Self-adaptive drug delivery microneedle patch as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a self-adaptive drug delivery microneedle patch as well as a preparation method and application thereof. Comprising a sulfhydrylation silk fibroin matrix used for preparing a microneedle matrix, a drug-loading liposome encapsulated in the microneedle matrix and cholesterol oxidase loaded in the microneedle matrix, the interior of the sulfhydrylated silk fibroin matrix comprises a three-dimensional space network structure formed by crosslinking disulfide bonds; a lipid-lowering drug is encapsulated in the drug-loaded liposome. According to the microneedle patch, the problem of excessive treatment or insufficient drug effect caused by individual metabolism difference in a traditional drug delivery mode is solved, and real-time synchronization of the drug release rate and the blood fat level change is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to an adaptive drug delivery microneedle patch and its preparation method and application. Background Art

[0002] With the development of social economy and the change of diet structure, the prevalence of dyslipidemia has shown a significant upward trend globally. Among them, hypercholesterolemia is the main type, which is significantly associated with atherosclerotic cardiovascular diseases (ASCVD), diabetes and other metabolic diseases. Current statin drugs regulate cholesterol synthesis by inhibiting HMG-CoA reductase, but the activity of this enzyme is affected by the circadian rhythm and usually reaches its peak at night, resulting in the need for the traditional oral administration method to follow a bedtime dosing regimen. However, clinical practice has shown that in some patients, the blood drug concentration fluctuates beyond the safety threshold due to forgotten medication, and the contradiction between this rigid dosing mode and individual metabolic differences exacerbates the risk of secondary complications.

[0003] Existing transdermal devices based on microneedle technology have achieved a technological iteration from passive diffusion to environmental response, but there are still significant limitations. For example, integrated sensing microneedle systems (such as electrochemical / colorimetric detection types) can only unidirectionally monitor blood lipid levels and cannot link with the drug delivery module to form a closed-loop regulation; pH-responsive / lipophilic microneedles can trigger drug release, but mostly show a single burst release and lack a termination mechanism, and cannot adapt to the dynamic fluctuations of cholesterol concentration (±18.7% within 24 hours); existing systems lack the ability of real-time feedback based on metabolic markers, and may cause abnormal reduction of total cholesterol due to over-dosing. This one-way and discrete regulation mechanism is difficult to meet the dynamic balance requirements of chronic disease management and severely restricts the implementation effect of precision medicine in chronic disease management. Therefore, it is urgently needed to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an adaptive drug delivery microneedle patch and its preparation method and application, which can solve the problems of over-treatment or insufficient drug efficacy caused by individual metabolic differences in traditional drug delivery methods, and achieve real-time synchronization of the drug release rate and the change of blood lipid level.

[0005] To achieve the above object, the technical solution of the present invention is realized as follows: an adaptive drug delivery microneedle patch and its preparation method and application, characterized in that it includes: a mercapto-functionalized silk fibroin matrix for making the microneedle matrix, drug-loaded liposomes encapsulated in the microneedle matrix, and cholesterol oxidase loaded in the microneedle matrix; the interior of the mercapto-functionalized silk fibroin matrix includes a three-dimensional network structure formed by disulfide bond cross-linking; the drug-loaded liposomes encapsulate lipid-lowering drugs.

[0006] Preferably, the three-dimensional spatial network structure undergoes a disulfide bond dynamic reduction-oxidation reaction under the action of the electron transfer generated by cholesterol oxidase catalyzing cholesterol.

[0007] When the cholesterol concentration increases, the disulfide bonds are reduced to thiol groups, resulting in partial dissociation of the three-dimensional spatial network structure, realizing the amplification of the three-dimensional spatial network structure to release liposomes and responding to cholesterol to release lipid-lowering drugs; when the cholesterol concentration decreases, the thiol groups spontaneously oxidize to reconstruct disulfide bonds, and the three-dimensional spatial network structure shrinks to stop releasing liposomes to block excessive drug release. Finally, the dynamic drug release ability of the whole system is realized.

[0008] Preferably, the drug-loaded liposome includes a lipid bilayer; the lipid bilayer is composed of dioleoylphosphatidylcholine (DOPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in a molar ratio of 1:1 - 5:1.

[0009] Preferably, the lipid-lowering drugs include one or more of simvastatin, atorvastatin, lovastatin, pravastatin, fluvastatin, and rosuvastatin.

[0010] Preferably, the loading amount of cholesterol oxidase is 0.5 - 5 U / mg of the microneedle matrix, and the electrons generated by its catalytic reaction trigger the dynamic reorganization of the three-dimensional spatial network structure through a thiol-disulfide exchange mechanism. That is, under the reducing condition after obtaining electrons, partial dissociation of the spatial network structure caused by the cleavage of disulfide bonds, and the spatial network structure restores after the oxidation of thiol groups to form disulfide bonds when losing electrons.

[0011] Preferably, the height of the needle body of the microneedle matrix is 500 - 1000 μm, the tip diameter is 0.5 - 5 μm, and the bottom diameter of the needle body is 100 - 300 μm; the disulfide bond crosslinking density is 0.2 - 0.8 mmol of sulfur groups per milligram of protein.

[0012] Preferably, it includes the following steps:

[0013] (1) Cut the silkworm cocoons into pieces and immerse them in a 0.01 - 0.05 M sodium carbonate solution, degum at 95 - 100 °C for 45 - 90 min, then rinse with deionized water at 40 - 50 °C for 3 - 5 times, and dry to obtain degummed silk; add the degummed silk to an 8 - 10 mol / L lithium bromide solution at a mass-to-volume ratio of 1:8 - 1:12, dissolve at a constant temperature of 55 - 65 °C for 1 - 3 h; load the dissolved solution into a dialysis bag with a molecular weight cut-off of 5 - 10 kDa, dialyze in flowing deionized water for 48 - 72 h and change the dialysis medium every 2 - 8 h; after the dialysis product is coarsely filtered through a 100 - 200 mesh filter cloth, centrifuge at 4 - 10 °C and 8000 - 10000 rpm for 15 - 25 min, and collect the supernatant and freeze-dry to obtain silk fibroin;

[0014] (2) Dissolve the silk fibroin obtained in step (1) in water to obtain a silk fibroin solution, and dilute the silk fibroin solution to 20 - 40 mg / mL, then cool it under the condition of 0 - 4 °C; adjust the pH of the solution to 5.0 - 6.0, and then add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), where the addition amount of NHS is 1 - 4% of the mass of silk fibroin and the addition amount of EDC is 2 - 6%; add cysteamine to the mixed solution to a final concentration of 50 - 70 mM, maintain the pH at 5.0 - 6.0 and react at 0 - 10 °C for 3 - 6 hours; load the reaction solution into a dialysis bag with a molecular weight cut-off of 6 - 15 kDa, and perform dialysis under inert gas protection in deionized water containing 0.5 - 2 mM sodium thiosulfate for 48 - 72 hours, and then change to pure deionized water for dialysis for 12 - 24 hours; after dialysis, filter through a 0.1 - 0.5 μm filter membrane and centrifuge at 5000 - 12000 rpm in sequence to obtain a thiolated silk fibroin solution, collect the supernatant and freeze-dry it to obtain thiolated silk fibroin;

[0015] (3) Dissolve DSPE-PEG2000 and DOPC in an organic solvent methanol according to a molar ratio of 1:3 - 1:5 to prepare a phospholipid mixed solution with a concentration of 2 - 8 mg / mL, and remove the solvent by rotary evaporation to form a lipid film; prepare a statin drug into a 1 - 10 mg / mL aqueous or ethanol solution, and stir it with the lipid film in the dark at 50 - 70 °C in a water bath at a rotation speed of 500 - 800 rpm for 20 - 60 min to form primary liposomes; perform 8 - 25 times of thermal cycling extrusion treatment on the mixed solution through a filter membrane with a pore size of 450 - 500 nm, and control the extrusion temperature at 55 - 65 °C; centrifuge the extruded solution at a rotation speed of 7000 - 9000 rpm for 2 - 4 times, and collect the precipitate to obtain drug-loaded liposomes with a particle size of 450 - 500 nm;

[0016] (4) Prepare a PDMS negative mold using a rigid microneedle mold with a needle height of 500 - 800 μm and a bottom diameter of the needle body of 200 - 300 μm; dissolve the thiolated silk fibroin obtained in step (2) in water to prepare an aqueous solution with a concentration of 3 - 8 wt%, and add drug-loaded liposomes and 0.1 - 10 U / mg cholesterol oxidase to the aqueous solution according to a mass-volume ratio of 1:5 - 1:15 to obtain a mixed solution; inject the mixed solution into the PDMS negative mold and perform staged vacuum degassing treatment, with the vacuum degree maintained at ≥0.09 MPa for 20 - 40 min each time, and cycle 3 - 5 times; then transfer it to a curing and drying condition at a relative humidity of 30 - 60% and a temperature of 25 - 40 °C for 4 - 8 h, and then demold to obtain a microneedle array.

[0017] Preferably, the drug-loaded liposomes described in step (3) are prepared by the thin film hydration method, with a particle size of 400 - 650 nm and a polydispersity index ≤0.2.

[0018] Preferably, the microneedle patch is used to prepare a transdermal drug delivery system for the treatment of hyperlipidemia, and dynamic regulation is achieved through the following mechanism: as the blood cholesterol level increases, the interstitial fluid cholesterol is decomposed by cholesterol oxidase to produce electron transfer, which promotes the breakage of disulfide bonds into thiol groups and releases liposome nanodrugs; when the cholesterol level drops below the threshold, the thiol groups spontaneously oxidize to rebuild the disulfide bond network, reducing the drug release rate by 70-95%.

[0019] Preferably, the response threshold of the transdermal drug delivery system to total cholesterol is 5.2-6.2 mmol / L, and the fluctuation range of blood drug concentration is maintained within ±15% during the treatment cycle.

[0020] The beneficial effects of the present invention are embodied in:

[0021] (1) The microneedle patch provided by the present invention can realize a dynamic closed-loop feedback treatment function: through the synergistic effect of enzyme catalysis and dynamic reconstruction of chemical bonds, the drug release rate is adaptively matched to the patient's cholesterol level. When the cholesterol concentration is detected to exceed the clinical safety threshold, the microneedle patch automatically increases the drug release to the effective treatment rate range; when the indicator returns to the normal physiological range, the release rate is intelligently lowered to the maintenance dose level. Animal experiments have shown that the microneedle patch can significantly improve the problem of unstable drug efficacy caused by diurnal fluctuations in blood lipids, and exhibits better blood lipid compliance rate and time maintenance characteristics compared to traditional therapies.

[0022] (2) The microneedle patch provided by the present invention has an individualized safety protection mechanism: based on the adaptive regulation characteristics of the dynamic network structure, after the blood lipid index reaches the preset safety threshold, the microneedle patch can autonomously inhibit excessive drug release, effectively reducing the incidence of common adverse reactions of statins. For special populations (such as patients with diabetes and hyperlipidemia), the microneedle patch preferentially responds to changes in specific pathological biomarkers, enhances the intensity of treatment response under abnormal conditions such as oxidative stress, and significantly improves the arterial inflammatory microenvironment. Preclinical studies have shown that it has a significant regulatory effect on the levels of key inflammatory factors.

[0023] (3) The microneedle patch provided by the present invention has clinical adaptability optimization: dynamically adjust drug release kinetic parameters according to the target range recommended by the international lipid management guidelines; adapt differentiated treatment intensity for patients with different cardiovascular risk levels; and improve the accuracy of treatment for complex metabolic disorders by cross-responding to pathological markers such as oxidatively modified lipoproteins. The above principle is to achieve the dynamic adjustment of drug release, adaptation of differentiated treatment intensity, and cross-response to pathological markers through the dissociation and self-repair of the spatial network structure of the microneedle patch in the redox state (the dissociation and self-repair speed can be controlled by controlling the intensity of the redox reaction), and use liposomes that adapt to the spatial changes as drug-carrying matrices, so as to achieve the dynamic adjustment of drug release, adaptation of differentiated treatment intensity, and cross-response to pathological markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the particle size distribution diagram of the drug-loaded liposomes of the present invention;

[0025] Figure 2 This is the TEM image of the drug-loaded liposomes of the present invention;

[0026] Figure 3 This is the drug encapsulation efficiency performance diagram of the drug-loaded liposomes of the present invention;

[0027] Figure 4 This is the selective drug release performance diagram of the drug-loaded liposomes of the present invention;

[0028] Figure 5 This is the scanning electron microscope image of the microneedle patch of the present invention;

[0029] Figure 6 This is the three-dimensional spatial network structure diagram of the microneedle patch of the present invention;

[0030] Figure 7 This is the responsive drug release performance diagram of the microneedle patch of the present invention to the cholesterol threshold (5.2 - 6.2 mmol / L). Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] Embodiment

[0033] As Figures 1 - 7 shown, the present invention provides an adaptive drug delivery microneedle patch and its preparation method and application, including the following steps:

[0034] A polymer negative mold is prepared using a microneedle mold with a predetermined size range, and the height of the needle body and the width of the base of the mold are adapted to the physiological structure of the skin cutin layer. A thiol-functionalized silk fibroin is dissolved in an aqueous medium to form a matrix solution within a specific concentration range, and a nano drug-loading unit and a cholesterol oxidase component with cholesterol-responsive activity are incorporated in an optimized ratio; the mixed system is subjected to multi-stage vacuum treatment to eliminate internal defects, and each treatment is maintained for a necessary duration under preset vacuum parameters and repeated multiple times; subsequently, matrix curing and molding are completed in a controllable temperature and humidity environment, and a smart microneedle array with a hierarchical structure is obtained after the demolding process. The nano drug-loading unit is a liposome loaded with statin drugs. The enzyme activity unit of cholesterol oxidase is 0.1 - 10 U / mg to achieve a response to the cholesterol concentration threshold.

[0035] The microneedle patch prepared by the present invention is used for the daily management of hyperlipidemia through self-adaptive lipid-lowering drug release.

[0036] The enzyme activity unit of cholesterol oxidase loaded in one dose of microneedle patch (15*15 array) is 0.1 - 10 U / mg, and the amount of statin drug loaded is 300 - 500 μg.

[0037] When the cholesterol concentration exceeds the safety threshold of 5.2 - 6.2 mmol / L, the disulfide bond is reduced to a thiol group, resulting in partial dissociation of the network structure and drug release; when the cholesterol concentration is within the safety threshold range, the thiol group spontaneously oxidizes to reconstruct the disulfide bond to block excessive drug release.

[0038] Example 1

[0039] The preparation of a smart microneedle patch for self-adaptive lipid-lowering drug delivery specifically includes the following steps:

[0040] Extraction of silk fibroin: Immerse silk cocoon fragments in a 0.02 M sodium carbonate solution, perform degumming treatment at a constant temperature of 98°C for 60 minutes, then rinse with deionized water at 45°C 4 times, and obtain degummed silk after vacuum drying at 40°C for 12 hours; take the degummed silk and add it to a 9 mol / L lithium bromide solution at a mass-to-volume ratio of 1:10, stir and dissolve at 60°C for 2 hours to form a uniform protein solution; load the dissolved solution into a dialysis bag with a molecular weight cut-off of 8 kDa, dialyze in flowing deionized water for 60 hours (changing the solution every 4 hours), and after dialysis, filter through a 150-mesh nylon filter cloth roughly, and centrifuge at 4°C and 9000 rpm for 20 minutes to remove impurities; collect the supernatant, pre-freeze it (-80°C / 4 h) and then freeze-dry it (-50°C / 0.5 Pa / 48 h) to finally obtain a white flocculent silk fibroin powder.

[0041] Thiolation modification of silk fibroin: At 4 °C and pH 5.5, 2.5% NHS and 5% EDC were added to a 30 mg / mL silk fibroin solution for activation, and then 60 mM cysteamine was introduced and reacted for 4 hours. After dialysis through a 10 kDa dialysis bag under the protection of 1 mM sodium thiosulfate and nitrogen for 60 hours, pure water was changed for continuous dialysis for 18 hours. Finally, a product with a high substitution degree (0.42 mmol / g) was obtained by filtration through a 0.22 μm filter membrane and centrifugation at 9000 rpm.

[0042] Preparation of drug-loaded liposomes: DSPE-PEG2000 and DOPC were mixed at a molar ratio of 1:4 and dissolved in methanol to prepare a 5 mg / mL phospholipid solution. A uniform lipid film was formed by rotary evaporation at 45 °C (vacuum degree 0.09 MPa). Pravastatin was dissolved in deionized water to prepare a 5 mg / mL drug solution, which was magnetically stirred (600 rpm) with the lipid film in a water bath at 60 °C in the dark for 40 minutes to form primary liposomes. The mixture was extruded 15 times through a 500 nm polycarbonate filter membrane preheated to 55 °C (extrusion pressure 3.5 MPa). The obtained extruded solution was centrifuged 3 times at 4 °C and 8000 rpm, and the precipitate was collected to obtain drug-loaded liposomes.

[0043] As Figure 1 shown, dynamic light scattering (DLS) detection showed that the average particle size of the drug-loaded liposomes was 500 ± 50 nm, and transmission electron microscopy (TEM, Figure 2 ) characterization showed that the vesicle structure was intact. The maximum drug encapsulation efficiency determined by ultraviolet spectrometer reached 82.3 ± 1.7% ( Figure 3 ). The stability test showed that the drug-loaded liposomes only responded to cholesterol and were not affected by interstitial fluid components including glucose and lactic acid ( Figure 4 ).

[0044] Preparation of intelligent microneedle patches: A stainless steel microneedle mold with a needle height of 600 μm and a bottom diameter of the needle body of 250 μm was used to prepare a PDMS negative mold by two-time casting and curing. The thiolated silk fibroin was prepared into a 5 wt% aqueous solution, and the liposomes loaded with pravastatin and 5 U / mg cholesterol oxidase were added at a mass-volume ratio of 1:10, and magnetically stirred (300 rpm) to form a homogeneous mixture. The mixture was injected into the PDMS negative mold and then placed in a vacuum degassing machine for three times of degassing treatment (vacuum degree 0.095 MPa, 30 minutes each time, and the normal pressure was restored after 10 minutes). Subsequently, it was transferred to a curing box with a humidity of 50% and a temperature of 35 °C for drying for 6 hours, and the microneedle patch was obtained after demolding.

[0045] Scanning electron microscopy characterization of the microneedle patches obtained in the examples showed ( Figure 5 ) that the needle body was in a regular conical shape without obvious collapse, and uniform nano-pores could be seen under high magnification ( Figure 6), with an average pore size of 500 ± 7 nm.

[0046] In vitro release experiments showed that when the cholesterol concentration was 6.2 mmol / L, the loaded pravastatin drug was significantly released, and after the cholesterol concentration decreased to 5.2 mmol / L, the drug release automatically attenuated by 90% ( Figure 7 ).

[0047] Example 2

[0048] Preparation of an intelligent microneedle patch for adaptive lipid-lowering drug delivery, specifically including the following steps:

[0049] The extraction method of silk fibroin is basically the same as step (1) in Example 1, except that the sodium carbonate concentration is 0.01 M, and the mass-volume ratio of degummed silk to lithium bromide solution (9 mol / L) is 1:5.

[0050] Thiol modification of silk fibroin: Adjust the pH of a 35 mg / mL silk fibroin solution to 6.5 at 4 °C, add 1% NHS and 2% EDC based on the mass of silk fibroin, and then slowly add cysteamine to a final concentration of 50 mM, stir the reaction in the dark for 3 hours; transfer the reaction solution to a dialysis bag with a molecular weight cut-off of 10 kDa, immerse it in nitrogen-protected deionized water containing 0.5 mM sodium thiosulfate, dialyze at 4 °C for 24 hours (change the solution every 6 hours), and then continue to dialyze with pure deionized water for 8 hours; the dialyzed solution is filtered through a 0.22 μm sterile filter membrane and centrifuged at 4 °C and 8000 rpm for 15 minutes, and the supernatant is collected to obtain the thiolated silk fibroin solution.

[0051] Preparation of drug-loaded liposomes: Mix DSPE-PEG2000 and DOPC in a molar ratio of 1:3, dissolve them in methanol to prepare a 3 mg / mL phospholipid solution, and form a lipid film by rotary evaporation at 60 °C; dissolve pravastatin in deionized water to prepare an 8 mg / mL drug solution, and magnetically stir (700 rpm) the drug solution and the lipid film in the dark at 55 °C in a water bath for 30 minutes to form primary liposomes; the mixture is thermally extruded 50 times through a 450 nm filter membrane preheated to 50 °C (extrusion pressure 3.0 MPa); the extruded solution is centrifuged 3 times at 4 °C and 8500 rpm, and the precipitate is collected to obtain the drug-loaded liposomes.

[0052] Preparation of intelligent microneedle patch: A stainless steel microneedle mold with a needle height of 600 μm and a needle body bottom diameter of 250 μm was used to prepare a PDMS negative mold through two casting-curing processes; The mercapto-functionalized silk fibroin was formulated into an 8 wt% aqueous solution, and the drug-loaded liposomes and 10 U / mg cholesterol oxidase were added according to a mass-volume ratio of 1:10; The mixed solution was injected into the PDMS negative mold and then subjected to staged vacuum degassing treatment. The vacuum was maintained at 0.095 MPa for 30 minutes each time, and the cycle was repeated 3 times; Subsequently, it was transferred to a condition of 45% relative humidity and 32 °C for gradient curing and drying for 6 hours. After demolding, a microneedle array was obtained.

[0053] Example 3

[0054] Extraction of silk fibroin: The extraction method of silk fibroin was basically the same as that in step (1) of Example 1, except that the mass-volume ratio of degummed silk to lithium bromide solution (9 mol / L) was 1:3.

[0055] Mercapto-functionalization modification of silk fibroin: The silk fibroin solution was adjusted to 25 mg / mL and cooled to 4 °C. After adjusting the pH to 5.5, 2% NHS and 4% EDC were added. After mixing evenly, cysteamine was added to a final concentration of 60 mM, and the reaction was carried out at 4 °C for 4 hours; The reaction solution was loaded into a 10 kDa dialysis bag and dialyzed in nitrogen-protected deionized water containing 1 mM sodium thiosulfate for 48 hours, and then continued to be dialyzed with pure water for 12 hours; After the dialysis solution was filtered through a 0.22 μm filter membrane, it was centrifuged at 9000 rpm for 20 minutes, and the supernatant was collected to obtain the mercapto-functionalized silk fibroin solution.

[0056] Preparation of drug-loaded liposomes: DSPE-PEG2000 and DOPC were mixed at a molar ratio of 1:5 and dissolved in methanol to prepare a 6 mg / mL phospholipid solution. A lipid film was formed by rotary evaporation at 40 °C; Pravastatin was dissolved in deionized water to prepare a 7 mg / mL drug solution, and it was magnetically stirred (550 rpm) in a 65 °C water bath with the lipid film in the dark for 50 minutes to form primary liposomes; The mixed solution was extruded through a preheated 480 nm filter membrane at 60 °C for 20 times (extrusion pressure 3.2 MPa); The extruded solution was centrifuged 3 times at 4 °C and 7500 rpm, and the precipitate was collected to obtain the drug-loaded liposomes.

[0057] Preparation of intelligent microneedle patch: A stainless steel microneedle mold with a needle height of 600 μm and a needle body bottom diameter of 250 μm was used to prepare a PDMS negative mold through two casting-curing processes; The mercapto-functionalized silk fibroin was formulated into a 6 wt% aqueous solution, and the drug-loaded liposomes and 3 U / mg cholesterol oxidase were added according to a mass-volume ratio of 1:12; The mixed solution was injected into the PDMS negative mold and then subjected to staged vacuum degassing treatment. The vacuum was maintained at 0.092 MPa for 25 minutes each time, and the cycle was repeated 4 times; Subsequently, it was transferred to a condition of 50% relative humidity and 35 °C for curing and drying for 5 hours. After demolding, a microneedle array was obtained.

[0058] Example 4

[0059] Extraction of silk fibroin: The extraction method of silk fibroin is basically the same as that in step (1) of Example 1, except that the concentration of sodium carbonate is 0.05 M.

[0060] Thiol modification of silk fibroin: Cool the 25 mg / mL silk fibroin solution to 4 °C, adjust the pH to 6.0, add 3% NHS and 5% ED, mix and then add cysteamine to a final concentration of 55 mM, and react at 4 °C for 3.5 hours; Dialyze the reaction solution in a nitrogen-protected solution containing 0.8 mM sodium thiosulfate for 60 hours, and continue dialysis with pure water for 18 hours; After dialysis, filter through a 0.3 μm filter membrane and centrifuge at 10000 rpm for 15 minutes to obtain a thiolated silk fibroin solution

[0061] Preparation of drug-loaded liposomes: Mix DSPE-PEG2000 and DOPC at a molar ratio of 1:4, dissolve in methanol to prepare a 4 mg / mL phospholipid solution, and form a lipid film by rotary evaporation at 42 °C; Dissolve pravastatin in deionized water to prepare a 3 mg / mL drug solution, and magnetically stir (750 rpm) with the lipid film in the dark at 58 °C in a water bath for 25 minutes to form primary liposomes; The mixture is extruded through a 470 nm filter membrane preheated at 52 °C 18 times (extrusion pressure 3.1 MPa); The extruded solution is centrifuged 4 times at 4 °C and 7200 rpm, and the precipitate is collected to obtain drug-loaded liposomes.

[0062] Preparation of intelligent microneedle patches: Use a stainless steel microneedle mold with a needle height of 600 μm and a needle body bottom diameter of 250 μm to prepare a PDMS negative mold by two-stage casting-curing; Prepare a 5 wt% aqueous solution of thiolated silk fibroin, and add drug-loaded liposomes and 2 U / mg cholesterol oxidase according to a mass-volume ratio of 1:8; Inject the mixed solution into the PDMS negative mold and perform staged vacuum degassing treatment, with each vacuum degree maintained at 0.093 MPa for 35 minutes, and cycle 4 times; Subsequently, transfer to a curing and drying condition of 55% relative humidity and 38 °C for 5 hours, and demold to obtain a microneedle array.

[0063] In summary, the preparation method of the adaptive lipid-lowering drug delivery intelligent microneedle patch provided by this patent systematically demonstrates the optimized combination and technical feasibility of adjusting key process parameters such as silk fibroin concentration, liposome ratio, and enzyme activity dose through four examples, and can achieve a cholesterol-responsive release threshold (5.2 - 6.2 mmol / L).

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An adaptive drug delivery microneedle patch, characterized in that: include: A thiolated silk fibroin matrix for making a microneedle matrix, a drug-loaded liposome encapsulated in the microneedle matrix, and a cholesterol oxidase loaded in the microneedle matrix; The interior of the thiolated silk fibroin matrix includes a three-dimensional spatial network structure formed by disulfide bond cross-linking; and the drug-loaded liposome encapsulates a lipid-lowering drug.

2. The adaptive drug delivery microneedle patch according to claim 1, characterized in that: The three-dimensional network structure undergoes a dynamic reduction-oxidation reaction of disulfide bonds under the action of electron transfer generated by cholesterol catalyzed by cholesterol oxidase.

3. The adaptive drug delivery microneedle patch according to claim 1, characterized in that: The drug-loaded liposome comprises a lipid bilayer; the lipid bilayer is composed of dioleoylphosphatidylcholine (DOPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in a molar ratio of 1:1-5:

1.

4. The adaptive drug delivery microneedle patch according to claim 1, characterized in that: The lipid-lowering drugs include statins; the statins include one or more of simvastatin, atorvastatin, lovastatin, pravastatin, fluvastatin, and rosuvastatin.

5. The adaptive drug delivery microneedle patch according to claim 1, characterized in that: The loading amount of the cholesterol oxidase is 0.5-5U / mg microneedle matrix, and the electrons generated by its catalytic reaction trigger the dynamic reorganization of the three-dimensional network structure through the thiol-disulfide bond exchange mechanism.

6. The adaptive drug delivery microneedle patch according to claim 1, characterized in that: The needle body height of the microneedle matrix is ​​500-1000 μm, the needle tip diameter is 0.5-5 μm, and the needle body bottom diameter is 100-300 μm; the density of the disulfide cross-linking is 0.2-0.8 mmol sulfur group per mg protein.

7. A method for preparing an adaptive drug delivery microneedle patch according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Cutting the silk cocoons into pieces and immersing them in a 0.01-0.05M sodium carbonate solution, degumming them at 95-100°C for 45-90 minutes, then rinsing them with deionized water at 40-50°C for 3-5 times, and drying them to obtain degummed silk; adding the degummed silk into an 8-10 mol / L lithium bromide solution at a mass volume ratio of 1:8-1:12, and dissolving them at a constant temperature of 55-65°C for 1-3 hours; placing the dissolved solution into a dialysis bag with a molecular weight cutoff of 5-10 kDa, dialysing them in flowing deionized water for 48-72 hours, and replacing the dialysis medium every 2-8 hours; filtering the dialyzed product through a 100-200 mesh filter cloth, centrifuging them at 4-10°C and 8000-10000 rpm for 15-25 minutes, collecting the supernatant and freeze-drying them to obtain silk fibroin; (2) dissolving the silk fibroin obtained in step (1) in water to obtain a silk fibroin solution, diluting the silk fibroin solution to 20-40 mg / mL, and cooling at 0-4° C.; adjusting the pH of the solution to 5.0-6.0, adding 1-4% N-hydroxysuccinimide (NHS) and 2-6% 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC); adding cysteamine to the mixed solution to a final concentration of 50-70 mM, maintaining the pH at 4 ° C. 5.0-6.0 and amidation reaction at 0-10°C for 3-6 hours; the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 6-15 kDa, and dialyzed in deionized water containing 0.5-2 mM sodium thiosulfate under inert gas protection for 48-72 hours, and then dialyzed with pure deionized water for 12-24 hours; after dialysis, it is filtered through a 0.1-0.5 μm filter membrane and centrifuged at 5000-12000 rpm to obtain a thiolated silk fibroin solution, and the supernatant is collected and freeze-dried to obtain thiolated silk fibroin; (3) DSPE-PEG2000 and DOPC are dissolved in an organic solvent, methanol, at a molar ratio of 1:3-1:5 to prepare a 2-8 mg / mL phospholipid mixed solution, and the solvent is removed by rotary evaporation to form a lipid film; a statin is prepared into a 1-10 mg / mL water or ethanol solution, and the statin is stirred with the lipid film in a water bath at 50-70°C and 500-800 rpm for 20-60 min under light-proof conditions to form primary liposomes; the mixed solution is subjected to 8-25 times of thermal cycle extrusion through a filter membrane with a pore size of 450-500 nm, and the extrusion temperature is controlled at 55-65°C; the extrudate is centrifuged at 7000-9000 rpm for 2-4 times, and the precipitate is collected to obtain drug-loaded liposomes with a particle size of 450-500 nm; (4) A PDMS negative mold is prepared using a hard microneedle mold with a needle height of 500-800 μm and a needle bottom diameter of 200-300 μm; the thiolated silk fibroin obtained in step (2) is prepared into a 3-8 wt % aqueous solution, and drug-loaded liposomes and 0.1-10 U / mg cholesterol oxidase are added to the aqueous solution at a mass volume ratio of 1:5-1:15 to obtain a mixed solution; the mixed solution is injected into the PDMS negative mold and subjected to a staged vacuum degassing treatment, each time the vacuum degree is maintained at ≥0.09 MPa for 20-40 min, and the cycle is repeated 3-5 times; then the microneedle patch is transferred to a relative humidity of 30-60% and a temperature of 25-40° C. for curing and drying for 4-8 h, and then demolded to obtain a microneedle patch.

8. The method for preparing an adaptive drug delivery microneedle patch according to claim 6, characterized in that: In step (3), the drug-loaded liposomes are prepared by a thin film hydration method; the particle size of the drug-loaded liposomes is 400-650nm, and the polydispersity index is ≤0.2; and the drug encapsulation rate of the drug-loaded liposomes is greater than 80%.

9. An application of the microneedle patch according to any one of claims 1 to 6, characterized in that: The microneedle patch is used for preparing a transdermal drug delivery system for treating hyperlipidemia.

10. The use of an adaptive drug delivery microneedle patch according to claim 8, characterized in that: The response threshold of the transdermal drug delivery system to total cholesterol is 5.2-6.2 mmol / L, and the fluctuation range of blood drug concentration is maintained within ±15% during the treatment cycle.

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