillin G, which has low solubility in water and usually needs to be made into sodium or potassium salts to increase its water solubility for clinical applications.

[0008] Cephalosporins: Some cephalosporins have relatively poor solubility. For example, cefazolin has limited solubility in water.

[0009] Macrolides: Erythromycin is a typical poorly soluble antibiotic with low solubility in water. Generally, it is made into enteric-coated tablets or esterified products and other dosage forms to improve its absorption and application. Azithromycin also has a similar situation, with poor solubility in water, and is often used in dosage forms such as granules and capsules.

[0010] Tetracyclines: Tetracycline has low solubility in water, and doxycycline also has certain poor solubility. These drugs usually improve their absorption and utilization in the body by making them into hydrochloride salts, etc.

[0011] Aminoglycosides: Aminoglycoside antibiotics such as gentamicin and tobramycin.

[0012] Among them, Equisetin is a secondary metabolite produced by the deep-sea fungus (Fusarium sp. 152) and has antibacterial effects against methicillin-resistant Staphylococcus aureus (MRSA).

[0013] According to the method for preparing microneedles according to the specific embodiment of the present invention, in step (1), the mass ratio of the antibiotic to bovine serum albumin is 1:5 to 20; and / or, Add the antibiotic into an organic solvent to obtain an antibiotic solution, and mix methanol and chloroform in a volume ratio of 1:1 to obtain an organic solvent; and / or, Add bovine serum albumin into water to obtain a bovine serum albumin solution.

[0014] According to the method for preparing microneedles according to the specific embodiment of the present invention, in step (2), rotary evaporation is carried out at 55 - 65 °C and 400 - 600 rpm.

[0015] According to the preparation method of microneedles in the specific embodiment of the present invention, in step (4), in the prepolymer solution, the concentration of methacrylated gelatin is 150-250 mg / mL; and / or, the concentration of the photoinitiator is 0.5-1 mg / mL; and / or, the concentration of bovine serum albumin / antibiotic nanoparticles is 30-40 mg / mL.

[0016] The present invention uses methacrylated gelatin (GelMA) as the substrate of the microneedles. Methacrylated gelatin is formed by the reaction of gelatin molecules with a concentration of 95% and methacrylic anhydride. The amino group on the lysine residue of the gelatin molecule undergoes a grafting reaction with methacrylic anhydride, and the grafting rate is 95%.

[0017] Preferably, in step (4), the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate.

[0018] According to the preparation method of microneedles in the specific embodiment of the present invention, in step (5), the prepolymer solution is added to the microneedle mold in two portions, 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.

[0019] Preferably, by adding samples to the PDMS mold twice, the single addition is 0.5 mL, and after each addition, it is concentrated in a forced air drying oven for 1 hour, ensuring the uniformity and strength of the microneedle substrate, thereby obtaining microneedles with good mechanical properties.

[0020] According to the preparation method of microneedles in the specific embodiment of the present invention, in step (5), after the prepolymer solution is added to the mold, it is centrifuged at 37 °C and 3500 rpm, and then dried.

[0021] According to the preparation method of microneedles in the specific embodiment of the present invention, in step (6), the microneedles are irradiated with 1 W, 405 nm ultraviolet light, and then dried in the dark.

[0022] The GelMA molecule contains methacryloyl groups, and its carbon-carbon double bonds are activated under the action of free radicals generated by photoinitiation, and a chain polymerization reaction occurs. The double bonds on different GelMA molecules are connected to each other through polymerization reactions to form a three-dimensional network structure, thereby curing the material to obtain hydrogel microneedles. A single microneedle of the drug-loaded microneedles can withstand a force of at least 0.1 N without deformation.

[0023] The present invention also provides microneedles loaded with poorly soluble antibiotics prepared by the above preparation method.

[0024] Furthermore, load-bearing microneedles were prepared using a PDMS microneedle mold. Residual air in the solution was removed by centrifugation at a rate of 3500 rpm. The resulting microneedle structure was a regular square pyramid with a needle height of 750 μm, a bottom side length of 350 μm × 350 μm, a needle spacing of 720 μm, and a microneedle quantity of 15 × 15. The overall size was 14.5 mm × 14.5 mm.

[0025] Advantages of the present invention: The microneedles of the present invention are based on methacrylated gelatin (GelMA). The poorly soluble antibiotic is encapsulated by bovine serum albumin and evenly distributed in the microneedles. The microneedles are obtained through steps such as preparing a GelMA solution, preparing BSA-encapsulated antibiotic nanoparticles, mixing the solutions, microneedle mold forming, crosslinking and curing, and demolding and drying.

[0026] The release behavior of the microneedles of the present invention is that 43.6% is achieved within the first 1.5 hours, and then the release rate gradually slows down, reaching 69% at 11 hours. After that, the drug release tends to be stable, and at 71 h, the cumulative drug release rate is 77.4%. Description of the drawings

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is a transmission electron micrograph of nanoparticles formed by bovine serum albumin encapsulating Equisetin.

[0029] Figure 2 It is the result of dynamic light scattering of BSA / EQ nanoparticles Figure 3 It is the morphology of microneedles loaded with BSA / EQ nanoparticles under the electron microscope; where the scale bar in (A) is 100 μm and the scale bar in (B) is 200 μm.

[0030] Figure 4 It is the morphology of microneedles loaded with BSA / EQ nanoparticles under white light.

[0031] Figure 5 It is the antibacterial effect of Equisetin, BSA / EQ, microneedle material (GelMA), microneedle material containing BSA / EQ nanoparticles, and blank control on MRSA. Figure 6The release efficiency of Equisetin in the drug-loaded microneedles was calculated from Franz diffusion cell experiments using mouse skin as a medium.

[0032] Figure 7 It is the displacement change of a single microneedle after being stressed.

[0033] Figure 8 It is the survival rate of L929 cells in the microneedle material containing BSA / EQ nanoparticles at BSA / EQ concentrations of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, and 40 mg / mL. The leftmost bar graph is the survival rate of the control group (containing only the culture medium). Detailed implementation manners

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the scope protected by the present invention.

[0035] The present invention provides a method for preparing microneedles loaded with the poorly soluble antibiotic Equisetin, comprising the following steps: (1) Add the Equisetin solution to the bovine serum albumin solution and mix well by ultrasonic treatment; (2) Rotate and evaporate the mixture obtained in step (1) to remove the solvent, and obtain a bovine serum albumin / Equisetin (BSA / EQ) inclusion; (3) Dissolve the inclusion obtained in step (2) in water, centrifuge, and rotate and evaporate to obtain bovine serum albumin / antibiotic nanoparticles; (4) Add the bovine serum albumin / antibiotic nanoparticles and a photoinitiator obtained in step (3) to a methacrylated gelatin solution, and mix well by ultrasonic treatment to obtain a prepolymer solution; the concentration of methacrylated gelatin is 150 - 250 mg / mL, the concentration of bovine serum albumin / antibiotic nanoparticles is 30 - 40 mg / mL, and the concentration of the photoinitiator is 0.5 - 1 mg / mL; (5) Add the prepolymer solution obtained in step (4) to a microneedle mold to prepare microneedles; (6) Irradiate the microneedles obtained in step (5) with a 405 nm ultraviolet lamp for 0.5 - 5 minutes, and after curing, obtain microneedles loaded with the poorly soluble antibiotic.

[0036] In step (1), the mass ratio of antibiotic equisetin to bovine serum albumin is 1:5 to 20. Preferably, the mass ratio of equisetin to bovine serum albumin is 1:8 to 12, or the mass ratio of equisetin 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.

[0037] Among them, equisetin is dissolved in a solvent (the volume ratio of methanol to chloroform is 1:1) to obtain an equisetin solution.

[0038] Bovine serum albumin is added to water to obtain a bovine serum albumin solution.

[0039] In step (3), rotary evaporation with water can remove EQ particles remaining in the wrapping.

[0040] The encapsulation efficiency of EQ in BSA / EQ nanoparticles is 94%. The minimum inhibitory concentration of equisetin against methicillin-resistant Staphylococcus aureus (MRSA) is 1 μg / mL, and the antibacterial concentration of BSA / EQ nanoparticles against MRSA is 4 μg / mL.

[0041] In step (4), in the prepolymer solution, the concentration of methacrylated gelatin 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.

[0042] The concentration of the photoinitiator is 0.5 - 1 mg / mL. Among them, the photoinitiator can be lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate (LAP). Preferably, the concentration of the photoinitiator is 0.5 - 0.7 mg / mL. More preferably, the prepolymer solution contains GelMA with a concentration of 200 mg / mL, LAP with a concentration of 0.625 mg / mL, and BSA / EQ nanoparticles with a concentration of 33 mg / mL; The microneedles of the present invention are based on methacrylated gelatin (GelMA), in which the antibiotic equisetin is encapsulated by bovine serum albumin and evenly distributed in the microneedles. The microneedles are obtained through steps such as preparing a GelMA solution, preparing BSA - encapsulated equisetin nanoparticles, a prepolymer solution, microneedle mold forming, cross - linking and curing, and demolding and drying. The equisetin (equisetin) - loaded microneedles encapsulated by bovine serum albumin consist of methacrylated gelatin (GelMA) forming the microneedle body, and lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate (LAP) that initiates photo - cross - linking triggers the cross - linking and curing of methacrylated gelatin to form a hydrogel needle body. After the microneedles penetrate the skin, equisetin can be slowly released.

[0043] Example 1 Preparation of microneedles loaded with antibiotics (Equisetin) The method for preparing the microneedle loaded with antibiotic (Equisetin) of this embodiment comprises the following steps: (1) Take 20 mg of Equisetin and add it to 700 μL of a 1:1 methanol to chloroform solution. Mix by ultrasonication at 25 °C for 5 min. (2) Add 200 mg of bovine serum albumin to 4 mL of deionized water and mix by ultrasonication at 25 °C for 5 min. (3) Add solution (1) to solution (2) and mix by ultrasonication at 25 °C for 10 min; (4) removing water, methanol and chloroform from the solution of step (3) by rotary evaporation at 60°C and 500 rpm to obtain solid BSA and inclusion BSA / EQ; (5) The BSA / EQ nanoparticles were redissolved in ultrapure water, placed in a centrifuge, centrifuged at 11,000 rpm for 10 min, and the rotary evaporation was repeated to obtain the BSA / EQ nanoparticles; (6) Take 0.4 g of methacryloyl gelatin and add it to a test tube containing 1.95 mL of deionized water; (7) Weigh 66 mg of the inclusion particles prepared in step (5) and add them to the solution in step (6); (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 a dark place. (9) Take 0.5 mL of 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 surface of the mold; (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; (11) Take out the PDMS and expose it to 1W, 405nm ultraviolet light for 1 min, dry it in the dark for 24 h, and then demold it.

[0044] Example 2 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 as shown).

[0045] Dynamic light scattering further confirmed that three parallel experiments were carried out by dynamic light scattering and the average value was taken. To ensure the accuracy of the measurement data, the signals with the highest 5% light intensity and the lowest 1% light intensity were excluded. The average diameter of BSA-EQ nanoparticles was about 176.98 nm (as Figure 2 shown).

[0046] Taking the microneedles obtained in Example 1, it was more carefully observed in the field emission electron microscope scanning image that the microneedles had no obvious collapse, broken needles, or distortion, the needle tips were sharp, the width of the needle bottom was about 220 μm, the height of the needle was about 567 μm, and the needle spacing was about 520 μm (as Figure 3 shown).

[0047] Under the white light image, the microneedles showed a 15 × 15 array distribution, and there was no obvious collapse, broken needles, or distortion of the microneedles. The overall size of the microneedles was about 12 mm × 12 mm (as Figure 4 shown).

[0048] Equisetin, BSA / EQ, the microneedle material (GelMA), and the microneedle material containing BSA / EQ nanoparticles were prepared into tablet forms. At the same time, a blank control was added and placed on the agar medium coated with MRSA to observe their respective antibacterial effects. The areas of the antibacterial zones from large to small were Equisetin, BSA / EQ, and the microneedle material containing BSA / EQ nanoparticles, among which the antibacterial zones of GelMA and the blank control were 0. It was proved that BSA / EQ and the microneedle material containing BSA / EQ nanoparticles had antibacterial ability (as Figure 5 shown).

[0049] The Franz diffusion cell was used to calculate the release rate of Equisetin from the microneedles. During the whole experiment, the Franz diffusion cell was placed under a water bath heating device to maintain a constant temperature of 37°C. The diffusion phase was mouse skin, and the receiving cell was filled with phosphate buffer solution.

[0050] The results were as Figure 6 shown. It reached 43.6% within the first 1.5 hours, and then the release rate gradually slowed down, reaching 69% at 11 hours. After that, the drug release tended to be stable. By 71 h, the cumulative drug release rate was 77.4%.

[0051] The displacement change of a single microneedle under force was as Figure 7 shown. The calculation formula was the total force divided by the total number of microneedles. When the microneedle was under a force of 3 N, the displacement was about 0.6 mm, and there was no breakage phenomenon, indicating that it had the ability to penetrate the skin surface layer.

[0052] The biocompatibility of the microneedle material containing BSA / EQ nanoparticles was as Figure 8As shown, when the BSA / EQ concentration is within 20 mg / mL, the material has no obvious inhibitory effect on cells, and 20 mg / mL is much lower than the actual use concentration (0.02 mg / mL).

[0053] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. Preparation method of microneedles carrying 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 well by ultrasonic treatment; (2) Rotate and evaporate the mixture obtained in step (1) to remove the solvent, obtaining bovine serum albumin / antibiotic inclusion complexes; (3) Dissolve the inclusion complexes obtained in step (2) in water, centrifuge, and then rotate and evaporate to obtain bovine serum albumin / antibiotic nanoparticles; (4) Add the bovine serum albumin / antibiotic nanoparticles and photoinitiator obtained in step (3) to the methacrylated gelatin solution and mix well by ultrasonic treatment to obtain a prepolymer solution; (5) Add the prepolymer solution obtained in step (4) to a microneedle mold to prepare microneedles; (6) Irradiate the microneedles obtained in step (5) with ultraviolet light to obtain microneedles loaded with poorly soluble antibiotics.

2. The preparation method of the microneedles according to claim 1, wherein In step (1), the mass ratio of the antibiotic to the bovine serum albumin is 1:5 to 20; and / or, Add the antibiotic to an organic solvent to obtain an antibiotic solution, mix methanol and chloroform at a volume ratio of 1:1 to obtain the organic solvent; and / or, Add the bovine serum albumin solution to water to obtain the bovine serum albumin solution.

3. The preparation method of the microneedles according to claim 1, wherein, In step (2), the rotation evaporation is carried out at 55 - 65 °C and 400 - 600 rpm.

4. The preparation method of the microneedles according to claim 1, wherein In step (4), in the prepolymer solution, the concentration of methacrylated gelatin is 150 - 250 mg / mL; and / or, the concentration of the photoinitiator is 0.5 - 1 mg / mL; and / or, the concentration of the bovine serum albumin / antibiotic nanoparticles is 30 - 40 mg / mL.

5. The preparation method of the microneedle according to claim 1, characterized in that, In step (4), the photoinitiator is lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate.

6. The preparation method of the microneedles according to claim 1, characterized in that, In step (5), the prepolymer solution is added to the microneedle mold in two equal - volume portions. After each addition of the prepolymer solution, it is dried for 0.5 - 2 hours.

7. The preparation method of the microneedle according to claim 1 or 6, characterized in that, In step (5), after the prepolymer solution is added to the mold, it is centrifuged at 37 °C and 3500 rpm, and then dried.

8. The method for preparing the microneedles according to claim 1 or 6, characterized in that, In step (6), the microneedles are irradiated with 1W, 405 nm ultraviolet light and then dried in the dark.

9. Microneedles loaded with poorly soluble antibiotics prepared by the preparation method according to any one of claims 1 - 8.

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