A porous structure soluble microneedle based on freeze-drying technology and a preparation method and application thereof

Abstract

The present application belongs to the technical field of microneedle, and particularly relates to a porous structure soluble microneedle patch prepared by freeze-drying technology and a preparation method and application thereof. The present application is prepared by preparing a microneedle preparation solution, filling the microneedle preparation solution into a microneedle mold, and then treating the microneedle mold by freeze-drying technology to obtain a porous structure soluble microneedle. The porous structure freeze-drying microneedle patch and the preparation method thereof have the advantages of convenient operation, no heat treatment, long-term preservation of active pharmaceutical ingredients, and are expected to realize industrial application and large-scale manufacturing. The present application realizes the preparation of microneedles by freeze-drying technology for the first time, and simultaneously develops two drug loading methods based on this. The drug-loaded microneedles prepared by the drug loading method have simple and fast process, and effectively avoid the waste of materials. In addition, the freeze-drying process does not affect the performance of the material, significantly improves the utilization efficiency of the material, simplifies the production process, and has a wide market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microneedle technology, specifically relating to a porous soluble microneedle patch prepared by freeze-drying technology, its preparation method and application, and two drug delivery methods within the microneedles. This soluble microneedle patch can effectively deliver drugs into the skin. Background Technology

[0002] Soluble microneedles are made from soluble polymers that dissolve and release drugs within the skin after penetrating the stratum corneum without irritating subcutaneous pain receptors and blood vessels. Commonly used soluble microneedle materials are mostly various polymers or sugars, including: sodium chondroitin sulfate, chitosan, polyvinylpyrrolidone, carboxymethyl cellulose, hyaluronic acid, silk fibroin, and other biodegradable polymers. Due to their excellent biocompatibility, biodegradability, high drug loading efficiency, and wide variety, soluble microneedles have a broad research and application prospect.

[0003] Currently, the main methods for preparing soluble microneedles include micromolding, photolithography, droplet air blowing, 3D printing, stretch lithography, hot pressing, and ultrasonic welding. Among these, micromolding is the most commonly used method for preparing soluble polymer microneedles, and it can be divided into casting, thermoforming, injection molding, and melt molding. Casting has low temperature requirements and a simple and convenient preparation process; however, the drying temperature and drying time of the microneedles are the main factors affecting their mechanical properties and drug stability. Curing at high temperatures can lead to uneven evaporation rates of moisture on the surface and in the needle body, thus affecting the shapeability of the microneedles and usually requiring very strict screening. At low temperatures, the drying process of the solution takes several hours to several days, resulting in low drying efficiency. Thermoforming, injection molding, and melt molding are mainly used to prepare biodegradable and insoluble microneedles, but the processing temperatures are generally relatively high, which can easily affect drug stability and activity.

[0004] Micromolding has been widely studied and applied in microneedle preparation. However, its industrial-scale application still faces several drawbacks, including sterilization issues, low dosage, instability in different systems and transport environments, and low drug utilization efficiency. Furthermore, this traditional method is not suitable for all drug-loaded microneedles in industrial production, especially those containing active drugs and nanomaterials. Additionally, this method often involves stepwise preparation of the microneedle body and backing material. While dry powder loading is frequently used to ensure drug stability, it can lead to unstable connections between the needle body and backing, resulting in breakage and further material waste. One-step microneedle preparation typically results in poor needle shape and numerous empty needles, further reducing drug loading and introducing air bubbles on the backing, affecting morphology and mechanical properties. To expand the drug applications of microneedles, such as overcoming the stability issues of nano-formulations and the application of unstable peptide and protein drugs, existing technologies require further improvement and development. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of existing technologies and provide a porous soluble microneedle based on freeze-drying technology, its preparation method, and its applications. The porous freeze-dried microneedle patch and its preparation method provided by this invention have advantages such as convenient operation, no heat treatment required, and long-term preservation of active pharmaceutical ingredients. This technology provides a new and promising method for microneedle preparation, with the potential for industrial scale-up and large-scale manufacturing. Simultaneously, this invention is the first to achieve integrated microneedle preparation through freeze-drying technology, and based on this, two drug loading methods have been developed: 1. a one-step drug loading method, and 2. a two-step drug loading method. The drug-loaded microneedles prepared through this method are simple and rapid, effectively avoiding material waste. Furthermore, freeze-drying does not affect the material's performance, significantly improving material utilization efficiency and greatly simplifying the production process. Moreover, the (drug-loaded) soluble microneedles prepared by this invention have foreseeable application prospects in multiple fields and a broad market application prospect.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] One objective of this invention is to provide a method for preparing porous soluble microneedles based on freeze-drying technology, the method comprising the following steps:

[0008] (1) Prepare the microneedle preparation solution and fill the microneedle preparation solution into the microneedle mold;

[0009] (2) Pre-freeze the microneedle mold filled with microneedle preparation solution;

[0010] (3) The pre-frozen microneedle mold is freeze-dried and demolded to obtain porous soluble microneedles.

[0011] Preferably, the matrix material used to form the microneedle in the microneedle preparation solution in step (1) includes at least one of chitosan, S-97, sodium chondroitin sulfate, and sodium carboxymethyl cellulose, more preferably sodium chondroitin sulfate. The microneedle comprises a microneedle body and a microneedle backing. The microneedle preparation solution containing the matrix material is filled into the microneedle mold, and after subsequent processing, a microneedle comprising both a body and a backing is formed in one step. This eliminates the need for stepwise preparation of the microneedle body and the microneedle backing, thereby effectively saving materials while ensuring the corresponding properties of the microneedle and / or the drug it may carry. The operation is simple, and the materials of the body and backing are the same, providing good compatibility. Furthermore, the one-step manufacturing process ensures stable connection between the body and backing. More preferably, the solvent for preparing the microneedle preparation solution is water. More preferably, the mass-volume concentration (g / mL) of the matrix material in the microneedle preparation solution is 10%-60%, more preferably 40%-60%, and more preferably 50%. More preferably, the microneedle preparation solution is prepared by mixing the matrix material with water until homogeneous.

[0012] Preferably, the microneedle preparation solution includes, in addition to the matrix material, a drug, which is at least one of the following: unstable drugs such as water-soluble peptides and proteins, poorly soluble drugs, other common soluble drugs, and corresponding nanosystems. More preferably, the water-soluble peptides and proteins include at least one of INS, EX4, etc.; the poorly soluble drugs include at least one of asiatic acid, curcumin, paclitaxel, and emodin, etc. More preferably, the drug is an asiatic acid nanostructure lipid carrier. More preferably, the mass ratio of drug to matrix material is 0-1:100, which can be adjusted according to the drug and the required drug loading, and is not limited to the data listed in this invention. More preferably, the microneedle preparation solution is prepared by adding the matrix material to water and then adding the drug and mixing them evenly.

[0013] Preferably, after the microneedle preparation liquid is filled into the microneedle mold in step (1), a vacuum treatment is performed, more preferably the vacuum degree range is -0.01Mpa to -0.09Mpa, more preferably -0.08Mpa; the microneedle preparation liquid is placed in a vacuum drying oven for vacuum treatment for no less than 5 minutes, more preferably 5-30 minutes, more preferably 10 minutes, so that the microneedle preparation liquid poured into the microneedle template under vacuum negative pressure can completely fill the pores, and can further eliminate air bubbles by standing.

[0014] Preferably, the pre-freezing temperature in step (2) is -20℃ to -80℃, and the time is not less than 2 hours, more preferably 2 hours to 24 hours. More preferably, the pre-freezing treatment is carried out in a refrigerator at -80℃ to freeze the microneedle preparation solution in the template.

[0015] Preferably, in step (3), the freeze-drying pressure range is 0.001 mbar to 6.1 mbar, more preferably 0.1 mbar, the freeze-drying temperature is -20 to -80°C, more preferably -80°C, and the time is not less than 4 hours. More preferably, the freeze-drying process is carried out in a freeze dryer.

[0016] Preferably, the porous soluble microneedles obtained after freeze-drying and demembraning in step (3) can be further subjected to drug loading treatment, which includes the following steps:

[0017] (3.1) Prepare a drug solution; preferably, the solvent for the drug solution is a volatile solvent; more preferably, the solvent is ethanol; more preferably, the concentration of the drug solution is 1 mg / mL;

[0018] (3.2) Immerse the entire microneedle obtained in step (3) in the drug solution and perform vacuum treatment. More preferably, perform vacuum drying in a vacuum drying oven for at least 5 minutes to absorb the drug solution using the porous structure and capillary force of the microneedle tip. Then, allow it to air dry at room temperature. ， As volatile solvents such as ethanol evaporate, the drug adheres to the surface and pore structure of the microneedles, further achieving stable drug loading. More preferably, the drug is at least one of unstable drugs such as water-soluble peptides and proteins, poorly soluble drugs, other common soluble drugs, and corresponding nanosystems. More preferably, water-soluble peptides and proteins include at least one of INS, EX4, etc.; poorly soluble drugs include at least one of asiatic acid, curcumin, paclitaxel, and emodin, etc. More preferably, the drug is asiatic acid.

[0019] By adopting the above technical solution, when the microneedle preparation solution does not contain drugs, the microneedles obtained in step (3) are blank microneedles. Drug loading can be performed on the microneedles through this drug loading process to further expand the drug loading methods of microneedles. When the microneedle preparation solution also contains drugs, the microneedles obtained in step (3) are drug-loaded microneedles. When drug loading is performed again, the same drug or another drug can be combined to achieve the increase of drug loading or the combination of multiple drugs to obtain composite drug-loaded microneedles, further expanding the drug loading methods and drug loading applications of microneedles.

[0020] Preferably, in this invention, the microneedle mold has the following microneedle dimensions: needle length 800 μm, bottom diameter 320 μm, needle tip 10 μm, needle tip distance 700 μm, and a quantity array of 10*10. The microneedle patch size is 10.3*10.3 mm. In practice, the microneedle mold dimensions can have reasonable variations and are not limited to the dimensions listed in this invention. More preferably, the microneedle mold of this invention uses PDMS material, but the microneedle mold material does not affect the method of this invention.

[0021] A second objective of this invention is to provide a porous soluble microneedle prepared by any of the above-mentioned methods, which can be a blank microneedle or a drug-loaded microneedle. More preferably, the porous structure of the microneedle has pores that are mainly spherical and elongated; the pore size is 5-10 μm; and the porosity is 35%-40%. The failure force of the microneedle tip, measured by a texture analyzer, is 0.0687 N / needle, indicating sufficient mechanical strength to pierce the skin.

[0022] More preferably, the microneedles of the present invention can achieve drug loading through two methods:

[0023] Method 1 involves mixing the drug into the microneedle preparation solution to achieve integrated preparation of drug-loaded microneedles and drugs. Taking the drug-loaded asiatic acid nanolipid carrier as an example, the drug loading of the one-step drug-loaded asiatic acid nanolipid carrier microneedle is (398.15±3.20)μg, and the backing of the microneedle contains the drug. After the backing dissolves on the skin surface, the nano-system drug it carries can also penetrate and be absorbed with the help of the permeation-promoting effect of the nanolipid carrier.

[0024] Method 2 involves preparing microneedles (which can be blank microneedles or drug-loaded microneedles), immersing the needle body of the microneedle in a drug solution, and drawing the drug solution into the pores through vacuum in the presence of the porous structure. As the solvent in the drug solution evaporates, the drug adheres to the pores, thus obtaining drug-loaded microneedles in two steps. Taking asiatic acid as an example, the drug loading of the two-step drug-loaded asiatic acid microneedles is (488.08±20.10) μg.

[0025] Compared with traditional microneedles, the microneedles prepared by the method of this invention have the significant advantages of one-step molding and abundant porosity. In the actual application of microneedles, the interconnected porous structure generated during freeze-drying absorbs water evaporated from the skin, promotes the dissolution of the backing, enhances drug release from the entire microneedle, and greatly improves the drug loading capacity and drug utilization rate of the microneedles.

[0026] A third objective of this invention is to provide an application of porous soluble microneedles prepared by any of the above-mentioned preparation methods.

[0027] Specifically, microneedles have applications in areas such as drug delivery, medical aesthetics, and tissue fluid extraction, and can be used to deliver vaccines, genes, proteins, and hydrophilic and hydrophobic drugs.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] (1) The present invention is based on the freeze-drying technology to prepare soluble microneedles. Due to the unique pore structure of freeze-dried microneedles, poorly soluble drugs can be directly loaded into the pore structure of the microneedles by means of capillary force. The preparation process of the microneedles is simple and has excellent universality for loading poorly soluble drugs, laying a good foundation for the application of soluble microneedles to hydrophobic drugs.

[0030] (2) Freeze-drying microneedles eliminates the need to consider the impact of moisture evaporation on microneedle shape at different drying temperatures, thus maximizing the preservation of drug activity and stability. The needle tip and backing can use the same concentration and material, making the process simpler and more stable compared to most needle-loaded microneedle formulations. Furthermore, it overcomes the limitation of nanosystems not being able to be stably stored in aqueous environments for extended periods, making it simpler, more efficient, more flexible, and more economical, with broad prospects for industrial applications.

[0031] (3) The microneedles prepared by the method of the present invention can be loaded with drugs in one step or two steps. The one-step drug loading can also be loaded with drugs in the backing, further improving the drug loading capacity and avoiding waste. The two-step drug loading is achieved through the porous structure of the microneedles of the present invention, which is a breakthrough in the field of microneedle drug loading methods and further expands the drug loading methods of microneedles. At the same time, the drug loading method of the present invention can be applied to all poorly soluble drugs, soluble drugs and unstable drugs and their corresponding nanosystems, which has a broader application prospect and research significance.

[0032] (4) The soluble microneedles prepared by this invention have good dissolving properties. The needle tip penetrates the stratum corneum and makes full use of the pore structure generated by the freeze-drying of the microneedles. It can absorb the interstitial fluid of the skin, promote the release of drugs from the microneedle tip and backing, and increase the drug utilization rate and drug loading of the microneedles.

[0033] (5) The microneedles prepared by this invention are simple to manufacture, have good mechanical properties, can effectively penetrate into the skin, are not prone to brittleness, and can meet the purpose of transdermal drug delivery.

[0034] (6) The microneedles prepared by this invention will not cause obvious adverse reactions such as skin inflammation, redness, swelling, bleeding, pain, and itching. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the morphology of the soluble microneedles prepared in Example 1 of the present invention.

[0036] Figure 2 The images show the appearance and microscopic morphology of the soluble microneedles at different pre-freezing temperatures of this invention.

[0037] Figure 3 The hardness test curve of the freeze-dried microneedles prepared in this invention;

[0038] Figure 4 This is an overall FESEM image of the freeze-dried microneedles prepared in this invention;

[0039] Figure 5 This is a side view of a single freeze-dried microneedle prepared according to the present invention via FESEM.

[0040] Figure 6 This is a FESEM image of multiple needles of the freeze-dried microneedles prepared in this invention;

[0041] Figure 7 This is a water solubility test diagram of the freeze-dried microneedles prepared in this invention;

[0042] Figure 8 The image shows the results of trypan blue staining after microneedle patches were applied to mouse skin.

[0043] Figure 9 Figure 1 shows the skin healing process after microneedle patches are applied to human skin.

[0044] Figure 10 TEM images of the asiatic acid nanolipid carrier before and after freeze-drying;

[0045] Figure 11 This is a schematic diagram of the transdermal release of microneedle patches in vitro. Detailed Implementation

[0046] The technical solution of the present invention will be further described in detail below through specific embodiments and with reference to the accompanying drawings. The room temperature mentioned in the present invention is 10-40℃, more preferably 20-25℃.

[0047] Example 1: Preparation of blank microneedles:

[0048] This embodiment provides a blank soluble microneedle array, the preparation method of which includes the following steps:

[0049] The microneedle mold is made of polydimethylsiloxane (PDMS) and consists of 10×10 conical MNs (with a bottom diameter of 320 μm, a tip diameter of 10 μm, and a needle length of 800 μm).

[0050] Soluble microneedles were prepared according to the following steps: A 50% (w / v, g / mL) chondroitin sulfate sodium gel (prepared with chondroitin sulfate and water) was used as the microneedle preparation solution, spread throughout a microneedle negative mold (placed in a six-well plate), and placed in a vacuum drying oven for 10 minutes. Under negative pressure, the microneedles filled the cavities in the negative mold. The microneedles were then pre-frozen at -80°C for 4 hours and then lyophilized in a freeze dryer. When the freeze dryer temperature dropped to approximately -80°C, the pre-frozen sample was placed in the freeze dryer, and the lyophilization program was started for 4 hours. After drying, the microneedles were removed from the mold, and the intact microneedle patch was carefully obtained using pointed forceps, yielding a porous, soluble blank microneedle patch.

[0051] Figure 1 The image shows the physical appearance of the freeze-dried soluble microneedle array prepared in this embodiment. It can be seen that the microneedles have a smooth and dense surface, a clean texture, no breaks, intact shape, and no air bubbles.

[0052] Example 2: Evaluation of the physical properties of the matrix material for freeze-dried microneedles:

[0053] Various materials can be used to prepare soluble microneedles, such as chitosan (CS), S-97, sodium chondroitin sulfate, polyvinylpyrrolidone (PVP), and sodium carboxymethyl cellulose (CMC). Traditional drying methods for microneedle preparation include constant temperature and humidity drying, room temperature drying, and oven drying. However, the use of freeze-drying technology for microneedle drying has not been reported. Therefore, there are no clear evaluation indicators for matrix materials suitable for freeze-drying technology in microneedle preparation. To verify the feasibility of freeze-drying for various materials, in this embodiment:

[0054] Different concentrations (w / v) of CS gel, S-97 gel, chondroitin sulfate sodium gel, PVP gel, and CMC gel were prepared by mixing the matrix material with deionized water at weight-to-volume ratios. Each prepared matrix gel was placed in a 24-well plate and placed in a vacuum drying oven for 10 min, followed by freeze-drying and then drying at room temperature and pressure. Table 1 summarizes the evaluation of the physical properties of microneedles using different matrix materials. The experimental results show that CS, S-97, chondroitin sulfate sodium, and CMC exhibited better mechanical strength after freeze-drying among the different concentrations of single matrix materials, and can be further prepared into microneedles.

[0055] Table 1 Evaluation of the physical properties of different microneedle matrix materials

[0056]

[0057]

[0058] Example 3: Optimization of the freeze-drying process for microneedles:

[0059] To investigate the effects of microneedle matrix material concentration, vacuum drying time, pre-freezing temperature, and freeze-drying time on the performance of the prepared microneedles, this invention uses sodium chondroitin sulfate as the microneedle matrix material and conducts performance influence experiments on the corresponding parameters.

[0060] In this embodiment, different concentrations of chondroitin A sodium sulfate (40%, 50%, and 60%) were prepared for the fabrication of corresponding microneedles. The results showed that the microneedles maintained their morphology intact. At a 40% concentration, the microneedles were relatively soft and easily compressed under vertical force. At a 60% concentration, the microneedles exhibited high toughness and were prone to bending. At a 50% concentration, the material showed good flowability and mechanical properties; therefore, a 50% chondroitin sulfate concentration is preferred.

[0061] In this embodiment, the prepared microneedles were placed in a vacuum drying oven for 5 min, 10 min, and 15 min of vacuum evacuation. Vacuum drying has a certain concentration effect. As the vacuum drying time increases, the viscosity of the microneedle matrix material increases, and the flowability decreases. Vacuum drying time: 5 min > 10 min > 15 min. Vacuum drying for 5 min resulted in the best material flowability, but the prepared microneedles were relatively brittle and prone to cracking after the backing was applied. Vacuum drying for 15 min resulted in excessive matrix viscosity, making it difficult to remove the air bubbles generated after vacuum drying. Therefore, a vacuum drying time of 10 min is preferred.

[0062] In this embodiment, the prepared microneedles were pre-frozen at two different temperatures: ① -20℃ and ② -80℃. The effects of these two methods on the microneedle's shapeability and mechanical properties were investigated. The results are as follows: Figure 2 As shown, Figure 2 The images show the appearance and microscopic morphology of soluble microneedles at different pre-freezing temperatures. The freeze-dried microneedles obtained by method ② have a smooth and dense surface, while those obtained by method ① are loose and uneven, containing numerous air bubbles. This may be because the dissolved solutes in the unfrozen water cannot be released in situ during the drying process, causing the material to melt and resulting in gas expansion and bottle spraying. Therefore, a pre-freezing temperature of -80℃ is preferred.

[0063] In this embodiment, the prepared microneedles were freeze-dried in a freeze dryer for 2, 4, 8, and 12 hours, respectively, and the appearance and mechanical strength of the obtained microneedles were examined. After 2 hours, the microneedles were completely dried, with a moisture content of 3.48% ± 2.76%. Generally, the moisture content standard for freeze-dried products is strictly controlled; for freeze-dried products, the moisture content should be ≤3%. High moisture content in freeze-dried products will cause product decomposition and shrinkage, thus reducing the product's shelf life. The results showed that the moisture content decreased to <1% after 4 hours of freeze-drying, and after 4 hours, the moisture content change was not significant and remained <1%, meeting the freeze-drying requirements. There was no significant difference in the penetration mechanical strength at different drying times. Considering the experimental cost and machine wear and tear, a freeze-drying time of not less than 4 hours is preferred.

[0064] Example 4: Characterization of blank soluble microneedles:

[0065] The freeze-dried porous soluble blank microneedles prepared in Example 1 were used for characterization tests:

[0066] (1) Mechanical property testing

[0067] The microneedle patch was cut into four pieces and fixed to the aluminum base of the analyzer with tape. Using the compression mode of the texture analyzer, the downward speed of the p / 6 probe was set to 0.5 mm / s, and the maximum compression distance was 0.8 mm. The trigger force was 0.015 N, and the data acquisition rate was 50. The microneedles were kept parallel to the probe axis. During probe displacement, the analyzer recorded the pressure on the microneedles until the microneedles were fully deformed. The results are as follows: Figure 3 As shown, Figure 3 The graph shows the hardness test results for freeze-dried microneedles. A single 800μm blank microneedle can withstand a maximum pressure of 0.0687 N / needle (far exceeding the force required to penetrate the skin, 3.596 × 10⁻⁶). -4 The microneedles (N) meet the requirements for transdermal delivery. This indicates that the microneedles have sufficient mechanical strength to pierce the skin and deliver the drug.

[0068] (2) Microneedle morphological characterization

[0069] The morphology of the lyophilized microneedles was observed using field emission scanning electron microscopy (FE-SEM, HITACHI Regulus 8100) under high vacuum conditions with an accelerating voltage of 10 kV. The distribution of relevant elements was analyzed using energy-dispersive X-ray spectroscopy (EDS, Oxford Ultim Max 65). Results are as follows: Figure 4-6 As shown, Figure 4 This is an overall FESEM image of the freeze-dried microneedles. Figure 5 This is a side view of a single freeze-dried microneedle via FESEM. Figure 6 The image shows a multi-needle FESEM test image of the freeze-dried microneedles. As can be seen from the image, the microneedles prepared by this invention are conical in shape, uniform in size, and have a large number of pore structures.

[0070] (3) Microneedle solubility test

[0071] After drying, the microneedles were placed tip-down on a pre-perforated polyethylene (PE) membrane, ensuring only the tip was exposed to PBS buffer (pH 6.0, simulating skin pH). The membrane was then placed in a constant-temperature shaking incubator at 37°C and 50 rpm for a solubility experiment. The microneedles were removed periodically, their surface moisture was blotted dry with filter paper, and the solubility was observed and photographed under a microscope. The results are shown below. Figure 7 As shown, Figure 7 The image shows the water solubility test results of the freeze-dried microneedles. It can be seen that the microneedles prepared by this invention dissolve rapidly, starting to dissolve in about 10 seconds and basically completely dissolving in 90 seconds. They have good solubility, less skin irritation, and good safety.

[0072] (4) Microneedle puncture rate experiment

[0073] The prepared microneedles were inserted into the skin on the back of mice. After 1 minute, the needles were removed, stained with trypan blue, and excess stain was removed. Figure 8 As shown, the number of pores was recorded, and the skin penetration rate was calculated. The higher the value, the better the mechanical properties. The formula for calculating the penetration rate is as follows: Penetration rate = (Number of pores / Number of soluble microneedles) * 100%. After trypan blue staining, the pores were clearly visible, indicating a penetration rate of over 90%. This demonstrates that the lyophilized microneedle patch has sufficient mechanical strength to penetrate the skin.

[0074] (5) Evaluation of the skin recovery performance of microneedles

[0075] Place MNs on the skin for a skin puncture test. After pressing the MNs for 1 minute, remove them and observe and photograph their recovery process. Figure 9 As shown in the image, when the MNs were first removed, the indentation of the microneedle patch and the micropores in the center were faintly visible, and the color was relatively light. Over time, redness appeared at the needle tips, and the color gradually deepened, becoming most pronounced at 12 minutes. The redness gradually faded, and the skin fully recovered at 28 minutes. This indicates that the freeze-dried microneedles have minimal skin irritation and high safety.

[0076] Mechanical property testing results indicate that the microneedles possess sufficient mechanical strength to pierce the skin and deliver drugs. Microscopic images show that the microneedles are conical in shape, uniform in size, and have a large number of porous structures. Furthermore, the lyophilized microneedles exhibit good solubility, minimal skin irritation, and good safety.

[0077] Example 5: Preparation method of drug-loaded microneedles (I)

[0078] Soluble microneedle patches loaded with the poorly soluble drug asiatic acid using a porous microneedle structure drug loading method are prepared by the following steps:

[0079] 1. Prepare blank microneedles according to the steps described in Example 1;

[0080] 2. Accurately weigh 20 mg of asiatic acid and dissolve it in 2 mL of anhydrous ethanol to obtain an alcoholic solution of asiatic acid.

[0081] 3. Take the prepared blank lyophilized microneedle patch, immerse the needle body in an alcoholic solution of asiatic acid, vacuum-dry in a vacuum oven at -0.08 MPa for 5 min, and then allow it to air dry at room temperature for about 8 h. The drug loading of the prepared drug-loaded microneedles AA MNs needle tip (needle body) is (93.56±5.25) ug, and the drug loading of the entire microneedle patch is (488.08±20.10) ug.

[0082] The drug loading method described in this invention is applicable to poorly soluble drugs that are soluble in volatile solvents such as ethanol and methanol, such as asiatic acid, curcumin, paclitaxel, and emodin.

[0083] Example 6: Method 2 for preparing drug-loaded microneedles:

[0084] Preparation of microneedles loaded with asiatic acid nanostructured lipid carriers (AA-NLC):

[0085] AA-NLC was prepared using an emulsification-ultrasonic method. 60 mg of glyceryl monostearate, 40 mg of oleic acid, and 20 mg of the prescribed amount of asiatic acid were dissolved in 5 mL of anhydrous ethanol and heated to (75±2) °C. Separately, 25 mL of a 1% (w / v) poloxamer and lecithin solution was used as the aqueous phase. The organic phase was slowly injected into the aqueous phase using a syringe under magnetic stirring at 1200 r / min. The mixture was stirred at 75 °C for 5 min to form a primary emulsion. The emulsion was then processed using an ultrasonic cell disruptor at a power of 300 W, with a 2-second operation followed by a 3-second pause, for a total of 5 min. Residual ethanol was removed by rotary evaporation. The resulting turbid liquid was ultrasonicated for 5 min in an ice bath to obtain the asiatic acid nanostructured lipid carrier dispersion. The particle size was (171.4±2.14) nm, and the zeta potential was (-21.1±0.32) mV. The encapsulation efficiency was 84.2%, and the drug loading was 4.55%. The NLC dispersion was concentrated by rotary evaporation to obtain NLC concentrate (drug content 4 mg / mL).

[0086] Weigh 0.5g of sodium chondroitin sulfate and dissolve it in 1mL of the prepared NLC concentrate. Mix them evenly to obtain a microneedle matrix solution. The mass ratio of drug to microneedle matrix material is 1:100.

[0087] The prepared microneedle matrix solution was spread into the microneedle negative mold (placed in a six-well plate), and placed in a vacuum drying oven for 10 minutes. Under negative pressure, the cavity of the microneedle negative mold was filled.

[0088] The microneedles were pre-frozen at -80°C for 4 hours and then freeze-dried in a freeze dryer.

[0089] When the freeze dryer temperature dropped to approximately -80℃, the pre-frozen sample was placed in the freeze dryer, and the freeze-drying program was started for 4 hours. After completion, the intact microneedle patch was carefully removed using pointed tweezers. The drug loading at the tip of the AA-NLC MNs microneedle was (19.27±1.17)ug, and the drug loading of the entire microneedle patch was (398.15±3.20)ug. TEM images of the asiatic acid nanolipid carrier before and after freeze-drying are shown below. Figure 10 As shown, Figure 10 TEM images of the asiatic acid nanolipid carrier before and after freeze-drying. After freeze-drying and reconstitution of the microneedles, the particle size of the nanolipid carrier did not increase significantly, remaining <200 nm.

[0090] Therefore, the drug delivery method described in this invention is suitable for nano-formulations and can further enhance their stability.

[0091] Example 7: Transdermal effect of drug-loaded microneedles:

[0092] In vitro transdermal studies were conducted using asiatic acid-loaded asiatic acid nanolipid carrier microneedles (AA-NLC MNs, prepared in Example 6) and directly drug-loaded asiatic acid microneedles (AA MNs, prepared in Example 5):

[0093] Existing research indicates that microneedle arrays pierce the stratum corneum of the skin, creating numerous pores on the skin surface, disrupting the barrier effect of the stratum corneum. This pore formation facilitates the interaction of information between the body and the external environment and increases transepidermal water loss. The microneedles of this invention, produced through a freeze-drying process, possess an interconnected porous structure that absorbs water evaporated from the skin, further promoting the dissolution of the backing and thus enhancing drug release throughout the entire microneedle, not just at the needle tip. This significantly improves the drug-carrying capacity and drug release effect of the microneedles.

[0094] In this embodiment, to study the transdermal diffusion of freeze-dried microneedles, a modified Franz diffusion cell was used, and in vitro transdermal activity was studied using isolated mouse skin. Figure 11 As shown, Figure 11 This diagram illustrates the transdermal release of the microneedle patch in vitro. Using AA-NLC MNs increases the in vitro skin permeability of the drug by several orders of magnitude. Within 3 hours, the amount of drug permeated by AA exceeds the amount of drug at the microneedle tip, and the release rate is >80% within 48 hours. The microneedle group releases not only the drug at the tip but also the drug in the backing layer. This is likely due to the porous structure of the microneedles, which absorbs interstitial fluid from the skin, promoting drug release from the backing layer. After absorbing interstitial fluid, the backing layer swells and dissolves, forming a hydrogel, which then slowly releases the drug at a certain rate. While AA MNs achieve physical permeation, the permeation rate is slower than that of AA-NLC MNs, only starting after 6 hours. This may be because AA and MNs achieve physical adsorption, with the hydrophobic drug adsorbed onto the pores and surface of the microneedles, reducing their solubility and resulting in a slower release rate within 24 hours. The amount of drug permeated by AA in 24 hours has exceeded the amount of drug at the tip of the microneedle, and has a certain degree of sustained release effect.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. A method for preparing porous soluble microneedles based on freeze-drying technology, characterized in that, The preparation method includes the following steps: (1) Prepare microneedle preparation solution, fill the microneedle preparation solution into the microneedle mold, and perform vacuum treatment for 10 minutes. (2) Pre-freeze the microneedle mold filled with microneedle preparation solution; (3) The pre-frozen microneedle mold is freeze-dried and demolded to obtain porous soluble microneedles; In step (1), the matrix material used to form microneedles in the microneedle preparation solution includes at least one of S-97, sodium chondroitin sulfate, and sodium carboxymethyl cellulose. The mass-volume concentrations of the matrix materials in the microneedle preparation solution are as follows: S-97 is 20%-40% (g / mL), sodium chondroitin sulfate is 50% (g / mL), and sodium carboxymethyl cellulose is 10%-30% (g / mL). And / or, in addition to the matrix material, the microneedle preparation solution also includes a drug, wherein the drug is at least one of an unstable drug, a poorly soluble drug, a soluble drug, and a corresponding nanosystem; In step (2), the pre-freezing temperature is -80℃ and the time is not less than 4 hours; The porous soluble microneedles obtained in step (3) have a pore size of 5-10 μm and a porosity of 35%-40%.

2. The method for preparing porous soluble microneedles based on freeze-drying technology according to claim 1, characterized in that, In step (3), the freeze-drying pressure range is 0.001 mbar to 6.1 mbar, the freeze-drying temperature is -20 to -80℃, and the time is not less than 4 hours.

3. The method for preparing porous soluble microneedles based on freeze-drying technology according to claim 1, characterized in that, The porous soluble microneedles obtained after freeze-drying and demembraning in step (3) further include a drug loading treatment, which includes the following steps: (3.1) Prepare the drug solution; (3.2) Immerse the microneedle body obtained in step (3) into the drug solution and perform vacuum treatment.

4. A porous soluble microneedle based on freeze-drying technology prepared by the preparation method according to any one of claims 1-3.

5. The application of the porous soluble microneedles based on freeze-drying technology as described in claim 4 in the preparation of microneedle formulations.

Images