A dissolvable microneedle and a method of making the same

Abstract

This invention relates to the field of microneedle drug delivery technology, specifically to a soluble microneedle and its preparation method. The soluble microneedle includes a needle hub and at least one needle body located on the needle hub. The needle body includes a first intermediate segment, a second intermediate segment, and a needle tip. The first intermediate segment is located on the needle hub, and the second intermediate segment is located between the first intermediate segment and the needle tip. The density of the second intermediate segment is greater than that of the first intermediate segment. The needle tip contains a drug active ingredient. The soluble microneedle provided by this invention has the drug active ingredient concentrated in the needle tip. Furthermore, the second intermediate segment, which is in contact with the needle tip, is a high-density segment, which can prevent the drug active ingredient in the needle tip from diffusing into the second intermediate segment and the first intermediate segment. Therefore, the soluble microneedle of this invention can significantly increase the local drug delivery concentration, achieving precise, targeted, and quantitative drug delivery.

Description

Technical Field

[0001] This invention relates to the field of microneedle drug delivery technology, specifically to a soluble microneedle and its preparation method. Background Technology

[0002] Dissolving microneedles are microneedles prepared by mixing biodegradable polymer materials with active pharmaceutical ingredients. After insertion into the skin, the biodegradable polymer material in the dissolving microneedle gradually degrades in the microenvironment, releasing the active pharmaceutical ingredient. This ingredient then crosses the stratum corneum barrier and is absorbed into the body through the subcutaneous tissue. When delivering drugs using dissolving microneedles, there is no need to form microchannels for drug delivery or remove the needle after delivery. This greatly improves patient compliance and reduces the risk of cross-infection from reusing microneedles.

[0003] Existing soluble microneedles are generally one-piece molded microneedles, in which the active pharmaceutical ingredient is mixed with a biodegradable polymer material to form the microneedle body. The inventors of this invention have discovered that existing soluble microneedles have at least the following problems: because the active pharmaceutical ingredient is uniformly dispersed in the biodegradable polymer material, and the active pharmaceutical ingredient diffuses within the microneedle, the local concentration of the drug during administration is low. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of low local drug concentration in existing soluble microneedles, thereby providing a soluble microneedle and its preparation method.

[0005] Therefore, the present invention provides a soluble microneedle, comprising: a needle hub and at least one needle body located on the needle hub, the needle body comprising a first intermediate segment, a second intermediate segment and a needle tip segment, the first intermediate segment being located on the needle hub, the second intermediate segment being located between the first intermediate segment and the needle tip segment, the density of the second intermediate segment being greater than the density of the needle tip segment and the first intermediate segment, and the needle tip segment containing a pharmaceutically active ingredient.

[0006] Optionally, the first intermediate segment can be formed by curing a first polymer material slurry. The first polymer material can be selected from a certain range. For example, the first polymer material can be selected from polyvinylpyrrolidone and / or sodium carboxymethyl cellulose, preferably polyvinylpyrrolidone.

[0007] Optionally, the second intermediate segment can be formed by curing a second polymer material slurry. The second polymer material can be selected from a certain range. For example, the second polymer material can be selected from hyaluronic acid and / or chitosan, preferably hyaluronic acid.

[0008] Optionally, the needle seat can be formed by curing a third polymer material slurry. The third polymer material can be selected from a certain range. For example, the third polymer material can be selected from polyvinyl alcohol and / or polyvinyl acetate, preferably polyvinyl alcohol.

[0009] Optionally, the needle tip can be formed by solidifying a solution of active pharmaceutical ingredient. The active pharmaceutical ingredient can be selected from a certain range. For example, the active pharmaceutical ingredient may include at least one of peptide drugs, protein drugs, antibodies, vaccines, probiotics, and nucleic acid drugs. The solvent used in the solution of active pharmaceutical ingredient can be selected from physiological saline and / or buffer salt solutions.

[0010] Optionally, the content of the active pharmaceutical ingredient in the needle tip segment can vary within a certain range. For example, based on the total weight of the needle tip segment, the weight percentage of the active pharmaceutical ingredient in the needle tip segment can be 10-20%.

[0011] Preferably, the active pharmaceutical ingredient is a vaccine, and the needle tip also contains an aluminum adjuvant, wherein the weight ratio of the vaccine to the aluminum adjuvant is 1:(1-2).

[0012] More preferably, the vaccine is a recombinant subunit protein of the novel coronavirus, and the aluminum adjuvant is aluminum hydroxide adjuvant and / or aluminum phosphate adjuvant.

[0013] Optionally, the novel coronavirus subunit recombinant protein can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed, or it can be obtained by purchase.

[0014] Optionally, the first intermediate segment may also contain nanomaterials.

[0015] Optionally, the nano-antibacterial function may be selected from at least one of nano silver, MXene, graphene and polydopamine.

[0016] Optionally, based on the total weight of the first intermediate segment, the weight percentage of the nanofunctional material in the first intermediate segment can be 20-30%.

[0017] Optionally, the needle body is pyramidal or conical. The dimensions of each part of the needle body can vary within a certain range; for example, the thickness of the needle base can be 1500 μm; the length of the first intermediate section can be 200 μm, and the volume can be 9.94 × 10^6 μm. 3 The second intermediate segment can be 300 μm long and have a volume of 4.36 × 10^6 μm. 3 The length of the needle tip can be 170 μm, and the volume is 1.6 × 10^5 μm. 3 .

[0018] The present invention also provides a method for preparing the soluble microneedles according to any one of the present invention, comprising the following operations: placing a solution of the active pharmaceutical ingredient into a microneedle mold, applying a vacuum, and performing a first curing to obtain a microneedle mold loaded with a needle tip segment; pouring a second polymer material slurry onto the microneedle mold loaded with the needle tip segment, applying a vacuum, and performing a second curing to obtain a microneedle mold loaded with a needle tip segment and a second intermediate segment; pouring a first polymer material slurry onto the microneedle mold loaded with the needle tip segment and the second intermediate segment, applying a vacuum, and performing a third curing to obtain a microneedle mold loaded with a needle tip segment, a second intermediate segment, and a first intermediate segment; pouring a third polymer material slurry onto the microneedle mold loaded with the needle tip segment, the second intermediate segment, and the first intermediate segment, applying a vacuum, and performing a fourth curing to obtain the soluble microneedles.

[0019] Optionally, the concentration of the active pharmaceutical ingredient in the solution can be 0.5–5 μg / μl, the viscosity of the solution is not higher than 5000 mPa, and the solvent used is selected from physiological saline and / or buffer salt solution. The concentration of sodium chloride in the physiological saline can be 0.9% (weight / volume), and the buffer salt solution can be selected from at least one of phosphate buffer, carbonate buffer, and acetate buffer.

[0020] Optionally, the content of the first polymer material in the first polymer material slurry is 900-1100 μg / μl, and the solvent used is deionized water.

[0021] Optionally, the content of the second polymer material in the second polymer material slurry is 140-160 μg / μl, and the solvent used is deionized water.

[0022] Optionally, the content of the third polymer material in the third polymer material slurry is 450-550 μg / μl, and the solvent used is deionized water.

[0023] Optionally, the microneedle mold is a hydrophobic mold.

[0024] Optionally, the first curing is freeze-drying at a temperature of -30 to 10°C for 5 to 10 hours; the second curing is freeze-drying at a temperature of -50 to 10°C for 5 to 10 hours; the third curing is freeze-drying at a temperature of -30 to 10°C for 5 to 10 hours; and the fourth curing is freeze-drying at a temperature of -30 to 10°C for 5 to 10 hours.

[0025] Optionally, the amount of the active pharmaceutical ingredient solution used is 5–10 μl.

[0026] Optionally, the amount of the first polymer material slurry is 2 to 8 μl.

[0027] Optionally, the amount of the second polymer material slurry is 5 to 20 μl.

[0028] Optionally, the amount of the third polymer material slurry is 300-500 μl.

[0029] Optionally, before pouring the first polymer slurry onto the microneedle mold containing the needle tip and the second intermediate section, the method may further include adding nanomaterials to the first polymer slurry.

[0030] The technical solution of this invention has the following advantages:

[0031] 1. The soluble microneedles provided by the present invention have the active pharmaceutical ingredient concentrated in the tip segment. Moreover, the second intermediate segment in contact with the tip segment is a high-density segment, which can prevent the active pharmaceutical ingredient in the tip segment from diffusing into the second intermediate segment and the first intermediate segment. Therefore, the soluble microneedles of the present invention can significantly improve the drug delivery depth and significantly improve the local drug delivery concentration, so as to achieve precise drug delivery at fixed points and in fixed quantities.

[0032] 2. The soluble microneedles provided by the present invention have a tip segment formed by solidification of a solution of active pharmaceutical ingredients. A large amount of active pharmaceutical ingredients can be dissolved in the solution. Compared with the case where the active pharmaceutical ingredients and polymer materials are co-solidified, the present invention can effectively avoid the limitation of the concentration of polymer materials on the content of active pharmaceutical ingredients. Therefore, the soluble microneedles of the present invention can have a high drug loading capacity.

[0033] 3. The soluble microneedles provided by the present invention contain nano-functional materials in the first intermediate segment. On the one hand, this can enhance the antibacterial or immune cell chemotaxis capabilities of the soluble microneedle products, making the soluble microneedles less susceptible to contamination. On the other hand, after the soluble microneedles dissolve, the nano-functional materials can form an antibacterial protective film on the skin surface, reducing the risk of bacterial infection at the site of application of the soluble microneedles.

[0034] 4. The soluble microneedle preparation method provided by the present invention adopts vacuum and in-situ solidification treatment, which can enrich the active pharmaceutical ingredient in the needle tip segment. This can not only increase the drug loading capacity of the microneedle, but also improve the recovery rate of the active pharmaceutical ingredient.

[0035] 5. The soluble microneedle preparation method provided by the present invention uses physiological saline or buffer salt solution as the solvent for the drug active ingredient solution, which has a low viscosity. This can effectively avoid the situation where the drug active ingredient recovery rate is low due to the excessive viscosity of the drug active ingredient solution. Therefore, the method of the present invention has a high drug active ingredient recovery rate.

[0036] 6. The soluble microneedle preparation method provided by the present invention uses freeze-drying as the curing method. The freeze-drying preparation conditions do not damage the stability of protein-based active substances and can improve the quality of the prepared soluble microneedles in terms of protein purity and content. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of the preparation method of Embodiment 1 of the present invention;

[0039] Figure 2 This is the fluorescence distribution result of the soluble microneedles prepared in Example 1, measured in Experimental Example 2 of this invention;

[0040] Figure 3 This is a molecular structure diagram of BH1 used in the embodiments of the present invention;

[0041] Figure 4 This is the test result diagram corresponding to the soluble microneedle S in Experimental Example 3 of this invention;

[0042] Figure 5 This is the test result diagram corresponding to soluble microneedles D in Experimental Example 3 of this invention;

[0043] Figure 6 This is a graph showing the test results after storage at 4°C for 3 days in Experiment Example 4 of this invention;

[0044] Figure 7 This is a graph showing the test results after storage at 4°C for 7 days in Experiment Example 4 of this invention;

[0045] Figure 8 This is a graph showing the test results after storage at 40°C for 3 days in Experiment Example 4 of this invention;

[0046] Figure 9 This is a graph showing the test results after storage at 40°C for 7 days in Experiment Example 4 of this invention. Detailed Implementation

[0047] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0048] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0049] The recombinant novel coronavirus subunit protein BH1 used in the embodiments and comparative examples of this invention can be a homodimer synthesized from the polypeptide shown in SEQ ID NO.1 using the FmoC solid-phase polypeptide synthesis method. Specifically, two polypeptides shown in SEQ ID NO.1 are linked by a lysine residue to form a homodimer with a molecular weight of 5699.12 Da, and its molecular structure is as follows. Figure 3 As shown. Among them, the amino acid sequence shown in SEQ ID NO.1 is YNSASFSTFKCYGVSPTKLNDLCFT, and the amino acid sequence of BH1 is (YNSASFSTFKCYGVSPTKLNDLCFT)2-K.

[0050] Example 1

[0051] This embodiment adopts Figure 1 The method shown for preparing soluble microneedles includes the following steps:

[0052] (1) Take the drug active ingredient solution and place it in the microneedle mold. The amount added is 8 μl. Vacuum is drawn and the microneedle mold is freeze-dried in situ at -30℃ for 7 h to form the needle tip segment. The microneedle mold loaded with the needle tip segment is obtained. The drug active ingredient solution contains a combination of novel coronavirus subunit recombinant protein BH1 and aluminum hydroxide adjuvant. The concentration of novel coronavirus subunit recombinant protein BH1 is 2.5 μg / μl, the concentration of aluminum hydroxide adjuvant is 3.75 μg / μl, and the solvent used is physiological saline with a sodium chloride concentration of 0.9% (weight / volume).

[0053] (2) Take the second polymer material slurry and pour it onto the microneedle mold loaded with the needle tip segment obtained in step (1), with a pouring amount of 10 μl; vacuum and freeze-dry at -30℃ for 7 h to form the second intermediate segment, and obtain the microneedle mold loaded with the needle tip segment and the second intermediate segment; wherein, the second polymer material is hyaluronic acid, the content of hyaluronic acid is 150 μg / μl, and the solvent used is deionized water.

[0054] (3) Take the first polymer material slurry, add 25wt% of the nano-functional material Mxene, and pour it onto the microneedle mold loaded with the needle tip segment and the second intermediate segment obtained in step (2). The pouring amount is 5μl. Vacuum is drawn and freeze-dried at -30℃ for 7h to form the first intermediate segment, and a microneedle mold loaded with the needle tip segment, the second intermediate segment and the first intermediate segment is obtained. Among them, the first polymer material is polyvinylpyrrolidone, the content of polyvinylpyrrolidone is 1000μg / μl, and the solvent used is deionized water.

[0055] (4) Take the third polymer material slurry and pour it onto the microneedle mold containing the needle tip, the second intermediate section and the first intermediate section obtained in step (3). The pouring amount is 400 μl. Vacuum is drawn and freeze-dried at -30℃ for 7 h to form a needle seat, and the soluble microneedles are obtained. The third polymer material is polyvinyl alcohol, the content of polyvinyl alcohol is 500 μg / μl, and the solvent used is deionized water.

[0056] The soluble microneedles prepared in this embodiment include a needle hub and at least one needle body located on the needle hub. The needle body includes a first intermediate section, a second intermediate section, and a needle tip. The first intermediate section is located on the needle hub, and the second intermediate section is located between the first intermediate section and the needle tip.

[0057] The needle tip contains a recombinant subunit protein of the novel coronavirus and aluminum hydroxide; the second intermediate segment is formed by curing hyaluronic acid slurry; the first intermediate segment is formed by curing polyvinylpyrrolidone slurry, which contains the nano-functional material Mxene; and the needle seat is formed by curing polyvinyl alcohol. Because the density of hyaluronic acid is greater than that of polyvinylpyrrolidone, the density of the second intermediate segment is greater than that of the first intermediate segment.

[0058] Example 2

[0059] Soluble microneedles were prepared according to the method of Example 1, except that the hyaluronic acid content in the second polymer slurry used in this example was 140 μg / μl, and the polyvinylpyrrolidone content in the first polymer slurry was 900 μg / μl.

[0060] Example 3

[0061] Soluble microneedles were prepared according to the method of Example 1, except that the hyaluronic acid content in the second polymer slurry used in this example was 160 μg / μl, and the polyvinylpyrrolidone content in the first polymer slurry was 1100 μg / μl.

[0062] Example 4

[0063] Soluble microneedles were prepared according to the method in Example 1, except that the concentration of the novel coronavirus subunit recombinant protein and the concentration of aluminum hydroxide adjuvant in the drug active ingredient solution used in this example was 0.5 μg / μl.

[0064] Example 5

[0065] Soluble microneedles were prepared according to the method in Example 1, except that the concentration of the novel coronavirus subunit recombinant protein and the concentration of aluminum hydroxide adjuvant in the drug active ingredient solution used in this example was 5 μg / μl.

[0066] Example 6

[0067] Soluble microneedles were prepared according to the method of Example 1, except that the first polymer material used in this example was sodium carboxymethyl cellulose, the second polymer material was chitosan, and the third polymer material was polyvinyl acetate.

[0068] Example 7

[0069] Soluble microneedles were prepared according to the method of Example 1, except that in step (3) of this example, no nano-functional materials were added to the first polymer slurry.

[0070] Comparative Example 1

[0071] Soluble microneedles were prepared according to the method of Example 1, except that the second polymer material used in this comparative example was polyvinylpyrrolidone, and the content of polyvinylpyrrolidone in the slurry of the second polymer material was 1000 μg / μl; the first polymer material was hyaluronic acid, and the content of hyaluronic acid in the slurry of the first polymer material was 150 μg / μl.

[0072] In the soluble microneedles prepared in this comparative example, the second intermediate segment is formed by solidifying polyvinylpyrrolidone slurry, the first intermediate segment is formed by solidifying hyaluronic acid slurry, and the density of the second intermediate segment is less than that of the first intermediate segment.

[0073] Comparative Example 2

[0074] Soluble microneedles were prepared using the following method:

[0075] (1) Take a mixed solution of the active pharmaceutical ingredient and the polymer material and place it in a microneedle mold with at least one needle-shaped die hole. The amount added to each needle-shaped die hole is 23 μl. Vacuum is drawn and the mixture is placed in an in-situ freeze dryer at -30°C for 7 h to form a needle body. The microneedle mold loaded with the needle body is obtained. The active pharmaceutical ingredient is a combination of novel coronavirus subunit recombinant protein and aluminum hydroxide adjuvant. The polymer material is polyvinylpyrrolidone. The concentration of novel coronavirus subunit recombinant protein BH1 in the mixed solution is 2.5 μg / μl, the concentration of aluminum hydroxide adjuvant is 3.75 μg / μl, the content of polyvinylpyrrolidone is 1000 μg / μl, and the solvent used is physiological saline with a sodium chloride concentration of 0.9% (weight / volume).

[0076] (2) Take polyvinyl alcohol slurry and pour it onto the microneedle mold loaded with needles obtained in step (1), with a pouring amount of 400 μl; vacuum and freeze-dry at -30℃ for 7 h to form needle seat, and obtain the soluble microneedle; wherein, the polyvinyl alcohol content in the polyvinyl alcohol slurry is 500 μg / μl, and the solvent used is deionized water.

[0077] The soluble microneedles prepared in this comparative example include a needle hub and at least one needle body located on the needle hub, wherein the needle body contains recombinant subunit protein of novel coronavirus and aluminum hydroxide adjuvant dispersed in it.

[0078] Experimental Example 1

[0079] The recombinant subunit proteins of the novel coronavirus in Example 1 and Comparative Examples 1-2 were labeled with FITC, and microneedles were made again according to the methods of Example 1 and Comparative Examples 1-2, respectively. The distribution of fluorescence in the microneedles was then observed.

[0080] The fluorescence distribution results of the soluble microneedles prepared by the method in Example 1 are as follows: Figure 2 As shown, fluorescence is mainly concentrated within a 170 μm range from the needle tip. Using PVP to encapsulate 555 fluorescence, the PVP layer was found to be concentrated within approximately 200 μm of the microneedle relative to the backing. Simultaneously, no fluorescence was observed on the backing, indicating that no recombinant protein diffused into the backing.

[0081] The fluorescence distribution results of the soluble microneedles prepared by the method of Comparative Example 1 showed that fluorescence appeared in the range from the needle tip to the needle seat, and the fluorescence gradually weakened as it moved away from the needle tip.

[0082] The fluorescence distribution results of the soluble microneedles prepared by the method in Comparative Example 2 showed that the fluorescence was uniformly dispersed throughout the needle body.

[0083] Experimental Example 2

[0084] This experimental example is used to verify the immune effects of various soluble microbes on mice.

[0085] Immunization experiments were conducted on experimental animals using the soluble microparticles from Examples 1-7 and Comparative Examples 1 and 2, respectively. The entire immunization process consisted of three immunizations, including one immunization and two booster immunizations, as follows: the first immunization was performed in week one; the second immunization (the first booster immunization) was performed in week four; the third immunization (the second booster immunization) was performed in week six; and in week nine, blood samples were collected from the experimental mice to separate serum and detect their neutralizing antibody titer against the novel coronavirus.

[0086] The experimental subjects were 6-week-old male Balb / c mice, with 5 mice in each group. During the experiment, the mice needed to have their backs and sides shaved to facilitate microneedle insertion. Therefore, the mice were first anesthetized, and the hair on the back was trimmed with a hair clipper. Depilatory cream was then applied to the areas where hair had been present. After 2 minutes, the cream was wiped off with a cotton ball soaked in water, at which point the back hair was completely removed. The skin was then dried with absorbent paper towels to prevent the microneedles from dissolving before penetration. The mouse skin was lifted with fingers or tweezers, and the microneedle patch array was placed on the skin, positioned between two fingers. Pressure was applied to insert the microneedles into the skin. This procedure was to avoid death caused by excessive downward pressure on the microneedle patches. Each mouse was immunized in this manner, for a total of three immunizations according to the experimental cycle. After the microneedle patch is inserted into the skin, it can remain on the skin for tens of minutes to tens of hours, allowing the microneedle array to completely dissolve and fully release the BH1 and adjuvant it contains.

[0087] Three weeks after the third immunization, blood was collected to separate serum and test its neutralizing titer against the novel coronavirus. Blood was collected from the tail vein of experimental mice by cutting the tail vein with a blade and allowing the blood to flow into centrifuge tubes without anticoagulants. Approximately 150 μL of blood was collected from each mouse and allowed to stand naturally at room temperature for 30-60 minutes to allow the blood to clot. After the blood had clotted, it was centrifuged at 5000 rpm for 10 minutes. The resulting pale yellow supernatant was the serum. The serum was carefully aspirated with a pipette, aliquoted, labeled, and then sent for testing.

[0088] The presence and relative concentration of antibodies in serum were determined using a mouse anti-2019-nCoV S-RBD protein IgG antibody detection kit (enzyme-linked immunosorbent assay). Before the experiment, to ensure accurate results, the kit and samples were allowed to equilibrate at room temperature for at least 30 minutes. The following steps were then followed for the detection experiment:

[0089] (1) Solution preparation: Take out the 20x concentrated washing solution from the kit, use a pipette to take out an appropriate amount and dilute it 20 times with deionized water;

[0090] (2) Numbering: Number the corresponding enzyme-labeled plates for the samples. At the same time, 3 negative control groups, 2 positive control groups and 1 blank control group need to be set up.

[0091] (3) Dilution: Dilute the sample 10 times in a blank 96-well plate and gently shake to mix thoroughly;

[0092] (4) Sample addition: According to the arrangement of each group of samples in the blank plate, use a pipette to transfer the samples in the blank plate to the ELISA plate;

[0093] (5) Incubation: After sealing the ELISA plate with the sealing film, place it in a constant temperature incubator at 37°C for 30 min;

[0094] (6) Washing the plate: Carefully peel off the sealing film, pour out the sample in the well, add 300uL of diluted washing solution to each well, soak for 30-60s and then pour out the liquid. Repeat 5 times, and finally drain the liquid in the well on the last time.

[0095] (7) Add enzyme: Pour the enzyme labeling reagent into the sample well, and use a pipette to add 100uL of enzyme labeling reagent to the other wells of the enzyme labeling plate except for the blank well;

[0096] (8) Incubation: The procedure is the same as step 5;

[0097] (9) Washing the plate: The operation is the same as step 6;

[0098] (10) Color development: Pour the color development solution into a new sample cell. First, add 50 μL of color development solution A to each well using a pipette, then add 50 μL of color development solution B. Gently tap the side of the microplate and shake to mix evenly. Then, keep it at 37°C in the dark for 15 minutes before taking it out.

[0099] (11) Measurement: Add 50 μL of stop solution to each well using a multi-channel pipette, and immediately detect the results using an ELISA reader. The ELISA reader is set to dual wavelengths, 450 nm and 650 nm, to measure the OD value of each well. The 650 nm wavelength is set for comparison.

[0100] To more clearly observe the experimental results, the detection results at a wavelength of 450nm were standardized according to the formula. The OD values ​​of each well after standardization are shown in the formula:

[0101] OD normalized =(OD-OD) blank ) / Mean(Neg)

[0102] Among them OD normalizedis the absorbance value after standardization, OD is the absorbance value measured by the microplate reader, OD blank is the absorbance value of the blank group, Mean(Neg) is the average absorbance value of the negative control group. That is, first zero the blank control group, then calculate the average values of the negative and positive control groups, and divide the values of each well after zeroing by the average value of the negative control group to obtain the standardized result. The neutralizing antibody titers of the experimental mice in each group are shown in Table 2.

[0103] Table 2 Neutralizing antibody titers of experimental mice in each group

[0104]

[0105]

[0106] Experimental Example 3

[0107] Take an equal amount of rhodamine B fluorescent dye to replace the drug active ingredient in Example 1, and prepare soluble microneedles S according to the method of Example 1. Take an equal amount of rhodamine B fluorescent dye to replace the drug active ingredient in Comparative Example 2, and prepare soluble microneedles D according to the method of Comparative Example 2.

[0108] Use soluble microneedles S and soluble microneedles D to pierce ex vivo porcine skin respectively. After 5 minutes, remove the microneedle patches. The microneedle arrays on the backing have all dissolved. Then, perform transverse slicing on the porcine skin respectively to determine the depth of microneedle penetration and the relative amount of rhodamine delivered at different depths.

[0109] In the slicing experiment, first place the pierced porcine skin at -18°C and freeze it overnight. Then place the frozen porcine skin on the freezing sample stage, put it on the ultra-fast freezing stage of the slicing machine, cover it with OCT compound, wait for it to freeze again, then fix the sample stage to the slicing machine, adjust the appropriate position and angle, slice the porcine skin, and set the thickness of each slice to 20um. Place the sliced porcine skin with OCT compound neatly on the adhesive-free slide. Wash it with 1×PBS buffer for 10 minutes before observation to remove the attached OCT compound on the slices. Finally, observe and image using a fluorescence inverted microscope.

[0110] Use the IVIS Lumina III software supporting the small animal fluorescence imaging system to count the average fluorescence intensity of each transverse slice, count the fluorescence intensity up to a depth of 400um, and plot the average fluorescence intensity at different skin depths, as Figure 4 and Figure 5 shown, where Figure 4 is the test result corresponding to soluble microneedle S, Figure 5 is the test result corresponding to soluble microneedle D.

[0111] From Figure 4 and Figure 5As can be seen, compared with soluble microneedles D, soluble microneedles S can deliver rhodamine to a deeper layer of skin, and the skin depth where high-intensity fluorescence values ​​appear is more concentrated. It is evident that the soluble microneedles of the present invention can significantly improve the depth of drug administration and significantly improve the local drug concentration, achieving precise drug administration at fixed points and in fixed quantities.

[0112] Experiment Example 4

[0113] This experimental example is used to verify the effect of freeze-drying preparation conditions on the stability of protein-based active substances.

[0114] A 150 mg / ml HA solution, a 1 g / ml PVP solution, and BSA powder were mixed in a ratio of 1 ml: 1 ml: 600 mg and then cured to obtain a microneedle cured block; a 150 mg / ml HA solution and a 1 g / ml PVP solution were mixed in a volume ratio of 1:1 and then cured to obtain a matrix cured block.

[0115] (1) The microneedle solidification block and matrix solidification block were stored at 4℃ for 3 days, and then diluted to prepare microneedle working solution and matrix solution, respectively. Simultaneously, fresh protein stock solution was prepared, and the three solutions were tested using Maurice (Protein Simple) CE-SDS assay. The results are as follows: Figure 6 As shown;

[0116] (2) The microneedle solidification block and matrix solidification block were stored at 4℃ for 7 days, and then diluted to prepare microneedle working solution and matrix solution, respectively. Simultaneously, fresh protein stock solution was prepared, and the three solutions were tested using Maurice (Protein Simple) CE-SDS assay. The results are as follows: Figure 7 As shown;

[0117] (3) The microneedle solidification block and matrix solidification block were stored at 40℃ for 3 days, and then diluted to prepare microneedle working solution and matrix solution, respectively. Simultaneously, fresh protein stock solution was prepared, and the three solutions were tested using Maurice (Protein Simple) CE-SDS assay. The test results are as follows: Figure 8 As shown;

[0118] (4) The microneedle solidification block and matrix solidification block were stored at 40℃ for 7 days, and then diluted to prepare microneedle working solution and matrix solution, respectively. Simultaneously, fresh protein stock solution was prepared, and the three solutions were tested using Maurice (Protein Simple) CE-SDS assay. The test results are as follows: Figure 9 As shown.

[0119] The microneedle working solution was prepared by diluting the microneedle solidification block to 1×Sample Buffer plus, with a final protein concentration of 1 mg / mL; replacing the microneedle solidification block with an equal amount of matrix solidification block, and preparing the matrix solution with an equal amount of 1×Sample Buffer plus; the protein stock solution contained 1 mg / mL BSA (bovine serum albumin).

[0120] The sample preparation method for Maurice CE-SDS assay is as follows: Add 2.4 μL of dissolved 25× internal standard and 7.5 μL of 75 mM NEM, vortex to mix, centrifuge, heat in a 70℃ metal bath for 10 min, place on ice for 5 min, vortex to mix again, centrifuge, take out 50 μL of sample, add to a 96-well plate, centrifuge at 1000×g for 10 min, observe each well after centrifugation, if there are still air bubbles, puncture them with a clean pipette tip. Store on ice for later use. The detection conditions are: sample load: 20 s, 4600 V; separation: 30 min, 5750 V. Follow the Maurice (Protein Simple) operating procedures.

[0121] Depend on Figure 6 and Figure 7 It can be seen that when stored at 4℃ for 3-7 days, the protein elution time of the microneedle-solidified block is similar to that of the original solution, indicating that the protein in the microneedle-solidified block has not changed; Figure 8 and 9 It can be seen that when stored at 40℃ for 3-7 days, the protein elution time of the microneedle-solidified block is earlier or later than that of the original solution, indicating that the protein in the microneedle-solidified block has changed; Figure 6 and 7 as well as Figure 8 and 9 It can be seen that compared with the storage condition at 4℃, the protein content is significantly reduced (the peak height is lower) under the storage condition at 40℃, which further illustrates that the protein has changed.

[0122] Therefore, the freeze-drying preparation conditions do not damage the stability of protein-based active substances and can improve the quality of the prepared soluble microneedles.

[0123] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A soluble microneedle, characterized in that, include: A needle hub and at least one needle body located on the needle hub, the needle body comprising a first intermediate section, a second intermediate section and a needle tip, the first intermediate section being located on the needle hub, the second intermediate section being located between the first intermediate section and the needle tip, the density of the second intermediate section being greater than the density of the needle tip and the first intermediate section, the needle tip containing a pharmaceutical active ingredient; the needle tip being formed by solidifying a solution of the pharmaceutical active ingredient. The active pharmaceutical ingredients are a vaccine and an aluminum adjuvant; The first intermediate segment is formed by curing a first polymer material slurry, wherein the first polymer material is selected from polyvinylpyrrolidone and / or sodium carboxymethyl cellulose; The second intermediate segment is formed by curing a second polymer material slurry, wherein the second polymer material is selected from hyaluronic acid and / or chitosan; The first intermediate segment also contains the nano-functional material MXene; The method for preparing the soluble microneedles comprises the following steps: composition: The active ingredient solution of the drug is placed in a microneedle mold, vacuum is applied, and the mold is solidified for the first time to obtain a microneedle mold loaded with needle tips. The second polymer material slurry is poured onto the microneedle mold containing the needle tip segment, vacuumed, and cured a second time to obtain the microneedle mold containing the needle tip segment and the second intermediate segment. The first polymer material slurry is poured onto a microneedle mold containing the needle tip segment and the second intermediate segment. Vacuum is applied, and the mold is cured for the third time to obtain a microneedle mold containing the needle tip segment, the second intermediate segment, and the first intermediate segment. The third polymer material slurry is poured onto a microneedle mold containing the tip segment, the second intermediate segment and the first intermediate segment, vacuumed, and cured for the fourth time to obtain the soluble microneedles; The first curing process is freeze-drying, with a temperature of -30 to 10°C and a time of 5 to 10 hours. The second curing process is freeze-drying, with a temperature of -50 to 10°C and a time of 5 to 10 hours. The third curing process is freeze-drying, with a temperature of -30 to 10°C and a time of 5 to 10 hours. The fourth curing process is freeze-drying, with a temperature of -30 to 10°C and a time of 5 to 10 hours.

2. The soluble microneedles according to claim 1, characterized in that, The needle hub is formed by curing a third polymer material slurry, which is selected from polyvinyl alcohol and / or polyvinyl acetate.

3. The soluble microneedles according to claim 2, characterized in that, The active pharmaceutical ingredient includes at least one of polypeptide drugs, protein drugs, antibodies, vaccines, probiotics, and nucleic acid drugs, and the solvent used in the active pharmaceutical ingredient solution is selected from physiological saline and / or buffer salt solution; And / or, based on the total weight of the needle tip segment, the weight percentage of the active pharmaceutical ingredient in the needle tip segment is 10-20%.

4. The soluble microneedles according to claim 3, characterized in that, The weight ratio of the vaccine to the aluminum adjuvant is 1:(1-2).

5. The soluble microneedles according to claim 4, characterized in that, The vaccine is a recombinant subunit protein of the novel coronavirus, and the aluminum adjuvant is aluminum hydroxide adjuvant and / or aluminum phosphate adjuvant.

6. The soluble microneedles according to claim 1, characterized in that, Based on the total weight of the first intermediate segment, the weight percentage of the nanofunctional material in the first intermediate segment is 20-30%.

7. The soluble microneedles according to claim 1, characterized in that, The concentration of the active pharmaceutical ingredient in the solution is 0.5–5 μg / μl, and the solvent used is selected from physiological saline and / or buffer solution. And / or, the content of the first polymer material in the first polymer slurry is 900-1100 μg / μl, and the solvent used is deionized water; And / or, the content of the second polymer material in the second polymer material slurry is 140-160 μg / μl, and the solvent used is deionized water; And / or, the content of the third polymer material in the third polymer material slurry is 450-550 μg / μl, and the solvent used is deionized water; And / or, the microneedle mold is a hydrophobic mold.

8. The soluble microneedles according to claim 1, characterized in that, The amount of the active pharmaceutical ingredient solution used is 5–10 μl; And / or, the amount of the first polymer material slurry is 2 to 8 μl; And / or, the amount of the second polymer material slurry used is 5 to 20 μl; And / or, the amount of the third polymer material slurry is 300-500 μl.

9. The soluble microneedles according to claim 1, characterized in that, Before pouring the first polymer slurry onto the microneedle mold containing the tip segment and the second intermediate segment, the method further includes: Nanomaterials are added to the first polymer slurry.

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