Biomolecular-soluble glass microneedles and manufacturing method

Abstract

This invention provides a biomolecular glass-soluble microneedle and a method for manufacturing the same. The microneedle comprises a microneedle array and a substrate, wherein at least a portion of the microneedle array is made of biomolecular glass, and the biomolecular glass consists of biomolecules, an inducer, and a trace amount of solvent. The microneedle of this invention has excellent mechanical strength, can be smoothly and completely inserted into the skin or mucous membranes, and can rapidly and controlly release biomolecules, thereby effectively improving the bioavailability of biomolecules. Because the tip of the microneedle is made entirely of biomolecules, once released, it can be completely absorbed into the body without increasing the metabolic burden on the body. The microneedle of this invention is suitable for industrial production because it has a large load capacity and its manufacturing method is simple and rapid.

Description

Technical Field

[0001] The present invention relates to a biomolecule glass-soluble microneedle, a manufacturing method, and its use, and belongs to the technical field of pharmaceutical preparations and their manufacturing methods.

Background Art

[0002] The technologies of transdermal absorption and transdermal administration have a long history dating back to ancient Rome. Technological innovations in this field have continued to evolve through chemical permeation enhancers, iontophoresis, microdermabrasion, ultrasonic cavitation, microneedle technology, and the like. In recent years, microneedle technology has been selected as one of the top 10 emerging technologies in the world in 2020 by both Scientific American magazine and the World Economic Forum due to its unique advantages and potential application possibilities.

[0003] A microneedle is a micrometer-sized (length < 1000 μm) conical, tapered, or polyhedral piercing protrusion that brings many advantages to intradermal administration. When using a microneedle, a temporary path is formed in the outer layer of the skin, avoiding the barrier function and enabling the delivery of various bioactive molecules, especially biopolymer drugs such as polypeptides, nucleic acids, proteins, and stem cells. Otherwise, these bioactive molecules cannot be delivered via the transdermal route. The short axis of the microneedle is long enough to penetrate the stratum corneum but does not reach the underlying nerve endings. Therefore, the use of microneedles is essentially painless, which improves patient compliance, especially for patients with needle phobia.

[0004] Microneedles can be classified into soluble and insoluble microneedles based on the dissolution properties of their tips after insertion into the stratum corneum of the skin. Insoluble microneedles are generally made of metal, single-crystal silicon, or cross-linked polymer materials. When assisting the transdermal delivery of bioactive molecules, it is necessary to apply or attach the bioactive molecules after the microneedles are removed, allowing the bioactive molecules to pass through the skin pathways formed by the microneedles. Once the microneedles are removed, the microscopic pathways in the skin begin to heal. Because the duration of the pathways created by the microneedles is limited by the skin's self-repair, it is difficult to further improve the overall efficiency of transdermal absorption of bioactive molecules. There is also a risk that the tips may break and remain in the skin. Soluble microneedles are generally made of biocompatible polymers, including water-soluble polymers such as hyaluronic acid, chitosan, polyvinyl alcohol, chondroitin sulfate, polyvinylpyrrolidone, trehalose, dextran, maltose, sucrose, and carboxymethylcellulose. These polymer-based matrix tips can support bioactive molecules, increasing the drug-carrying capacity of the microneedles and resulting in the most promising types of microneedles. For example, reference (Chinese Patent CN104027324B) provides a soluble microneedle vaccine patch suitable for transdermal vaccine administration, manufactured using a water-soluble polymer material as the matrix material. This patch enables effective combined use of vaccine and adjuvant in soluble microneedles, enhancing the effect of the Th1 immune response and maintaining a balance between Th1 and Th2 immune responses. US Patent US2023015942A1 discloses a microneedle delivery device with a removable hybrid microneedle reservoir for delivering mesenchymal stem cells to various tissues and organs for tissue regeneration. Patent CN110769812A describes a polyvinylpyrrolidone (PVP)-based microneedle system that supports and releases glucagon-like peptide analogs.U.S. Patent US9320878B2 discloses a polymer microneedle patch that enables controlled transdermal release of hydrophilic polymers such as proteins, polypeptides, DNA, RNA, and other drugs.

[0005] A common feature of the soluble microneedles and / or microneedle devices disclosed above for biomolecule delivery is that the microneedle body is composed primarily of inert auxiliary material components, which function only as carriers for biomolecules or plasticizers for the microneedles. For this reason, the current use of soluble microneedles still faces several urgent challenges that need to be addressed. On the one hand, for preclinical applications, it is necessary to study the metabolic and excretion pathways of skin-soluble components and confirm the biological safety of the materials, which inevitably leads to delays in the commercialization process. For this reason, microneedle devices currently in the clinical trial and clinical approval stages are mainly insoluble microneedles. On the other hand, in the use of microneedles, existing polymer tips may partially deposit in the skin after dissolving in the body, causing granulation tissue, localized erythema, or accumulation in internal organs, potentially leading to material accumulation and metabolic burden of the materials. Furthermore, due to their small size, microneedles have a limited number of active molecules that can be delivered, limiting applications that require high doses or sustained release of bioactive molecules.

[0006] As described above, the development of soluble microneedles composed of bioactive molecules themselves, without relying on excipients such as water-soluble polymers, can improve the dosage and safety of microneedles and lay the foundation for expanding the clinical application of soluble microneedles. [Overview of the project] [Problems that the invention aims to solve]

[0007] This invention provides biomolecular glass soluble microneedles and a method for producing the same to solve problems present in the background art. It provides a general strategy of introducing multiple non-covalent bonds and trace amounts of solvent to obtain processable biomolecular glass, and this strategy was used in the production of biomolecular glass microneedles. The resulting biomolecular glass network exhibits extremely high compressibility and high mechanical strength, with no fracture observed even under 90% strain.

[0008] Existing literature has shown that the salt form, crystal form, crystalline state, and crystallization process of active pharmaceutical ingredients (APIs) significantly affect their stability, solubility, dissolution rate, bioavailability, and in vivo absorption and distribution characteristics. Therefore, the development of various solid states is necessary to improve the inherent properties of APIs, such as dissolution, solubility, hygroscopicity, and stability. CN113754556B and CN114014908A disclose methods for producing amino acid-based biomolecular glass, cyclic peptide glass, and pharmaceutical composition glass containing cyclic peptides, respectively, by melt-quenching, thereby expanding the range of applications for such biomolecules. [Means for solving the problem]

[0009] Because the tip of the microneedle of the present invention is made of biomolecular glass, it can not only be smoothly and completely inserted into the skin or mucous membranes, but can also regulate the release rate of bioactive molecules, effectively accelerate the elution rate of bioactive molecules, and improve the bioavailability of drugs. The tip of the microneedle is made entirely of biomolecules and can be completely absorbed into the body after drug administration without increasing the metabolic burden on the body. The microneedle of the present invention is suitable for industrial production because it has a large capacity for carrying bioactive molecules and its manufacturing method is simple and rapid.

[0010] In the first embodiment, a biomolecular glass-soluble microneedle is provided, the microneedle comprising a microneedle array and a substrate, At least a portion of the microneedle array is made of biomolecular glass. The biomolecular glass comprises a biomolecule, an inducer, and a trace amount of solvent, wherein the biomolecule is an amino acid and its derivatives and / or peptide, the trace amount of solvent means that the solvent content in the biomolecular glass is 0.1 to 5 wt%, preferably 0.1 to 2 wt%, and the inducer is selected from nucleotides, nucleotide polymers, RNA, DNA and / or pH adjusters.

[0011] In the second embodiment, a biomolecular glass soluble microneedle is provided, wherein the biomolecular glass refers to an amorphous (non-crystalline) biomolecular matrix that forms a thermodynamically stable liquid, that is, a liquid with extremely high viscosity and equivalent to a physical solid.

[0012] The aforementioned biomolecular glass is a glass in a broad sense, referring to an amorphous solid structure composed of biomolecules that have short-range order and long-range disorder, and its solid structure exhibits a glass transition phenomenon (Tg > 0°C).

[0013] Preferably, the Tg range of the biomolecular glass is 0°C <Tg<200℃である。

[0014] More preferably, the Tg range of the biomolecular glass is 20°C <Tg<160℃である。

[0015] The biomolecular glass has a relatively good glass-forming ability (GFA), that is, the ratio of Tg (glass transition temperature) to Tm (melting point temperature) is distributed between 0.55 and 0.75, preferably between 0.66 and 0.75.

[0016] In a third embodiment, a biomolecular glass soluble microneedle is provided, wherein the biomolecular glass consists of a biomolecule, an inducer, and a trace amount of solvent. The biomolecule is an amino acid and its derivatives and / or a peptide therapeutic agent. The aforementioned amino acids include one or more of the following: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine, and pyrrolicin. The derivative is an amino acid or peptide having a protecting group, and the protecting group includes Trt, Boc, Fmoc, Cbz / Z, Allyl, C2-C18 acyl, benzoyl, naphthoyl, OFm, Otbu, OBzl, OAll, OMe, and OEt. Preferably, the peptide therapeutic agent has biological activity and / or pharmacological activity, and the peptide therapeutic agent may be a linear peptide, a cyclic peptide, or a peptide derivative or conjugate.

[0017] The peptide therapeutic agent includes antitumor peptides, cytokine-mimicking peptides, cardiovascular peptides, host defense peptides, immunomodulatory peptides, metabolic peptides, antiviral polypeptides, and diagnostic polypeptides.

[0018] Examples of peptide therapies include octreotide acetate, lanreotide acetate, tetracosactide acetate, leuprorelin, buserelin, goserelin, gonadrelin, nafarelin, triptorelin, histrelin, cetrorelix, degarelix, abarelix, octreotide, lutasera, somatostatin, lanreotide, pasireotide, romidepsin, mifamultide, recombinant human insulin, thymopentin, recombinant human interferon, glutathione, teriparatide acetate, semaglutide, and liraglutide. This includes one or more of the following: lixisenatide, exenatide, venaglutide, trunkisine, albiglutide, thymopentin, thymalfacin, salkatonin, teriparatide, bacitracin, octreotide, carperitide, neuropeptide Y, brain natriuretic peptide (BNP), islet amyloid polypeptide (IAPP), vasoactive intestinal peptide (VIP), nesilitide, eptifibatide, oxytocin, bivalirudin, antiphagocytic peptide, vancomycin, methotrexate, and cyclosporine.

[0019] The introduction of an inducer can modify multiple non-covalent interactions between biomolecules.

[0020] Preferably, the inducer is selected from nucleotides, nucleotide polymers, RNA, DNA and / or pH adjusters. Exemplary nucleotides may include, but are not limited to, adenosine-5′-monophosphate (AMP), nicotinamide mononucleotide, guanosine-5′-monophosphate (GMP), uridine-5′-monophosphate (UMP), cytidine-5′-monophosphate (CMP), and adenosine-3′-monophosphate (3-AMP).

[0021] The pH adjusting agent is selected from DL-tartaric acid, hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, lactobionic acid, citric acid, tartaric acid, oxalic acid, DL-malic acid, maleic acid, quinic acid, adipic acid, fumaric acid, hexanoic acid, heptanoic acid, caprylic acid, valeric acid, butyric acid, propionic acid, and glacial acetic acid. Preferably, it is citric acid, nucleotide, or malic acid.

[0022] The solvent is water or physiological saline. The mass fraction of the solvent is between 0% and 5%, preferably 0% to 2%.

[0023] In the fourth aspect, a biomolecule glass soluble microneedle is provided, and the biomolecule glass microneedle is characterized by having good mechanical properties and processability. Considering the skin permeability of the glass soluble microneedle, the hardness of the tip of the glass microneedle is greater than 50 MPa, and the elastic modulus of the tip of the glass microneedle is greater than 1 GPa, which is helpful for smooth puncture of the skin.

[0024] In the fifth aspect, a biomolecule glass soluble microneedle is provided, and the substrate of the biomolecule glass soluble microneedle can be manufactured with a biomolecule glass and / or a polymer auxiliary material.

[0025] Here, the polymer auxiliary material is one or more of hydroxypropyl cellulose, alginic acid, methyl cellulose, chitosan, tragacanth gum, propylene glycol alginate, maltose, hyaluronic acid, chitosan, polyvinyl alcohol, chondroitin sulfate, polyvinyl pyrrolidone, trehalose, dextran, polylactic acid, carrageenan, cellulose acetate, hydroxyethyl methyl cellulose, ethyl hydroxy cellulose, sucrose, carboxymethyl cellulose, poly γ-glutamic acid, pullulan, gelatin, polydopamine, and polyacrylamide.

[0026] In the sixth aspect, a method for manufacturing a biomolecule glass soluble microneedle is provided, and the manufacturing method includes: Manufacturing of biomolecule glass by hydrothermal method: Dissolving a certain amount of biomolecule raw material in water or a mixed solvent, adding an appropriate amount of inducer, adjusting the pH of the solution system, keeping the temperature constant in the hydrothermal method, and adjusting the constant temperature time to volatilize the solvent step by step to obtain a biomolecule glass in step (1); Preheating the biomolecule glass and manufacturing a microneedle array by a casting method, a stretching method, an atomization spray method, a microfluidic method, or 3D printing (step (2)); Finally, based on the microneedle array, performing casting and defoaming to form a microneedle substrate (step (3)).

[0027] In the above step (1), the pH of the system is adjusted to 1-9, preferably 3-7. The constant temperature is 20-120°C, preferably 40-100°C. The constant temperature time is 5 min-6 h, preferably 30 min-3 h. The preheating temperature is 20-200°C, preferably 40-160°C, more preferably 40-100°C.

[0028] In the seventh aspect, in some embodiments, the manufacturing process of the biomolecule glass by the hydrothermal method can be combined with the casting method process for manufacturing microneedles, that is, it can be manufactured by the following steps. (1) Preparation of a microneedle pre-casting solution: Dissolving the biomolecule raw material in water or a mixed solvent, adding an appropriate amount of inducer, and adjusting the pH of the solution system. (2) Injecting the above pre-casting solution into a microneedle mold and performing a defoaming treatment. (3) Drying and demolding to obtain microneedles.

[0029] The injection methods include, but are not limited to, self-leveling, high-pressure injection, and microfluidic methods.

[0030] The defoaming methods include, but are not limited to, centrifugation, reduced pressure, or vacuum adsorption.

[0031] In step (3), the drying conditions are a temperature of 20-100°C, a relative humidity of 1-60%, and a drying time of 2 h or more, more preferably a temperature of 20-50°C, a relative humidity of 1-30%, and a drying time of 2 h or more.

[0032] In the eighth aspect, when the microneedle substrate to be manufactured is a polymer auxiliary material, step (3) for manufacturing the biomolecular glass-soluble microneedle manufactured as described in the sixth aspect is improved as follows. (3) A polymer auxiliary material solution is prepared and cast onto the biomolecular glass microneedle array prepared in step (2) to form a microneedle substrate.

[0033] In the ninth aspect, the method for producing biomolecular glass-soluble microneedles described in the seventh and eighth aspects can also be combined with other common known manufacturing processes to produce types such as multilayer microneedles, bubble microneedles, and porous microneedles.

[0034] The physical shape and size of the biomolecular glass-soluble microneedles of the present invention are not particularly limited, and any conventionally known size can be used.

[0035] For example, the height of the soluble microneedle body is 100-1000 μm, and the angle of the tip is 30-40 degrees.

[0036] After drying and peeling, the biomolecular glass-soluble microneedles may be further cut into patch shapes and / or lined with an adhesive support for use.

[0037] In the tenth embodiment, the biomolecular glass-soluble microneedle described above may have other active substances supported on it during manufacturing, and the active substances may be supported on the microneedle array and / or the substrate, and when the active substance is supported on the microneedle array, its mass ratio is less than 5% of the microneedle array, and when the active substance is supported on both the microneedle array and the substrate, its mass ratio is less than 10% of the total mass.

[0038] The aforementioned active substances include, but are not limited to, small molecule drugs such as analgesics, nerve agents, anti-allergic drugs, anxiolytics, and anti-inflammatory drugs, as well as biomolecular drugs such as antigen peptides, peptide vaccines, and monoclonal antibodies. Exemplary small molecule drugs include lidocaine, fentanyl, aspirin, ibuprofen, cetirizine, loratadine, rofecoxib, celecoxib, diclofenac sodium, pipothiazine, perphenazine, chlorpromazine, fluphenazine decanoate, thioridazine, sulpiride, penfluridol, clozapine, risperidone, olanzapine, clomipramine, amitriptyline, doxepin, fluoxetine, paroxetine, and cerutine. It contains larine, fluvoxamine, citalopram, carbamazepine, sodium valproate, magnesium salicylate, sodium salicylate, diflunisal, disalsalate, naproxen, fenbufen, sulindac, piroxicam, celecoxib, magnesium choline salicylate, paracetamol, indomethacin, naproxen, nabumetone, diclofenac, nimeslide, ibuprofen, and diclofenac. Exemplary large-molecule drugs include cancer vaccines, anthrax vaccines, influenza vaccines, Lyme disease vaccines, rabies vaccines, measles vaccines, mumps vaccines, varicella vaccines, smallpox vaccines, hepatitis vaccines, hepatitis A vaccines, hepatitis B vaccines, hepatitis C vaccines, pertussis vaccines, rubella vaccines, diphtheria vaccines, encephalitis vaccines, Japanese encephalitis vaccines, respiratory syncytial virus (RSV) vaccines, yellow fever vaccines, polio vaccines, herpes vaccines, human papillomavirus vaccines, rotavirus vaccines, pneumococcal vaccines, meningitis vaccines, pertussis vaccines, tetanus vaccines, typhoid vaccines, cholera vaccines, pulmonary tuberculosis vaccines, severe acute respiratory syndrome (SARS) vaccines, HSV-1 vaccines, HSV-2 vaccines, HIV vaccines, and combinations thereof.

[0039] In the eleventh embodiment, the use of a biomolecular glass-soluble microneedle is provided, characterized in that the glass-soluble microneedle can be used for puncturing the skin and / or mucous membranes, the blood-eye barrier, the blood-brain barrier, etc., for pharmaceutical or cosmetic purposes.

[0040] The aforementioned pharmaceutical applications refer to bioactive molecule delivery platforms, biosensors, or stimulus-responsive delivery systems.

[0041] The aforementioned cosmetic uses refer to removing dead skin cells and improving the appearance, smoothness, and brightness of the skin.

[0042] In this invention, terms such as “one,” “one type,” and “the foregoing” do not refer to a single entity, but rather include general classes that can be illustrated with specific examples. The terms “one,” “one type,” and “the foregoing” are interchangeable with the term “at least one type.” The phrases “at least one type (one)” and “including at least one type (one)” following a list refer to any single item in the list, and any combination of two or more items in the list. Unless otherwise specified, all numerical ranges include non-integer values ​​between their endpoints.

[0043] In this invention, the terms "microneedle," "microneedle array," or "microneedle array" refer to structures related to arrays that puncture the stratum corneum to facilitate transdermal delivery of bioactive molecules to the skin. In many cases, these can be used interchangeably. [Effects of the Invention]

[0044] The biomolecular glass microneedle according to the present invention has the following major advantages. (1) The body of the biomolecular glass microneedle of the present invention is composed of the therapeutic agent itself and can be completely absorbed and utilized in the body without causing any metabolic burden. (2) The therapeutic agent in the biomolecular glass microneedle of the present invention has high photostability and thermal stability, which is advantageous for long-term storage and use of the therapeutic agent. (3) The microneedles of the present invention have a large load capacity and can rapidly release biomolecules, thereby effectively improving the bioavailability of biomolecules. (4) The method for producing biomolecular glass microneedles of the present invention is simple, environmentally friendly, and highly reproducible in batches, making it suitable for industrial production. [Brief explanation of the drawing]

[0045] [Figure 1] This is a photograph of the thymopentine glass in Example 1. [Figure 2] This is a photograph of the vancomycin glass used in Example 2. [Figure 3] These are photographs of soluble glass microneedles manufactured in Example 3 (left) and Example 5 (right). [Figure 4] This is a photograph of the soluble glass microneedles produced in Example 4. [Figure 5] These are photographs of soluble glass microneedles manufactured in Example 6 (left) and Example 7 (right). [Figure 6] These are photographs of soluble glass microneedles manufactured in Example 8 (left) and Example 9 (right). [Figure 7] These are scanning electron microscope images of soluble glass microneedles manufactured in Example 10 (left) and Example 11 (right). [Figure 8] These are micrographs of soluble glass microneedles produced in Example 10 (left) and Example 11 (right). [Figure 9] This is a photograph of the soluble glass microneedle produced in Example 10 piercing Parafilm. [Figure 10] This is a photograph of a soluble glass microneedle, manufactured in Example 1, being used to puncture the skin of a fresh pig's ear. [Figure 11]This is an in vivo pharmacodynamic evaluation of thymopentin-soluble glass microneedles in rats in Example 1. [Figure 12] This is an evaluation of the blood glucose control effect of insulin-soluble glass microneedles manufactured in Example 9. [Figure 13] These are time-course curves of neurological function scores in various groups of mice with autoimmune encephalomyelitis (EAE). [Figure 14] These are the release curves of the drug-carrying microneedles in Examples 19 and 20. [Modes for carrying out the invention]

[0046] The present invention will be described in more detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0047] Microneedle molds and material sources The PDMS microneedle female mold of the present invention was purchased from Taizhou Weixin Pharmaceutical Technology Co., Ltd. The working portion of the microneedle array comes in two types: conical and square pyramidal, with a height of approximately 300 or 600 microns and an aspect ratio of approximately 2:1. The microneedles are arranged in a square pattern consisting of approximately 10 × 10 microneedles, with equal spacing of 500 to 600 μm between each microneedle. All reagents and equipment without manufacturer information are commercially available products obtained through legitimate sales channels. Where specific techniques and conditions are not shown in the examples, the techniques and conditions described in the relevant art literature or product descriptions shall be followed.

[0048] In the following examples, the solvent content in the obtained glass is measured using a thermogravimetric analyzer.

[0049] Manufacturing of biomolecularly soluble glass microneedles

[0050] Example 1

[0051] 100 mg of thymopentin powder was weighed and added to 10 mL of ultrapure water to obtain a thymopentin solution. When 20 mL of 0.01 M citric acid aqueous solution was added, the pH of the measured solution was 6.0. The above solution system was heat-treated, heated to a temperature of 50 °C at a heating rate of 10 °C / min, and then subjected to constant temperature treatment at this temperature for 1 hour to evaporate the solvent. When the solvent content was less than 2%, thymopentin glass was obtained.

[0052] The obtained thymopentine glass is shown in Figure 1. As shown in the figure, the thymopentine glass possesses the properties of glass and exhibits good transparency and stretching performance.

[0053] After preheating the above thymopentine glass to 60°C, it was injected into a PDMS microneedle female mold to fill all parts of the tip and substrate of the microneedle mold. Degassing was performed by centrifugation at a speed of 3000 rpm for 5 minutes, and the mold was cooled. After the microneedle body hardened, the microneedle glass was released from the mold to obtain thymopentine-soluble glass microneedles.

[0054] Example 2

[0055] 100 mg of vancomycin powder was weighed and added to 20 mL of ultrapure water and 1 mL of glycerol to obtain a vancomycin solution. When 10 mL of 0.01 M hydrochloric acid aqueous solution was added, the pH of the measured solution was 4.0. The above solution system was heat-treated by heating it to a temperature of 60°C at a heating rate of 10°C / min, and then subjected to constant temperature treatment at this temperature for 3 hours to evaporate the solvent. When the solvent content was less than 1%, vancomycin glass was obtained. The obtained vancomycin glass is shown in Figure 2.

[0056] The vancomycin glass described above was preheated to 60°C and then injected into a PDMS microneedle female mold to fill all parts of the tip and substrate of the microneedle mold. Degassing was performed by centrifugation at a speed of 2000 rpm for 5 minutes, and the mold was cooled. After the microneedle body hardened, the microneedle glass was released from the mold to obtain vancomycin-soluble glass microneedles.

[0057] Example 3

[0058] 500 mg of L-histidine powder was weighed and added to 20 mL of ultrapure water and 1 mL of ethanol to obtain a histidine solution. When 10 mL of 0.01 M hydrochloric acid aqueous solution was added, the pH of the measured solution was 5.0. The above solution system was heat-treated by heating it to a temperature of 100 °C at a heating rate of 50 °C / min, and then subjected to constant temperature treatment at this temperature for 0.5 hours to evaporate the solvent. When the solvent content was less than 3%, histidine glass was obtained.

[0059] After preheating the above histidine glass to 60°C, it was injected into a PDMS microneedle female mold to fill all parts of the tip and substrate of the microneedle mold. The pressure was rapidly reduced using a vacuum pump for 10 minutes to remove bubbles, and the mold was cooled to room temperature. After the microneedle body hardened, the microneedle glass was released from the mold to obtain histidine-soluble glass microneedles.

[0060] The resulting histidine-soluble glass microneedles are shown in Figure 3 (left).

[0061] Example 4

[0062] 100 mg of aspartic acid powder was weighed and added to 20 mL of ultrapure water and 1 mL of glycerol to obtain an aspartic acid solution. When 10 mL of 0.01 M hydrochloric acid aqueous solution was added, the pH of the measured solution was 4.0. The above solution system was heat-treated by heating it to a temperature of 60°C at a heating rate of 10°C / min, and then subjected to constant temperature treatment at this temperature for 3 hours to evaporate the solvent. When the solvent content became less than 1%, aspartic acid glass was obtained.

[0063] After preheating the above-mentioned aspartic acid glass to 80°C, it was injected into a PDMS microneedle female mold to fill all parts of the tip and substrate of the microneedle mold. Centrifugal degassing was performed at a speed of 2000 rpm, and the mold was cooled to harden the microneedle body. After that, the microneedle glass was released from the mold to obtain aspartic acid-soluble glass microneedles. The obtained aspartic acid-soluble glass microneedles are shown in Figure 4.

[0064] Example 5

[0065] 20 mg of tuftosine powder was weighed and added to 20 mL of ultrapure water to obtain a tuftosine solution. When 10 mL of 0.01 M malic acid aqueous solution was added, the pH of the measured solution was 6.0. The above solution system was heat-treated, heated to a temperature of 60 °C at a heating rate of 10 °C / min, and then subjected to constant temperature treatment at this temperature for 2 hours to evaporate the solvent. When the solvent content was less than 1%, tuftosine glass was obtained.

[0066] The above tuftosine glass was preheated to 50°C and then injected into a PDMS microneedle female mold. Degassing was performed by centrifugation at a speed of 2000 rpm for 5 minutes, and the mold was cooled. After the microneedle body hardened, the microneedle glass was released from the mold to obtain tuftosine-soluble glass microneedles.

[0067] The resulting histidine-soluble glass microneedles are shown in Figure 3 (right).

[0068] Example 6

[0069] 10 mg of semaglutide powder was weighed and added to 20 mL of ultrapure water and 1 mL of glycerol to obtain a semaglutide solution. When 10 mL of 0.01 M hydrochloric acid aqueous solution was added, the pH of the measured solution was 4.0. The above solution system was heat-treated, heated to a temperature of 60°C at a heating rate of 10°C / min, and then subjected to constant temperature treatment at this temperature for 0.5 hours to evaporate the solvent. When the solvent content was less than 1%, semaglutide glass was obtained.

[0070] After preheating the above semaglutide glass to 60°C, it was filled into a PDMS microneedle female mold by microfluidics, ensuring that only the tip portion of the microneedle mold was filled. Centrifugal degassing was performed at 2000 rpm for 5 minutes, and the mold was cooled. After the microneedle tips hardened, a 5% polyvinylpyrrolidone solution was cast onto the base material portion of the PDMS microneedle mold, and centrifugal degassing was performed at 2000 rpm for 5 minutes. Furthermore, it was dried for 6 hours under conditions of 60°C and 50% relative humidity. After hardening, the microneedle glass was released from the mold to obtain semaglutide-soluble glass microneedles.

[0071] The resulting semaglutide-soluble glass microneedles are shown in Figure 5 (left).

[0072] Example 7

[0073] 20 mg of methotrexate powder was weighed and added to 20 mL of ultrapure water to obtain a methotrexate solution. When 10 mL of 0.01 M citric acid aqueous solution was added, the pH of the measured solution system was 3.0. The above solution system was heat-treated, heated to a temperature of 60 °C at a heating rate of 10 °C / min, and then subjected to constant temperature treatment at this temperature for 2 hours to evaporate the solvent. When the solvent content was less than 1%, methotrexate glass was obtained.

[0074] The methotrexate glass described above was preheated to 50°C and then injected into a PDMS microneedle female mold so as to fill only the tip portion of the microneedle mold. After centrifuging at 2000 rpm for 5 minutes to remove air bubbles and cooling the mold, the microneedle tips hardened. Then, a 5% hyaluronic acid solution was cast into the base material portion of the PDMS microneedle mold and centrifuged at 2000 rpm for 5 minutes to remove air bubbles. Furthermore, it was dried for 6 hours under conditions of 80°C and 30% relative humidity. After hardening, the microneedle glass was demolded to obtain methotrexate-soluble glass microneedles.

[0075] The resulting methotrexate-soluble glass microneedles are shown in Figure 5 (right).

[0076] Example 8

[0077] 20 mg of cyclosporine powder was weighed and added to 20 mL of ultrapure water and 1 mL of ethanol to obtain a cyclosporine solution. When 10 mL of 0.01 M lactobionic acid aqueous solution was added, the pH of the measured solution system was 6.0. The above solution system was heat-treated by heating it to a temperature of 100 °C / min to a temperature of 100 °C, and then subjected to constant temperature treatment at this temperature for 2 hours to evaporate the solvent. When the solvent content became less than 1%, cyclosporine glass was obtained.

[0078] The cyclosporine glass described above was preheated to 120°C and then injected into a PDMS microneedle female mold so as to fill only the tip portion of the microneedle mold. After centrifuging at 2000 rpm for 5 minutes to remove air bubbles and cooling the mold, the microneedle tip portion hardened. Then, a 5% polyvinyl alcohol solution was cast onto the base portion of the PDMS microneedle mold and centrifuged at 2000 rpm for 5 minutes to remove air bubbles. Furthermore, it was dried for 6 hours under conditions of 80°C and 50% relative humidity. After hardening, the microneedle glass was released from the mold to obtain cyclosporine-soluble glass microneedles.

[0079] The resulting cyclosporine-soluble glass microneedles are shown in Figure 6 (left).

[0080] Example 9

[0081] 10 mg of recombinant human insulin powder was weighed and added to 20 mL of physiological saline to obtain a recombinant human insulin solution. When 10 mL of 0.01 M adenosine-5′-monophosphate (AMP) aqueous solution was added, the pH of the measured solution was 7.0. The above solution system was heat-treated, heated to 40 °C at a heating rate of 10 °C / min, and then subjected to constant temperature treatment at this temperature for 6 hours to evaporate the solvent. When the solvent content was less than 1%, a recombinant human insulin glass was obtained.

[0082] The recombinant human insulin glass described above was preheated at 40°C and then injected into a PDMS microneedle female mold to fill all parts of the microneedle mold, including the tip and base material. After centrifuging at 2000 rpm and cooling the mold, the microneedle tip portion hardened. Then, a 5% hydroxypropyl methylcellulose solution was cast into the base material portion of the PDMS microneedle mold and centrifuged at 2000 rpm for 5 minutes. Furthermore, it was dried for 12 hours under conditions of 30°C and 1% relative humidity. After hardening, the microneedle glass was demolded to obtain recombinant human insulin-soluble glass microneedles.

[0083] The resulting recombinant human insulin-soluble glass microneedles are shown in Figure 6 (right).

[0084] Example 10

[0085] 100 mg of arginine powder was weighed and added to 20 mL of ultrapure water to obtain an arginine solution. When 10 mL of 0.01 M citric acid aqueous solution was added, the pH of the measured solution was 5.0. The above solution system was heat-treated by heating it to a temperature of 120 °C at a heating rate of 10 °C / min, and then subjected to constant temperature treatment at this temperature for 1 hour to evaporate the solvent. When the solvent content was less than 1%, arginine glass was obtained.

[0086] After preheating the arginine glass described above to 120°C, it was injected into a PDMS microneedle female mold to fill all parts of the tip and substrate of the microneedle mold. Centrifugal degassing was performed at a speed of 2000 rpm, and the mold was cooled to harden the microneedle body. After that, the microneedle glass was released from the mold to obtain arginine-soluble glass microneedles.

[0087] The morphology of the obtained arginine-soluble glass microneedles was observed macroscopically using a 3D microscope, and the microneedle body was observed microscopically using a scanning electron microscope (SEM). The results are shown in Figures 7 (left) and 8 (left).

[0088] Example 11

[0089] The arginine-soluble glass microneedles in Example 10 described above can be manufactured by a combination of a hydrothermal biomolecular glass manufacturing process and a casting process for manufacturing microneedles, i.e., by the following steps.

[0090] 100 mg of arginine powder was weighed and added to 20 mL of ultrapure water to obtain an arginine solution. When 10 mL of 0.01 M citric acid aqueous solution was added, the pH of the measured solution was 5.0.

[0091] After injecting the above arginine-citric acid mixed casting solution into a PDMS microneedle mold, the mold was heated to 120°C at a heating rate of 10°C / min and subjected to constant temperature treatment at this temperature for 1 hour to evaporate the solvent. Subsequently, the mold was cooled by centrifugal degassing at a speed of 2000 rpm. After the microneedle body hardened, the microneedle glass was released from the mold to obtain arginine-soluble glass microneedles.

[0092] The morphology of the obtained arginine-soluble glass microneedles was observed macroscopically using a 3D microscope, and the microneedle body was observed microscopically using a scanning electron microscope (SEM). The results are shown in Figures 7 (right) and 8 (right).

[0093] Scanning electron micrographs and optical micrographs of arginine-soluble glass microneedles revealed that the manufacturing methods in both Examples 10 and 11 yielded soluble glass microneedles with undamaged tips. Macro and micro imaging results showed that the needle shape and tip sharpness of the microneedles, whether conical or pyramidal, were good and consistent with the PDMS mold design.

[0094] Example 12: Elastic modulus and hardness of biomolecular-soluble glass microneedles

[0095] The elastic modulus and hardness of biomolecular-soluble glass microneedles were measured using a nanoindenter (Nano Indenter G200, Agilent). The results are shown in Table 1. These results indicate that the biomolecular-soluble glass microneedles produced in Examples 1 to 11 all possess excellent elastic modulus and high hardness, laying the foundation for further applications in skin puncture.

[0096] Example 13: Experiment to evaluate the puncture performance of biomolecule-soluble glass microneedles.

[0097] The puncture performance of soluble microneedles was measured using the Parafilm (Parafilm M Laboratory Film) puncture method. Artificial skin was simulated by attaching eight layers of Parafilm to a foam board, and the manufactured soluble microneedles were inserted into the artificial skin using an applicator. The results shown in Figure 9 indicate that the Parafilm was completely punctured without tip breakage. The punctured Parafilm was placed under a microscope, the number of holes was observed, and the puncture depth was calculated (depth = ΣD × a / A (a is the number of holes, A is the total number of microneedles, D is the thickness of a single layer of Parafilm, 125 μm)). The results are shown in Table 1. The tip lengths of Examples 1, 2, and 11 were 600 μm, while the tip lengths of the remaining glass microneedles were 300 μm. From these results, it was found that the biomolecular-soluble glass microneedles in each example all showed good puncture performance.

[0098] Strength testing and puncture depth of biomolecularly soluble glass microneedles [Table 1]

[0099] Example 14: Method for puncturing fresh pigskin

[0100] Fresh pig ear skin, 800 μm thick, was taken, and the soluble microneedles prepared in Example 1 were inserted into the pig ear skin using an applicator. The results are shown in Figure 10. Clear microneedle holes were observed in the pig ear skin, and the number and arrangement of the holes were consistent with the microneedle array, indicating that the microneedles possessed sufficient mechanical properties to penetrate in vitro pig skin.

[0101] Example 15 Evaluation of the stability of biomolecules in glass microneedles

[0102] The microneedle arrays prepared in Example 9 were placed in a storage chamber maintained at 40°C and 96% relative humidity (RH) with an equivalent volume of insulin solution. After storage in the chamber for 1, 3, 7, and 14 days, the insulin content in the microneedle arrays was measured. At the specified time points, the arrays were removed from the chamber and washed with 0.1 mol / L acetic acid (1 mL) to obtain insulin solutions. The insulin content in the obtained washing solutions was analyzed by high-performance liquid chromatography (HPLC). The stability of insulin in the prepared microneedles was compared and analyzed against insulin solutions stored under the same conditions. The percentage of undegraded insulin remaining in the microneedles or solution at each time point was determined by measuring the peak area of ​​insulin in the selected sample and dividing it by the peak area of ​​insulin measured initially. The results are shown in Table 2.

[0103] [Table 2]

[0104] The results above demonstrate that the stability of insulin in glass microneedles is significantly superior to that of the solution group in a 14-day accelerated stability experiment, indicating that the manufactured soluble glass microneedles effectively protect the activity of bioactive molecules and are advantageous for long-term storage.

[0105] Evaluation of the effectiveness of biomolecules in glass microneedles

[0106] Example 16: Evaluation of the blood glucose control effect of recombinant human insulin-soluble glass microneedles prepared in Example 9.

[0107] Spontaneously developing type 1 diabetes model mice (male, 8 weeks old) were purchased and divided into three groups of six mice each. The experimental group mice received transdermal administration of the drug at a dose of 4 IU / kg using a glass microneedle. The blank group mice received no treatment. The control group mice were injected with recombinant insulin at a dose of 4 IU / kg via the tail vein. Blood glucose levels were measured at different time points. Blood was collected from the mice's tails every hour, and the fasting blood glucose levels of the mice were measured using a blood glucose meter. A curve was plotted with time on the x-axis and the fasting blood glucose level of the experimental mice on the y-axis. The results are shown in Figure 11. From the blood glucose control curve of spontaneously developing type 1 diabetes mice, it was confirmed that the recombinant human insulin-soluble glass microneedle manufactured in Example 9 could effectively control the blood glucose levels of mice, and that the blood glucose control effect was more stable than that of insulin injection.

[0108] Example 17: In vivo pharmacodynamic evaluation of thymopentin-soluble glass microneedles in rats.

[0109] Experimental Method: Twenty-eight SPF-grade male SD rats weighing 180-220g were used. No other drugs were administered before the experiment. A thymopentin solution was prepared by dissolving thymopentin powder in physiological saline, and this was used as the control formulation. The test formulation was the thymopentin-soluble glass microneedle manufactured in Example 1. The 28 SD rats were randomly divided into four groups (Groups 1-4) of seven rats each, and in vivo pharmacodynamic studies were conducted.

[0110] An immunosuppressive model was constructed before the start of the experiment. Cyclophosphamide powder, an immunosuppressant, was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd., and prepared as a 1 mg / L cyclophosphamide solution in physiological saline. Rats in the three groups excluding Group 1 were intraperitoneally injected with 35 mg / (kg*d) of the immunosuppressant for three consecutive days to suppress the rats' immune function. Group 1 was used as a blank control group and was intraperitoneally injected with 35 mL / kg of physiological saline.

[0111] After establishing a model by administering injections for three consecutive days, pharmacodynamic evaluations were performed. Group 1 rats served as the blank control group, and Group 2 rats served as the negative control group. Both groups received subcutaneous intravenous injections of 1 mL / kg of physiological saline for seven consecutive days. Group 3 rats served as the positive control group and received subcutaneous injections of thymopentin solution at a dose of 100 μg / kg. Group 4 rats were administered thymopentin-soluble glass microneedles prepared in Example 1 at a dose of 100 μg / kg for seven consecutive days. Rats from all three groups were sacrificed by cervical dislocation. The thymus and spleen of each rat were collected, weighed, and the organ index was calculated. The formula for calculating the organ index is as follows:

number

[0112] Here, W0 is the mass of the thymus or spleen, and W is the body weight of the rat. As shown in Figure 12, the spleen and thymus indices of the negative control group (group 2) were both smaller than those of the blank control group (group 1), confirming the establishment of an immunosuppressive model. The spleen and thymus indices of the administered groups (groups 3 and 4) were both higher than those of the negative control group in the model. The organ indices of the immunosuppressed rats were all elevated, and the difference was statistically significant. This demonstrated that the efficacy of thymopentin-soluble glass microneedles was superior to that of the solution formulation.

[0113] Example 18: Neurological function score of microneedles produced in Example 5 in mice with autoimmune encephalomyelitis (EAE).

[0114] Specifically, in the EAE group, 200 μg of MOG35-55 was dissolved in 200 μl of PBS buffer and thoroughly mixed with 200 μl of complete Freund's adjuvant (CFA) to form an emulsion (final concentration of inactivated tuberculin bacteria was 5 mg / mL). In the normal control group, 200 μl of PBS was directly thoroughly mixed with 200 μl of CFA. 200 μg of the emulsion was subcutaneously injected into two locations on the back of the mice. 500 ng of pertussis toxin was intraperitoneally injected at 0 and 48 hours after immunization. On day 1 after the EAE model was established, the drug was subcutaneously administered to the back of the mice at a dose of 100 μg / kg for 7 consecutive days via a glass microneedle; this group was designated as the glass microneedle intervention group.

[0115] Neurological function scores for experimental mice in each group were scored daily after immunization and observed continuously for 30 days. Neurological function was scored according to the following criteria: 0 points: no symptoms, 1 point: hypotonia of the tail (complete weakness, inability to bend the tip of the tail) or weakness of the hind limbs (unstable gait), 2 points: weakness of the tail and hind limbs, 3 points: hind limb paralysis (some movement of one or both hind limbs), 4 points: complete hind limb paralysis (complete immobility of the hind limbs, or forelimbs dragging the hind limbs), and 5 points: death.

[0116] The results are shown in Figure 13. The results of the neurological function scores in EAE mice demonstrate that the tuftosin-soluble glass microneedles produced in Example 5 can effectively restore neurological function in EAE mice.

[0117] Example 19

[0118] As described in the tenth aspect, the microneedle according to the present invention can carry an active drug for rapid and convenient transdermal administration of the drug. The specific method is as follows: 100 mg of arginine powder and 0.5 mg of lidocaine hydrochloride powder were weighed and added to 20 mL of ultrapure water to obtain an arginine-lidocaine solution. When 10 mL of 0.01 M citric acid aqueous solution was added, the pH of the measured solution was 5.0.

[0119] After injecting the above mixed solution casting liquid into a PDMS microneedle mold, the mold was heated to 40°C at a heating rate of 10°C / min and subjected to constant temperature treatment at this temperature for 12 hours to evaporate the solvent. Subsequently, the mold was degassed by centrifugation at a speed of 2000 rpm and cooled. After the microneedle body hardened, the microneedle glass was released from the mold to obtain arginine-lidocaine soluble glass microneedles.

[0120] Example 20

[0121] 100 mg of arginine powder and 0.2 mg of loratadine were weighed and added to 20 mL of ultrapure water to obtain an arginine-loratadine solution. When 10 mL of 0.01 M citric acid aqueous solution was added, the pH of the measured solution was 5.0.

[0122] After injecting the above mixed solution casting liquid into a PDMS microneedle mold, the mold was heated to 40°C at a heating rate of 10°C / min and subjected to constant temperature treatment at this temperature for 12 hours to evaporate the solvent. Subsequently, the mold was degassed by centrifugation at a speed of 2000 rpm and cooled. After the microneedle body hardened, the microneedle glass was released from the mold to obtain arginine-loratadine soluble glass microneedles.

[0123] Example 21: Measurement of the release curve of drug-carrying soluble microneedles

[0124] The kinetic characteristics of in vitro drug release from the manufactured drug-loaded microneedles were measured. The drug-loaded microneedles manufactured in Examples 19 and 20 above were sealed in dialysis bags (molecular weight cutoff 3 kDa) and immersed in phosphate buffer (PBS, pH=7.4) at 37°C. The drug-loaded microneedles dissolved in the dialysis bags, and the drug was released into the PBS through the dialysis bags. Samples (0.2 mL each) were taken at predetermined time intervals, and the same amount of PBS solution was replenished to measure the cumulative drug release rate from the microneedles. As shown in the results in Figure 14, the cumulative drug release amount exceeded 80% within 1 hour in all cases, indicating that the microneedles can rapidly release the loaded drug.

[0125] While the applicant stated that the present invention illustrates the detailed methods of the present invention by the above embodiments, the present invention is not limited to the above detailed methods; that is, it is not necessary to rely on the above detailed methods to carry out the present invention. It will be apparent to those skilled in the art that any improvements to the present invention, equivalent substitutions of each raw material of the present invention product and addition of auxiliary components, and selection of specific methods are also included within the scope of protection and disclosure of the present invention.

Claims

1. A biomolecular glass microneedle, wherein the microneedle comprises a microneedle array and a substrate. At least a portion of the microneedle array is made of biomolecular glass. The biomolecular glass microneedle comprises a biomolecule, an inducer, and a trace amount of solvent, wherein the biomolecule is an amino acid and its derivatives and / or peptide, the trace amount of solvent means that the solvent content in the biomolecular glass is 0.1 to 5 wt%, preferably 0.1 to 2 wt%, the inducer is selected from nucleotides, nucleotide polymers, RNA, DNA and / or pH adjusters, and the pH adjuster is one or more selected from DL-tartaric acid, hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, lactobionic acid, citric acid, tartaric acid, oxalic acid, DL-malic acid, maleic acid, quinic acid, adipic acid, fumaric acid, hexanoic acid, heptanoic acid, caprylic acid, valeric acid, butyric acid, propionic acid and glacial acetic acid.

2. The biomolecular glass microneedle according to claim 1, characterized in that the ratio of Tg (glass transition temperature) / Tm (melting point temperature) of the biomolecular glass is distributed between 0.55 and 0.

75.

3. The biomolecular glass microneedle according to claim 1, characterized in that the ratio of Tg (glass transition temperature) / Tm (melting point temperature) of the biomolecular glass is distributed between 0.66 and 0.

75.

4. The biomolecular glass microneedle according to claims 1 to 3, characterized in that the solvent is water, physiological saline, a mixed solvent of water and ethanol, or a mixed solvent of water and glycerol, and the proportion of solvents other than water in the mixed solvent is between 0 and 50 v / v%, preferably between 5 and 10 v / v%.

5. The biomolecular glass microneedle according to claims 1 to 4, characterized in that the hardness of the biomolecular glass microneedle is greater than 50 MPa, and the elastic modulus of the biomolecular glass microneedle is greater than 1 GPa.

6. The biomolecular glass microneedle according to claims 1 to 5, characterized in that the substrate of the biomolecular glass soluble microneedle can be manufactured from biomolecular glass and / or polymer.

7. A method for producing biomolecular glass microneedles according to claims 1 to 6, Manufacturing of biomolecular glass by hydrothermal method: Step (1) involves dissolving a certain amount of biomolecular raw material in water or a mixed solvent, adding an appropriate amount of inducer to adjust the pH of the solution system, maintaining a constant temperature in the hydrothermal method, and gradually evaporating the solvent by adjusting the constant temperature time to obtain biomolecular glass, and Step (2) involves preheating biomolecular glass and manufacturing a microneedle array by casting, stretching, atomizing spray, microfluidics, or 3D printing, A method for producing biomolecular glass microneedles, characterized by comprising the step (3) of forming a microneedle substrate by casting and degassing based on a microneedle array.

8. A method for producing biomolecular glass microneedles according to any one of claim 7, characterized in that, in the step of producing biomolecular glass by hydrothermal method, the pH of the system is adjusted to 1 to 9, preferably 3 to 7; the constant temperature is 20 to 120°C, preferably 40 to 100°C; the constant temperature time is 5 min to 6 h, preferably 30 min to 3 h; and the preheating temperature is 20 to 200°C, preferably 40 to 160°C, more preferably 40 to 100°C.

9. A method for producing a biomolecular glass-soluble microneedle according to any one of claims 7 to 8, characterized in that the casting method includes self-leveling, high-pressure injection, and microfluidic methods, and the degassing method includes centrifugation, reduced pressure, or vacuum adsorption.

10. Use of biomolecular glass microneedles according to any one of claims 1 to 6 as a drug carrier or in the manufacture of cosmetic products.

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