阅读说明：本技术 一种耐湿的螨变应原可溶性微针 (Moisture-proof mite allergen soluble microneedle ) 是由 不公告发明人 于 2020-04-30 设计创作，主要内容包括：本发明公开了一种耐湿的螨变应原可溶性微针,包括针体和背衬,针体包括螨变应原和复合基质材料,其中,复合基质材料为甲基乙烯基醚-顺丁烯酸酐共聚物与聚乙烯醇的混合,甲基乙烯基醚-顺丁烯酸酐共聚物与聚乙烯醇的含量比为(1:1.5)～(2:1)。本发明通过筛选适合的基质材料种类与含量(Gantrez S-97+PVA)、适合的药载比,使制得的螨变应原可溶性微针在高湿条件下仍能具有良好的机械强度,易于刺入皮肤给药,且不影响螨变应原活性。(The invention discloses a moisture-proof mite allergen soluble microneedle, which comprises a needle body and a back lining, wherein the needle body comprises mite allergen and a composite matrix material, the composite matrix material is a mixture of a methyl vinyl ether-maleic anhydride copolymer and polyvinyl alcohol, and the content ratio of the methyl vinyl ether-maleic anhydride copolymer to the polyvinyl alcohol is (1:1.5) - (2: 1). According to the invention, through screening the proper type and content of the matrix material (Gantrez S-97+ PVA) and the proper drug loading ratio, the prepared mite allergen soluble microneedle still has good mechanical strength under a high-humidity condition, is easy to penetrate into the skin for administration, and does not influence the activity of the mite allergen.)

1. A moisture-resistant mite allergen soluble microneedle comprises a needle body and a back lining, and is characterized in that the needle body comprises mite allergen and a composite matrix material, wherein the composite matrix material is a mixture of a methyl vinyl ether-maleic anhydride copolymer and polyvinyl alcohol, and the content ratio of the methyl vinyl ether-maleic anhydride copolymer to the polyvinyl alcohol is (1:1.5) - (2: 1).

2. The moisture-resistant mite allergen soluble microneedle according to claim 1, wherein the content ratio of the methyl vinyl ether-maleic anhydride copolymer to the polyvinyl alcohol is (1: 1).

3. The moisture-resistant mite allergen soluble microneedle according to claim 1, wherein the content ratio of the mite allergen to the composite base material is (1:4) to (2: 3).

4. The moisture-resistant mite allergen soluble microneedle according to claim 3, wherein the content ratio of the mite allergen to the composite base material is 2: 3.

5. The moisture resistant mite allergen soluble microneedle of claim 1, wherein said methylvinyl ether-maleic anhydride copolymer is of the type Gantrez S-97.

6. The moisture-resistant mite allergen soluble microneedle according to any one of claims 1 to 5, wherein the needle body comprises the following components in percentage by weight: the mass ratio of the mite allergen is 40%, the mass ratio of the methyl vinyl ether-maleic anhydride copolymer is 30%, and the mass ratio of the polyvinyl alcohol is 30%.

7. The method for preparing moisture-resistant mite allergen soluble microneedle according to any one of claims 1 to 5, comprising the steps of:

(1) negative mold preparation of microneedles

Preparing a polydimethylsiloxane negative mould required by the soluble micro needle of the mite allergen vaccine by adopting a reverse mould method;

(2) preparation of mite allergen soluble microneedle

The components in the prescription amount are mixed and dissolved into a uniform matrix solution, a proper amount of the matrix solution is taken to be coated on a polydimethylsiloxane mold, and the matrix solution is repeatedly extruded by a roller to enter the holes of a female mold. Finally, drying at 37 ℃, and demoulding to obtain the product.

Technical Field

The invention relates to the technical field of administration of mite allergen, in particular to a moisture-resistant mite allergen soluble microneedle.

Background

The incidence of allergy is high and about one third of people reported in the literature have suffered from allergic disease during their lifetime. According to the statistics of Beijing areas in China, the incidence rate of allergy in general population is about 15%. Allergic diseases in parts of the pacific region can even reach as high as 50%. Since the 21 st century, the disease has become one of the most common global diseases. Data of the world health organization show that 22-25% of people worldwide suffer from allergic diseases at present, the speed of the people increases by 23 times every 10 years, and two hundred million people suffer from the diseases in China only at present. Allergy and anti-allergy are therefore serious problems facing modern people that cannot be avoided. The antigenic substances causing allergy are called allergens, and mite allergens belong to a large group of allergens. The existing mite allergen is mostly administrated by injection, and the frequent injection of a needle easily causes poor compliance of patients. Because the mite allergen contains antigen active protein, oral administration is easily decomposed and inactivated by protease in gastrointestinal tract, resulting in poor patient compliance; the mite allergen belongs to biological macromolecules, and the medicament is difficult to permeate into the skin due to the existence of stratum corneum obstacle of the skin. Thus, a new route of delivery of mite allergens is highly desirable.

Microneedles, particularly soluble microneedles, have rapidly developed in recent years as a novel drug delivery system. The microneedle can break the stratum corneum barrier of the skin, reach the epidermis layer without touching the nerve endings of the dermis layer, achieve painless minimally invasive administration, and the administration effect far breaks through the traditional transdermal administration preparation. Soluble microneedles are one type of microneedle, also the most studied type at present, which dissolve itself after penetrating into the skin and do not cause harmful residues. The matrix material constructed by the soluble micro-needle is generally natural polymer or synthetic polymer material with good water solubility, and it is known from the literature (Wang Q L, Ren J W, Chen B Z, et al. Effect of humidity on mechanical properties of dissolving microrods for transdermaldriver [ J ]. Journal of industrial and engineering chemistry,2018,59: 251) that the mechanical strength of the soluble micro-needle made of many polymer materials is reduced after being stored for a certain time under certain humidity condition, so that the soluble micro-needle can not penetrate into the skin. In southern China, particularly summer, the humidity in the air is high, even though the microneedles are packaged in vacuum, the hidden danger that the mechanical performance is reduced due to moisture absorption exists in the process from taking out to using, so that the moisture-resistant soluble microneedles are prepared, and the technical problem to be solved in the future industrialization process of the soluble microneedles of the mite allergen is high.

Through search, the relevant documents and patents relating to the loading of mite allergen into soluble microneedles are as follows:

soluble microneedles are prepared from a single material of hyaluronic acid by loading mite allergen into soluble microneedles as in the literature (Kim J H, Shin J U, Kim S H, et al. Successful transerdal allergen delivery and allergen-specific immunological use biomedical patches [ J ]. Biomaterials,2018,150:38-48), but no mention is made of whether humidity affects the mechanical strength of microneedles prepared from hyaluronic acid.

For example, chinese patent publication No. CN109715075A provides a formulation idea of loading a composition containing at least one allergen into microneedles, and a specific matrix material hyaluronic acid is selected for research, but the technical solution has disadvantages in that: the patent focuses on the change of the preparation process of allergen-loaded microneedles, and does not examine the influence factors of humidity, different allergens and different substrate materials, and does not examine the prescription screening when a specific allergen (such as mite allergen) is loaded into the soluble microneedles. Therefore, the difference in mechanical properties of soluble microneedles due to the change in the formulation when a mite allergen is loaded into the soluble microneedles under a specific humidity condition cannot be known from the patent.

Also, as in japanese patent publication JP2014156433A, the possibility of allergen loading into microneedles is only generally described, and the change in the structure of the microneedles is emphasized, but the technical solution has disadvantages in that: the examples do not show the difference in mechanical properties of different matrix materials for developing mite allergen soluble microneedles, nor can it be determined whether moisture resistance is present.

Soluble microneedles were prepared from 4 single materials (hyaluronic acid, polyvinyl alcohol, chitosan and gelatin) as in the literature (Wang Q L, Ren J W, Chen B Z, et al. Effect of humidity on mechanical properties of dispersed microrods for transdermal drug delivery [ J ]. Journal of industrial and engineering chemistry,2018,59: 251) and the effect on the mechanical properties of soluble microneedles was found when left for 30 minutes under 20%, 40%, 60%, 80% humidity conditions and dry conditions: under the conditions of drying and 20% relative humidity, the soluble microneedles prepared from the four materials have good mechanical properties and 100% perforation rate; secondly, under the conditions of 40% and 60% relative humidity, the perforation rate of the soluble micro-needle prepared by the polyvinyl alcohol is reduced to 90%, and the hyaluronic acid, the chitosan and the gelatin still keep the mechanical properties equivalent to those of the hyaluronic acid, the chitosan and the gelatin during drying; thirdly, under the condition of 80 percent relative humidity, the mechanical properties of the four materials are greatly reduced, and the skin can not be penetrated.

The preparation of vitamin K-carrying microneedles using Gantrez S-97, which have better mechanical properties at 86% relative humidity than at 43% humidity, is reported in the literature (Hutton A R J, Quinn H L, McCague P J, et al. Transdermal delivery of vitamin K using deforming microrods for the provision of vitamin K defining coverage [ J ]. International journal of pharmaceutical, 2018,541(1-2): 56-63). The conclusion of this document is quite different from the common general knowledge of "the mechanical properties are worse the greater the relative humidity is", but the technical disadvantage of this document is that: the relative humidity of air is greatly different due to different climates and regions in daily life, and the literature only considers the relative humidity conditions of 43 percent RH and 86 percent RH and is not enough to predict that the mechanical property of the microneedle of Gantrez S-97 meets the requirement of a preparation under other humidity conditions; furthermore, because the physicochemical properties of the mite allergen are different from that of vitamin K, whether the soluble microneedle prepared from the Gantrez S-97 drug-loaded mite allergen still meets the rule that the mechanical property of the microneedle is superior to 43% relative humidity under the condition of 86% relative humidity is unknown.

Therefore, under different humidity conditions, the soluble microneedles prepared by loading different drugs with different materials have different influences on the mechanical properties of the microneedles, which brings uncertainty to the selection of the substrate material when the soluble microneedles for the mite allergen are developed.

In addition, the influence of the type and concentration of the polymer, the preparation and storage temperature and the drying conditions on the activity of the protein drug Lysozyme (Lysozyme) was investigated in the literature (Shayan Fakhsae i Lahiji, Yoojung Jang, et al. effects of dissolution and microorganism degradation parameters on the activity of encapsulated Lysozyme [ J ]. European Journal of Pharmaceutical Sciences,2018,117: 290-. Therefore, it is uncertain which polymer is selected as the matrix material to keep the mite allergen in a better activity as another protein drug different from lysozyme.

In conclusion, it is an urgent need for those skilled in the art to develop a soluble mite allergen microneedle which has a suitable formulation, moderate mechanical strength, moisture resistance, and no influence on the activity of mite allergen.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the moisture-proof mite allergen soluble microneedle, and the defects of the prior art are overcome by selecting the proper types and content of the matrix materials and the drug loading ratio.

Therefore, the invention adopts the following technical scheme:

a moisture-resistant mite allergen soluble microneedle comprises a needle body and a back lining, wherein the needle body comprises mite allergen and a composite matrix material, the composite matrix material is a mixture of methyl vinyl ether-maleic anhydride copolymer (Gantrez) and polyvinyl alcohol (PVA), and the content ratio of the methyl vinyl ether-maleic anhydride copolymer to the polyvinyl alcohol is (1:1.5) - (2: 1).

Preferably, the content ratio of the methyl vinyl ether-maleic anhydride copolymer to the polyvinyl alcohol is (1: 1).

Preferably, the content ratio of the mite allergen to the composite matrix material is (1:4) - (2: 3).

More preferably, the content ratio of the mite allergen to the composite matrix material is 2: 3.

Preferably, the methyl vinyl ether-maleic anhydride copolymer is of the type Gantrez S-97.

Preferably, the needle body comprises the following components in percentage by weight: the mass ratio of the mite allergen is 40%, the mass ratio of the methyl vinyl ether-maleic anhydride copolymer is 30%, and the mass ratio of the polyvinyl alcohol is 30%.

The invention also provides a preparation method of the moisture-resistant mite allergen soluble microneedle, which comprises the following steps:

(1) negative mold preparation of microneedles

Preparing a polydimethylsiloxane negative mould required by the soluble micro needle of the mite allergen vaccine by adopting a reverse mould method;

(2) preparation of mite allergen soluble microneedle

The components in the prescription amount are mixed and dissolved into a uniform matrix solution, a proper amount of the matrix solution is taken to be coated on a polydimethylsiloxane mold, and the matrix solution is repeatedly extruded by a roller to enter the holes of a female mold. Finally, drying at 37 ℃, and demoulding to obtain the product.

Compared with the prior art, the moisture-proof mite allergen soluble microneedle provided by the invention has the following beneficial effects:

the applicant of the invention initially finds that when the Gantrez S-97 and PVA are combined to be used as a composite matrix material to prepare the mite allergen soluble microneedle, the mechanical property of the mite allergen soluble microneedle tends to increase firstly and then decrease with the increase of humidity, which is different from the common general knowledge that the mechanical property of the microneedle is worse when the relative humidity is higher.

Secondly, the invention also screens proper types and contents (Gantrez + PVA) of matrix materials and proper drug loading ratio to ensure that the prepared mite allergen soluble microneedle still has good mechanical strength under high humidity conditions, is easy to penetrate into skin for administration, does not influence the activity of mite allergen and obtains unexpected and different effects from common knowledge.

Drawings

Fig. 1 is a schematic flow chart of preparing a microneedle negative mold by a reverse molding method.

FIG. 2 is a schematic flow chart of the method for preparing mite allergen soluble microneedle by extrusion molding.

Fig. 3 shows the mechanical property changes of mite allergen soluble microneedles under different humidity conditions in example 3 of the present invention.

Fig. 4 shows the mechanical property changes of mite allergen soluble microneedles under different humidity conditions in example 4 of the present invention.

FIG. 5 shows the change of mechanical properties of mite allergen soluble microneedles in groups No.1, No.2 and No.3 under different humidity conditions in example 5 of the present invention.

FIG. 6 shows the change of mechanical properties of mite allergen soluble microneedles in groups No.4, No.5 and No.6 under different humidity conditions in example 5 of the present invention.

FIG. 7 is a graph showing the changes in mechanical properties of mite allergen-soluble microneedles in groups No.7, No.8 and No.9 in example 5 under different humidity conditions.

Fig. 8 shows the mechanical property changes of mite allergen soluble microneedles under different humidity conditions in example 6 of the present invention.

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.

Example 1 preparation of negative mold of mite allergen soluble microneedle

Referring to fig. 1, the invention adopts the MEMS process and the reverse mold method to prepare the microneedle negative mold, and the steps are as follows:

1) preparation of monocrystalline silicon male mold microneedle

The MEMS process is adopted to prepare the male mold micro-needle, the row of the micro-needle is 10 x 10, the height of the micro-needle is 600 mu m, the diameter of the bottom of the micro-needle is 300 mu m, and the diameter of the needle point is less than or equal to 10 mu m.

2) Mould for preparing polydimethylsiloxane

Mixing polydimethylsiloxane and a curing agent according to the mass ratio of 8:1, and pouring the mixture into a cuboid container provided with a monocrystalline silicon male mold microneedle; placing the container in a vacuum drying oven, and vacuumizing for 3min under the vacuum degree of 0.1MPa to remove bubbles in the mixed solution; and then putting the mould into an oven, drying the mould at 80 ℃ for 3h, and taking out the mould to obtain the formed polydimethylsiloxane micro-needle array mould.

Example 2 preparation of mite allergen soluble microneedles

Referring to fig. 2, the method for preparing mite allergen soluble microneedle by using an extrusion molding method comprises the following steps:

1) mixing the matrix material and the mite allergen according to the formula proportion, dissolving the mixture into uniform mixed solution (namely matrix solution) by Phosphate Buffered Saline (PBS) with the pH value of 7.4, putting a proper amount of the matrix solution into a centrifuge tube, placing the centrifuge tube in a centrifugal precipitator, centrifuging to remove air bubbles in the matrix solution, and standing for later use.

2) Pouring the matrix solution obtained after centrifugation in the step 1) on a polydimethylsiloxane mold prepared in the embodiment 1 of the invention, placing the polydimethylsiloxane mold on a plane, extruding the matrix solution above the mold by using a roller, and repeating the extruding and the extruding for many times to inject the matrix solution into holes of the mold. Then, the mould is placed in a 37 ℃ oven, dried for 4h and taken out, and demoulding is carried out, thus obtaining the finished product.

Example 3 Effect of different humidity conditions on mechanical Properties and Activity of mite allergen soluble microneedles

In the present invention, a high molecular polymer is selected as a matrix material, based on the research of the above documents, the methyl vinyl ether-maleic anhydride copolymer (Gantrez S-97) is loaded with the mite allergen to prepare the mite allergen soluble microneedle, and the influence of the humidity condition on the mechanical properties and the activity of the mite allergen soluble microneedle is investigated by measuring the mechanical strength and the activity change of the mite allergen under different humidity conditions. The humidity condition is 10-90% relative humidity (10-90% RH). Provides a precondition for judging the influence of the subsequent microneedle material composition, the combination with the allergen and the mutual relationship on the microneedle penetration performance.

The mite allergen soluble microneedle according to the present example was prepared by referring to examples 1 and 2 of the present invention.

Setting a humidity condition: due to the climate characteristics of the moisture regain weather in south China, the relative humidity of air is more than 90%, and the exposure time of the microneedle in the natural environment is about 20min when the microneedle is used generally, so that the relevant performance of the microneedle is investigated by simulating a real use scene and strictly selecting experimental conditions (namely the humidity is 90% RH and the storage time is 40 min). The investigation method comprises the following steps: drug stability test chambers were used to simulate different humidity environments. The storage temperature was fixed at 25 ℃, the relative humidity was set at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, and the sample was taken out after standing for 40 min.

The skin puncture performance of the mite allergen soluble microneedle is investigated: the prepared soluble microneedle of the mite allergen vaccine is penetrated into the skin of the in-vitro pig ear for 1min by a doser with the force of 15N, and then the microneedle patch is taken down. The microneedle administration site was stained with methylene blue dye, excess dye at the edge of the hole was wiped off, and the puncture rate (the ratio of the number of blue spots on the skin to the number of needles on the microneedle) was calculated in parallel for 6 groups. The perforation rate of more than 80% is determined to be satisfactory.

Measurement of relative biological activity of mite allergen: (UniCAP method): sample and standard preparation: the sample is a mixed solution of a mite allergen solution and a matrix material, the standard substance is a mite allergen solution, and after the sample and the corresponding standard substance are respectively mixed with 0.1mol/L PBS (pH 7.4) with the same volume, the mixture is respectively diluted to 6 points in a 3-time series manner. Samples of each dilution were mixed with an equal volume of dust mite positive serum (4-fold dilution) and a negative control (50. mu.L of LPBS buffer + 50. mu.L of diluted dust mite positive serum) and a 100% inhibition control (50. mu.L of undiluted standard + 50. mu.L of diluted dust mite positive serum) were set. And (3) biological activity determination: incubating the samples, the standard substance and the control sample at 37 ℃ for 2h, measuring the specific slgE concentration in each sample, the standard substance and the control sample on an ImmunoCAP 100E full-automatic allergen analyzer, repeatedly measuring for 2 times at each dilution, and calculating the inhibition rate by taking the average value of the fluorescence intensity.

And (4) calculating a result: the inhibition rate (%) × 100% (negative control measurement value-sample measurement value)/(negative control measurement value-measurement value of 100% inhibition control).

Curves were drawn with each dilution as abscissa and inhibition as ordinate. And (4) obtaining the relative biological activity of the sample by adopting a parallel line analysis method for the sample and the standard substance. The assay was performed 6 times in parallel.

According to the instruction in technical guidelines for the quality control of allergen (allergen) products issued by CFDA: the biological activity of the product is 50-200% of the marked amount, namely, when the relative biological activity is lower than 50%, the test requirement is not met.

First, the applicant examined the change of mechanical strength of mite allergen soluble microneedles manufactured using Gantrez S-97 as a base material under different humidity conditions, and the results are shown in fig. 3.

As can be seen from fig. 3, the mechanical properties of the microneedles tend to increase first and then decrease with the increase in humidity, which is different from the common general knowledge that "the mechanical properties of the microneedles are worse the greater the relative humidity is"; secondly, when the RH is 70 percent, the puncture rate of the micro-needle is more than 80 percent and reaches the peak value, and the mechanical property is the best at the moment; ③ when the puncture rate of the microneedle is lower than 80 percent at 90 percent RH, the microneedle does not meet the test requirement; that is, in a humid weather condition such as 90% of relative humidity, the mite allergen soluble microneedle prepared by using the Gantrez S-97 as a substrate material may not completely penetrate into the skin due to the decrease of mechanical properties in use; the mechanical properties of the microneedles at 83% RH are comparable or slightly inferior to 43% RH, as can be seen from the curves in FIG. 3, which is contrary to the conclusions in the literature described in the background of the invention (Hutton A R J, Quinn H L, McCague P J, et al. Transdermal delivery of vitamin K using a dispensing microelectronic systems for the prevention of vitamin K configuring the visualization [ J ]. International journel of pharmaceuticals, 2018,541(1-2): 56-63).

Next, the applicant examined the relative bioactivity change of mite allergen soluble microneedles prepared by using Gantrez S-97 as a matrix material under different humidity conditions, and the results are shown in Table 1.

TABLE 1 comparison of relative biological Activity of mite allergens under different humidity conditions (mean + -SD,%)

Matrix material	10％RH	20％RH	30％RH	40％RH	50％RH
						Gantrez S-97	75.4±1.5	74.6±1.8	75.6±2.1	76.8±1.6	73.6±1.4
Matrix material	60％RH	70％RH	80％RH	90％RH
						Gantrez S-97	72.3±1.8	78.7±1.2	76.9±2.0	74.9±1.5

As can be seen from Table 1: firstly, a substrate material is Gantrez S-97, and the biological activity of the mite allergen under different humidity conditions is not obviously different; ② at 90% RH, the relative biological activity of the mite allergen is 74.9% +/-1.5%, although meeting the test requirement, it is close to the unqualified standard line.

In conclusion, since the Gantrez S-97 is a brittle material, which tough material is selected to be combined with the Gantrez S-97 to be used as a composite matrix material for carrying the mite allergen, the mechanical property of the microneedle is improved at 90% RH so that the microneedle meets the test requirement, and the developed mite allergen soluble microneedle can be ensured, the relative biological activity of the mite allergen is not less than 50%, and uncertainty is provided. And the literature "matrix material and composite material for constructing the soluble microneedle" (octogen, mafengsen, great wealth and wisdom, material guidance, 2017,31(19): 129-. Exploratory studies are required.

Example 4 Effect of the type of composite matrix Material on the mechanical Properties and Activity of mite allergen soluble microneedles

Based on the preliminary experimental study of this example 3, it is necessary to perform an exploratory study on the selection of composite matrix materials under different humidity conditions to solve the technical confusion of developing mite allergen soluble microneedles.

Therefore, the invention researches the influence of the types of matrix materials shown in table 2 on the mechanical property and the activity of the mite allergen soluble microneedle under different humidity conditions by setting a gradient humidity condition and investigating the mechanical strength of the mite allergen soluble microneedle and the activity change of the mite allergen under 10% RH-90% RH.

In this embodiment, the tough material is selected from one of PVA, polyvinylpyrrolidone (PVP) and polyacrylic acid (PAA), and is combined with the brittle material Gantrez S-97 to form a composite matrix material.

The preparation method of the mite allergen soluble microneedle related to the embodiment refers to the embodiment 1 and the embodiment 2; humidity conditions and methods for measuring mechanical properties and activity of microneedles are described in example 3 of the present invention.

First, the applicant measured the puncture rates of mite allergen-soluble microneedles shown in table 2 under different humidity conditions, and the measurement results are shown in fig. 4.

TABLE 2 microneedle formulations consisting of different matrix material components

No.	Composite matrix material (wt%)	Mite allergen (wt%)
			1	30％Gantrez S-97+30％PVP	40％
2	30％Gantrez S-97+30％PAA	40％
			3	30％Gantrez S-97+30％PVA	40％

As can be seen from table 2 and fig. 4:

firstly, the mechanical performance of the microneedle of the No.3 group (Gantrez S-97+ PVP) is gradually reduced along with the increase of relative humidity, and is reduced from qualified to unqualified, so that the microneedle does not meet the test requirement;

② under any humidity condition of 10 percent RH-90 percent RH, the perforation rate of the NO.2 group (Gantrez S-97+ PAA) and the NO.3 group (Gantrez S-97+ PVA) is more than 80 percent, which accords with the test requirement.

Further, the activities of the mite allergens were measured for the No.2 group and the No.3 group, and since it was shown in example 3 that the change in the humidity condition had no significant influence on the relative biological activities of the mite allergens, the relative biological activities of the mite allergens at 90% RH were measured here, and the measurement results are shown in table 3.

TABLE 3 Effect of Polymer species on the biological Activity of mite allergens (mean. + -. SD, n ═ 6)

No.	Relative biological Activity (%)
		2	47.3±2.8
3	88.6±4.3

From table 3, it can be seen that:

firstly, when the RH is 90 percent, the relative bioactivity of the NO.2 group (Gantrez S-97+ PAA) is less than 50 percent, and the test requirement is not met;

② at 90 percent RH, the relative biological activity of NO.3 group (Gantrez S-97+ PVA) is more than 50 percent and still meets the test requirement.

In conclusion, the combination of Gantrez S-97 and PVA was selected as the matrix material for developing mite allergen soluble microneedles, taking into consideration the mechanical properties of the microneedles and the activity of mite allergen.

Example 5 influence of the content ratio of each component in the matrix material on the mechanical properties and activity of mite allergen soluble microneedles

On the basis of example 4, assuming that the mass ratio of the matrix material in the needle body is a certain value and the components of the matrix material are fixed (combination of Gantrez S-97 and PVA), the content ratio of each component of the matrix material is changed only, and the influence of the components of the matrix material with different content ratios on the mechanical properties of mite allergen-soluble microneedles under the humidity conditions of 10% RH to 90% RH and the influence of mite allergen activity at 90% RH as shown in table 4 are examined similarly.

The preparation method of the mite allergen soluble microneedle related to the embodiment refers to the embodiment 1 and the embodiment 2; humidity conditions and methods for measuring mechanical properties and activity of microneedles are described in example 3 of the present invention.

First, the applicant measured the puncture rates of mite allergen-soluble microneedles shown in table 4 under different humidity conditions, and the measurement results are shown in fig. 5, 6 and 7.

TABLE 4 microneedle formulations consisting of matrix material components in different content ratios

No.	Gantrez S-97：PVA(w/w)	Mite allergen (wt%)
			1	1:3	40％
2	1:2.5	40％
			3	1:2	40％
4	1:1.5	40％
			5	1:1	40％
6	1.5:1	40％
			7	2:1	40％
8	2.5:1	40％
			9	3:1	40％

As can be seen from table 5, fig. 6 and fig. 7:

firstly, as the relative humidity increases, the perforation rates of all 9 groups tend to rise firstly and then fall;

and the perforation rates of only the No.4 group, the No.5 group, the No.6 group and the No.7 group are all more than 80 percent under any humidity condition, and the test requirements are met. Among them, the No.5 group had the largest perforation rate.

Next, the applicant measured the relative change in bioactivity of the mite allergen-soluble microneedles at 90% RH as shown in table 4, and the measurement results are shown in table 5.

TABLE 5 influence of the base material components in different content ratios on the mite allergen activity (mean + -SD, n ═ 6)

No.	Relative biological Activity (%)
		1	88.8±4.2
2	86.5±3.7
		3	90.6±2.8
4	87.6±3.1
		5	88.6±4.3
6	89.7±3.1
		7	90.8±1.9
8	87.6±2.4
		9	88.4±1.5

In conclusion, the combination of Gantrez S-97 and PVA with the content ratio of (1:1.5) - (2:1) is selected as a composite matrix material for developing mite allergen soluble microneedles by comprehensively considering the mechanical properties of the microneedles and the activity of the mite allergen. Wherein, the optimal content ratio is 1:1.

Example 6 Effect of drug Loading on mechanical Properties of mite allergen-soluble microneedles

In example 5, assuming that the content ratio of each component in the composite matrix material is constant (the content ratio of Gantrez S-97 to PVA is 1:1), the effect of different drug loading ratios on the mechanical properties of mite allergen-soluble microneedles under humidity conditions of 10% RH to 90% RH as shown in table 6 was examined by changing only the drug loading ratio (i.e., the content ratio of mite allergen to composite matrix material).

The preparation method of the mite allergen soluble microneedle related to the embodiment refers to the embodiment 1 and the embodiment 2; method for measuring mechanical properties of microneedles, refer to example 3 of the present invention. The test results are shown in FIG. 8.

TABLE 6 microneedle formulations with different drug loading ratios

As can be seen from FIG. 8, the puncture rates of the No.2 group, the No.3 group and the No.4 group under any humidity condition are all greater than 80%, and the test requirements are met, that is, when the drug loading ratio is (1:4) - (2:3), the mechanical properties of the mite allergen soluble microneedles meet the requirements. The influence of the drug loading on the therapeutic effect of the mite allergen soluble microneedle is comprehensively considered, and a NO.2 group (namely, the drug loading ratio is 2:3) is selected as an optimal prescription.

In summary, in the mite allergen soluble microneedle provided by the present invention, the optimal prescription of the needle body is as follows: the mass ratio of the mite allergen is 40%, the mass ratio of the methyl vinyl ether-maleic anhydride copolymer is 30%, and the mass ratio of the polyvinyl alcohol is 30%.

In the mite allergen soluble microneedle provided by the invention, a suitable prescription of the needle body is as follows: the content ratio of the mite allergen to the composite matrix material is (1:4) - (2:3), and the content ratio of the methyl vinyl ether-maleic anhydride copolymer to the polyvinyl alcohol in the composite matrix material is (1:1.5) - (2: 1).