Microneedle array and method for producing same

Abstract

Provided is a microneedle array in which a plurality of microneedles formed by a crosslinked body of photoreactive hyaluronic acid having a photocrosslinking group are arranged on the surface of a substrate.

Description

Microneedle array and method for manufacturing the same

[0001] The present invention relates to a microneedle array comprising a crosslinked product of photoreactive hyaluronic acid having a photocrosslinking group, and a method for producing the same. This application claims priority based on Japanese Patent Application No. 2024-130199, filed on August 6, 2024, the contents of which are incorporated herein by reference.

[0002] Generally, hyaluronic acid is a water-soluble polymer with high biocompatibility, and is used for the purpose of being injected into the body in medical or cosmetic surgery.The hyaluronic acid injected into the body is absorbed and decomposed in a relatively short time, so that in order to maintain the effect of injection, there is a need for poorly soluble hyaluronic acid.Patent document 1 discloses the technology of introducing photoreactive crosslinking group into hyaluronic acid, and making poorly soluble hyaluronic acid by causing crosslinking reaction by irradiation with light such as ultraviolet light.

[0003] Patent No. 4172176

[0004] The microneedles are, for example, 2 mm or less in height and cone-shaped needles with sharp tips. A large number of microneedles are aligned on the adhesive surface of a patch seal, and the patch seal is then applied to the skin, allowing the microneedles to puncture the skin. When a patch seal equipped with microneedles pre-applied with a drug is applied to the skin, the microneedles can be fixed in a punctured state in the skin, and a sustained release effect can be expected, in which the drug is gradually released into the skin.

[0005] The present inventors have focused on the biocompatibility of hyaluronic acid and have intensively studied the use of hyaluronic acid as a material for microneedles.Microneedles are required to have a sharp tip that can be punctured into the skin and have strength that will not break.Therefore, the inventors have studied the introduction of photocrosslinking groups, which have conventionally been introduced to adjust the viscosity and solubility of hyaluronic acid, in order to realize the properties required for microneedles, and have completed the present invention.

[0006] The present invention provides a microneedle formed from a crosslinked product of photoreactive hyaluronic acid having a photocrosslinking group and a method for producing the same, as well as a microneedle array and a method for producing the same.

[0007] [1] A microneedle formed from a crosslinked product of photoreactive hyaluronic acid having a photocrosslinking group. [2] The microneedle according to [1], wherein the needle portion of the microneedle is immersed from tip to base in PBS (room temperature) for 10 seconds, and when removed, 80% or more of the height from tip to base before immersion remains. [3] The microneedle according to [1] or [2], wherein the needle portion of the microneedle is punctured into the surface of pig skin under the following conditions, and when the microneedle is left to stand at 36°C for 17 hours and removed, the height from tip to base before puncturing is 90% or less of the height. <Conditions> After fat peeling and surface hair removal, pig skin is stretched from all sides, and the needle portion of the microneedle is punctured into the skin surface while maintaining that state. The pig skin and the microneedle are sandwiched between two plates, and both ends of the plates are fastened with clips. One plate has a through-hole drilled in advance and is placed in contact with the fat side of the pig skin. The pig skin is placed flat in a container with the fat side facing downwards, and a small amount of PBS is poured into it to prevent the pig skin from drying. With only the fat side of the pig skin in contact with the PBS, the plate is left standing at 36°C for 17 hours. The microneedles are then removed from the pig skin, dried, and the height of the needle portion is measured. The average value of the measurements for five or more microneedles is used as the result. [4] The microneedle according to any one of [1] to [3], wherein the photoreactive hyaluronic acid has an average molecular weight of 50,000 to less than 1,000,000. [5] The microneedle according to any one of [1] to [4], wherein the modification rate of the photoreactive hyaluronic acid with photocrosslinking groups is 2% to 15%. [6] The microneedle according to any one of [1] to [5], wherein the height of the microneedle is 150 μm to 2,000 μm. [7] A method for producing a microneedle, comprising the steps of injecting a solution of photoreactive hyaluronic acid having a photocrosslinking group into a mold having a recess corresponding to the shape of the microneedle, drying the solution in the recess, demolding the solidified microneedle, and irradiating the photoreactive hyaluronic acid in the solidified microneedle with light to crosslink it, thereby obtaining a microneedle formed from a crosslinked product of the photoreactive hyaluronic acid.[8] The method for producing a microneedle according to [7], wherein the photoreactive hyaluronic acid has an average molecular weight of 50,000 or more and less than 1,000,000. [9] The method for producing a microneedle according to [7] or [8], wherein the modification rate of photocrosslinking groups possessed by the photoreactive hyaluronic acid is 2% or more and 15% or less.

[10] The method for producing a microneedle according to any of [7] to [9], wherein the depth of the recess corresponding to the shape of the microneedle is 150 μm or more and 2,000 μm or less.

[0008] A preferred embodiment of the microneedle described in [1] above is in the form of a microneedle array in which a plurality of the microneedles are arranged on the surface of a substrate. The number of the microneedles arranged in the microneedle array is preferably 5 or more. There is no particular upper limit to the number, and examples include 100 or less. The present invention includes the following aspects

[11] to

[20] .

[0009]

[11] A microneedle array in which a plurality of microneedles formed from a crosslinked product of photoreactive hyaluronic acid having a photocrosslinking group are arranged on the surface of a substrate.

[12] The microneedle array according to

[11] , wherein the plurality of microneedles are immersed from tip to base in PBS at 15°C to 30°C, and the substrate is left to stand for 10 seconds without being entirely immersed in the PBS, and then removed and dried to remove moisture on the surface. When the heights of five or more microneedles randomly selected from the remaining microneedles, excluding those broken among the plurality of microneedles, are measured, 80% or more of the height from tip to base before immersion remains.

[13] The microneedle array according to

[11] or

[12] , wherein the plurality of microneedles are punctured into the surface of pig skin under the following conditions, left to stand at 36°C for 17 hours, removed, and dried to remove moisture on the surface, and dissolved to a height of 90% or less of the height from tip to base before puncturing. <Conditions> After fat peeling and removal of the hair from the skin surface, the pig skin is pulled from all sides. While in this state, the microneedles are inserted into the skin surface to their bases. The pig skin and the microneedle array are sandwiched between two plates, and both ends of the plates are fastened with clips. One plate is pre-drilled with through-holes, which are in contact with the fat side of the pig skin. The pig skin is placed flat in a container with the fat side facing downwards. A small amount of PBS is poured into the plate to prevent the pig skin from drying out. The plate is then left to stand at 36°C for 17 hours, with only the fat side of the pig skin immersed in PBS. Ensure that the PBS does not run out during this standing time. The microneedle array is then removed from the pig skin and dried to remove any moisture on the surface. The height of five or more microneedles randomly selected from the remaining microneedles, excluding any broken ones, is measured. The average value of the measurements of the five or more microneedles is used as the result.

[14] The microneedle array according to any one of

[11] to

[13] , wherein the photoreactive hyaluronic acid has an average molecular weight of 50,000 or more and less than 1,000,000.

[15] The microneedle array according to any one of

[11] to

[14] , wherein the modification rate of the photoreactive hyaluronic acid with photocrosslinking groups is 2% or more and 15% or less.

[16] The microneedle array according to any one of

[11] to

[15] , wherein the height of each of the plurality of microneedles is 150 μm or more and 2000 μm or less.

[17] A method for producing a microneedle array, comprising the steps of: injecting a solution of photoreactive hyaluronic acid having photocrosslinking groups into a mold having a plurality of recesses formed therein corresponding to the shapes of the plurality of microneedles; drying the solution in the recesses; demolding the solidified plurality of microneedles; and irradiating the photoreactive hyaluronic acid in the solidified plurality of microneedles with light to crosslink them, thereby obtaining a microneedle array in which a plurality of microneedles formed of crosslinked products of the photoreactive hyaluronic acid are arranged on the surface of a substrate.

[18] The method for producing a microneedle array according to

[17] , wherein the photoreactive hyaluronic acid has an average molecular weight of 50,000 or more and less than 1,000,000.

[19] The method for producing a microneedle array according to

[17] or

[18] , wherein the modification rate of the photoreactive hyaluronic acid with photocrosslinking groups is 2% or more and 15% or less.

[20] The method for producing a microneedle array according to any one of

[17] to

[19] , wherein the depth of each of the plurality of recesses is 150 μm or more and 2000 μm or less.

[0010] According to the present invention, it is possible to provide a microneedle formed of a crosslinked product of photoreactive hyaluronic acid having a photocrosslinking group, which has the properties required of a microneedle, and a method for producing the same. Similarly, it is possible to provide a microneedle array and a method for producing the same.

[0011] <Microneedle> A first aspect of the present invention is a microneedle formed from a crosslinked product of photoreactive hyaluronic acid having photocrosslinking groups (hereinafter, may be abbreviated as "photocrosslinked HA").

[0012] The hyaluronic acid that constitutes photocrosslinked HA has a chemical structure in which disaccharide units of N-acetylglucosamine and D-glucuronic acid are linked in a linear chain. The origin of the hyaluronic acid before the introduction of photocrosslinking groups is not particularly limited, and hyaluronic acid derived from a natural organism that is easily available can be used. The carboxyl group of the hyaluronic acid may form an alkali metal salt or alkaline earth metal salt.

[0013] The average molecular weight of the photocrosslinked HA of this embodiment is not particularly limited, but from the viewpoint of providing sufficient strength and poor solubility to the microneedle, it is preferably 50,000 or more, more preferably 100,000 or more, even more preferably 150,000 or more, and particularly preferably 200,000 or more.In addition, from the viewpoint of easily sharpening the tip of the microneedle, it is preferably less than 1 million, more preferably 800,000 or less, even more preferably 600,000 or less, particularly preferably 400,000 or less, particularly preferably 300,000 or less, and most preferably 250,000 or less.In other words, it is most preferably 200,000 or more and 250,000 or less.The average molecular weight of the photocrosslinked HA of this embodiment can be measured by the following method.Using a gel filtration column, a plurality of hyaluronic acids (reference substances) with known average molecular weights are analyzed by liquid chromatography, and a calibration curve is created based on their retention times.Similarly, the modified hyaluronic acid to be measured is analyzed by liquid chromatography, and the average molecular weight is determined using the calibration curve, so that the average molecular weight of the modified hyaluronic acid can be determined. Examples of liquid chromatography analyzers that can be used for the liquid chromatography analysis include the Waters Alliance 2690 HPLC Separations Module (manufactured by Waters) and the Waters Alliance 2695 HPLC Separations Module (manufactured by Waters). Examples of columns that can be used for the liquid chromatography analysis include Shodex ligand exchange chromatography columns (ligand exchange mode + size exclusion mode) with model names "SUGAR KS-801," "SUGAR KS-802," "SUGAR KS-804," "SUGAR KS-805," "SUGAR KS-806," and "SUGAR KS-807" and TOSOH size chromatography columns with model name "TSKgel GMPW."

[0014] <Method for Measuring Average Molecular Weight> The "average molecular weight" of the hyaluronic acid reference material refers to the viscosity-average molecular weight. The average molecular weight is calculated according to the method described in the Japanese Pharmacopoeia (18th Edition) for "Purified Sodium Hyaluronate" average molecular weight. Specifically, an amount of hyaluronic acid powder dissolved in 100 mL of 0.2 mol / L sodium chloride test solution was precisely measured so that the flow time of the solution was 2.0 to 2.4 times that of the 0.2 mol / L sodium chloride test solution, and this was dissolved in 0.2 mol / L sodium chloride test solution to make exactly 100 mL, which was designated as sample solution (1). 16 mL, 12 mL, and 8 mL of sample solution (1) were accurately measured, and 0.2 mol / L sodium chloride test solution was added to each to make exactly 20 mL, which were designated as sample solutions (2), (3), and (4). For the above sample solutions (1), (2), (3), and (4), a Ubbelohde viscometer with a flow time of 200-300 seconds for a 0.2 mol / L sodium chloride test solution was used to measure the time required for a certain volume of liquid to flow down through a capillary tube at 30°C ± 0.1°C using the general viscosity measurement method (capillary viscometer method), and the specific viscosity (Equation (1)) and reduced viscosity at each concentration were calculated (Equation (2)). A graph was drawn with the reduced viscosity on the vertical axis and the concentration (g / 100mL) of hyaluronic acid converted to dry matter on the horizontal axis, and the intrinsic viscosity was determined from the intersection of the line connecting each point and the vertical axis. The average molecular weight was calculated from the intrinsic viscosity determined here using Equation (3). Specific viscosity = {(number of seconds required for sample solution to flow down) / (number of seconds required for 0.2 mol / L sodium chloride solution to flow down)} - 1 (1) Reduced viscosity (dL / g) = specific viscosity / (concentration of this product relative to the dry matter in g / 100 mL) (2)

[0015]

[0016] The photocrosslinking group of the photocrosslinked HA of this embodiment is a photoreactive crosslinking group.In hyaluronic acid, the functional group to which the crosslinking group is introduced can be a carboxyl group or a hydroxyl group, and the carboxyl group is preferred because it is easy to introduce.

[0017] The photocrosslinking group (photoreactive residue) is preferably a residue of a compound that undergoes a photodimerization reaction or photopolymerization reaction upon ultraviolet irradiation. Specific examples include cinnamic acid, substituted cinnamic acid (e.g., aminocinnamic acid (a cinnamic acid in which one of the hydrogen atoms on the benzene ring is substituted with an amino group: preferably p-aminocinnamic acid), acrylic acid, maleic acid, fumaric acid, sorbic acid, coumarin, and thymine. A monovalent group obtained by removing one of the hydrogen atoms contained in these compounds can be used as a photocrosslinking group. Among these, compounds having a vinylene group capable of forming a cyclobutane ring upon exposure to light are preferred, with cinnamic acid or substituted cinnamic acid (particularly aminocinnamic acid) being more preferred. A spacer (any divalent linking group) may be present between the functional group of hyaluronic acid and the photocrosslinking group.

[0018] Specific preferred photocrosslinking groups include, for example, an amino alcohol (H ２ N-(CH ２ ) ｎ -OH; n=1-18, H ２ N-(CH ２ -O) ｍ -CH ２ cinnamic acid amino alkyl ester derivatives in which —OH; m=1 to 9) is ester-linked, (Ph-CH═CH—CO—O—(CH ２ ) ｎ -NH ２ , Ph-CH=CH-CO-O-CH ２ -(OCH ２ )m-NH ２ n and m are the same as above, and -Ph represents a phenyl group), diamine (H ２ N-(CH ２ )l-NH ２ ; l = 1 to 10), diol (HO-(CH ２ ) ｋ -OH; k=1 to 10)-introduced derivatives, (Ph-CH=CH-CO-NH-(CH ２ ) ｌ -NH ２ , Ph-CH=CH-CO-O-(CH ２ ) ｋ -OH: l, k, -Ph are the same as above), amino acid (HOOC-(CHR) ｊ -NH２ ; j = 1 to 10, R represents the side chain of an amino acid), derivatives in which peptides or the like are introduced into substituted cinnamic acids (aminocinnamic acids) (OC-CH=CH-Ph-NH-CO-(CHR) ｊ , OC-CH=CH-Ph-NH- (peptide): R and j are the same as above, -Ph- represents a phenylene group). Of these, derivatives in which an amino alcohol is introduced into the carboxyl group of cinnamic acid (amino alkyl cinnamate esters) are preferred. The amino alcohol is preferably one represented by the above general formula, and in the above general formula, n is preferably 1 to 18, more preferably 3 to 6, and even more preferably 3 to 4. When an amino alkyl cinnamate ester is used as a photocrosslinking group, the amino group of the amino alkyl and the carboxyl group of hyaluronic acid form an amide bond, thereby binding the photocrosslinking group to hyaluronic acid.

[0019] The introduction rate of photocrosslinking group into hyaluronic acid is defined as the number of photocrosslinking groups introduced per repeating disaccharide unit of hyaluronic acid, expressed as a percentage.For example, when one photocrosslinking group is introduced into 100 repeating disaccharide units of hyaluronic acid, the introduction rate of this photocrosslinking group is 1.0%.The introduction rate (modification rate) in the photocrosslinked HA of this embodiment can be, for example, 1.0% or more and 20% or less, preferably 2.0% or more and 15% or less, more preferably 2.5% or more and 15% or less, and even more preferably 3.0% or more and 10% or less.The introduction rate can be appropriately adjusted by adjusting the mole number of hyaluronic acid and the mole number of the compound having photocrosslinking group to be reacted.The measurement method of the introduction rate is as described in the examples below.

[0020] The shape of the microneedle of this embodiment may be any shape that protrudes toward the tip, and preferably has a pointed tip.Specifically, in addition to polygonal pyramids such as triangular pyramids, quadrangular pyramids, and pentagonal pyramids, cones are also preferred shapes.These pyramids may have truncated polygonal pyramids or truncated cones with the tips cut off.From the viewpoint of facilitating puncture into the skin, it is preferable that the tip has a sharp, pointed shape.

[0021] The microneedles of this embodiment are preferably supported at their bases by a substrate. In one embodiment, for example, a microneedle array can be formed in which a plurality of microneedles are arranged regularly or randomly on the surface of a substrate. The substrate is not particularly limited as long as it is capable of supporting microneedles on its surface, and may be made of, for example, resins such as PMMA, cellulose acetate, ethyl cellulose, polyethylene, polypropylene, ethylene-propylene copolymer, polyester, polyurethane, and various rubbers, as well as inorganic materials such as glass and metal. Alternatively, a crosslinked body of photocrosslinked HA constituting the microneedles may form the substrate. The substrate may be in a thin form, generally referred to as a film or sheet, or may be a member of any thickness, which may be referred to as a base material.

[0022] The diameter of the base of the microneedle of this embodiment may be, for example, 100 μm to 800 μm, 200 μm to 600 μm, or 300 μm to 500 μm. The diameter of the base is defined as the diameter of the smallest circle surrounding the base. The diameter of the tip of the microneedle of this embodiment may be, for example, 5 μm to 80 μm, 10 μm to 60 μm, 15 μm to 40 μm, or 20 μm to 30 μm. The tip diameter is defined as the diameter of the smallest circle surrounding the periphery of the microneedle at a position 5 μm down in the height direction from the apex of the tip. The height of the microneedle of this embodiment may be, for example, from 150 μm to 2000 μm, from 300 μm to 1600 μm, from 500 μm to 1400 μm, from 700 μm to 1200 μm, or from 800 μm to 1000 μm. The height is defined as the length of the line segment connecting the center of the base (the region in contact with the substrate) and the apex of the tip. The aspect ratio obtained by dividing the height of the microneedle of this embodiment by the diameter of the base may be, for example, from 1 to 5, from 1 to 3, or from 1.5 to 2. When a plurality of microneedles of this embodiment are regularly arranged on the substrate, the distance (pitch) between the tips of adjacent microneedles may be, for example, from 300 μm to 3000 μm, from 400 μm to 2000 μm, or from 500 μm to 1000 μm. The height and diameter of the microneedles, and the distance between adjacent microneedles can be measured based on images acquired using a known magnification observation means such as a digital microscope.

[0023] The microneedle of this embodiment preferably contains 60% by mass or more of the crosslinked body of photocrosslinked HA relative to its total mass, more preferably 80% by mass or more, even more preferably 90% by mass or more, and may even be 100% by mass.The materials other than the crosslinked body that may be contained in the microneedle include, for example, sugars such as maltose and pullulan; proteins such as collagen and gelatin; synthetic polymers such as polyvinyl alcohol; various drugs intended to penetrate into punctured skin; pH buffer and water.It should be noted that, in the form in which the above-mentioned drug is applied to the outer surface of the microneedle, the mass of the drug is not included in the mass of the microneedle.

[0024] In this embodiment, the microneedle is immersed in PBS (phosphate buffered saline) at room temperature (15-30°C) from the tip to the base of the needle portion of the microneedle (excluding the substrate) for 10 seconds, and when removed, it is preferable that 80% or more of the height remains, with the height from the tip to the base (total length) before immersion being 100% of the reference height. By remaining 80% or more, the microneedle does not dissolve in a short time, and can maintain its punctured state in the skin for a while, allowing, for example, sufficient injection of a drug. Note that if the microneedle is removed from the PBS and left with PBS droplets attached, dissolution may progress. Therefore, it is desirable to remove the PBS droplets by blowing dry air onto the microneedle immediately after removal.

[0025] When testing the microneedle array using the above method, the plurality of microneedles are immersed from tip to base in PBS at 15°C to 30°C, and the substrate is left to stand for 10 seconds without being entirely immersed in the PBS. After removal, the substrate is dried to remove moisture from the surface. When the heights of five or more microneedles randomly selected from the remaining microneedles, excluding those that are broken, are measured, it is preferable that 80% or more of the height remains, based on the height from tip to base before immersion. In this test, microneedles that are severely deformed or broken due to the application of physical stress during operation are considered to be broken microneedles, and are not used to measure the height.

[0026] The microneedles of this embodiment are preferably dissolved to a predetermined height, with the height from the tip to the base before puncturing being 100% of the reference height, under the following conditions: the needle of the microneedle is punctured into the surface of pig skin, left at 36°C for 17 hours, and then removed. Here, the predetermined height is preferably 90% or less, more preferably 80% or less, and even more preferably 70% or less. Depending on the application, the lower limit may be, for example, 10% or more, 20% or more, or 30% or more. Microneedles exhibiting the above solubility can reduce the burden on the skin caused by the microneedle puncturing the skin, and if a drug is contained in the microneedle, the drug inside the microneedle can be dissolved and gradually released. It is preferable to use the average value of the measurements of multiple microneedles (e.g., 5 or more) as the result.

[0027] <Conditions> After fat peeling and removal of skin surface hair, the pig skin is pulled from all sides, and while in this state, the needle of the microneedle is punctured into the skin surface. The pig skin and microneedle are sandwiched between two plates (e.g., polycarbonate plates), and both ends of the plates are fastened with clips. A through-hole is pre-drilled in one plate, which is in contact with the fat side of the pig skin. The pig skin is placed flat in a container with the fat side facing downwards, and a small amount of PBS (which may contain sodium azide to prevent spoilage) is poured in to prevent the pig skin from drying out. The pig skin is then left to stand at 36°C for 17 hours with only the fat side of the pig skin in contact with the PBS (with the PBS in contact with the fat side of the pig skin through the through-hole). Here, to ensure that the pig skin does not dry out, it is preferable to immerse the fat side of the pig skin in PBS. The microneedle is then removed from the pig skin and allowed to dry naturally, after which the height of the needle is measured. The average value of the measurements of five or more microneedles is used as the result. The pig skin used in this test is a test skin tissue piece excised from the trunk of an adult pig, with the subcutaneous tissue removed. The size of the test piece is preferably, for example, 3 cm to 6 cm in length, 3 cm to 6 cm in width, and 5 mm to 15 mm in thickness.

[0028] When testing the microneedle array using the above method, the microneedles are preferably punctured into the surface of pig skin under the following conditions, left at 36°C for 17 hours, removed, and then dried to remove moisture from the surface. The microneedles are preferably dissolved to a predetermined height, based on the height from the tip to the base before puncturing. The preferred range of the predetermined height is the same as when testing individual microneedles described above. <Conditions> After fat peeling and hair removal from the skin surface, the pig skin is pulled from all sides. While in this state, the microneedles are punctured into the skin surface to the base. The pig skin and the microneedle array are sandwiched between two plates, and both ends of the plates are fastened with clips. A through-hole is pre-drilled in one plate, which is in contact with the fat side of the pig skin. The pig skin is placed flat in a container with the fat side facing downwards. A small amount of PBS is poured into the pig skin to prevent it from drying out. With only the fat side of the pig skin immersed in the PBS, the skin is left to stand at 36°C for 17 hours. Add PBS as needed to prevent the PBS from running out during this resting time. The microneedle array is then removed from the pig skin and dried to remove moisture from the surface. The heights of five or more microneedles randomly selected from the remaining microneedles, excluding any broken ones, are then measured. The average value of the measurements of the five or more microneedles measured is used as the result. In this test, microneedles that are severely deformed or broken due to physical stress during operation are considered broken microneedles and are not included in the height measurement.

[0029] After removing the microneedle array that has been punctured into the pig skin, the array is dried before measurement by leaving it to stand for 1 hour in a laboratory at a temperature of 20 to 25°C and a relative humidity of 40 to 60% to dry naturally.

[0030] <<Method for manufacturing microneedles>> A second aspect of the present invention is a method for manufacturing microneedles, comprising the steps of injecting a solution of photoreactive hyaluronic acid having a photocrosslinking group into a mold having a recess corresponding to the shape of the microneedle, drying the solution in the recess, demolding the solidified microneedle (uncrosslinked body), and irradiating the photoreactive hyaluronic acid in the solidified microneedle with light to crosslink it, thereby obtaining a microneedle formed by a crosslinked body of the photoreactive hyaluronic acid.By using the manufacturing method of this aspect, the microneedle of the first aspect described above can be manufactured.In the manufacturing method of this aspect, by using a mold having a plurality of the recesses arranged, the microneedle array of one aspect of the present invention can be manufactured.

[0031] The mold can be produced by a known microfabrication method. The base material of the mold may be a polymer such as plastic, or an inorganic material such as glass or metal. The recesses provided in the mold correspond to a shape that includes at least the needle portion of the microneedle to be formed, and for example, recesses in which the apex of a cone or polygonal pyramid is inverted to form a bottom can be used. By regularly arranging the recesses, a regularly arranged microneedle array can be produced. The recesses may have a shape that includes not only the needle portion of the microneedle but also the aforementioned substrate.

[0032] Specific examples and preferred embodiments of the shape of the recesses of the mold are the same as those of the microneedles in the first embodiment, and therefore, redundant explanations will be omitted. The height of the microneedles can be the same as the depth of the recesses of the mold.

[0033] The hyaluronic acid having photocrosslinking groups (photocrosslinked HA) to be poured into the recesses of the mold can be obtained by a known method. Examples of the method include a method using a water-soluble condensing agent such as a water-soluble carbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI.HCl), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate, 1-cyclohexyl-3-(2-morpholinoethylcarbodiimide hydrochloride, etc.); a method using the above-mentioned condensing agent together with a condensation auxiliary such as N-hydroxysuccinimide (HOSu) or N-hydroxybenzotriazole (HOBt); the active ester method; and the acid anhydride method. Among these, for the reaction in the presence of an aqueous medium, a method using a water-soluble condensing agent or a method using a reaction auxiliary and a water-soluble condensing agent is preferred. Examples of the aqueous medium include water alone, as well as mixed solvents of water and a water-soluble organic solvent such as dioxane, dimethylformamide (DMF), acetone, and alcohols (methanol, ethanol, etc.).

[0034] After the introduction of photocrosslinking groups into hyaluronic acid, the hyaluronic acid may be treated with alkali to enhance its dispersibility in an aqueous dispersion medium. The alkali treatment can be carried out according to the method described in Patent Document 1.

[0035] Specific examples and preferred embodiments (average molecular weight, modification rate, etc.) of the photocrosslinked HA of this embodiment are the same as those of the photocrosslinked HA of the first embodiment, and therefore, redundant explanations will be omitted.

[0036] The photocrosslinked HA of this embodiment can be poured into a mold, optionally dried, and then irradiated with white light to cause a photocrosslinking reaction and cure. Ultraviolet light may be used instead of white light. Examples of ultraviolet light sources include a high-pressure mercury lamp and a metal halide lamp.

[0037] The solution of photoreactive hyaluronic acid to be injected into the recess of the mold may be, for example, an aqueous solution in which photoreactive hyaluronic acid is dissolved in an aqueous medium containing water or a pH buffer at a content of 1 to 5 wt %.

[0038] The method of drying the solution of photoreactive hyaluronic acid in the cavity of the mold is not particularly limited, for example, can be air-dried or heated.The degree of drying the solution is not particularly limited, and it is preferable to solidify it to the degree that maintains the shape of the target microneedle when it is taken out from the cavity (removed from the mold).

[0039] The method for demolding the plurality of microneedles solidified in the plurality of recesses of the mold is not particularly limited.As an example, a separately prepared substrate is attached to the base portion of each microneedle exposed at the opening of each recess, and this substrate is separated from the mold, thereby demolding the microneedles and obtaining a microneedle array in which a desired number of microneedles are arranged on the surface of the substrate.Another example is a method in which not only a plurality of recesses corresponding to the shape of a plurality of microneedles are provided in the mold, but also recesses corresponding to the shape of the substrate located at the base portion of each microneedle, and the substrate integrated with the microneedles is formed in the mold using photoreactive hyaluronic acid.According to this latter method, the substrate can be demolded at the same time as the plurality of microneedles integrated with the substrate are demolded.

[0040] Example 1: Hyaluronic acid (photocrosslinked HA) introduced with 3-aminopropyl cinnamate ester having an average molecular weight of 75,000 was obtained by the method described in Patent Document 1. The introduction rate (modification rate) of the cinnamic acid derivative per hyaluronic acid repeating disaccharide unit was 6.9%.

[0041] Here, the introduction rate means the number of photocrosslinking groups introduced per repeating disaccharide unit of hyaluronic acid, expressed as a percentage. The modification rate was measured by dissolving a 0.5% aqueous solution of the hyaluronic acid derivative according to this example in the following manner. 1 The values ​​were obtained by H-NMR spectrum analysis. 1 Measurement of modification rate by H-NMR> Sample concentration: 0.5% (D2O) Equipment: Varian NM system 400NB (Varian Technologies Japan Limited) Observation frequency: 400MHz Temperature: 30℃ Reference: DSS (0ppm) Pulse width: 45° Number of accumulations: 64 times 1In H-NMR spectrum, the peak appearing at about 2.0 ppm is considered to represent the three protons in the hyaluronic acid skeleton, and the peak appearing at about 7.5 ppm is considered to represent the one proton contained in the structure of aminocinnamic acid derivative.From these two peaks, the proportion of aminocinnamic acid derivatives bonded to the disaccharide unit of hyaluronic acid skeleton (modification rate (%)) is calculated based on the following formula: Modification rate (%) = (integrated value of the specific peak derived from aminocinnamic acid / the number of protons of the specific peak derived from aminocinnamic acid) / (integrated value of the specific peak derived from hyaluronic acid / the number of protons of the specific peak derived from hyaluronic acid) x 100

[0042] Next, the photocrosslinked HA was dissolved in 5 mM phosphate-buffered saline (PBS) to achieve hyaluronic acid equivalent concentrations of 1 wt% and 5 wt%. First, a 1 wt% photocrosslinked HA solution was poured into the recesses at the needle tips, followed by a 5 wt% photocrosslinked HA solution poured into the recesses in the sheet. The solution was left to dry at room temperature for 12 hours, demolded, and then irradiated under white fluorescent light for approximately one day to harden. This resulted in a microneedle array in which photocrosslinked HA microneedles with a square pyramidal shape (tip diameter approximately 20 μm, height approximately 1000 μm) reflecting the shape of the recesses in the mold were arranged in a two-dimensional array (16 x 16) on the sheet surface. The content of photocrosslinked HA relative to the total mass of each microneedle was 80-90% by mass. The shape of each microneedle arranged in the microneedle array obtained above was measured using a digital microscope, and the results were as follows.・Base diameter: 400 to 550 μm ・Tip diameter: 20 to 30 μm ・Height: 800 to 1000 μm ・Aspect ratio: 1.5 to 2.0 ・Distance between the tips of adjacent microneedles: 700 μm

[0043] <Short-time solubility test> The prepared microneedle array was immersed in PBS (room temperature) for 10 seconds. At this time, the entire sheet portion, which is the substrate, was not immersed in PBS. Then, the microneedle array was lifted from the PBS, and air was blown to remove the moisture on the surface of each microneedle, and then the change rate (remaining rate) of the tip diameter and height of each microneedle was measured, with the value before immersion being 100% as the reference. The results are shown in Table 1.

[0044] <Long-term solubility test> The prepared microneedle array was punctured into pig skin (36 ° C) for 17 hours, then removed, and the changes in the tip diameter and height of each microneedle were measured, with the values ​​before puncturing being 100%. The average values ​​of the results are shown in Table 1. The specific test method is as follows. After fat peeling and hair removal from the skin surface, pig skin was pulled from all sides, and in this state, the needles of the microneedle array were sufficiently pressed into the surface of the pig skin to puncture it. The pig skin and microneedle array were sandwiched between two polycarbonate plates and fastened with clips on both sides. One plate had many through-holes and was in contact with the fat side of the pig skin (the opposite side of the punctured skin surface). The pig skin was placed flat in a container with the fat side facing downwards, and a small amount of PBS was poured in to prevent the pig skin from drying out. With only the fat side of the pig skin immersed in PBS, it was left to stand at 36 ° C for 17 hours. The PBS was not depleted during this standing time. Thereafter, the microneedle array was removed from the pig skin, and left to stand for 1 hour in a laboratory at a temperature of 25 ° C and a relative humidity of 50%, and then allowed to dry naturally. Then, five or more microneedles were randomly selected from the remaining microneedle group, excluding those that were physically damaged when removed from the pig skin, and the change in needle height (residual rate) was measured based on the value before puncture. The average value of the results is shown in Table 1. In Table 1, a residual rate of 0% means that the microneedle was completely dissolved and the height became 0. A residual rate of 100% means that there was no change at all. A residual rate of more than 100% means that the microneedle had swelled and become longer than before.

[0045] [Example 2] A microneedle array was prepared in the same manner as in Example 1, except that the average molecular weight of the photocrosslinked HA was changed to 220,000 and the modification rate was changed to 2.3%, and the same test was carried out. The results are shown in Table 1.

[0046] [Examples 3 to 6] Microneedle arrays were prepared and tested in the same manner as in Example 2, except that the average molecular weight and modification rate of the photocrosslinked HA were changed as shown in Table 1. The results are shown in Table 1.

[0047] [Comparative Example 1] A microneedle array was prepared in the same manner as in Example 2, except that the average molecular weight and modification rate of the photocrosslinked HA were changed as shown in Table 1, and the same test was carried out. The results are shown in Table 1.

[0048]

[0049] From the above results, it can be seen that the lower the modification rate of photocrosslinked HA, the higher the solubility of the microneedles, and the smaller the average molecular weight of photocrosslinked HA, the higher the solubility of the microneedles.In Examples 3 and 4, the modification rate of photocrosslinked HA is 2.5% or more and 10.0% or less, and the average molecular weight is 150,000 or more and 300,000 or less, so the microneedles showed moderate solubility in the long-term solubility test.Such moderate solubility is preferable because it can release the drug held in the microneedle at a moderate speed.In addition, the results of the short-term solubility test in Examples 3 and 4 are also excellent, which shows that the microneedles do not dissolve when puncturing the skin, and can be punctured into the skin to the base of the microneedle.

Claims

1. A microneedle array in which multiple microneedles formed by crosslinking photoreactive hyaluronic acid having photocrosslinking groups are arranged on the surface of a substrate.

2. The microneedle array according to claim 1, wherein the plurality of microneedles are immersed from tip to base in PBS at 15°C or higher and 30°C or lower, and the substrate is left to stand for 10 seconds without the entire substrate being immersed in the PBS, and then removed and dried to remove moisture from the surface. When the heights of five or more microneedles randomly selected from the plurality of microneedles remaining after excluding any broken ones are measured, 80% or more of the height from tip to base before immersion remains.

3. The microneedle array according to claim 1 or 2, wherein the microneedles are punctured into the surface of pig skin under the following conditions, left at 36°C for 17 hours, removed, and dried to remove surface moisture. The microneedles are dissolved to a height of 90% or less, based on the height from tip to base before puncturing. <Conditions> After fat peeling and hair removal from the skin surface, pig skin is stretched from all sides. While in this state, the microneedles are punctured into the skin surface to the base. The pig skin and the microneedle array are sandwiched between two plates, and both ends of the plates are fastened with clips. One plate has a through-hole pre-drilled, which contacts the fat side of the pig skin. The pig skin is placed flat in a container with the fat side facing downwards. A small amount of PBS is poured into the skin to prevent it from drying out. The skin is then left at 36°C for 17 hours, with only the fat side of the skin immersed in the PBS. Ensure that the PBS does not run out during this time. Thereafter, the microneedle array is removed from the pig skin and dried to remove moisture on the surface, and then the heights of five or more microneedles randomly selected from the remaining microneedles, excluding the broken ones, are measured, and the average value of the measured values ​​of the five or more microneedles is used as the result.

4. The microneedle array according to claim 1, wherein the photoreactive hyaluronic acid has an average molecular weight of 50,000 or more and less than 1,000,000.

5. The microneedle array described in claim 1, wherein the modification rate of the photocrosslinking groups possessed by the photoreactive hyaluronic acid is 2% or more and 15% or less.

6. The microneedle array according to claim 1, wherein the height of each of the plurality of microneedles is 150 μm or more and 2000 μm or less.

7. A method for producing a microneedle array, comprising the steps of injecting a solution of photoreactive hyaluronic acid having a photocrosslinking group into a mold having a plurality of recesses formed therein corresponding to the shapes of a plurality of microneedles, drying the solution in the recesses, demolding the solidified microneedles, and irradiating the photoreactive hyaluronic acid in the solidified microneedles with light to crosslink them, thereby obtaining a microneedle array in which a plurality of microneedles formed from crosslinked photoreactive hyaluronic acid are arranged on the surface of a substrate.

8. The method for producing a microneedle array according to claim 7, wherein the photoreactive hyaluronic acid has an average molecular weight of 50,000 or more and less than 1,000,000.

9. A method for producing a microneedle array according to claim 7 or 8, wherein the modification rate of the photocrosslinking group possessed by the photoreactive hyaluronic acid is 2% or more and 15% or less.

10. The method for manufacturing a microneedle array according to claim 7, wherein the depth of each of the plurality of recesses is 150 μm or more and 2000 μm or less.

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