Microneedles, methods of making, microneedle patches, and uses

Abstract

The application discloses a microneedle, a preparation method, a microneedle patch and application. The microneedle comprises a shell of a cross-linked hydrophilic polymer, a negatively charged substance filled in a cavity surrounded by the shell, and verteporfin arranged on the outer surface of the shell and / or the shell layer. The cross-linked hydrophilic polymer comprises a plurality of inflammation-responsive linkers. The microneedle patch containing the microneedle is programmed to promote faster wound healing, repair normal skin function, prevent or reduce scar formation, and has a significant alleviating effect on the formed hyperplastic scar.

Description

TECHNICAL FIELD

[0001] The present application relates to a microneedle, a preparation method, a microneedle patch and an application. BACKGROUND

[0002] The repair of wounds has always been the focus of medical development and clinical treatment. The classic wound healing can be divided into four stages: hemostasis period, inflammation period, proliferation period and remodeling period, and each stage is accompanied by immune regulation. Immune regulation disorder often leads to aggravated inflammation, prolonged inflammation period, and even formation of biofilm due to bacterial infection, so that the wound cannot heal, and finally leads to impaired skin function and scar formation. Traditional wound treatment programs mainly regulate the immune microenvironment in the inflammation period, however, biological barriers such as bacterial biofilm limit the therapeutic effect of drugs.

[0003] Microneedle systems have been increasingly applied to transdermal drug delivery to treat various diseases such as cancer, obesity, infection, etc. At present, there are various types and functions of microneedles, but none of them can simultaneously regulate the immune system at different stages of wound healing and repair skin function, resulting in slow or difficult wound healing and scar formation after wound healing. Therefore, there is an urgent need for a new microneedle patch that can regulate each stage of wound healing, promote faster wound healing, repair normal skin function, and prevent or reduce scar formation. SUMMARY

[0004] The technical problem solved by the present application is to overcome the defects of existing microneedles that cannot regulate the immune system at different stages of wound healing and repair skin function, resulting in slow or difficult wound healing and scar formation after wound healing. The present application provides a microneedle, a preparation method, a microneedle patch and an application. The microneedle patch of the present application is programmed to promote faster wound healing, repair normal skin function, prevent or reduce scar formation, and significantly reduce the effect of existing hypertrophic scars.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a microneedle comprising a shell of crosslinked hydrophilic polymer, a negatively charged substance filled in a cavity surrounded by the shell, and verteporfin provided in the outer surface and / or shell layer of the shell; the crosslinked hydrophilic polymer comprises a plurality of inflammation-responsive linkers.

[0007] In the present application, the inflammation-responsive linker refers to a chemical bond that is cleavable under inflammation-related conditions, which include but are not limited to one or more of the following conditions: increase in reactive oxygen species (ROS) concentration, pH lower than 6, hypoxia, and increase in the concentration of inflammation-related enzymes and / or small molecules, preferably increase in reactive oxygen species (ROS) concentration.

[0008] wherein, when the inflammation-related condition is an increase in reactive oxygen species (ROS) concentration, the inflammation-responsive linker is preferably one or more of aryl boronic ester, phenyl boronic acid, phenyl boronic ester, thioether, seleno, telluro, thioketone, and oxalic acid aryl ester.

[0009] In a preferred embodiment of the present application, the inflammation-responsive linker is a group formed by cross-linking reaction of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA) and polyvinyl alcohol (PVA).

[0010] In the present application, it is understood by those skilled in the art that the cross-linked hydrophilic polymer refers to a cross-linking reaction of a hydrophilic polymer and a cross-linking agent to form the inflammation-responsive linker.

[0011] wherein, the hydrophilic polymer can be conventional in the art, preferably one or more of polyvinyl alcohol (PVA), cellulose, hyaluronic acid, polylysine, and polyethylene glycol, more preferably PVA, and more preferably PVA type 1795.

[0012] The weight average molecular weight of the PVA can be conventional in the art, preferably 70-80 kDa, and more preferably 75 kDa.

[0013] The alcoholysis degree of the PVA can be conventional in the art, preferably 92-94%.

[0014] wherein, the cross-linking agent can be a cross-linking agent capable of forming the inflammation-responsive linker as described above, and is preferably N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA).

[0015] wherein, the mass ratio of the hydrophilic polymer and the cross-linking agent is preferably set such that the shell degrades within 10-30 h, and more preferably such that the shell degrades within 20-28 h, for example 24 h.

[0016] The inventors have found through creative research that degradation of the microneedle shell within the above-mentioned time can better achieve targeted regulation of healing by verteporfin and negatively charged gel at different stages of wound healing, achieving better wound healing and preventing scar formation.

[0017] wherein, in some preferred embodiments, the mass ratio of the hydrophilic polymer and the cross-linking agent is (1-3):1, and more preferably 2:1.

[0018] In some preferred embodiments of the present application, the cross-linked hydrophilic polymer is obtained from a cross-linking reaction of PVA and N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA), wherein the mass ratio of the PVA to the TSPBA is 2:1.

[0019] In the present application, the thickness of the shell is preferably set to allow the shell to degrade within 10-30h, more preferably within 20-28h, for example 24h.

[0020] In some preferred embodiments of the present application, the thickness of the shell is 10-20μm.

[0021] In some preferred embodiments of the present application, the thickness of the shell is 10-20μm, and the cross-linked hydrophilic polymer is obtained from a cross-linking reaction of PVA and N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA), wherein the mass ratio of the PVA to the TSPBA is 2:1.

[0022] In the present application, the verteporfin is conventional in the art, and has the molecular formula of C 41 H 42 N4O8, and the chemical name of 9-methyl (I) and 13-methyl (II) trans-(±)-18-ethenyl-4,4a-dihydro-3,4-bis(methylcarbonyl)-4a,8,14,19-tetramethyl-23H,25H-benzoporphyrin-9,13-dipropionate. The chemical formula of verteporfin can be shown as formula (1) or formula (2) as follows:

[0023]

[0024]

[0025] In the present application, the verteporfin is preferably loaded on the outer surface of the shell.

[0026] In the present application, it is known to those skilled in the art that part of the inflammatory factors are positively charged, and the negatively charged substance can adsorb and neutralize the inflammatory factors after exposure.

[0027] In the present application, the negatively charged substance is preferably in the form of a gel.

[0028] In the present application, the negatively charged substance can be a substance with a negative charge that can adsorb and neutralize inflammatory factors, and preferably includes a negatively charged monomer and a non-inflammatory response linker connecting the negatively charged monomer.

[0029] The negatively charged monomer can be conventional in the art, preferably one or more selected from heparin, heparin derivatives and glycosaminoglycans, more preferably heparin.

[0030] The heparin is a conventional mucopolysaccharide sulfate in the art, which is composed of glucosamine, L-iduronic acid, N-acetylglucosamine and D-glucuronic acid alternately, and the weight average molecular weight is 1200-40000.

[0031] The non-inflammatory response linker can be conventional in the art, which is not cleaved under the inflammatory condition as described above, but can be cleaved under other conditions, preferably a photo-responsive linker.

[0032] The photo-responsive linker can be conventional in the art, which is cleaved under light irradiation, preferably a group obtained by cross-linking reaction of the double bond modified negatively charged monomer and a photo-initiator under light irradiation.

[0033] When the negatively charged substance contains a non-inflammatory response linker such as a photo-responsive linker, it is not degraded under the inflammatory condition, but can be degraded under external conditions such as light irradiation, so that the negatively charged substance has enhanced flowability after degradation, and is more easily removed.

[0034] The double bond modified negatively charged monomer can be obtained by reaction of the negatively charged monomer and a double bond modifier.

[0035] The double bond modifier can be conventional in the art, preferably ethylene glycol dimethacrylate (EGDMA).

[0036] The double bond modification rate in the double bond modified negatively charged monomer can be conventional in the art, preferably 15-30%, more preferably 20%, the double bond modification rate referring to the molar ratio of grafted double bonds to total graftable carboxyl groups.

[0037] The mass ratio of the double bond modifier to the negatively charged monomer can be conventional in the art, preferably 0.5-4%, more preferably 1-2%.

[0038] In the photo-responsive linker, the photo-initiator can be conventional in the art, preferably one or more selected from photo-initiator 2959, photo-initiator MBF, TPO photo-initiator, 907 photo-initiator, 184 photo-initiator, ITX photo-initiator, more preferably photo-initiator 2959.

[0039] In the photo-responsive linker, the mass ratio of the photo-initiator to the negatively charged monomer can be conventional in the art, preferably 0.5-4%, more preferably 1-2%.

[0040] The light in the photoresponsive linker can be light of a wavelength conventional in the art, preferably ultraviolet light.

[0041] In a preferred embodiment of the present application, the negatively charged substance is obtained by cross-linking reaction of heparin and EGDMA after reaction, and then cross-linking reaction of the product with photoinitiator 2959 under ultraviolet light irradiation, the mass ratio of the EGDMA to the heparin is 1-2%, and the mass ratio of the photoinitiator 2959 to the heparin is 1-2%.

[0042] In the present application, the mass ratio of the hydrophilic polymer, the negatively charged substance and the verteporfin is preferably (0.15-0.35):1:(0.0008-0.0025), more preferably 0.26:1:0.0012.

[0043] In a preferred embodiment of the present application, the hydrophilic polymer is PVA, the negatively charged substance comprises heparin, and the mass ratio of the PVA, the heparin and the verteporfin is 0.26:1:0.0012.

[0044] In the present application, the shape of the microneedle can be conventional in the art, preferably a conical microneedle.

[0045] The length of the conical microneedle can be conventional in the art, preferably 400-800 μm, more preferably 500-700 μm, and further more preferably 600 μm.

[0046] The base radius of the conical microneedle can be conventional in the art, preferably 200-400 μm, and more preferably 300 μm.

[0047] The tip diameter of the conical microneedle can be conventional in the art, preferably 7-15 μm, and more preferably 10 μm.

[0048] The present application also provides a preparation method of the microneedle as described above, comprising the following steps:

[0049] S1: filling the negatively charged substance into the cavity of the shell;

[0050] S2: applying the verteporfin to the outer surface of the shell;

[0051] The order of S1 and S2 is not limited.

[0052] In the present application, preferably, S1 is performed first and then S2 is performed.

[0053] In S1, the filling can be performed by a method conventional in the art, preferably vacuum filling.

[0054] In S1, when the negatively charged substance includes a negatively charged monomer and a photoresponsive linker connecting the negatively charged monomer, preferably, the negatively charged monomer and the double bond modifier are mixed, and then the photoinitiator is added, the obtained mixture is filled into the cavity of the shell, and then cross-linked by light irradiation to form a gel.

[0055] Preferably, the step of drying is performed before the light irradiation.

[0056] Preferably, the step of drying is performed naturally without light.

[0057] In S2, the method of application can be conventional in the art, and preferably, a solution containing verteporfin is applied to the outer surface of the shell, and then dried.

[0058] As understood by those skilled in the art, the solution of verteporfin applied to the shell has permeability, and when applied to the outer surface of the shell, part of the verteporfin will eventually penetrate into the shell layer.

[0059] Preferably, the solvent in the solution containing verteporfin is tetrahydrofuran.

[0060] Preferably, the concentration of verteporfin in the solution containing verteporfin is 1-5 g / L, and more preferably 3 g / L.

[0061] Preferably, the application is dropwise.

[0062] In the present application, the preparation method of the shell can be conventional in the art, and preferably, the hydrophilic polymer and the crosslinking agent containing the inflammation-responsive linker are mixed and then filled into a microneedle mold, and after cross-linking reaction, the shell is obtained.

[0063] Preferably, the hydrophilic polymer is dissolved in water to form a solution of the hydrophilic polymer, and then mixed with the crosslinking agent.

[0064] The concentration of the hydrophilic polymer in the solution of the hydrophilic polymer is 1-5%, and more preferably 2%, wherein the percentage is the mass percentage of the hydrophilic polymer in the solution of the hydrophilic polymer.

[0065] Preferably, the crosslinking agent is first dissolved in water to form a solution containing the crosslinking agent, and then mixed with the hydrophilic polymer.

[0066] The concentration of the crosslinking agent in the solution of the crosslinking agent can be conventional in the art, and preferably, 1-5%, and more preferably 2%, wherein the percentage is the mass percentage of the crosslinking agent in the solution of the crosslinking agent.

[0067] The present application also provides a microneedle patch comprising a plurality of microneedles as described above.

[0068] In the present application, the microneedle patch preferably further comprises a backing, and the microneedles are arranged on one side of the backing.

[0069] Preferably, the material of the backing is a negatively charged substance as described above.

[0070] Preferably, the other side of the backing is further covered with a sticker or tape.

[0071] In the present application, the arrangement of the microneedles can be conventional in the art, preferably in an array, more preferably in a matrix.

[0072] Preferably, the matrix arrangement is 32x32.

[0073] Preferably, the center-to-center distance of the array arrangement can be conventional in the art, preferably 400-800 μm, more preferably 600 μm.

[0074] In the present application, the total content of verteporfin in one microneedle patch is preferably 80-160 μg, more preferably 100-140 μg, and further more preferably 120 μg.

[0075] In the present application, the content of the negatively charged substance in one microneedle patch is preferably 0.05-0.15 g, more preferably 0.1 g.

[0076] In the present application, the content of the hydrophilic polymer in one microneedle patch is preferably 0.015-0.035 g, more preferably 0.0267 g.

[0077] The present application also provides the use of a microneedle or a microneedle patch as described above in the preparation of a wound healing medicament.

[0078] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, thereby obtaining various preferred examples of the present application.

[0079] The reagents and raw materials used in the present application are commercially available.

[0080] The positive progress effect of the present application is that the microneedles and the microneedle patches containing the microneedles of the present application can promote faster healing of the wound surface, reduce the wound surface area to less than 25% on the 7th day, and basically completely heal in 14 days under light conditions; repair the normal function of the skin, prevent or reduce the formation of scars; have a remarkable treatment effect on diabetic wound surfaces, promote faster and scar-free healing of the wound surface; and have a remarkable alleviating effect on the formed hypertrophic scars, and almost approach normal skin after treatment. BRIEF DESCRIPTION OF DRAWINGS

[0081] Figure 1 Structure schematic of core-shell microneedle patch of Example 1 (front view);

[0082] Figure 2 Structure schematic of core-shell microneedle patch of Example 1 (top view);

[0083] Figure 3 Flow chart of preparation method of core-shell microneedle patch of Example 1;

[0084] Figure 4 Photo of core-shell microneedle patch of Example 1;

[0085] Figure 5 Graph of mechanical property test results of core-shell microneedle patch of Example 1;

[0086] Figure 6 Graph of early inhibition of biofilm formation by MRSA by PF-MNs patch of Example 1;

[0087] Figure 7 Graph of late destruction of biofilm formed by MRSA by PF-MNs patch of Example 1;

[0088] Figure 8 Graph of wound treatment of healthy mice by PF-MNs patch of Example 1;

[0089] Figure 9 Graph of IFN-γ concentration at wound site of healthy mice on day 3 after different treatments;

[0090] Figure 10 Graph of relative contents of M1 macrophages, M2 macrophages and regulatory T cells at wound site on day 3 after different treatments;

[0091] Figure 11 Graph of epithelial thickness of wound of healthy mice on day 7 after different treatments;

[0092] Figure 12 Graph of treatment process of diabetic mice after excision of skin on back infected by MRSA;

[0093] Figure 13 Photos of wounds of diabetic mice on day 3, 7 and 14 after different treatments;

[0094] Figure 14 Graph of bacterial content at wound site of diabetic mice on day 3 after different treatments;

[0095] Figure 15 Graph of contents of different inflammatory factors at wound site of diabetic mice on day 3 after different treatments;

[0096] Figure 16 Figure 8. Relative amount of Ml macrophages, M2 macrophages and regulatory T cells in the wound of diabetic mice on day 3 after different treatments;

[0097] Figure 17 Figure 9. Epithelial thickness of the wound of diabetic mice on day 7 after different treatments;

[0098] Figure 18 Figure 10. Hair follicle number of diabetic mice on day 14 after different treatments;

[0099] Figure 19 Figure 11. Ratio of collagen type I and collagen type III of diabetic mice on day 14 after different treatments;

[0100] Figure 20 Figure 12. Process of the treatment of rabbit ear wound by PF-MNs patch;

[0101] Figure 21 Figure 13. Pictures of the rabbit ear wound on day 0, 5, 10, 20 and 30 after different treatments;

[0102] Figure 22 Figure 14. Thickness of the hypertrophic scar of the rabbit ear on day 30 after different treatments of the rabbit ear wound;

[0103] Figure 23 Figure 15. Collagen content of the rabbit ear tissue after different treatments of the rabbit ear wound;

[0104] Figure 24 Figure 16. Ratio of collagen type I and collagen type III in the scar tissue on day 30 after different treatments of the rabbit ear wound;

[0105] Figure 25 Figure 17. Process of the treatment of the rabbit ear scar by microneedle patch plus light irradiation;

[0106] Figure 26 Figure 18. Pictures of the rabbit ear scar on day 0, 7, 15, 25 and 35 after different treatments;

[0107] Figure 27 Figure 19. Thickness of the hypertrophic scar of the rabbit ear on day 15 after different treatments of the rabbit ear scar;

[0108] Figure 28 Figure 20. Collagen content of the scar tissue on day 15 after different treatments of the rabbit ear scar;

[0109] Figure 29 Figure 21. Ratio of collagen type I and collagen type III in the scar tissue on day 15 after different treatments of the rabbit ear scar.

[0110] Reference signs

[0111] 1-Microneedle; 11-Shell; 12-Photocrosslinked heparin; 13-Verteporfen; 2-Cloaking. Detailed Implementation

[0112] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0113] Heparin sodium (Mw 15 k-19 kDa), verteporfen (97%), 4-bromomethylphenylboronic acid (98%), ampicillin, streptozotocin (98%, STZ), and crystal violet (98%) were purchased from Maclean's; PVA (Mw ~ 75 kDa, degree of hydrolysis 92.0-94.0%) from Example 1, PVA (Mw ~ 47 kDa, degree of hydrolysis 99%) from Comparative Example 4, photoinitiator 2959, and EGDMA (98%) were purchased from Aladdin. N,N,N',N'-tetramethyl-1,3-ethylenediamine (≥99%), 2-aminoethyl methacrylate hydrochloride (90%, AEMA), and polyethylene glycol (PEG) were purchased from Sigma.

[0114] Example 1

[0115] like Figure 3 As shown, the preparation method of the microneedle patch in this embodiment includes:

[0116] S1: First, prepare an aqueous solution (2wt%) of polyvinyl alcohol (PVA) (Mw ~ 75kDa, degree of hydrolysis 92.0-94.0%) and N... 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 N 1 N 3 N 3 A 2 wt% aqueous solution of tetramethylpropane-1,3-diamine (TSPBA) was mixed at a mass ratio of 2:1 and then coated onto a hollow microneedle mold (32×32 matrix, conical hollow microneedles, 600 μm in length, 300 μm in radius of the base circle, 10 μm in diameter at the tip, and 600 μm in center-to-center distance). The microneedles were then vacuum-filled and allowed to air dry overnight to obtain a microneedle shell with a thickness of 20 μm and a PVA content of 0.0267 g.

[0117] S2: Then 20 parts of heparin and 0.3 parts of ethylene glycol dimethacrylate (EGDMA) were dissolved into 80 parts of water, and then 0.4 parts of photoinitiator 2959 was added, and after mixing, a mixed solution was formed, which was filled into the cavity of the above-mentioned microneedle shell, and after natural drying in the dark, it was crosslinked by ultraviolet light, and the inner core of the photo-crosslinked heparin was obtained. The overflowed photo-crosslinked heparin forms a lining. Among them, the amount of heparin is 0.1 g. By nuclear magnetic resonance characterization, the double bond modification rate of heparin is 20%.

[0118] S3: Finally, 120 μg of verteporfin was dissolved in 40 μL of tetrahydrofuran to form a verteporfin solution, which was uniformly dropped onto the microneedle shell. After the evaporation of tetrahydrofuran, the partial verteporfin penetrated into the shell layer from the outer surface of the shell, and the core-shell microneedle patch (PF-MN) of the present embodiment was demolded, and the structure is as shown in Figure 1 and Figure 2 . Figure 1 is a front view, Figure 2 is a top view.

[0119] As shown in Figure 1 , the core-shell microneedle patch of the present embodiment includes a lining 2 and microneedles 1 arranged in an array on one side of the lining 2. The microneedle 1 includes a TSPBA-crosslinked PVA shell 11, and photo-crosslinked heparin 12 filled in the cavity surrounded by the shell. The photo-crosslinked heparin is a double-bond modified heparin obtained by reacting heparin and a double-bond modifier ethylene glycol dimethacrylate (EGDMA), and the product obtained by the crosslinking reaction of the double-bond modified heparin and a photoinitiator 2529 under ultraviolet light irradiation; The shell layer and / or the outer surface of the shell 11 are provided with verteporfin 13.

[0120] The above-mentioned core-shell microneedle patch can be cut to a size suitable for a wound when in use, and then a medical adhesive tape (such as a 3M adhesive tape) is attached to the other side of the lining, and then covered on the wound, and the microneedle side is pressed into the subcutaneous tissue and fixed, and then the remaining microneedle patch is removed after a specified time.

[0121] The photo of the core-shell microneedle patch (PF-MN) of the present embodiment is shown in Figure 4 .

[0122] Comparative Example 1

[0123] Compared with Example 1, S3 is not performed, and the blank PF-MN is obtained after demolding.

[0124] Comparative Example 2

[0125] Subsequently, 20 parts of heparin and 0.3 parts of ethylene glycol dimethacrylate (EGDMA) were dissolved into 80 parts of water, 0.4 parts of a photoinitiator 2959 was added, and the mixture was uniformly mixed to form a mixed solution, which was vacuum filled into a microneedle mold (32x32 matrix, conical solid microneedles, length 600 μm, base circle radius 300 μm, tip diameter 10 μm, center distance 600 μm), and then naturally dried in the dark and crosslinked by ultraviolet light. After the shell was removed, the solid heparin microneedles HP-MNs were obtained.

[0126] Comparative Example 3

[0127] Compared with Comparative Example 2, the heparin therein was replaced by polyethylene glycol PEG, and EGDMA was not added, and the other steps were the same as those in Comparative Example 2, to obtain the PEG microneedle patch PEG-MNs.

[0128] Comparative Example 4

[0129] Compared with Example 1, only the PVA with an alcoholysis degree of 92.0-94.0% therein was replaced by PVA with an alcoholysis degree of 99%, and the other steps and conditions were the same, to obtain the non-degradable microneedle patch NMNs.

[0130] Comparative Example 5

[0131] 10 μL of a 3 mg / mL verteporfin / PBS solution (VP group) (PBS as a solvent).

[0132] Comparative Example 6

[0133] 10 μL of a 200 μg / mL ampicillin (antibiotic group).

[0134] Comparative Example 7

[0135] 10 μL of PBS (control group).

[0136] Effect Example

[0137] 1. Mechanical property test

[0138] A microneedle strength tester was used to measure the mechanical properties of the microneedles to ensure that the microneedles have sufficient mechanical strength to penetrate the skin. The specific method was as follows: 1 core-shell microneedle obtained in Example 1 and 1 hollow microneedle obtained in S1 of Example 1 were fixed on a flat metal block of the instrument as samples, and then a metal block above moved towards the microneedles at a predetermined speed, and the force-displacement curve was recorded by a computer. The change in the slope of the curve indicated the fracture of the microneedles, and the results are shown in Figure 5 Figure 5 ​It can be seen that with the increase of displacement, the force borne by the microneedle also increases, and the slope of the force-displacement curve changes in the middle time, tends to be flat, which is the force of the microneedle fracture, and the force represents the mechanical strength of the microneedle. Figure 5 It can be seen that the mechanical strength of the core-shell microneedle of the embodiment 1 of the present application is 2.7N, and the mechanical strength of the hollow microneedle is 0.5N, the core-shell microneedle can penetrate the skin, and has good mechanical strength.

[0139] 2, Effect of PF-MNs on biofilm formation

[0140] 1mL, 10 7 The staphylococcus aureus MRSA liquid of 1mL, 10 CFU / mL LB liquid medium is added to a 24-well plate with a round cover glass, and after 6h culture at 37℃, the culture medium is replaced with the following: group 1 is a control group (LB medium), group 2 is an illumination group (LB medium and 690nm illumination for 10min), group 3 is an antibiotic group (adding 1mL, 2μg / mL ampicillin LB medium), group 4 is a VP+L group (adding 1mL, 30μg / mL verteporfin LB medium and 690nm illumination for 10min), group 5 is a PF-MNs group (PF-MNs and LB medium), and group 6 is a PF-MNs+L group (PF-MNs and LB medium and 690nm illumination for 10min). After treatment, the plate is continued to be cultured at 37℃ for 36h, then the supernatant is discarded, PBS is washed for 3 times, air-dried, methanol is fixed for 10min, the methanol is discarded, air-dried, 500μL, 1g / L crystal violet solution is added for dyeing for 10min, PBS is washed for 3 times, air-dried, photographed, dissolved in 500μL, 95% ethanol solution, and the absorbance at 570nm is read by an enzyme-labeled instrument to represent the degree of damage of the biofilm. At the same time, the biofilm after the above treatment steps is dyed with a bacterial live and dead dyeing kit for 20min, and then photographed by a confocal microscope and analyzed for the fluorescence intensity of the living bacteria by image J software. Figure 6 The early inhibition of MRSA biofilm formation by each group is shown (n=3). Figure 6 It is shown that the early inhibition of biofilm by the PF-MNs group and the PF-MNs+L group is significantly better than that of other groups, and has better early inhibition of biofilm, and the early inhibition effect of the PF-MNs+L group is the best.

[0141] Figure 7 The late damage of MRSA biofilm by the microneedle is shown (n=3). The late biofilm needs to be cultured for 48h first, and then the same treatment method as the above grouping is used, and after 12h, crystal violet and DMAO are used for dyeing. The dyeing of crystal violet and DMAO on the biofilm shows that the late damage of the biofilm by the PF-MNs under illumination has an ideal effect.Figure 6-7 In particular, data are expressed as mean ± standard deviation, and statistical significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0142] 4. Test of the therapeutic effect of PF-MNs patches on wounds of healthy mice

[0143] Figure 8 Figure 9 Figure 10 and Figure 11 Figures 1-4 show the therapeutic effect of PF-MNs patches on an acute wound model of healthy mice. In particular, the test was performed as follows: after the mice were anesthetized with isoflurane, a punch was used to create two 8-mm circular wounds on the back skin, and after the skin of the mice was excised, 10 μL of PBS (control group), 10 μL of a 3 mg / mL verteporfin / PBS solution (VP group), blank PF-MNs, and PF-MNs were administered for treatment. Figure 8 Figures 1-4 show the therapeutic effect of PF-MNs patches on an acute wound model of healthy mice. In particular, the test was performed as follows: after the mice were anesthetized with isoflurane, a punch was used to create two 8-mm circular wounds on the back skin, and after the skin of the mice was excised, 10 μL of PBS (control group), 10 μL of a 3 mg / mL verteporfin / PBS solution (VP group), blank PF-MNs, and PF-MNs were administered for treatment.

[0144] Table 1

[0145] Group / Days Control VP Blank PF-MNs PF-MNs 3 days / % 110.90 112.77 90.83 81.52 7 days / % 67.76 69.93 46.51 38.83 14 days / % 21.70 15.65 15.40 16.29

[0146] Table 1 shows that the blank PF-MNs group and the PF-MNs group can promote wound healing, and the wound area is significantly reduced, and the PF-MNs have a better effect on wound healing than the blank PF-MNs on the 3rd day and the 7th day.

[0147] The skin at the wound site on the 3rd day was taken in 0.5 mL of a PBS solution, and the skin was treated using a tissue homogenizer, and then centrifuged (10,000 rpm, 10 min, 4°C) to take the supernatant for ELISA analysis of the concentration of IFN-γ. Figure 9 Figure 6 shows the concentration of IFN-γ at the wound site on the 3rd day after different treatments (n = 3), and it can be seen that the PF-MNs group has the lowest content of IFN-γ, and the heparin in the PF-MNs significantly reduces the content of IFN-γ in the wound by adsorption, which will be conducive to the healing of the wound.

[0148] ​​The skin at the wound site on day 3 was taken and cut into pieces, then digested with 5 mL of collagenase II at 37 °C for 30 min, and then the digestion reaction was terminated by adding FBS (fetal bovine serum) buffer (2% FBS dissolved in PBS). The solution was then centrifuged (350 g, 5 min, 4 °C) and filtered with a 40 pm mesh to obtain a single cell solution, which was then subjected to flow cytometry analysis, and the results are shown in Figure 10 . Figure 10 The relative contents of Ml macrophages, M2 macrophages and regulatory T cells at the wound site on day 3 after different treatments are shown (n = 4). In the PF-MNs group, the content of Ml macrophages was significantly reduced, and the contents of M2 macrophages and regulatory T cells were significantly increased, and were significantly better than those in other groups, which was most conducive to wound healing.

[0149] The intact skin at the wound site on day 7 was fixed in paraformaldehyde, and then hematoxylin-eosin staining (H&E staining) was used to analyze the tissue and measure the epithelial thickness of the wound, and the results are shown in Figure 11 . Figure 11 The epithelial thickness of the wound on day 7 after different treatments is shown (n = 3). The epithelial thickness was significantly increased after PF-MNs treatment, and a continuous and complete epithelium was formed, indicating an ideal treatment effect, which was the best in each group. In Figure 9-11 , the data are expressed as mean ± standard deviation, and the statistical significance was determined by one-way analysis of variance. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0150] 5. Test of the treatment effect of PF-MNs patches on wounds of diabetic mice

[0151] Figure 12 , Figure 13 , Figure 14 , Figure 15 , Figure 16 , Figure 17 and Figure 18 show the treatment effect of PF-MNs in a STZ-induced diabetic mouse model infected with methicillin-resistant Staphylococcus aureus MRSA. STZ (10 mg / mL dissolved in a citric acid buffer at pH 4.4) was injected intraperitoneally (50 mg / kg) into BALB / c mice after fasting for 12 h, for 5 consecutive days, and diabetic mice (blood glucose value greater than 16.7 mmol / L) were obtained one week later. Two 8 mm circular skin excision wounds were obtained on the back of the diabetic mice after anesthesia with isoflurane, and 10 7 CFU of MRSA / PBS solution (10 μL, 10 9 CFU / mL) was added, and two days later, the model was obtained. Figure 12 The treatment process after the excised skin on the back of the mouse was infected with MRSA is shown.Figure 13 Pictures of the wounds at day 3, 7 and 14 after treatment with 10 μL PBS (control), 10 μL of 200 μg / mL ampicillin (antibiotic), 10 μL of 3 mg / mL verteporfin solution at 690 nm for 10 min (VP+L), NMNs patch at 690 nm for 10 min (NMN+L), PF-MNs and PF-MNs+L (PF-MNs patch at 690 nm for 10 min) are shown. Table 2 is the percentage of the wound area at day 3, 7 and 14 relative to day 0 after treatment with the above-mentioned treatments.

[0152] Table 2

[0153]

[0154] As can be seen from Table 2, at day 3 and day 7, the wound area of the PF-MNs group and the PF-MNs+L group are significantly smaller than the other groups, and the wound area of the PF-MNs+L group is smaller than that of the PF-MNs, indicating that PF-MNs have a very good effect on promoting wound healing in diabetic mice, and they have a better effect on promoting wound healing under visible light irradiation.

[0155] The skin at the wound site at day 3 was taken in 0.5 mL PBS solution, and the sample after treatment with a tissue homogenizer was divided into two parts, one part was used for bacterial colony counting (10 μL was taken after gradient dilution and added to LB agar solid medium at 37°C overnight), and the other part was centrifuged (10,000 rpm, 10 min, 4°C) to take the supernatant and analyze the concentration of each factor using a multi-factor kit. Figure 14 The bacterial content at the wound site at day 3 (n=3) is shown. The bacterial activity of the PF-MN+L group is only 18.6% of the control group, showing an ideal antibacterial effect.

[0156] Figure 15 The content of different inflammatory factors at the wound site at day 3 is shown, including TNF-α, IFN-γ, MCP-1, IL-1β and IL-6 (n=3). Various inflammatory factors were significantly reduced after microneedle treatment. The skin at the wound site at day 3 was taken and cut into pieces, then 5 mL of collagenase II was used to digest at 37°C for 30 min, then FBS buffer (2% FBS solution in PBS) was added to terminate the digestion reaction, then the solution was centrifuged (350g, 5 min, 4°C) and filtered with a 40 μm screen to obtain a single cell solution, followed by flow cytometry analysis.

[0157] Figure 16The relative amount of M1 macrophages, M2 macrophages and regulatory T cells at the wound site on day 3 after different treatments (n=5) is shown. The amount of M1 macrophages is significantly reduced, and the amount of M2 macrophages and regulatory T cells is significantly increased, both of which are beneficial to the healing of the wound, by flow cytometry analysis.

[0158] The intact skin at the wound site on day 7 and day 14 was fixed in paraformaldehyde, and then the tissue was analyzed by H&E staining, and the epithelial thickness of the wound was measured. Figure 17 The epithelial thickness of the wound on day 7 after different treatments (n=3) is shown. The epithelial thickness is significantly increased after microneedle treatment, and a continuous and complete epithelium is formed, indicating an ideal treatment effect.

[0159] Figure 18 The number of hair follicles on day 14 after treatment (n=3) is shown. As an indicator of scar formation, the number of hair follicles in the treatment group containing verteporfin is significantly increased, and the number of hair follicles in the PF-MNs group and the PF-MNs+L group is more than that in the VP+L group, indicating that PF-MNs can better inhibit the formation of scars than verteporfin solution.

[0160] The intact skin at the wound site on day 14 was fixed in paraformaldehyde, and then the tissue was analyzed by Sirius red staining. Figure 19 The ratio of type I collagen to type III collagen on day 14 (n=3) is shown. After Sirius red staining, type I collagen can be stained red, and type III collagen is green. Type I collagen is more present in hypertrophic scars, while type III collagen is more present in normal tissue. Therefore, the significant decrease in the ratio in the PF-MNs group and the PF-MNs+L group, and the lower ratio than that in the VP+L group, shows an ideal anti-hypertrophic scar treatment effect, and the effect is better than that of verteporfin solution. Figure 14-19 In the above, the data is expressed as mean ± standard deviation, and the statistical significance is determined by one-way analysis of variance. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0161] 6. Treatment effect of PF-MNs patches in New Zealand white rabbit ear scar models

[0162] Figure 20 , Figure 21 , Figure 22 , Figure 23 and Figure 24 The treatment effect of PF-MNs patches in New Zealand white rabbit ear scar models is shown. Rabbits anesthetized with pentobarbital (30 mg / kg) were given three 8 mm circular wounds on the inner side of the ear with a punch, and the fascia layer was removed, and then treated. Figure 20The process of the treatment of the rabbit ear wound by the PF-MNs patch is shown. Figure 21 The pictures of the wounds at 0, 5, 10, 20 and 30 days after the treatment with 10 μL of PBS solution (control group), 10 μL of 3 mg / mL of verteporfin solution (VP group), blank PF-MNs group and PF-MNs group are shown. It can be seen from the pictures that the PF-MNs group promotes the fastest healing of the New Zealand rabbit ear wound, and the wound is basically completely healed at the 10th day, and the healing effect is significantly better than those of the other groups.

[0163] The above-mentioned 30-day rabbit ear tissues and normal rabbit ear tissues were fixed in paraformaldehyde and subjected to histological staining (H&E staining, Masson staining and Sirius red staining), and the thickness of the hypertrophic scar was measured. Figure 22 The thickness of the 30-day rabbit ear hypertrophic scar (n = 3) is shown. SEI is the ratio of the scar thickness after the treatment to the skin thickness of the normal rabbit ear, and the greater the ratio, the more serious the scar. After the treatment, the SEI of the VP group and the PF-MNs group is significantly reduced, indicating that the scar can be prevented.

[0164] The 30-day rabbit ear tissues and normal rabbit ear tissues were fixed in paraformaldehyde and subjected to histological staining (Masson staining). After the Masson staining, the collagen can be dyed blue, and the collagen content can be shown by comparing the size of the blue area, and the results are shown in Figure 23 The results show that after the treatment, the collagen content of the VP group and the PF-MNs group is close to that of the normal tissue, and the collagen content of the PF-MNs group is closer to that of the normal ear tissue than that of the VP group.

[0165] The 30-day rabbit ear tissues and normal rabbit ear tissues were fixed in paraformaldehyde and subjected to histological staining (Sirius red staining). After the Sirius red histological staining, type I collagen is dyed red, and type III collagen is green, Figure 24 The ratio of type I collagen to type III collagen in the 30-day scar tissue (n = 3) is shown. The ratio of the two is close to that of the normal tissue in the VP group and the PF-MNs group, and the above results all show that the PF-MNs can effectively prevent the formation of the scar. In Figure 22-24 The data are expressed as mean ± standard deviation, and the statistical significance is determined by one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0166] 7、PF-MNs patch in the effect of photodynamic therapy on the formed hypertrophic scar of the rabbit ear

[0167] Figure 25 , Figure 26 , Figure 27 , Figure 28 and Figure 29The effect of PF-MNs patch on the established hypertrophic scar of rabbit ear after photodynamic therapy was shown. After the rabbit was anesthetized with pentobarbital (30 mg / kg), three 8 mm round wounds were punched on the inner side of the ear with a puncher, and the fascia layer was removed. After 30 days, the obvious hypertrophic scar was formed, and then the treatment was given. Figure 25 The process of the rabbit ear scar treated by microneedle patch plus light was shown.

[0168] Figure 26 The scar pictures of the 0th, 7th, 15th, 25th and 35th day after treatment without any treatment (control group), 690 nm light for 10 min (light group), PF-MNs and PF-MNs treated by 690 nm light for 10 min (PF-MNs+L group) were shown. It can be seen from the pictures that the PF-MNs group and the PF-MNs+L group have obvious alleviating effect on the established hypertrophic scar, especially the PF-MNs+L group, which is almost close to normal skin after 35 days of treatment.

[0169] The 15th day rabbit ear tissue was taken for histological staining and analysis. Figure 27 The thickness of the 15th day rabbit ear hypertrophic scar (n=3) was shown. SEI is the ratio of the scar thickness after treatment to the skin thickness of normal rabbit ear. The larger the ratio, the more serious the scar. The results show that after treatment by PF-MNs and PF-MNs+L, the SEI is significantly reduced, indicating that the established hypertrophic scar can be reduced, and the reduction effect of PF-MNs+L is the most significant.

[0170] The 15th day rabbit ear tissue was taken for histological staining and analysis. Figure 28 The collagen content in the 15th day scar tissue (n=3) was shown. After treatment, the collagen content of the PF-MNs group and the PF-MNs+L group had no significant difference with the normal tissue, indicating that they had significant effect on reducing the scar.

[0171] The 15th day rabbit ear tissue was taken for histological staining and analysis. Figure 29 The ratio of type I collagen to type III collagen in the 15th day scar tissue (n=3) was shown. After picrosirius red histological staining, type I collagen was dyed red, and type III collagen was green. The ratio of the two had no significant difference with the normal tissue in the PF-MNs group and the PF-MNs+L group.

[0172] The above results all show that PF-MNs can effectively remove the established scar. In the Figure 27-29 In the above results, the data is expressed as mean ± standard deviation, and the statistical significance is determined by one-way analysis of variance. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Claims

1. A microneedle, characterized in that, It includes a shell of a cross-linked hydrophilic polymer, a negatively charged substance filling a cavity enclosed by the shell, and verteporfen disposed on the outer surface and / or shell layer of the shell; the cross-linked hydrophilic polymer includes a plurality of inflammatory-responsive linkers; (1) The crosslinked hydrophilic polymer is obtained by a crosslinking reaction between a hydrophilic polymer and a crosslinking agent to form the inflammatory-responsive linker; The inflammatory-responsive linker is a group formed by the cross-linking reaction of TSPBA and polyvinyl alcohol PVA; The hydrophilic polymer is PVA; The crosslinking agent is TSPBA, and the TSPBA is N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine; The mass ratio of the hydrophilic polymer to the crosslinking agent is set such that the shell degrades within 10 to 30 hours. (2) The negatively charged substance includes a negatively charged monomer and a non-inflammatory responsive linker connecting the negatively charged monomer; The negatively charged monomer is heparin; The non-inflammatory responsive linker is a photoresponsive linker, which is a group obtained by photo-initiated crosslinking reaction of the negatively charged monomer modified with double bonds and a photoinitiator. The negatively charged monomer modified with double bonds is obtained by reacting the negatively charged monomer with a double bond modifier. The double bond modifier is EGDMA. The double bond modification rate in the negatively charged monomer modified with the double bond is 20-30%; (3) The thickness of the outer shell is set such that the outer shell degrades within 10 to 30 hours.

2. The microneedle as described in claim 1, characterized in that, The inflammatory-responsive linker is a chemical bond that can be broken in response to inflammation-related conditions, namely, an increase in reactive oxygen species concentration. And / or, the PVA is a type 1795 PVA; And / or, the weight-average molecular weight of the PVA is 70~80kDa; And / or, the degree of alcoholysis of the PVA is 92-94%; And / or, the mass ratio of the hydrophilic polymer to the crosslinking agent is set to allow the shell to degrade in 20-28 hours; And / or, the mass ratio of the hydrophilic polymer to the crosslinking agent is (1~3):1; And / or, the thickness of the outer shell is set such that the outer shell degrades in 20 to 28 hours.

3. The microneedle as described in claim 2, characterized in that, The weight-average molecular weight of the PVA is 75 kDa; And / or, the mass ratio of the hydrophilic polymer to the crosslinking agent is 2:

1.

4. The microneedle as described in claim 1, characterized in that, The mass ratio of the hydrophilic polymer to the crosslinking agent is set to allow the shell to degrade in 24 hours; And / or, the thickness of the outer casing is set such that the outer casing degrades in 24 hours.

5. The microneedle as described in claim 1, characterized in that, The thickness of the outer shell is 10~20 μm.

6. The microneedle as described in claim 2, characterized in that, The thickness of the outer shell is 10~20 μm, and the cross-linked hydrophilic polymer is obtained by cross-linking PVA and TSPBA, wherein the mass ratio of PVA to TSPBA is 2:

1.

7. The microneedle as described in claim 1, characterized in that, The negatively charged substance is in the form of a gel; And / or, the mass ratio of the double bond modifier to the negatively charged monomer is 0.5-4%; And / or, the photoinitiator is one or more of photoinitiator 2959, photoinitiator MBF, TPO photoinitiator, 907 photoinitiator, 184 photoinitiator, and ITX photoinitiator; And / or, the mass ratio of the photoinitiator to the negatively charged monomer is 0.5-4%; And / or, the illumination is ultraviolet light.

8. The microneedle as described in claim 7, characterized in that, The mass ratio of the double bond modifier to the negatively charged monomer is 1-2%; And / or, the photoinitiator is photoinitiator 2959; And / or, the mass ratio of the photoinitiator to the negatively charged monomer is 1~2%.

9. The microneedle as described in claim 7, characterized in that, The negatively charged substance is a compound obtained by reacting heparin and EGDMA and then cross-linking it with photoinitiator 2959 under ultraviolet light irradiation. The mass ratio of EGDMA to heparin is 1~2%, and the mass ratio of photoinitiator 2959 to heparin is 1~2%.

10. The microneedle as described in claim 1, characterized in that, The microneedle is conical in shape; The length of the conical microneedle is 400~800 μm; The radius of the bottom circle of the conical microneedle is 200~400 μm; The tip diameter of the conical microneedle is 7~15 μm.

11. The microneedle as described in claim 10, characterized in that, The length of the conical microneedle is 500~700 μm; And / or, the radius of the bottom circle of the conical microneedle is 300 μm; And / or, the tip diameter of the conical microneedle is 10 μm.

12. The microneedle as described in claim 10, characterized in that, The length of the conical microneedle is 600 μm.

13. A method for preparing microneedles as described in any one of claims 1 to 12, characterized in that, It includes the following steps: S1: The negatively charged material is filled into the cavity enclosed by the shell; the negatively charged material includes a negatively charged monomer and a photoresponsive linker connecting the negatively charged monomer; the shell is prepared by mixing the hydrophilic polymer and a crosslinking agent containing the inflammatory-responsive linker and filling it into a microneedle mold, and then obtaining it after a crosslinking reaction; S2: Apply the vertipofen to the outer surface of the shell; The order of S1 and S2 is not important.

14. The method for preparing microneedles as described in claim 13, characterized in that, Perform S1 first, then S2; And / or, in S1, the filling atmosphere is vacuum filling; And / or, in S1, the negatively charged monomer and the double bond modifier are mixed and then the photoinitiator as described in claim 7 is added. The resulting mixture is filled into the cavity of the shell and then crosslinked by light to form a gel. And / or, in S2, the method of application is to apply a solution containing vertiporfin to the outer surface of the shell and then dry it; And / or, the hydrophilic polymer is dissolved in water to form a solution of the hydrophilic polymer, and then mixed with the crosslinking agent.

15. The method for preparing microneedles as described in claim 14, characterized in that, In S1, a drying step is performed before the light exposure; the drying step is natural drying under no-light conditions. And / or, the solvent in the verteporfen-containing solution is tetrahydrofuran; And / or, the concentration of verteporfen in the verteporfen-containing solution is 1~5 g / L; And / or, the application is by dripping; And / or, in the solution of the hydrophilic polymer, the concentration of the hydrophilic polymer is 1-5%; And / or, the concentration of the crosslinking agent in the solution of the crosslinking agent is 1~5%.

16. The method for preparing microneedles as described in claim 15, characterized in that, The concentration of verteporfen in the solution is 3 g / L; And / or, in the solution of the hydrophilic polymer, the concentration of the hydrophilic polymer is 2%; And / or, the concentration of the crosslinking agent in the solution of the crosslinking agent is 2%.

17. A microneedle prepared by any one of claims 13 to 16.

18. A microneedle patch, characterized in that, It includes microneedles as described in any one of claims 1 to 12 and 17.

19. The microneedle patch as described in claim 18, characterized in that, The microneedle patch also includes a substrate, and the microneedles are disposed on one side of the substrate; And / or, the microneedles are arranged in an array; And / or, the total content of verteporfen in one of the microneedle patches is 80~160 μg; And / or, in one of the microneedle patches, the content of the negatively charged substance is 0.05~0.15 g; And / or, in one of the microneedle patches, the content of the hydrophilic polymer is 0.015~0.035 g.

20. The microneedle patch as described in claim 19, characterized in that, The other side of the liner is also covered with stickers or tape; And / or, the lining material is a negatively charged substance as described in any one of claims 1, 7 to 9; And / or, the microneedles are arranged in a matrix; And / or, the center-to-center spacing of the array arrangement is 400~800 μm; And / or, the total content of verteporfen in one of the microneedle patches is 100~140 μg; And / or, in one of the microneedle patches, the content of the negatively charged substance is 0.1 g; And / or, in one of the microneedle patches, the content of the hydrophilic polymer is 0.0267 g.

21. The microneedle patch as described in claim 20, characterized in that, The center-to-center spacing of the array arrangement is 600 μm; And / or, the total content of vertepofen in one of the microneedle patches is 120 μg.

22. The use of a microneedle as described in any one of claims 1 to 12 and 17 or a microneedle patch as described in any one of claims 19 to 21 in the preparation of a wound healing medical device.

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