CN116942592A

CN116942592A - Water-soluble biomolecular glass microneedle and preparation method thereof

Info

Publication number: CN116942592A
Application number: CN202310497211.XA
Authority: CN
Inventors: 闫学海; 邢蕊蕊
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2023-05-05
Filing date: 2023-05-05
Publication date: 2023-10-27

Abstract

The invention provides a biological molecular glass soluble microneedle and a preparation method thereof, wherein the microneedle is provided with a microneedle array and a substrate; wherein at least a portion of the microneedle array is comprised of biomolecular glass; the biomolecular glass consists of biomolecules, an inducer and a trace amount of solvent. The microneedle has good mechanical strength, can completely and smoothly penetrate into skin and mucous membrane, can regulate and rapidly release biological molecules, and effectively improves the bioavailability of the biological molecules; the microneedle tip is completely composed of biomolecules, and can be completely absorbed by organism after release is completed, without increasing metabolic burden in vivo. The microneedle has the advantages of large load capacity, simple and quick preparation method and suitability for industrial production.

Description

Water-soluble biomolecular glass microneedle and preparation method thereof

Technical Field

The invention relates to a biomolecular glass soluble microneedle, a preparation method and application thereof, belonging to the technical field of pharmaceutical preparations and preparation methods thereof.

Background

Percutaneous absorption and transdermal delivery techniques have a long history, even going back to the ancient roman period. The technology in this field is also continually evolving, such as chemical permeability enhancers, iontophoresis, microdermabrasion, ultrasonic cavitation and microneedle technology. In recent years, due to its unique advantages and application potential, microneedle technology has been co-selected by the scientific americans and world economic forum as a globally ten emerging technology in 2020.

Microneedles are micron-sized (length <1000 μm), conical, tapered or multi-faceted piercing projections, providing a number of advantages for intradermal administration. The use of microneedles creates temporary channels in the outer layers of the skin, thereby bypassing the barrier function and allowing the delivery of different bioactive molecules, especially the delivery of biomacromolecule drugs or stem cells such as polypeptides, nucleic acids, proteins, etc., which would otherwise not be delivered by the transdermal route. The short axis of the microneedle is long enough to penetrate the stratum corneum but not to the underlying nerve endings, so that the microneedle application is essentially painless, improving patient compliance, especially in needle phobia patients.

The dissolution performance of microneedles after insertion of tips into the stratum corneum of skin is classified into dissolution microneedles and non-dissolution microneedles. The insoluble microneedles are mostly made of metal, monocrystalline silicon or cross-linked polymer, and when the skin permeation of the bioactive molecules is assisted, the bioactive molecules need to be smeared or applied after the microneedles are removed, so that the bioactive molecules pass through skin tunnels formed by the microneedles. The micro-tunnels on the skin begin to heal when the microneedles are removed, and the residence time of the microneedle tunnels is limited by skin self-repair, making it difficult to further increase the overall efficiency of transdermal absorption of bioactive molecules. In addition, there is also a risk that the needle tip breaks and stays in the skin. The soluble microneedle is usually made of biocompatible polymers, including water-soluble polymers such as hyaluronic acid, chitosan, polyvinyl alcohol, chondroitin sulfate, polyvinylpyrrolidone, trehalose, dextran, maltose, sucrose, and carboxymethyl cellulose. The polymer needle tip matrixes can load bioactive molecules, so that the drug loading capacity of the microneedles is improved, and the microneedles with the most application prospect are formed. For example: in the reference (Chinese patent CN 104027324B), a soluble microneedle vaccine patch which is prepared by taking a water-soluble polymer material as a matrix material and is suitable for transdermal administration of a vaccine is provided, and the effective combined use of the vaccine and an adjuvant in the soluble microneedle is realized, so that the effect of Th1 type immune response is improved, and the relatively balanced Th1 and Th2 type immune responses are obtained. US2023015942A1 discloses a microneedle delivery device with a detachable mixed microneedle library for delivering mesenchymal stem cells to various tissues and organs for tissue regeneration. Patent CN 110769812A describes microneedle systems based on polyvinylpyrrolidone (PVP) loaded release of glucagon-like peptide analogues. U.S. patent No. 9320878B2 discloses a polymeric microneedle patch that can achieve transdermal controlled release of hydrophilic macromolecules such as proteins, polypeptides, DNA, RNA, and other drugs.

The above disclosed soluble microneedles and/or microneedle devices for delivering biomolecules have in common that the needle bodies of the microneedles are mainly composed of inactive auxiliary ingredients, and the components are only used as carriers of the biomolecules or plastic agents of the microneedles, so that the application of the soluble microneedles still faces some problems to be solved: on the one hand, before application in clinic, it is necessary to conduct research on metabolism and elimination pathways of components soluble in the skin, judging the biosafety of the material, and thus inevitably delaying the commercialization process. Thus, microneedle devices currently in clinical trials and clinical trials remain predominantly insoluble microneedles. On the other hand, in microneedle applications, existing polymeric needle tips may partially deposit in the skin after dissolution in vivo, forming granulation, localized erythema or accumulation in organs in the body, creating material accumulation or material metabolic burden. Furthermore, due to its small size, microneedles can only deliver a limited number of active molecules, limiting the application of microneedles when large doses or continuous release of bioactive molecules are required.

In conclusion, the development of the soluble microneedle which is independent of excipients such as water-soluble polymers and consists of bioactive molecules can enlarge the dosage and safety of the microneedle, and lay a foundation for expanding the clinical application of the soluble microneedle.

Disclosure of Invention

In order to solve the problems in the background art, the invention provides a biomolecular glass soluble microneedle and a preparation method thereof. We have successfully provided a general strategy to introduce multiple non-covalent bonds, trace amounts of solvents to obtain a biomolecular glass that can have processability, and use it to make biomolecular glass microneedles. By the above-mentioned kinetic stabilization technique, the obtained biomolecular glass network has extremely strong compressibility, exhibits high mechanical strength, and no breakage is observed at a strain of 90%.

The prior literature shows that the salt form, crystal form, crystalline state, crystallization process and the like of the raw material medicine have obvious influence on the stability, solubility, dissolution rate, bioavailability, in-vivo absorption and distribution properties. Therefore, various solid-state developments are required for the raw material drugs to improve their properties such as dissolution, solubility, hygroscopicity and stability. In CN113754556B and CN114014908A, there are disclosed a glass for preparing biomolecules based on amino acids, a glass for preparing cyclopeptides and a glass for preparing pharmaceutical compositions containing cyclopeptides by a melting-quenching method, respectively, and the application range of the biomolecules is widened.

The microneedle tip is composed of the biomolecular glass, so that not only can the skin and mucous membrane be completely and smoothly penetrated, but also the rate of releasing bioactive molecules can be regulated and controlled, the dissolution rate of the bioactive molecules can be effectively increased, and the bioavailability of the medicine can be improved; the microneedle tip is entirely composed of biomolecules, and can be completely absorbed by the body after the administration is finished, so that the metabolic burden in the body is not increased. The microneedle bioactive molecular load is large, the preparation method is simple and quick, and the microneedle bioactive molecular load is suitable for industrial production;

first aspect: providing a biomolecular glass soluble microneedle, wherein the microneedle is provided with a microneedle array and a substrate;

wherein at least a portion of the microneedle array is comprised of biomolecular glass;

the biomolecular glass consists of biomolecules, an inducer and a trace amount of solvent; the biological molecule is amino acid, derivative and/or peptide; the trace amount of solvent refers to the content of the solvent in the biomolecular glass of 0.1-5wt%; preferably 0.1 to 2wt%; the inducer is selected from the group consisting of nucleotides, nucleotide polymers, RNA, DNA and/or pH modifiers.

In a second aspect, a soluble microneedle of biomolecular glass is provided, wherein the biomolecular glass is an amorphous (non-crystalline) biomolecular matrix that forms a thermodynamic liquid, i.e. a liquid with extremely high viscosity, corresponding to a physical solid.

The biomolecular glass is glass in a broad sense, and refers to an amorphous solid structure which consists of biomolecules and has short-range ordered and long-range unordered, and the solid structure has a glass transition phenomenon (Tg >0 ℃).

Preferably, the biomolecular glass has a Tg range of 0 ℃ < Tg <200 ℃.

More preferably, the biomolecular glass has a Tg range of 20 ℃ < Tg <160 ℃.

The biomolecular glass has a good Glass Forming Ability (GFA), i.e. the ratio of Tg (glass transition temperature)/Tm (melting point temperature) is distributed between 0.55 and 0.75, preferably between 0.66 and 0.75.

In a third aspect, a soluble microneedle of biomolecular glass is provided, which is characterized in that the biomolecular glass is composed of a biomolecule, an inducer and a trace amount of solvent;

the biological molecule is amino acid and its derivative and/or peptide therapeutic agent;

the amino acid comprises one or more of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine and pyrrolysine;

The derivative is an amino acid or peptide with a protecting group, and the protecting group comprises: trt, boc, fmoc, cbz/Z, allyl, C C18 acyl, benzoyl, naphthoyl, OFm, otbu, OBzl, OAll, OMe, OEt;

preferably, the peptide therapeutic agent has biological and/or pharmacological activity; the peptide therapeutic agent may be a linear peptide, a cyclic peptide or a derivative or conjugate of a peptide.

The peptide therapeutic agent comprises anti-tumor peptide, cytokine mimic peptide, cardiovascular peptide, host defense, immune regulation, metabolism peptide, antiviral polypeptide and diagnostic polypeptide.

Exemplary peptide therapeutic agents include octreotide acetate, lanreotide acetate, tikexin acetate, leuprorelin, buserelin, goserelin, nafarelin, triptorelin, histrelin, cil Qu Ruilin, degarelin, abarelix, octreotide, lutetium oxyoctreotide, somatostatin, lanreotide, pasireotide, romidepsin, mivariin, recombinant human insulin, thymopentapeptide, recombinant human interferon, glutathione, teriparatide acetate, cable Ma Lutai, liraglutide, exenatide, benraglutide, duloxetide, apride, thymopentine, salmon calcitonin, teriparatide, bacitracin, octreotide, capelin, neuropeptide Y, brain Natriuretic Peptide (BNP), farinacide (IAPP), vasoactive intestinal peptide (gliptin), ceripotide, ivatide, ivacin, vanadprin, cerulopterin, or several of the like.

The introduction of an inducer can modulate multiple non-covalent interactions between biomolecules.

Preferably, the inducer is selected from the group consisting of nucleotides, nucleotide polymers, RNA, DNA and/or pH modifiers;

exemplary nucleotides 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).

The pH regulator 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, caproic acid, heptanoic acid, caprylic acid, valeric acid, butyric acid, propionic acid and glacial acetic acid. Preferably citric acid, nucleotides, malic acid.

The solvent is water or physiological saline. The mass percentage of the solvent is between 0 and 5 percent, preferably between 0 and 2 percent.

In a fourth aspect, a soluble microneedle of biomolecular glass is provided, which is characterized in that the biomolecular glass microneedle has good mechanical properties and processability. Considering the skin permeability of the glass soluble microneedle, the hardness of the tip of the glass microneedle is more than 50MPa, and the elastic modulus of the tip of the glass microneedle is more than 1Gpa, so that the glass microneedle is beneficial to smoothly puncturing the skin.

In a fifth aspect, a biomolecular glass soluble microneedle is provided, wherein a substrate of the biomolecular glass soluble microneedle may be made of biomolecular glass and/or a polymer auxiliary material.

Wherein the polymer auxiliary material is one or more of hydroxypropyl cellulose, alginic acid, methylcellulose, chitosan, tragacanth, propylene glycol alginate, maltose, hyaluronic acid, chitosan, polyvinyl alcohol, chondroitin sulfate, polyvinylpyrrolidone, trehalose, dextran, polylactic acid, carrageenan, cellulose acetate, hydroxyethyl methylcellulose, hydroxyethyl cellulose, sucrose and carboxymethyl cellulose, poly gamma-glutamic acid, pullulan, gelatin, polydopamine and polyacrylamide.

In a sixth aspect, a method for preparing a biomolecular glass soluble microneedle is provided, the method comprising the steps of:

(1) Preparing biomolecular glass by a hydrothermal method: dissolving a certain amount of biological molecular raw materials in water or a mixed solvent, adding a proper amount of inducer and regulating the pH value of a solution system; keeping the constant temperature in a hydrothermal method, and regulating the constant temperature time to realize the step-by-step solvent volatilization to obtain the biomolecular glass;

(2) Preheating biomolecular glass, and preparing a microneedle array by a pouring method, a stretching method, an atomization spraying method, a microfluidic method or a 3D printing method;

(3) Finally, casting and defoaming are carried out on the basis of the microneedle array to form the microneedle substrate.

In the step (1), the pH of the system is adjusted to 1 to 9, preferably 3 to 7;

the constant temperature is 20-120 ℃, preferably 40-100 ℃;

the constant temperature time is 5 min-6 h, preferably 30 min-3 h;

the preheating temperature is 20 to 200 ℃, preferably 40 to 160 ℃, more preferably 40 to 100 ℃.

In a seventh aspect, in certain embodiments, the process of preparing biomolecular glass by a hydrothermal method may be combined with the casting process of preparing the biomolecular glass by a microneedle, i.e. prepared by the steps of:

(1) Preparing microneedle pre-casting liquid: dissolving the biological molecular raw material in water or a mixed solvent, adding a proper amount of inducer and regulating the pH value of a solution system;

(2) Pouring the precast liquid into a microneedle mould for defoaming treatment;

(3) Drying and demoulding to obtain the micro needle.

Methods of priming include, but are not limited to, self-leveling, high pressure jetting, and microfluidic.

The defoaming method includes but is not limited to centrifugation, decompression or vacuum adsorption.

The drying conditions in the step (3) are as follows: the temperature is 20-100 ℃, the relative humidity is 1-60%, and the drying time is more than 2 hours; further preferably 20-50 ℃, and the relative humidity is 1-30%, and the drying time is more than 2 hours.

In an eighth aspect, when the prepared microneedle substrate is a polymer adjuvant, the preparation step (3) of the biomolecular glass soluble microneedle prepared as described in the sixth aspect is modified as follows:

(3) And (3) preparing a polymer auxiliary material solution, and pouring the polymer auxiliary material solution onto the biomolecular glass microneedle array prepared in the step (2) to form a microneedle substrate.

The method for preparing the biomolecular glass soluble microneedle according to the ninth aspect, as described in the seventh aspect and the eighth aspect, may be combined with other conventionally known preparation processes to prepare a multi-layer microneedle, a bubble microneedle, a porous microneedle, or the like.

The physical shape and size of the biomolecular glass soluble microneedle of the present invention are not particularly limited, and any size known in the prior art may be used.

For example: the height of the soluble microneedle body is 100 to 1000 micrometers, and the angle of the tip is 30 to 40 degrees.

The dried and peeled biomolecular glass soluble microneedle can be further cut into a patch shape and/or further lined with an adhesive support for use.

In a tenth aspect, a biomolecular glass soluble microneedle as described above can carry other actives during the preparation process, the actives can be carried on the microneedle array or/and the substrate of the microneedle; when the active substance is loaded on the microneedle array, the mass ratio of the active substance is lower than 5% of that of the microneedle array; when the active substance is loaded on the microneedle array and the substrate, the mass ratio of the active substance is lower than 10% of the total mass.

The active substances include but are not limited to pain relieving medicines, nerve medicines, antiallergic medicines, anxiolytic medicines, anti-inflammatory medicines, micromolecular medicines, and biological macromolecule medicines such as antigenic peptides, peptide vaccines, monoclonal antibodies and the like. Exemplary small molecule drugs include lidocaine, fentanyl, aspirin, ibuprofen, cetirizine, loratadine, rofecoxib, celecoxib, diclofenac sodium, pipothiazine, perphenazine, chlorpromazine, fluphenazine maleate, thioridazine, sulpiride, penfluide, clozapine, risperidone, olanzapine, chlorimipramine, amitriptyline, doxepin, fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram, carbamazepine, sodium valproate, magnesium salicylate, sodium salicylate, diflunisal (diflunisal), bissalicylate, naproxen, fenbufen, sulindac, piroxicam, celecoxib, choline magnesium salicylate, acetaminophen, indomethacin, naproxen, diclofenac, nimesulide, fenide, and Futamide. Exemplary macromolecular drugs include therapeutic cancer vaccines, anthrax vaccines, influenza vaccines, lyme disease vaccines, rabies vaccines, measles vaccines, mumps vaccines, varicella vaccines, smallpox vaccines, hepatitis a vaccines, hepatitis b vaccines, hepatitis c vaccines, pertussis vaccines, rubella vaccines, diphtheria vaccines, encephalitis vaccines, japanese encephalitis vaccines, respiratory syncytial virus vaccines, yellow fever vaccines, cerebral poliosis vaccines, herpes vaccines, human papilloma virus vaccines, rotavirus vaccines, pneumococcal vaccines, meningitis vaccines, pertussis vaccines, tetanus vaccines, typhoid fever vaccines, cholera vaccines, tuberculosis vaccines, severe Acute Respiratory Syndrome (SARS) vaccines, HSV-1 vaccines, HSV-2 vaccines, HIV vaccines, and combinations thereof.

In an eleventh aspect, there is provided the use of biomolecular glass-soluble microneedles, wherein the glass-soluble microneedles can be used for pharmaceutical or aesthetic use as skin and/or mucous membranes, blood-ocular barriers, blood-brain barriers, etc.

The pharmaceutical use refers to a bioactive molecule delivery platform, a biosensing or stimulus responsive delivery system.

The aesthetic use refers to use for exfoliating or improving the appearance, smoothness or brightness of the skin.

In the present invention, terms such as "a", "an", and "the" do not refer to only a single entity, but include general categories, specific examples of which are used for illustration. The terms "a," an, "and" the "are used interchangeably with the term" at least one. The phrases "at least one" and "including at least one" of the following list refer to any one of the list and any combination of two or more of the list. Unless otherwise indicated, all numerical ranges include their endpoints and non-integer values between the endpoints.

In the present invention, the terms "microneedle", "microneedle array" or "array of microneedles" refer to structures associated with an array capable of piercing the stratum corneum to facilitate transdermal delivery of bioactive molecules to the skin. In many cases, they may be used interchangeably.

The biomolecular glass microneedle provided by the invention has the following remarkable advantages:

(1) The microneedle body of the biomolecular glass is constructed by the therapeutic agent, and the body can be completely absorbed and utilized without generating any metabolic burden;

(2) The biological molecular glass microneedle has high photostability and thermal stability of the therapeutic agent, and is favorable for long-time storage and application of the therapeutic agent;

(3) The microneedle of the invention has large load capacity, can rapidly release biological molecules, and effectively improves the bioavailability of the biological molecules;

(4) The biomolecular glass microneedle provided by the invention has the advantages of simple preparation process, environment friendliness, high batch repeatability and suitability for industrial production.

Description of the drawings:

FIG. 1 is a photograph of thymopentin glass of example 1;

FIG. 2 is a photograph of vancomycin glass of example 2;

FIG. 3 is a photograph of soluble glass micropins prepared in example 3 (left) and example 5 (right);

FIG. 4 is a photograph of a soluble glass microneedle prepared in example 4;

FIG. 5 is a photograph of soluble glass micropins prepared in example 6 (left) and example 7 (right);

FIG. 6 is a photograph of soluble glass micropins prepared in example 8 (left) and example 9 (right);

FIG. 7 is a scanning electron micrograph of the soluble glass microneedles prepared in example 10 (left) and example 11 (right);

FIG. 8 is a photomicrograph of the soluble glass microneedles prepared in example 10 (left) and example 11 (right);

FIG. 9 is a photograph of a soluble glass microneedle fabricated in example 10 to puncture a sealing film;

FIG. 10 is a photograph of a soluble glass microneedle prepared in example 1 piercing fresh porcine ear skin;

FIG. 11 is an in vivo pharmacodynamic evaluation of thymopentin soluble glass microneedles of example 1 in rats;

FIG. 12 is a graph showing the evaluation of the glucose controlling effect of the insulin soluble glass microneedle prepared in example 9;

FIG. 13 is a plot of the neural function score over time in various groupings of autoimmune encephalomyelitis (EAE) mice;

fig. 14 is a release profile for drug loaded microneedles of example 19 and example 20.

Detailed Description

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

Microneedle mould and material source

The PDMS microneedle negative mold in the present invention was purchased from the state microchip pharmaceutical technologies ltd. The microneedle array acts in part in two types, conical and tetragonal, having a height of about 300 or 600 microns and a height of about 2: an aspect ratio of 1. The microneedles are arranged in a square pattern of about 10 x 10 microneedles with an equal spacing of 500-600um between each microneedle. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications.

The solvent content of the resulting glass was determined by thermogravimetric analysis in the following examples.

Preparation of biomolecular soluble glass microneedle

Example 1

Weighing 100mg of thymus pentapeptide powder, and adding 10mL of ultrapure water to obtain thymus pentapeptide solution; after adding 20mL of 0.01M aqueous citric acid, the pH of the system solution was measured to be 6.0; heating the solution system to 50 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment for 1h to volatilize the solvent, and obtaining thymic pentapeptide glass when the solvent content is lower than 2%;

the thus-obtained thymic pentapeptide glass is shown in FIG. 1. As shown in the figure, the thymic pentapeptide glass has glass characteristics and good transparency and stretching processability.

Preheating the thymic pentapeptide glass at the temperature of 60 ℃, pouring the preheated thymic pentapeptide glass into a PDMS microneedle female die, and filling the tip of the microneedle mould and the base part; and (3) carrying out centrifugal defoaming at the speed of 3000rpm for 5min, cooling the die, and demoulding the microneedle glass after the microneedle body is solidified to obtain the thymic pentapeptide soluble glass microneedle.

Example 2

100mg of vancomycin powder is weighed and added into 20mL of ultrapure water and 1mL of glycerol to obtain a vancomycin solution; after adding 10ml of 0.01m aqueous hydrochloric acid, the pH of the system solution was measured to be 4.0; heating the solution system to 60 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment for 3 hours to volatilize the solvent, and obtaining vancomycin glass when the solvent content is lower than 1%; the obtained vancomycin glass is shown in fig. 2.

Preheating the vancomycin glass at 60 ℃, pouring the preheated vancomycin glass into a PDMS microneedle female die, and filling the microneedle tips and the base part of the microneedle die; and (3) carrying out centrifugal defoaming at 2000rpm for 5min, cooling the die, and demoulding the microneedle glass after the microneedle body is solidified to obtain the vancomycin soluble glass microneedle.

Example 3

Weighing 500mg of L-histidine powder, and adding into 20mL of ultrapure water and 1mL of ethanol to obtain histidine solution; after adding 10ml of 0.01m aqueous hydrochloric acid, the pH of the system solution was measured to be 5.0; heating the solution system, heating to 100 ℃ at a heating rate of 50 ℃/min, performing constant temperature treatment at the temperature for 0.5h to volatilize the solvent, and obtaining histidine glass when the solvent content is lower than 3%;

preheating the histidine glass at the temperature of 60 ℃, pouring the histidine glass into a PDMS microneedle female die, and filling the microneedle mould tip and a base part; and (3) rapidly decompressing for 10min under a vacuum pump to defoam, cooling the die to room temperature, and demoulding the microneedle glass after the microneedle body is solidified to obtain the histidine soluble glass microneedle.

The histidine-soluble glass microneedles obtained are shown in figure 3 (left).

Example 4

100mg of aspartic acid powder was weighed and added to 20mL of ultrapure water and 1mL of glycerol to obtain an aspartic acid solution; after adding 10ml of 0.01m aqueous hydrochloric acid, the pH of the system solution was measured to be 4.0; heating the solution system to 60 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment for 3 hours to volatilize the solvent, and obtaining aspartic glass when the solvent content is lower than 1%;

preheating the aspartic glass at 80 ℃, pouring the preheated aspartic glass into a PDMS microneedle female die, and filling the microneedle mould tip and a base part; and (3) performing centrifugal defoaming at the speed of 2000rpm, cooling the die, and demolding the microneedle glass after the microneedle body is solidified to obtain the aspartic acid soluble glass microneedle. The aspartic acid soluble glass microneedles obtained are shown in figure 4.

Example 5

Weighing 20mg of phagocytosis peptide powder, and adding 20mL of ultrapure water to obtain a phagocytosis peptide solution; after 10ml of 0.01m aqueous malic acid solution was added, pH of the system solution was measured to be 6.0; heating the solution system to 60 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment for 2 hours to volatilize the solvent, and obtaining the phagocytosis-promoting peptide glass when the solvent content is lower than 1%;

Preheating the phagocytotic peptide glass at 50 ℃, and pouring the preheated phagocytotic peptide glass into a PDMS microneedle female die; and (3) carrying out centrifugal defoaming at 2000rpm for 5min, cooling the die, and demolding the microneedle glass after the microneedle body is solidified to obtain the phagocytic peptide-promoting soluble glass microneedle.

The histidine-soluble glass microneedles obtained are shown in figure 3 (right).

Example 6

10mg of powder of Soxhlet Ma Lutai was weighed and added to 20mL of ultrapure water and 1mL of glycerol to obtain a Soxhlet Ma Lutai solution; after adding 10ml of 0.01m aqueous hydrochloric acid, the pH of the system solution was measured to be 4.0; heating the solution system to 60 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment at the temperature for 0.5h to volatilize the solvent, and obtaining the cable Ma Lutai glass when the solvent content is lower than 1%;

preheating the cable Ma Lutai glass at the temperature of 60 ℃, filling the glass into a PDMS microneedle female die through micro-flow control, and filling only the tip part of a microneedle mould; centrifuging at 2000rpm for 5min to remove foam, cooling the mold, and solidifying at the tip of the microneedle; pouring a 5% polyvinylpyrrolidone solution into a substrate part of a PDMS microneedle mould, and centrifuging at 2000rpm for 5min for defoaming; further, under the conditions of the temperature of 60 ℃ and the relative humidity of 50%, the drying time is 6 hours; after curing, the microneedle glass is demolded, and the soluble glass microneedle of the cable Ma Lutai is obtained.

The resulting cord Ma Lutai soluble glass microneedle is shown in fig. 5 (left).

Example 7

Weighing 20mg of methotrexate powder, and adding 20mL of ultrapure water to obtain a methotrexate solution; after adding 10mL of 0.01M aqueous citric acid, the pH of the system solution was measured to be 3.0; heating the solution system to 60 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment for 2 hours to volatilize the solvent, and obtaining the methotrexate glass when the solvent content is lower than 1%;

preheating the methotrexate glass at 50 ℃, pouring the preheated methotrexate glass into a PDMS microneedle female die, and filling only the tip part of the microneedle mould; centrifuging at 2000rpm for 5min to remove foam, cooling the mold, and solidifying at the tip of the microneedle; pouring a solution of 5% hyaluronic acid into a substrate part of a PDMS microneedle mould, and centrifuging at 2000rpm for 5min for defoaming; further, under the conditions of the temperature of 80 ℃ and the relative humidity of 30%, the drying time is 6 hours; after solidification, the microneedle glass is demoulded, and the methotrexate soluble glass microneedle is obtained.

The methotrexate soluble glass microneedles obtained are shown in figure 5 (right).

Example 8

Weighing 20mg of cyclosporine powder, and adding 20mL of ultrapure water and 1mL of ethanol to obtain a cyclosporine solution; after adding 10ml of 0.01M aqueous lactose acid, the pH of the system solution was measured to be 6.0; heating the solution system to 100 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment for 2 hours to volatilize the solvent, and obtaining cyclosporine glass when the solvent content is lower than 1%;

Preheating the cyclosporine glass at 120 ℃, pouring the cyclosporine glass into a PDMS microneedle female die, and filling only the tip part of the microneedle mould; centrifuging at 2000rpm for 5min to remove foam, cooling the mold, and solidifying at the tip of the microneedle; pouring a 5% polyvinyl alcohol solution into a substrate part of a PDMS microneedle mould, and centrifuging at 2000rpm for 5min for defoaming; further, under the conditions of the temperature of 80 ℃ and the relative humidity of 50%, the drying time is 6 hours; after solidification, the microneedle glass is demoulded, and the cyclosporine soluble glass microneedle is obtained.

The cyclosporine soluble glass microneedle obtained is shown in fig. 6 (left).

Example 9

Weighing 10mg of recombinant human insulin powder, and adding the powder into 20mL of physiological saline to obtain a recombinant human insulin solution; after adding 10ml of 0.01M aqueous adenosine-5' -monophosphate (AMP), the pH of the system solution was measured to be 7.0; heating the solution system to 40 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment at the temperature for 6 hours to volatilize the solvent, and obtaining the recombinant human insulin glass when the solvent content is lower than 1%;

preheating the recombinant human insulin glass at 40 ℃, pouring the preheated recombinant human insulin glass into a PDMS microneedle female die, and filling the microneedle tips and the base part of the microneedle die; centrifugal defoaming is carried out at the speed of 2000rpm, the mould is cooled, and after the microneedle tip part is solidified; pouring 5% hydroxypropyl methyl cellulose solution into the substrate part of a PDMS microneedle mould, and centrifuging at 2000rpm for 5min for defoaming; further, under the condition of the temperature of 30 ℃ and the relative humidity of 1%, the drying time is 12 hours; and after solidification, demolding the microneedle glass to obtain the recombinant human insulin soluble glass microneedle.

The recombinant human insulin-soluble glass microneedles obtained are shown in fig. 6 (right).

Example 10

Weighing 100mg of arginine powder, and adding the arginine powder into 20mL of ultrapure water to obtain an arginine solution; after 10ml of 0.01M aqueous citric acid solution was added, the pH of the system solution was measured to be 5.0; heating the solution system to 120 ℃ at a heating rate of 10 ℃/min, performing constant temperature treatment for 1h to volatilize the solvent, and obtaining arginine glass when the solvent content is lower than 1%;

preheating the arginine glass at 120 ℃, pouring the arginine glass into a PDMS microneedle female die, and filling the microneedle tips and the base part of the microneedle die; and (3) performing centrifugal defoaming at the speed of 2000rpm, cooling the die, and demolding the microneedle glass after the microneedle body is solidified to obtain the arginine soluble glass microneedle.

The morphology of the obtained arginine-soluble glass microneedle was observed macroscopically by a 3D microscope, and the microscopic observation of the microneedle body was performed by a Scanning Electron Microscope (SEM), and the results are shown in FIG. 7 (left) and FIG. 8 (left)

Example 11

The arginine-soluble glass microneedle of the above-mentioned example 10 can also be prepared by combining the process of preparing biomolecular glass by a hydrothermal method with the casting process of preparing the microneedle, namely, by the following steps:

Weighing 100mg of arginine powder, and adding the arginine powder into 20mL of ultrapure water to obtain an arginine solution; after 10ml of 0.01M aqueous citric acid solution was added, the pH of the system solution was measured to be 5.0;

pouring the pre-arginine-citric acid mixed casting solution into a microneedle mould of PDMS, heating the mould to 120 ℃ at a heating rate of 10 ℃/min, and performing constant temperature treatment for 1h to volatilize the solvent; then, centrifugal defoaming was performed at 2000rpm, and the mold was cooled. And after the microneedle body is solidified, demolding the microneedle glass to obtain the arginine soluble glass microneedle.

The morphology of the obtained arginine-soluble glass microneedle was observed macroscopically by a 3D microscope, and the microstructure of the microneedle body was observed microscopically by a Scanning Electron Microscope (SEM), the results of which are shown in FIG. 7 (right) and FIG. 8 (right)

As can be seen from the scanning electron microscope and the optical microscope photographs of arginine-soluble glass microneedles, the preparation methods of example 10 and example 11 can obtain soluble glass microneedles with complete needle tips. Macroscopic and microscopic picture results are displayed: whether conical or tetragonal cone needle type, the micro needle type and the needle point have good sharpness and are consistent with the design of the PDMS mould.

Example 12 elastic modulus and hardness of biomolecular soluble glass microneedles

The elastic modulus and hardness of the biomolecule soluble glass microneedles were measured using a nanoindenter (Nano indicator G200, agilent company), and the results are shown in table 1, which shows that the biomolecule soluble glass microneedles prepared in examples 1-11 all have excellent elastic modulus and higher hardness, thus laying a foundation for further skin penetration application.

EXAMPLE 13 experiments for evaluation of penetration Property of biomolecular soluble glass microneedles

The puncture performance of the soluble micro-needles was measured by using a sealing film (Parafilm M Laboratory Film) puncture method, 8 layers of sealing films were fixed on a foam plate to simulate artificial skin, and the prepared soluble micro-needles were inserted into the artificial skin using a drug applicator, and the results were shown in fig. 9, showing that the sealing films were completely punctured and that the needle tips were not broken. The puncture-finished sealing films were placed under a microscope, the number of holes was observed, and the puncture depth was calculated (depth = Σdχa/a (a is the number of holes; a is the total needle content of the microneedles; D is the single-layer sealing film thickness, 125 μm.) the results are shown in table 1, wherein the needle tip lengths of examples 1, 2 and 11 were 600 μm, and the remaining glass microneedle needle tip lengths were 300 μm.

Table 1: strength test and penetration depth of biomolecule soluble glass microneedle

EXAMPLE 14 fresh pigskin puncture test method

Fresh 800 μm thick pig ear skin was selected, and the soluble microneedle prepared in example 1 was inserted into the pig ear skin using a drug applicator, and the results are shown in fig. 10. Clear micro needle pinholes are visible on the skin of the pig ear, and the number and arrangement of the pinholes are consistent with those of the micro needle array, which indicates that the micro needle has enough mechanical property to be pricked into the skin of the isolated pig skin.

EXAMPLE 15 evaluation of stability of biomolecules in glass microneedles

The microneedle array prepared in example 9 was placed in a storage cell maintained at 40 ℃ and 96% Relative Humidity (RH) along with a comparable amount of insulin solution. After 1, 3, 7 and 14 days of storage in the chamber, the insulin content of the microneedle array was determined. At the indicated time point, the array was removed from the chamber and washed with 0.1mol/L acetic acid (1 mL) to obtain an insulin solution. The insulin content of the resulting wash solution was analyzed using High Performance Liquid Chromatography (HPLC) method. The stability of insulin in the prepared microneedles was analyzed by comparison by measuring insulin solutions under the same storage conditions as a reference. The percentage of undegraded insulin remaining in the microneedle or solution at each time point was determined by measuring the peak area of insulin in the selected sample and dividing by the peak area of the initially measured insulin. The results are shown in Table 2.

The results show that in the 14-day stability acceleration experiment, the stability of insulin in the glass microneedle is obviously superior to that of a solution group, and the prepared soluble glass microneedle can effectively protect the activity of bioactive molecules and is beneficial to long-term storage.

Evaluation of drug efficacy of biomolecules in glass microneedles

EXAMPLE 16 evaluation of the glucose controlling effect of the recombinant human insulin-soluble glass microneedle prepared in example 9

Ordered spontaneous type I diabetes model mice (male, 8 weeks old) were divided into three groups of 6. The mice in the experimental group are administrated transdermally through a glass microneedle at a dose of 4IU/Kg, the mice in the blank group are not treated, the mice in the control group are administrated with recombinant insulin through tail vein, the dose of administration is 4IU/Kg, and the blood glucose concentration is measured at different time points. Mice were bled from their tail every hour and their fasting blood glucose values were measured with a blood glucose tester. The abscissa is time, and the ordinate is fasting blood glucose of the experimental mice. The results are shown in FIG. 11. The regulation and control curve of spontaneous type I diabetes mouse blood sugar proves that the recombinant human insulin soluble glass microneedle prepared in the embodiment 9 can effectively control the mouse blood sugar and has a smoother sugar control effect than insulin injection.

Example 17 in vivo pharmacodynamic evaluation of thymopentin soluble glass microneedles in rats

The experimental method comprises the following steps: SD rats 28, SPF grade, male, weighing 180-220 g, were not dosed with any other drug prior to the experiment. The thymopentin powder is dissolved in physiological saline to prepare a thymopentin solution which is used as a reference preparation. The test formulation was thymopentin soluble glass microneedle prepared in example 1. 28 SD rats were randomly divided into 4 groups, designated 1-4 groups each, 7 for in vivo pharmacodynamics studies.

An immunosuppression model is established before the experiment begins. The immunosuppressant cyclophosphamide powder was purchased from Jiangsu Hengrui medicine Co., ltd, and a lmg/L cyclophosphamide solution was prepared with physiological saline. Except group 1, the remaining 3 rats were intraperitoneally injected with immunosuppressant at a dose of 35 mg/(kg x d) for three consecutive days to suppress immune function in the rats. Group 1 served as a blank, and 35mL/kg of physiological saline was intraperitoneally injected.

After the continuous injection three days of model establishment is successful, pharmacodynamics evaluation is carried out. Group 1 rats served as a blank control group and group 2 rats served as a negative control group, and were continuously injected for seven days by subcutaneous intravenous injection of 1mL/kg of physiological saline. Group 3 rats served as positive control groups and were subcutaneously injected with thymopentin solution at a dose of 100ug/kg. Group 4 rats were continuously dosed for 7 days at a dose of 100ug/kg using the thymopentin-soluble glass microneedle prepared in application example 1. Subsequently, 3 groups of rats were sacrificed by cervical dislocation, thymus and spleen of each rat were collected, weighed, and organ coefficients were calculated. The calculation formula of Organ coefficients (Organ index) is as follows:

Wherein W is ₀ The weight of the rat is W, which is the weight of thymus or spleen. As shown in fig. 12, the negative control group 2 had lower spleen and thymus coefficients than the blank control group 1, which confirmed successful establishment of the immunosuppression model. The spleen and thymus coefficient values of the 3 rd and 4 th groups of the administration group are higher than those of the negative control group of the model group, the organ coefficients of immune suppression rats are improved, the difference has statistical significance, and the efficacy of the thymus pentapeptide soluble glass microneedle is verified to be superior to that of a solution formulation.

Example 18 neurological score of microneedles prepared in example 5 in autoimmune encephalomyelitis (EAE) mice specifically, EAE groups were mixed well with 200 μl PBS buffer in 200 μg MOG35-55, and then with 200 μl freund's complete adjuvant (CFA) to form an emulsion (final inactivated tuberculin bacillus concentration of 5 mg/mL), and normal control groups were mixed well with 200 μl PBS directly with 200 μl CFA. 200 μg of the emulsion was injected subcutaneously in two points on the back of mice. 500ng pertussis toxin was intraperitoneally injected at 0 hours and 48 hours after immunization, respectively. The mice were given subcutaneous doses of 100ug/kg subcutaneously via glass microneedles on day 1 after EAE model establishment, for 7 days in series, as listed in the glass microneedle intervention group.

Neurological score of each group of experimental mice the neurological score was performed daily for 30 days of continuous observation. Neurological scoring was performed on the scale of 0 as asymptomatic; 1 is classified as tail tension decrease (complete weakness, tail tip unable to curl) or hindlimb weakness (gait instability); 2, tail and hind limb weakness; 3, hind limb paralysis (single-side or double-side hind limb can move to a certain extent); 4, the hind limb is completely paralyzed (the hind limb cannot move at all or the forelimb drags the hind limb to move); death was classified as 5.

Results as shown in fig. 13, the results of neurological function scoring in autoimmune encephalomyelitis (EAE) mice indicate that the phagocyte-promoting peptide soluble glass microneedles prepared in example 5 can effectively repair neurological function in autoimmune encephalomyelitis (EAE) mice.

Example 19

As described in the tenth aspect, the microneedles provided by the present invention may also be loaded with active agents for rapid and convenient transdermal delivery of the agents. The specific method comprises the following steps: weighing 100mg of arginine powder and 0.5mg of lidocaine hydrochloride powder, and adding into 20mL of ultrapure water to obtain an arginine-lidocaine solution; after adding 10mL of 0.01M aqueous citric acid, the pH of the system solution was measured to be 5.0;

Pouring the mixed solution casting solution into a microneedle mould of PDMS, heating the mould to 40 ℃ at a heating rate of 10 ℃/min, and performing constant temperature treatment for 12 hours to volatilize the solvent; then, centrifugal defoaming was performed at 2000rpm, and the mold was cooled. And after the microneedle body is solidified, demolding the microneedle glass to obtain the arginine-lidocaine soluble glass microneedle.

Example 20

Weighing 100mg of arginine powder and 0.2mg of loratadine, and adding the arginine powder and the loratadine into 20mL of ultrapure water to obtain an arginine-loratadine solution; after 10ml of 0.01M aqueous citric acid solution was added, the pH of the system solution was measured to be 5.0;

pouring the mixed solution casting solution into a microneedle mould of PDMS, heating the mould to 40 ℃ at a heating rate of 10 ℃/min, and performing constant temperature treatment for 12 hours to volatilize the solvent; then, centrifugal defoaming was performed at 2000rpm, and the mold was cooled. And after the microneedle body is solidified, demolding the microneedle glass to obtain the arginine-loratadine soluble glass microneedle.

Example 21 release profile determination of drug loaded soluble microneedles.

The kinetics of the drug release in vitro of the prepared drug-loaded microneedles were measured, the drug-loaded microneedles prepared in examples 19 and 20 above were encapsulated in a dialysis bag (molecular weight cut-off of 3 kDa), and then immersed in a phosphate buffer solution (PBS, ph=7.4) at 37 ℃. Samples were taken at preset time points (0.2 mL each) and an equal amount of PBS solution was added to determine the cumulative drug release rate from the microneedles. The results are shown in fig. 14, and it can be seen that the cumulative release of drug exceeded 80% over 1 hour, indicating that the microneedles can release the loaded drug rapidly.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A microneedle of biomolecular glass, the microneedle having a microneedle array and a substrate;

the biomolecular glass consists of biomolecules, an inducer and a trace amount of solvent; the biological molecule is amino acid, derivative and/or peptide; the trace amount of solvent refers to the content of the solvent in the biomolecular glass of 0.1-5wt%; preferably 0.1 to 2wt%; the inducer is selected from the group consisting of nucleotides, nucleotide polymers, RNA, DNA and/or pH modifiers; the pH regulator is selected from one or more of 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, caproic acid, heptanoic acid, caprylic acid, valeric acid, butyric acid, propionic acid and glacial acetic acid.

2. The biomolecular glass microneedle according to claim 1, wherein the ratio Tg (glass transition temperature)/Tm (melting point temperature) of the biomolecular glass is distributed between 0.55 and 0.75, preferably between 0.66 and 0.75.

3. The biomolecular glass microneedle according to claim 1 or 2, wherein the solvent is water, physiological saline, or a mixed solvent of water and ethanol, or a mixed solvent of water and glycerol, and the ratio of the solvent other than water in the mixed solvent is between 0 and 50v/v%, preferably between 5 and 10v/v%.

4. A biomolecular glass microneedle according to claims 1-3, wherein said biomolecular glass microneedle has a hardness of more than 50MPa and an elastic modulus of more than 1Gpa.

5. The biomolecular glass microneedle according to claims 1-4, wherein the substrate of the biomolecular glass soluble microneedle may be made of biomolecular glass and/or polymer.

6. The method for preparing a biomolecular glass needle according to claims 1-5, wherein the preparation comprises the steps of:

7. The method for preparing a biomolecular glass microneedle according to claim 6, wherein in the step of preparing a biomolecular glass by a hydrothermal method, the pH of the system is adjusted to 1-9, preferably, 3-7; the constant temperature is 20-120 ℃; preferably 40-100 ℃; the constant temperature time is 5 min-6 h, preferably 30 min-3 h. The preheating temperature is 20 to 200 ℃, preferably 40 to 160 ℃, more preferably 40 to 100 ℃.

8. The method for preparing the biomolecular glass soluble microneedle according to any one of claims 6 to 7, wherein the casting method comprises self-leveling, high-pressure spraying and micro-fluidic; the defoaming method comprises centrifugation, decompression or vacuum adsorption.

9. Use of the biomolecular glass microneedle according to any one of claims 1-5 as a drug carrier or in the preparation of a cosmetic product.