Double-layer microneedles with controllable transdermal performance and their preparation

Abstract

The present invention discloses a double-layer microneedle with controllable transdermal performance and its preparation, belonging to the field of biomedical technology. The lower layer of the double-layer microneedle is a hyaluronic acid-tyrosine hydrogel (HAT) bonded with tyramine prepared by enzymatic crosslinking method, and the transdermal performance and cell penetration of the microneedle can be strengthened after compounding collagenase (Coll) in HAT, and the present application is also further optimized by studying the structure-activity relationship between HAT crosslinking strength and microneedle transdermal performance, and the subcutaneous swelling of the microneedle and the sustained release behavior of the hydrogel are further optimized; the upper layer of the microneedle is the PcNP@Drug complex obtained after PcNP carries the corresponding drug, and the microneedle tip introduces mesoporous silica nanospheres (MSN) to solve the problems of low drug loading of the microneedle and difficult control of drug release, and the microneedle can be well applied to the photodynamic therapy of a class of diseases present on the skin surface after doping photosensitizer in MSN, and finally realizes the combined treatment of such diseases with microneedles.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a double-layer microneedle with controllable transdermal performance and a preparation method thereof. Background Art

[0002] The transdermal drug delivery system can directly penetrate the epidermal barrier through injection and other routes, and then transport the drug to the lesion site through the internal circulation. Compared with oral administration, this method can effectively avoid the damage of the gastrointestinal environment to the drug and the first-pass effect of the liver on the drug, achieving a drug efficacy similar to intravenous infusion. Traditional transdermal drug delivery is completed through subcutaneous needle injection, which is somewhat invasive. The injection wound may cause infection and is accompanied by obvious pain, causing discomfort to the patient. The discarded needle after injection also needs additional treatment.

[0003] In recent years, researchers in this field have developed a new type of microneedle technology to solve a series of problems caused by subcutaneous needle injections. Specifically, the needle is miniaturized and a series of microneedle arrays with a height of 50-900μm are prepared through template technology and other means. The microneedle has a sharp injection tip similar to that of a subcutaneous needle, which can penetrate the 10-15μm thick skin stratum corneum and avoid irritating the capillaries and nerve endings in the dermis. Therefore, it has lower invasiveness and can improve drug penetration.

[0004] The microneedle-based transdermal drug delivery system is particularly suitable for treating a type of disease that mainly exists on the surface of the skin, such as melanoma, which has the characteristics of high metastasis, high mortality rate, and strong drug resistance. The use of microneedles to treat such diseases is expected to increase the concentration of target drugs and enhance the efficacy. At the same time, local drug delivery can not only reduce systemic toxic side effects, but also reduce the risk of infection. Therefore, the development of microneedle technology provides new research methods and theoretical basis for exploring the diagnosis, intervention and treatment of diseases, and is therefore highly favored in the fields of biomaterials, medical engineering, etc.

[0005] However, in order to achieve the best transdermal drug delivery effect of microneedles, two key aspects need to be addressed in the design of microneedles: First, the shape and mechanical strength of the microneedles themselves need to be comprehensively considered and optimized. Although the sharp needle tip can easily penetrate the stratum corneum, it will deform and break if the mechanical strength is insufficient, affecting normal drug delivery; Second, the drug loading and release capacity of the microneedles needs to be precisely controlled. Due to its own miniaturized design, how to increase the drug loading capacity of the microneedles while avoiding sudden drug release is still one of the difficult problems facing the current research field.

[0006] Regarding how to slow down the rate of drug release in the body and extend the treatment cycle, Chinese patent CN106492220A has proposed a feasible solution, which provides a nano-mesoporous silica composite hydrogel with controlled release function, specifically, encapsulating drug-loaded mesoporous silica nanoparticles inside the hydrogel, slowly releasing the drug-loaded nanoparticles through the degradation of the hydrogel, and then releasing the drug encapsulated in the nanoparticles in the body fluid. By introducing mesoporous silica nanoparticles into natural polymer-based hydrogels, the mechanical properties of the hydrogels can be significantly increased, making up for the shortcomings of pure natural polymer hydrogels in mechanical properties.

[0007] The above scheme provides a feasible idea for effectively avoiding the sudden release of drugs, which has guiding significance for the application of gel materials in tissue engineering. However, for a type of disease existing on the surface of the skin, it is difficult for the gel material without further treatment to act directly on the lesion site, so the efficacy will be significantly affected. In order to solve this problem, combined with the understanding of microneedle technology, the applicant believes that it would be a good solution if such materials can be processed into microneedles. However, due to the limitations of microneedle size and material, how to successfully combine some feasible ideas for slowing down the sudden release of drugs provided in the prior art into the microneedle transdermal drug delivery system, so that the microneedle has both the characteristics of controllable mechanical strength and high drug loading and the ability to control drug sustained release, and then can use the idea of ​​combined treatment to carry out targeted and efficient treatment of such diseases, this still requires more efforts by technical personnel in this field to make further exploration. Summary of the invention

[0008] The purpose of the present invention is to solve the problems existing in the prior art and to provide a double-layer microneedle with controllable transdermal performance and a preparation method thereof. This application breaks through traditional thinking and utilizes microneedles for transdermal drug delivery while intending to exert the combined therapeutic efficacy of microneedle carriers through multifunctional mesoporous composite materials.

[0009] The present invention is achieved as follows: a double-layer microneedle with controllable transdermal performance, wherein the main part of the microneedle except the base is a double-layer structure with upper and lower layers, the upper layer is a PcNP@Drug complex obtained by loading PcNP with drugs, and the lower layer is a HAT@Coll hydrogel obtained by compounding hyaluronic acid-tyrosine hydrogel HAT and collagenase Coll; the hydrogel part is a tyramine-bonded HAT hydrogel prepared by an enzymatic crosslinking method, the HAT hydrogel undergoes an enzymatic crosslinking reaction under the catalysis of HRP and H2O2, and the addition of collagenase enhances the transdermal performance and cell penetration of the microneedle; the PcNP is obtained by doping the photosensitizer phthalocyanine Pc on the mesoporous silica nanospheres MSN.

[0010] The preparation method of the above-mentioned double-layer microneedle with controllable transdermal performance is as follows:

[0011] 1) Preparing a PDMS microneedle template;

[0012] 2) Preparation of HAT pre-gel solution:

[0013] ⅰ. Weigh a certain amount of HA-Tyr powder and dissolve it in phosphate buffer as HAT stock solution;

[0014] ii. Weigh a certain amount of Coll powder and dissolve it in phosphate buffer as Coll stock solution;

[0015] ⅲ, after dissolving the drug to be loaded, disperse it into the PcNP solution, continuously stir to achieve drug loading, wash, centrifuge, and disperse the resulting particle product in water as the PcNP@Drug stock solution;

[0016] iv. Mix H2O2, HRP, Coll stock solution, HAT stock solution and PcNP@Drug stock solution in a certain volume ratio, and vortex to obtain a HAT pre-gel solution;

[0017] 3) Preparation of microneedles: Add 100 μL of HAT pregel solution to the PDMS template, aspirate the supernatant after centrifugation, add 200 mg of 55kHA base to form the base of the microneedle, dry it in the dark at room temperature overnight, add another 200 mg of base, dry it in the dark at room temperature overnight, and peel it off from the mold to obtain the double-layer microneedle HAT@Colll / PcNP@Drug-MN.

[0018] Further, in step 2), in step 2), the mass concentration of HAT stock solution is 1.0wt%~6.0wt%, the mass concentration of Coll stock solution is 0.5wt%~3wt%, and the mass concentration of PcNP@Drug stock solution is 1wt%~10wt%; in step iv, the mass concentration of H2O2 used is 0.1wt%~1.0wt%, and the concentration of HRP used is 1U / mL~5U / mL.

[0019] Preferably, the mass concentration of HAT stock solution is 4.0wt%, the mass concentration of Coll stock solution is 2.0wt%, the mass concentration of PcNP@Drug stock solution is 6.0wt%, and in step iv, the mass concentration of H2O2 used is 0.5wt%, and the concentration of HRP used is 5U / mL.

[0020] Furthermore, in step 2), in step iv, the volume ratio of H2O2, HRP, Coll stock solution, HAT stock solution and PcNP@Drug stock solution is 1:1:50:50:100.

[0021] Furthermore, in step 1), the preparation process of the PDMS microneedle template is as follows: the PDMS monomer and the curing agent are mixed in a certain weight ratio and poured into a container containing a stainless steel microneedle mold, vacuumed in a vacuum drying oven at room temperature, heated and cured in an oven, and after cooling to room temperature, the mold is taken out, the PDMS is separated from the stainless steel microneedle, and the PDMS microneedle template is obtained.

[0022] Further, the preparation process of PcNP used in step iii in step 2) is as follows:

[0023] (1) Synthesis of Pc-4NO2: Ammonium molybdate was mixed with 4-nitrophthalic anhydride and urea, and then added to a nitrobenzene solution of zinc chloride; the mixture was stirred at 185°C for 4 hours under nitrogen protection, cooled, toluene was added, the precipitate was washed, centrifuged, washed with toluene, water, MeOH / ether mixture and ethyl acrylate / hexane mixture, and air-dried to obtain a dark green solid;

[0024] (2) Synthesis of Pc-4NH2: Pc-4NO2 was dissolved in DMF, and sodium sulfide nonahydrate was added thereto. The mixture was heated and stirred at 60°C for 1.5 hours under nitrogen protection. The reaction mixture was cooled to room temperature, ice water was added, and the precipitate was separated by centrifugation. The precipitate was repeatedly washed with a MeOH / ether mixture and EA, and air-dried to obtain a dark green solid.

[0025] (3) Synthesis of Pc-Si: Weigh a certain amount of Pc-4NH2 into a three-necked flask, add anhydrous DMF to dissolve it, dissolve 3-(triethoxysilyl)propyl isocyanate in anhydrous DMF and inject it into the three-necked flask, reflux at 120°C, protect with nitrogen, and stir overnight to obtain a Pc-Si solution;

[0026] (4) Synthesis of PcNPs: CTAB was dissolved in H2O, triethanolamine was added, and the mixture was vigorously stirred at 80°C for 30 minutes to obtain a CTAB solution; TMOS and Pc-Si were uniformly mixed and then added dropwise to the CTAB solution under vigorous stirring. After the reaction was completed, the temperature was lowered to 50°C, and 2-(methoxy(polyethoxy)-propyl)trimethoxysilane was added dropwise. The mixture was stirred overnight to complete the reaction, and the mixture was centrifuged. The precipitate was washed with water and ethanol to obtain the product PcNPs.

[0027] Furthermore, in step 3), before adding the HAT pre-gel solution into the PDMS template, the template needs to be cleaned with O2 plasma to enhance the hydrophilicity of its surface.

[0028] Furthermore, the drug loaded into the PcNP in step iii of step 2) is a drug for treating a class of diseases present on the surface of the skin, such as trametinib.

[0029] Beneficial effects:

[0030] 1. The hydrogel microneedle disclosed in the present application has a double-layer structure, the upper layer is a PcNP@Drug complex obtained by loading PcNP with drugs, and the lower layer is a HAT@Coll hydrogel obtained by compounding hyaluronic acid-tyrosine hydrogel HAT and collagenase Coll; wherein the hydrogel matrix material part is a tyramine-bonded hyaluronic acid hydrogel prepared by an enzymatic cross-linking method, and the addition of Coll further enhances the transdermal performance and cell penetration of the microneedle; the introduction of mesoporous silica nanospheres (MSN) at the tip of the microneedle can solve the problems of low drug loading and difficult drug release of the microneedle, and after doping the photosensitizer PC on the MSN, the microneedle can be used in photodynamic therapy, and the depth reached by the subcutaneous microneedle can be stimulated by infrared light to generate free radicals to kill pathogenic cells, thereby achieving the purpose of synergistic treatment with the chemical drugs embedded in the MSN;

[0031] 2. The MSN used in the double-layer microneedle prepared in the present application has a complementary effect with HAT in terms of function: the cross-linked HAT and the collagenase contained therein mainly control the skin penetration, subcutaneous swelling and other properties of the microneedle, while the MSN can increase the drug loading and promote sustained release. The photosensitizer bonded to the MSN can be further used in photodynamic therapy. Therefore, the microneedle disclosed in the present application can be loaded with the corresponding drug to achieve a combined treatment effect on a class of diseases present on the skin surface;

[0032] 3. The present application utilizes an enzymatic cross-linking method to prepare the base material HAT required for the microneedles. The cross-linking strength of the gel can be adjusted by changing the reaction concentration of hydrogen peroxide. Appropriate cross-linking strength can simultaneously ensure the mechanical properties and transdermal properties of the microneedles (such as skin penetration and subcutaneous swelling), and ultimately achieve the sustained release of collagenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 are the gelation photos of HAT@Coll hydrogel, where a is the gelation photo of HAT-Low@Coll hydrogel, b is the gelation photo of HAT-Medium@Coll hydrogel, and c is the gelation photo of HAT-High@Coll hydrogel;

[0034] Figure 2 are the in-situ gelation rheology diagrams of HAT@Coll hydrogels, wherein a is the in-situ gelation rheology diagram of HAT-Low@Coll hydrogel, b is the in-situ gelation rheology diagram of HAT-Medium@Coll hydrogel, and c is the in-situ gelation rheology diagram of HAT-High@Coll hydrogel;

[0035] Figure 3 Panel a in the middle is the standard curve of collagenase, and panel b is the enzyme release curve of HAT@Coll hydrogel with different cross-linking strengths;

[0036] Figure 4 Statistics of reactive oxygen generation data for HAT-Low@Coll / PcNP, HAT-Medium@Coll / PcNP, HAT-High@Coll / PcNP, PcNP and Pc-Si solutions in aqueous solution;

[0037] Figure 5 is the optical image of HAT@Coll / PcNP@Drug-MN; wherein, a is the optical image of HAT-low@Coll / PcNP@Drug-MN, b is the optical image of HAT-Medium@Coll / PcNP@Drug-MN, and c is the optical image of HAT-High@Coll / PcNP@Drug-MN;

[0038] Figure 6 Characterization of the mechanical properties of HAT@Coll / PcNP@Drug-MN with different cross-linking strengths. The left figure is the force-displacement curve of HAT@Coll / PcNP@Drug-MN with different cross-linking strengths, and the right figure is the force of HAT@Coll / PcNP@Drug-MN with different cross-linking strengths when the compression displacement is 0.5 mm;

[0039] Figure 7 The results of in vitro skin penetration test of HAT@Coll / PcNP@Drug-MN with different cross-linking strengths are shown in the figure on the left. The fluorescence microscopic images of pig skin before and after treatment with HAT@Coll / PcNP@Drug-MN with different cross-linking strengths are shown in the figure on the right. The average fluorescence intensity of pig skin after treatment with HAT@Coll / PcNP@Drug-MN with different cross-linking strengths is shown in the figure on the right. In the two figures, HAT-low@Coll / PcNP@Drug-MN, HAT-Medium@Coll / PcNP@Drug-MN and HAT-High@Coll / PcNP@Drug-MN are referred to as HAT-low, HAT-Medium and HAT-High, respectively.

[0040] Figure 8 The images show the morphological changes of HAT@Coll / PcNP@Drug-MN with different cross-linking strengths dissolving at different time periods on agarose simulated skin;

[0041] Fig. 9 Panels A and B in the middle are in vivo fluorescence imaging of mice at different time points after treatment with HA@Coll / PcNP@Drug-MN and HAT@Coll / PcNP@Drug-MN, respectively. Panel C is a statistical chart of the retention time of microneedles in mice. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0043] 1. Preparation of PcNPs:

[0044] (1) Synthesis of Pc-4NO2: Ammonium molybdate (6.5 mg, 0.005 mmol) was mixed with 4-nitrophthalic anhydride (1.0 g, 5 mmol) and urea (1.5 g, 25 mmol) and then zinc chloride (191

[0045] mg, 1.3 mmol) in nitrobenzene (7.5 mL); the mixture was stirred at 185 ° C for 4 hours under nitrogen protection, cooled, toluene (40 mL) was added, the precipitate was washed and centrifuged, and toluene, water, methanol (MeOH) / ether (V MeOH :V 醚 =1:9) mixed solution and ethyl acrylate (EA) / hexane (V EA :V 己烷 =2:1) ​​mixed solution was washed and air-dried to obtain a dark green solid (1.0 g, 98%);

[0046] (2) Synthesis of Pc-4NH2: Pc-4NO2 (0.95 g, 1.25 mmol) was dissolved in DMF (25

[0047] mL) was added with sodium sulfide nonahydrate (3.7 g, 15.5 mmol), heated, stirred and reacted at 60°C for 1.5 hours under nitrogen protection, the reaction mixture was cooled to room temperature, and ice water (75

[0048] mL), centrifuged to separate the precipitate, and washed with MeOH / ether (V MeOH :V 醚 =1:9) mixed solution and EA were repeatedly washed and air-dried to obtain a dark green solid (0.6 g, 75%);

[0049] (3) Synthesis of Pc-Si: Pc-4NH2 (9.0 mg) was weighed into a three-necked flask, anhydrous DMF (5 mL) was added to dissolve it, and 3-(triethoxysilyl)propyl isocyanate (13.8

[0050] μL) was dissolved in anhydrous DMF (0.1 mL) and injected into a three-necked flask for reflux reaction at 120°C under nitrogen protection and stirred overnight to obtain a Pc-Si solution;

[0051] (4) Synthesis of PcNPs: CTAB (1.0 g) was dissolved in H2O (120 mL), and triethanolamine (420 μL, w / w ratio with water = 1:1) was added, followed by vigorous stirring at 80 °C for 30 min to obtain a CTAB solution. Tetramethoxysilane (TMOS, 160 μL) and Pc-Si (800 μL) were uniformly mixed and then added dropwise to the CTAB solution under vigorous stirring. After reacting for 2 h, the temperature was lowered to 50 °C, and 2-(methoxy(polyethoxy)-propyl)trimethoxysilane (400 μL) was added dropwise.

[0052] μL), stirred overnight to complete the reaction, centrifuged at 15000 rpm for 30 min, and the precipitate was washed with water and ethanol to remove CTAB and unreacted compounds. The final product was PcNP.

[0053]

[0054] 2. Preparation of PcNP@Drug stock solution

[0055] Since 60% of melanomas have BRAF mutations (mainly BRAFV600E), which leads to high activation of intracellular MEK, small molecule inhibitors dabrafenib and trametinib targeting this target can significantly improve patient survival in Phase III clinical trials and have been approved by the FDA for clinical treatment. Therefore, trametinib is used as an example to illustrate the drug loading treatment of PcNP in this step.

[0056] Weigh 4 mg of trametinib and dissolve it in 2 mL of DMSO. After it is fully dissolved, disperse it into 2 mL of 4 mg / mL PcNP solution and stir continuously for 24 h to load the drug to obtain a drug-loaded solution. Then wash the nanoparticles with a large amount of ethanol and water to remove excess drug and DMSO, and then centrifuge at 9000 rpm for 45 minutes to collect the PcNP@Drug precipitate. Finally, the obtained particle product is dispersed in water as a PcNP@Drug stock solution (6 wt%).

[0057] The drug loading and encapsulation efficiency were calculated based on the calibration curve of the mixture of Dabrafenib and Trametinib (v / v = 1:1).

[0058]

[0059] 3. Preparation of HAT@Coll hydrogel

[0060] A certain amount of hyaluronic acid-tyrosine (HA-Tyr) powder was weighed and dissolved in a phosphate buffer solution with a pH of 7.40 to form a HAT stock solution with a mass concentration of 2.0 wt%, and the pH of the solution was adjusted to 7.4 with 0.1 M NaOH / HCl;

[0061] Weigh a certain amount of collagenase (Coll) powder and dissolve it in a phosphate buffer at pH = 7.40 to form a Coll stock solution with a mass concentration of 1.0 wt%;

[0062] 1 μL of H2O2 of different concentrations (0.3wt%, 0.5wt%, 0.8wt%) was measured respectively, and 1 μL of 5U / mL horseradish peroxidase (HRP), 100 μL of Coll stock solution and 100 μL of HAT stock solution were added to each of the H2O2 of different concentrations, and vortexed evenly. The final concentrations of Coll and HRP in the obtained mixed solution were 0.5wt% and 24.75 μU / mL respectively; the final concentrations of H2O2 in the three mixed solutions were 90 μM, 150 μM and 240 μM respectively, and the mixture was left to stand at room temperature to wait for gelation. The structural formula of the hydrogel is as follows:

[0063]

[0064] from Figure 1 It can be seen that the three concentrations of hydrogen peroxide can form hydrogels. However, due to the different contents of H2O2 in the three gels, the strength of the enzymatic cross-linking is different. As the concentration of H2O2 increases, the cross-linking strength increases. Therefore, the three gels obtained are recorded as HAT-Low@Coll (low cross-linking gel), HAT-Medium@Coll (medium cross-linking gel) and HAT-High@Coll (high cross-linking gel) according to the cross-linking strength.

[0065] Characterization experiments of HAT@Coll hydrogel:

[0066] Rheological experiments: A rheometer was used to characterize the rheological properties of the hydrogel. The experiment was carried out at 25°C using a Discovery HR-1A hybrid oscillation rheometer equipped with a 20mm aluminum parallel plate and a 0.8mm gap distance. 1.0wt% of HAT-Low@Coll, HAT-Medium@Coll and HAT-High@Coll pre-gel solutions were loaded onto Peltier, respectively, and dynamic time scans were performed at a fixed frequency of 1Hz and a fixed stress of 1.0%, and the changes in their storage modulus (G') and loss modulus (G") as well as the time required for gelation were recorded. All modes of each colloidal sample were scanned in parallel 3 times.

[0067] from Figure 2 It can be seen that with the increase of hydrogen peroxide concentration, that is, with the increase of cross-linking strength, the storage modulus (G') of the hydrogel gradually increases, and the mechanical strength of the hydrogel gradually increases.

[0068] In vitro drug release experiment of HAT@Coll hydrogel:

[0069] Prepare 500 μL of HAT-Low@Coll, HAT-Medium@Coll and HAT-High@Coll hydrogels (the final concentration of HAT is 1.0 wt%) in the gelling vials respectively. After the gelling is stabilized, slowly add 1000 μL of PBS solution (pH=7.4) on the surface of the colloid and place it in a 37°C constant temperature oscillating box with a rotation speed of 40 rpm. Take out 500 μL of the supernatant solution at 2, 4, 8, 12, 24, 48, 96, 168 and 216 h, and immediately add 500 μL of fresh blank PBS solution to keep the volume constant. The release amount of Coll is measured by an enzyme-labeled instrument and calculated according to the established standard curve. Each group of experiments is parallel to 3.

[0070] Figure 3 Figure 3 is the release difference curve of Coll in HAT@Coll hydrogels with different cross-linking strengths. It can be seen from the figure that the release percentage of Coll can be controlled by adjusting the mechanical strength of the hydrogel. At the same time, with the increase of the cross-linking strength, the release percentage of Coll will decrease to a certain extent. It is speculated that the high concentration of hydrogen peroxide may cause excessive cross-linking with the HAT gel after the reaction, which will have an adverse effect on the release of Coll. Therefore, the cross-linking strength of the HAT@Coll hydrogel needs to be reasonably selected.

[0071] 4. Preparation of PDMS microneedle template

[0072] The polydimethylsiloxane (PDMS) mixture (weight ratio of PDMS monomer and curing agent w / w = 10:1) was poured into a container containing a stainless steel microneedle mold, and vacuumed in a vacuum oven at room temperature for 10 minutes to remove bubbles in the PDMS. The mixed solution was heated in an oven at 80°C for 90 minutes to completely cure the PDMS. After cooling to room temperature, the mold was removed, and the PDMS was separated from the stainless steel microneedles to obtain a 10×10 array of PDMS microneedle templates.

[0073] 5. Preparation of HAT@Coll / PcNP@Drug microneedles (hereinafter referred to as HAT@Coll / PcNP@Drug-MN)

[0074] HAT@Coll / PcNP@Drug-MN was prepared by centrifugation method.

[0075] (1) Preparation of HAT pregel solution: Weigh a certain amount of HA-Tyr powder and dissolve it in a phosphate buffer solution at pH = 7.40 to form a HAT stock solution with a concentration of 4.0 wt%, and adjust the pH of the solution to 7.4 with 0.1 M NaOH / HCl;

[0076] Weigh a certain amount of Coll powder and dissolve it in a phosphate buffer solution with a pH of 7.40 to form a Coll stock solution with a concentration of 2.0 wt%;

[0077] Take 1 μL of each of three different concentrations of H2O2 (0.3wt%, 0.5wt%, 0.8wt%), add 1 μL of HRP (5U / mL), 50 μL of Coll stock solution, 50 μL of HAT stock solution and 100 μL of PcNP@Drug stock solution with a mass concentration of 6.0wt% prepared in step 2 to hydrogen peroxide of different concentrations, vortex evenly, and finally obtain three HAT pregel solutions with different degrees of cross-linking;

[0078] Generation of ROS in HAT@Coll / PcNP@Drug under near-infrared light irradiation

[0079] ABDA was used as a reactive oxygen species indicator to investigate the ROS production ability of HAT@Coll / PcNP@Drug in aqueous solution under NIR light irradiation. In aqueous solution, ABDA can effectively capture ROS and react with them, reducing their maximum UV absorbance at 401 nm.

[0080] The specific operation is as follows: HAT-Low@Coll / PcNP@Drug, HAT-Medium@Coll / PcNP@Drug, HAT-High@Coll / PcNP@Drug, PcNP and Pc-Si solutions were mixed with ABDA solution respectively, so that the final concentration of ABDA was 50μg / mL, the final concentration of PcNP and Pc-Si was 20μg / mL, and the final concentration of PcNP in the three composite gel solutions with different cross-linking degrees was 20μg / mL. Then, the mixed solutions were exposed to laser irradiation (730nm, 1W / cm 2 ) environment, the absorbance of the mixed solution at 401 nm was measured by UV-visible spectrophotometer at 0, 2, 4, 6, 8 and 10 min to determine the generation of ROS.

[0081] Figure 4 It was shown that HAT@Coll / PcNP@Drug hydrogels with three degrees of cross-linking can effectively generate ROS under near-infrared light irradiation, and their ability to generate ROS is stronger than that of Pc-Si alone. Therefore, this method will play an important role in the photodynamic therapy of cancer.

[0082] (2) Fabrication of microneedles: First, the PDMS microneedle template was cleaned with O2 plasma for 10 seconds to enhance the hydrophilicity of its surface. Then, 100 μL of HAT pregel solutions with different crosslinking degrees were added to the PDMS mold and centrifuged at 4,000 rpm for 10 minutes to fill the cavity of the mold; after aspirating the supernatant, 200 mg of 55kHA base with a mass concentration of 25% was added to form the base of the microneedle, and dried at room temperature in the dark overnight, and 200 mg of base was added, and dried at room temperature in the dark overnight; the obtained microneedle array was separated from the mold, and HAT-Low@Colll / PcNP@Drug-MN, HAT-Medium@Colll / PcNP@Drug-MN and HAT-High@Colll / PcNP@Drug-MN were placed in a desiccator containing color-changing silica gel and stored at room temperature in the dark.

[0083] Since the pre-gel solution prepared by mixing different materials will sediment at different rates when centrifuged in the mold, the three microneedles with different degrees of cross-linking finally obtained are all double-layer structures, with the upper layer being PcNP@Drug and the lower layer being HAT@Coll hydrogel. The hydrogel part of the double-layer microneedle is prepared by enzymatic cross-linking method with tyramine-bonded hyaluronic acid-tyrosine hydrogel (HAT). The addition of collagenase can enhance the transdermal performance and cell penetration of the microneedle. The introduction of mesoporous silica nanospheres at the tip of the double-layer microneedle can solve the problems of low drug loading and difficult to control drug release of the microneedle. The incorporation of photosensitizer Pc into the mesoporous silica nanospheres is helpful for photodynamic therapy of related diseases. The microneedle can be used for combined treatment of related diseases to enhance the efficacy.

[0084] Related performance tests of HAT@Coll / PcNP@Drug-MN

[0085] 1) Geometric structure and morphology of HAT@Coll / PcNP@Drug-MN

[0086] The specific operation is as follows: use a digital camera equipped with a macro lens to take optical images of HAT-low@Coll / PcNP@Drug-MN, HAT-Medium@Coll / PcNP@Drug-MN and HAT-High@Coll / PcNP@Drug-MN to observe and characterize the length, bottom width and needle tip spacing of the microneedles.

[0087] Study on the mechanical properties of HAT@Coll / PcNP@Drug-MN

[0088] The mechanical properties of HAT@Coll / PcNP@Drug-MN were determined by compression test, as follows: A universal testing machine equipped with a load cell (80N) was used to perform compression test, and the microneedles were pressed at a rate of 0.5 mm / min with a force perpendicular to the stainless steel disk at a distance of 700 μm. Then, the force-distance curve was plotted and the force of HAT-low@Coll / PcNP@Drug-MN, HAT-Medium@Coll / PcNP@Drug-MN, and HAT-High@Coll / PcNP@Drug-MN at a compression distance of 500 μm was compared.

[0089] from Figure 6 It can be seen that HAT-High@Coll / PcNP@Drug-MN has the best mechanical strength, but after comprehensively considering the effect of excessive cross-linking on the in vitro drug release of the gel, it is believed that HAT-Medium@Coll / PcNP@Drug-MN has better overall performance.

[0090] 2) In vitro skin penetration experiment of HAT@Coll / PcNP@Drug-MN

[0091] The in vitro skin penetration performance of HAT@Coll / PcNP@Drug-MN was evaluated by chemiluminescence imaging analyzer.

[0092] The specific operation is as follows: First, take the fat-free pig skin out of the -20℃ refrigerator, treat it with 0.9% saline and restore it to room temperature, and then use filter paper to absorb the moisture on the surface of the pig skin. Place the needle tip of HAT-low@Coll / PcNP@Drug-MN, HAT-Medium@Coll / PcNP@Drug-MN and HAT-High@Coll / PcNP@Drug-MN on the pig skin with the tip facing down, press down with the thumb for 5 minutes, and after pressing, use a cotton swab to remove excess liquid on the surface, then put the pig skin into the chemiluminescence imaging analyzer to image its fluorescence, with an excitation wavelength of 650nm, an emission wavelength of 699nm, and an exposure time of 100ms. Finally, use ImageJ software to analyze the average fluorescence intensity of the skin.

[0093] from Figure 7 It can be seen that among the three HAT@Coll / PcNP@Drug-MNs with different cross-linking degrees, HAT-Medium@Coll / PcNP@Drug-MN with medium mechanical strength exhibited the highest PcNP transdermal delivery efficiency in the porcine skin model.

[0094] 3) Determination of in vitro dissolution rate of HAT@Coll / PcNP@Drug-MN

[0095] Preparation of agarose gel: Weigh an appropriate amount of agarose powder and dissolve it in ultrapure water (2.0 wt%), heat the solution to 90°C until the agarose solution is completely dissolved, and stop heating. Pour the clear solution into a petri dish and cool it to room temperature to obtain agarose gel.

[0096] The in vitro dissolution rate of HAT@Coll / PcNP@Drug-MN with different degrees of cross-linking was evaluated by an agarose gel model. The specific operation is as follows: HAT-low@Coll / PcNP@Drug-MN, HAT-Medium@Coll / PcNP@Drug-MN and HAT-High@Coll / PcNP@Drug-MN were gently inserted into agarose gel (2.0wt%) for 5s, 10s, 30s and 1min respectively, and then the microneedles were removed from the agarose gel. HA@Coll / PcNP@Drug-MN was used as the control group. Then, the morphological changes of various types of microneedles were observed under a microscope.

[0097] HA@Coll / PcNP@Drug-MN was prepared by centrifugation method.

[0098] (1) Preparation of HA injection solution: Weigh a certain amount of hyaluronic acid (HA) powder and dissolve it in a phosphate buffer solution at pH = 7.40 to form a HA stock solution with a concentration of 10.0 wt%;

[0099] Weigh a certain amount of Coll powder and dissolve it in a phosphate buffer solution with a pH of 7.40 to form a Coll stock solution with a concentration of 2.0 wt%;

[0100] To prepare 5.0 wt% HA@Coll: 50 μL Coll stock solution, 50 μL HA stock solution and 100 μL 6.0 wt% PcNP@Drug stock solution were vortexed to obtain a mixed solution.

[0101] (2) Fabrication of microneedles: First, the PDMS microneedle template was cleaned with O2 plasma for 10 seconds to enhance the hydrophilicity of its surface. Then, 100 μL of HA needle-forming solution was added to the PDMS mold and centrifuged at 4,000 rpm for 10 minutes to fill the cavity of the mold. After the supernatant was aspirated, 200 mg of 55kHA base with a mass concentration of 25% was added to form the base of the microneedle, and dried at room temperature in the dark overnight. 200 mg of base was added and dried at room temperature in the dark overnight; the obtained microneedle array was separated from the mold, placed in a desiccator containing color-changing silica gel, and stored at room temperature in the dark.

[0102] from Figure 8It can be seen that HA@Coll / PcNP@Drug-MN and HAT@Coll / PcNP@Drug-MN with different mechanical strengths have rapid dissolution characteristics. Figure 8 It shows that the dissolution rate of HAT@Coll / PcNP@Drug-MN slightly slows down with the increase of mechanical strength, but it can basically be dissolved after 60s.

[0103] 4) In vivo imaging experiments of HAT-Medium@Coll / PcNP@Drug-MN

[0104] The transdermal delivery efficiency of HA@Coll / PcNP@Drug-MN and HAT-Medium@Coll / PcNP@Drug-MN was investigated in vivo using BALB / c nude mice. The specific operation was as follows: BALB / c nude mice weighing about 20 g (male, purchased from Nanjing Qinglongshan Animal Breeding Center) were selected and raised in an SPF environment for 6-8 weeks. The left back skin of the mouse was wiped with 70% ethanol, and one piece of HA@Coll / PcNP@Drug-MN and one piece of HAT-Medium@Coll / PcNP@Drug-MN were applied to the left back skin of the mouse with the needle tip facing down for 5 minutes. In order to monitor the transdermal delivery efficiency of the microneedles, a live animal imaging system with excitation / emission (Tanon, 4600SF, China) was used for fluorescence scanning at different time points (0, 1, 2, 4, 8, 24, 48 and 96 h), where the wavelength was 650 / 699 nm and the exposure time was fixed at 50 ms. The fluorescence intensity of the microneedle application area was then quantified using ImageJ software. All animal procedures were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of China Pharmaceutical University.

[0105] from Fig. 9 It can be seen that compared with HA@Coll / PcNP@Drug-MN, HAT-Medium@Coll / PcNP@Drug-MN has a longer retention time in the body and a better sustained-release effect.

[0106] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a double-layer microneedle with controllable transdermal performance, characterized in that: The main steps are as follows: 1) Preparation of PDMS microneedle template; 2) Preparation of HAT pre-gel solution: ⅰ. Weigh HA-Tyr powder and dissolve it in phosphate buffer as HAT stock solution; ii. Weigh Coll powder and dissolve it in phosphate buffer as Coll stock solution; ⅲ, after dissolving the drug to be loaded, disperse it into the PcNP solution, continuously stir to achieve drug loading, wash, centrifuge, and disperse the resulting particle product in water as the PcNP@Drug stock solution; iv. Mix H2O2, HRP, Coll stock solution, HAT stock solution and PcNP@Drug stock solution, and vortex to obtain HAT pre-gel solution; 3) Preparation of microneedles: 100 μL of HAT pregel solution was added to the PDMS template, the supernatant was aspirated after centrifugation, 200 mg of 55kHA base was added to form the base of the microneedle, and the base was dried at room temperature in the dark overnight. 200 mg of base was added, and the base was dried at room temperature in the dark overnight. The double-layer microneedle HAT@Colll / PcNP@Drug-MN was obtained by peeling it from the mold; The main part of the obtained double-layer microneedle except the base is a double-layer structure with upper and lower layers. The upper layer is the PcNP@Drug complex obtained by loading PcNP with drugs, and the lower layer is the HAT@Coll hydrogel obtained by combining hyaluronic acid-tyramine hydrogel HAT and collagenase Coll. PcNP is obtained by doping photosensitizer phthalocyanine Pc on mesoporous silica nanospheres MSN.

2. The method for preparing double-layer microneedles with controllable transdermal performance according to claim 1, characterized in that: In step 2), the mass concentration of HAT stock solution is 1.0 wt%~6.0 wt%, the mass concentration of Coll stock solution is 0.5 wt%~3 wt%, and the mass concentration of PcNP@Drug stock solution is 1 wt%~10 wt%; in step iv, the mass concentration of H2O2 used is 0.1 wt%~1.0 wt%, and the concentration of HRP used is 1 U / mL~5 U / mL.

3. The method for preparing a double-layer microneedle with controllable transdermal performance according to claim 1, characterized in that: In step 2), in step iv, the volume ratio of H2O2, HRP, Coll stock solution, HAT stock solution and PcNP@Drug stock solution is 1:1:50:50:

100.

4. The method for preparing double-layer microneedles with controllable transdermal performance according to claim 1, characterized in that: In step 1), the preparation process of the PDMS microneedle template is as follows: the PDMS monomer and the curing agent are mixed and poured into a container containing a stainless steel microneedle mold, vacuumed in a vacuum drying oven at room temperature, heated and cured in an oven, and after cooling to room temperature, the mold is taken out, and the PDMS is separated from the stainless steel microneedle to obtain a PDMS microneedle template.

5. The method for preparing double-layer microneedles with controllable transdermal performance according to claim 1, characterized in that: The preparation process of PcNP used in step iii of step 2) is as follows: (1) Synthesis of Pc-4NO2: Ammonium molybdate was mixed with 4-nitrophthalic anhydride and urea and then added to a nitrobenzene solution of zinc chloride; the mixture was stirred at 185°C for 4 hours under nitrogen protection, cooled, toluene was added, the precipitate was washed and centrifuged, and then washed with toluene, water, MeOH / ether mixture and ethyl acrylate / hexane mixture, and air-dried to obtain a dark green solid; (2) Synthesis of Pc-4NH2: Pc-4NO2 was dissolved in DMF, and sodium sulfide nonahydrate was added thereto. The mixture was heated and stirred at 60°C for 1.5 hours under nitrogen protection. The reaction mixture was cooled to room temperature, ice water was added, and the precipitate was separated by centrifugation, washed, and air-dried to obtain a dark green solid. (3) Synthesis of Pc-Si: Weigh a certain amount of Pc-4NH2 into a three-necked flask, add anhydrous DMF to dissolve it, dissolve 3-(triethoxysilyl)propyl isocyanate in anhydrous DMF and inject it into the three-necked flask, reflux at 120°C, protect with nitrogen, and stir overnight to obtain a Pc-Si solution; (4) Synthesis of PcNPs: CTAB was dissolved in H2O, and triethanolamine was added, followed by vigorous stirring at 80°C for 30 min to obtain a CTAB solution. TMOS and Pc-Si were uniformly mixed and then added dropwise to the CTAB solution under vigorous stirring. After the reaction was completed, the temperature was lowered to 50°C, and 2-(methoxy(polyethoxy)-propyl)trimethoxysilane was added dropwise. The mixture was stirred overnight to complete the reaction, and the mixture was centrifuged. The precipitate was washed with water and ethanol to obtain the product PcNPs.

6. The method for preparing double-layer microneedles with controllable transdermal performance according to claim 1, characterized in that: In step 3), the template needs to be cleaned with O2 plasma before adding the HAT pre-gel solution to the PDMS template to enhance the hydrophilicity of its surface.

7. The method for preparing double-layer microneedles with controllable transdermal performance according to claim 1, characterized in that: The drug loaded into PcNP in step iii of step 2) is a drug for treating a class of diseases present on the surface of the skin.

8. The method for preparing double-layer microneedles with controllable transdermal performance according to claim 2, characterized in that: In step 2), the mass concentration of HAT stock solution was 4.0 wt%, the mass concentration of Coll stock solution was 2.0 wt%, and the mass concentration of PcNP@Drug stock solution was 6.0 wt%. In step iv), the mass concentration of H2O2 used was 0.5 wt%, and the concentration of HRP used was 5 U / mL.

9. A double-layer microneedle with controllable transdermal performance, characterized in that: The invention is prepared based on the preparation method described in any one of claims 1 to 8.

Images