A soluble microneedle transdermal drug delivery system, a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and specifically provides a soluble microneedle transdermal drug delivery system and a preparation method thereof. The nanocapsule is PEGylated, so as to improve the biocompatibility of the nanocapsule. The obtained soluble microneedle transdermal drug delivery system comprises a substrate and a needle body, and both of them load the nanocapsule by using a biocompatible degradable polymer component. The system has strong tissue specificity, avoids the toxic and side effects of drugs on normal tissues, reduces the pain of patients in the treatment process, and improves the compliance of patients in the treatment process. The microneedle in the system can dissolve in the skin and release drugs, and will not cause damage to the human body. The system has low residual and low biological pollution risk, and has higher drug loading capacity and higher safety compared with the coating microneedle and the hollow microneedle. The system has excellent biocompatibility, high viscoelasticity, plasticity and permeability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a soluble microneedle transdermal drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Pain in the body is caused by disease, trauma, external noxious stimuli, accompanied by existing or potential tissue damage. Pain is a subjective unpleasant complex physiological and psychological activity, which can affect the body locally or as a whole, and is one of the most common symptoms in clinical practice. Therefore, pain relief is an important task for medical workers. Generally, after using related drugs, the pain can be within the range of the body. At present, there are various painkillers on the market, and such drugs have certain therapeutic effect in the treatment of pain, but the effect is not significant. Researchers have been exploring various drug delivery systems to deliver drugs into the body to achieve local and systemic prevention and treatment of pain. At present, the most common drug delivery method is oral administration and injection, and oral administration has slow effect and low drug utilization rate; but injection using a hard needle can cause bleeding and pain.

[0003] The microneedle transdermal drug delivery system is a research hotspot of transdermal drug delivery system in recent years. The microneedle transdermal drug delivery system forms a drug delivery channel by nanocrystal chip to dredge the skin surface layer, so that the active ingredients on the skin surface can quickly penetrate into the body, thereby effectively improving the therapeutic effect, having the double advantages of injection and transdermal drug delivery, and having many advantages such as rapidity, convenience and painlessness, which can significantly improve the transdermal rate and absorption amount of drugs, especially in the field of transdermal preparation of macromolecular substances such as proteins, polysomes, DNA and RNA, showing good effect and application prospect. Compared with the commonly used metal and silicon microneedles on the market, the polymer microneedle has higher biocompatibility and can be used as a transport carrier for growth factors, drugs and genes. SUMMARY

[0004] The purpose of the present application is to provide a soluble microneedle transdermal drug delivery system loaded with nanocystis for treating pain.

[0005] To this end, the present application provides a preparation method of a soluble microneedle transdermal drug delivery system, comprising the following steps:

[0006] (1) The raw material of the thylakoid membrane is stored in the dark, and the starch stored in the chloroplast matrix is consumed;

[0007] (2) The stored raw material is mixed with water, crushed, filtered, the filtrate is centrifuged, the supernatant is removed, and the precipitate is collected;

[0008] (3) Low-osmotic lysis solution is added to the precipitate, which is homogenized and then placed in the dark, and the precipitate obtained by centrifugation is the thylakoid;

[0009] (4) adding a buffer solution to the thylakoids, mixing uniformly after cell disruption and treatment, adding a PEG2000 aqueous solution, stirring, and ultrafiltration and concentration to obtain PEGylated nanometer thylakoids;

[0010] (5) dissolving the PEGylated nanometer thylakoids in water containing calcium ions to prepare a nanometer thylakoid solution, dissolving sodium alginate in the nanometer thylakoid solution to prepare a sodium alginate solution containing nanometer thylakoids;

[0011] (6) dissolving the PEGylated nanometer thylakoids in water, and then adding hyaluronic acid to prepare a hyaluronic acid solution containing nanometer thylakoids;

[0012] (7) injecting the sodium alginate solution containing nanometer thylakoids into a microneedle mold, centrifuging, freezing and vacuum drying the microneedle mold to obtain a needle body, then adding the hyaluronic acid solution containing nanometer thylakoids as a substrate into the microneedle mold, centrifuging, freezing and vacuum drying again, taking out the microneedle mold, and demolding to obtain a soluble microneedle transdermal drug delivery system loaded with nanometer thylakoids.

[0013] Specifically, the thylakoid membrane raw material in the above step (1) includes plants, green algae and photosynthetic bacteria; the plants include one or more of alfalfa, rape, sugar beet, dandelion, Arabidopsis thaliana, corn, tobacco, sunflower, lettuce, chamomile, seepweed, spinach, and grass.

[0014] Specifically, after homogenization treatment in the above step (3), the thylakoids are placed on ice in the dark for 0.5-2h, centrifuged at 4-30℃ and 2000-20000rpm for 5-30min to obtain the thylakoids.

[0015] Specifically, the mass ratio of thylakoids to PEG2000 in the above step (4) is 1:(1-20).

[0016] Specifically, the concentration of nanometer thylakoids in the sodium alginate solution containing nanometer thylakoids in the above step (5) is 3-50mg / mL, the concentration of calcium ions is 1-100mM, and the concentration of sodium alginate is 10-300mg / mL.

[0017] Specifically, the sodium alginate solution containing nanometer thylakoids in the above step (5) further includes a penetration enhancer.

[0018] Specifically, the concentration of nanometer thylakoids in the hyaluronic acid solution containing nanometer thylakoids in the above step (6) is 3-50mg / mL, and the concentration of hyaluronic acid is 10-300mg / mL.

[0019] Specifically, the thickness of the substrate in the above step (7) is 1-5mm.

[0020] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0021] 1、The preparation method of the soluble microneedle transdermal drug delivery system provided by the application improves the biocompatibility of the nanocapsule by PEGylation of the nanocapsule, and the obtained soluble microneedle transdermal drug delivery system comprises a substrate and a needle body, both of which are made of biocompatible and degradable polymer components, such as hyaluronic acid and sodium alginate, as main raw materials for loading the nanocapsule. The needle body contains sodium alginate and calcium ions, the carboxyl group of sodium alginate can be mediated by calcium ions to form a hydrogel, and the nanocapsule is wrapped in the gel to form a needle tip structure with good mechanical properties, which can quickly penetrate the stratum corneum of the skin. In addition, the needle body also contains a penetration enhancer that can increase the permeability of the skin, which is released in the body after the microneedle puncture, can increase the permeability of the body's biological barrier, promote the penetration of the nanocapsule, and improve the penetration efficiency and release speed of the nanocapsule. The main raw material of the substrate is hyaluronic acid, which is used to load the nanocapsule. Finally, under the irradiation of laser, the photosystem I, photosystem II, catalase, Cytb6 / f complex and ATPase complex on the nanocapsule membrane can remove oxygen free radicals in the tissue, improve the microenvironment of the tissue, and decompose water to continuously generate oxygen, ATP and NADPH, thereby eliminating or weakening the pain and realizing long-term and multiple light irradiation treatment of the painful tissue.

[0022] 2、The soluble microneedle transdermal drug delivery system provided by the application has strong tissue specificity, avoids the toxic and side effects of drugs on normal tissues throughout the body, reduces the pain of patients during treatment, and improves the compliance of patients during treatment. The microneedles in the system can dissolve and release drugs in the skin, and even if they are broken in the skin, they will not cause damage to the human body, have less residue, low risk of biological pollution, higher drug loading capacity and higher safety compared with coated microneedles and hollow microneedles, and the use of hyaluronic acid as a substrate material can rapidly dissolve in tissue fluid and degrade in the body, so that the soluble microneedle transdermal drug delivery system has excellent biocompatibility, high viscoelasticity, plasticity and permeability. It provides the possibility for simple, convenient and effective treatment and / or prevention of some long-term and chronic diseases, and has the characteristics of safety, high efficiency and non-invasiveness.

[0023] The application will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the particle size distribution of the PEGylated nanocapsule in the embodiment of the application.

[0025] Figure 2 is a schematic diagram of the in vitro oxygen production capacity of the PEGylated nanocapsule provided by the application.

[0026] Figure 3is the characterization of the oxygen production capacity of hydrogen peroxidase and photosynthetic organelle on the PEGylated nanocystidium in vitro with calcium ions.

[0027] Figure 4 is the quantitative measurement result chart of NADPH production capacity of the PEGylated nanocystidium provided by the present application.

[0028] Figure 5 is the back fluorescence imaging chart of a mouse treated with the soluble microneedle transdermal drug delivery system.

[0029] Figure 6 is the HE staining result chart of the PEGylated nanocystidium. DETAILED DESCRIPTION

[0030] The technical solutions in the present application will be clearly and completely described below with embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Although the representative embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present application without departing from the scope of the present application. Therefore, the scope of the present application should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0031] The present application provides a preparation method of a soluble microneedle transdermal drug delivery system, comprising the following steps:

[0032] (1) Put the thylakoid membrane raw material into a sealed bag, cover it with tin paper to avoid light, and store it at 4℃ overnight to consume the stored starch in the chloroplast matrix.

[0033] The thylakoid raw material includes plants, green algae and photosynthetic bacteria; the photosynthetic bacteria include blue-green bacteria and purple bacteria; the plants include one or more of alfalfa, rape, sugar beet, dandelion, Arabidopsis thaliana, corn, tobacco, sunflower, lettuce, Salsola collniana, Atriplex, spinach, and grass.

[0034] (2) The sealed raw material is mixed with water at a mass ratio of 1:1-10 using a screw press and a mixer, then crushed and filtered with 8 layers of gauze, and the filtrate is centrifuged at 4-30℃ and 2000-20000rpm for 5-30min, and the supernatant is removed to collect the intact and broken chloroplasts, and the precipitate is collected.

[0035] (3) Add a hypotonic lysis solution to the precipitate, process it in a homogenizer for 1-10min, then stand it on ice in the dark for 0.5-4h, and centrifuge it at 4-30℃ and 2000-20000rpm for 5-30min to obtain thylakoids.

[0036] (4) Add a buffer solution, preferably a PBS solution, to the thylakoids, and treat with an ultrasonic cell disruptor for 0.1-2 h. Then add a PEG2000 aqueous solution, stir at 37°C for 2 h, and concentrate using a 10 kDa MWCO ultrafiltration tube at 3000 x g to obtain the PEGylated nanometer thylakoids. The mass ratio of thylakoids to PEG2000 is 1:1-20.

[0037] (5) Dissolve the PEGylated nanometer thylakoids in water containing calcium ions to obtain a nanometer thylakoid solution. Dissolve sodium alginate in the nanometer thylakoid solution to obtain a sodium alginate solution containing nanometer thylakoids.

[0038] The concentration of nanometer thylakoids in the sodium alginate solution containing nanometer thylakoids is 3-50 mg / mL, the concentration of calcium ions is 1-100 mM, and the concentration of sodium alginate is 10-300 mg / mL.

[0039] The sodium alginate solution containing nanometer thylakoids also includes a penetration enhancer. The principle of chemical penetration enhancer is to change the arrangement structure of the lipid in the stratum corneum by interacting with it, thereby enhancing the permeability, making the transdermal effect of drugs better, and the targeted drug delivery effect better, providing solid technical support for more extensive use in clinical practice. Penetration enhancers can be subdivided into chemical penetration enhancers (such as ethanol, propylene glycol, azone, organic acids, surfactants, etc.), traditional Chinese medicine penetration enhancers (such as borneol, eucalyptus, peppermint, clove, etc.). Preparation of combined penetration enhancer: mix chemical penetration enhancer and traditional Chinese medicine penetration enhancer according to a certain proportion, such as 1% azone, 2% borneol, 2% menthol, 2% glycerol, and the rest is water.

[0040] (6) Dissolve the PEGylated nanometer thylakoids in water, then add hyaluronic acid to obtain a hyaluronic acid solution containing nanometer thylakoids.

[0041] The concentration of nanometer thylakoids in the hyaluronic acid solution containing nanometer thylakoids is 3-50 mg / mL, and the concentration of hyaluronic acid is 10-300 mg / mL.

[0042] (7) Inject the sodium alginate solution containing nanometer thylakoids into a microneedle mold, centrifuge at 2000-15000 rpm for 5-20 min, then place the microneedle mold in a freeze vacuum drying box at 4°C for 4-24 h, vacuumize to remove air bubbles in the microneedle mold, and discharge air and moisture in the microneedle mold to obtain a needle body. Then add the hyaluronic acid solution containing nanometer thylakoids as a substrate into the microneedle mold, centrifuge at 2000-15000 rpm for 5-20 min, freeze and vacuumize again (4°C, 4-24 h), take out the microneedle model, and demold to obtain a soluble microneedle transdermal drug delivery system loaded with nanometer thylakoids. The thickness of the substrate of the final transdermal drug delivery microneedle is 1-5 mm.

[0043] The biocompatible materials used for the substrate and the needle body are mainly sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, hyaluronic acid, sodium carboxymethyl cellulose and polylactic acid-glycolic acid copolymer.

[0044] The effect of the soluble microneedle transdermal drug delivery system is studied through specific examples as follows.

[0045] Example 1

[0046] This example provides a soluble microneedle transdermal drug delivery system, which is prepared by the following steps:

[0047] (1) Remove the hard veins on the fresh spinach leaves and wash them with deionized water for 4 times. Dry the surface water of the leaves with a clean paper towel, put them into a sealed bag, cover them with tin foil to avoid light, and store them at 4°C overnight to consume the starch stored in the chloroplast matrix.

[0048] (2) Use a screw press and a mixer to mix the frozen spinach with water at a ratio of 1:1, and crush the spinach leaves.

[0049] (3) Filter with 8 layers of gauze, and centrifuge the filtrate at 6000 rpm in a centrifuge at 4°C for 15 min to collect intact and broken chloroplasts.

[0050] (4) Remove the supernatant, add a hypotonic lysis solution (10 mM HEPES, pH 8.0) to the precipitate, and process it in a homogenizer for 5 min, then store it on ice in the dark for 30 min to break the chloroplast membrane using osmotic pressure.

[0051] (5) Centrifuge the precipitate at 10000 rpm in a centrifuge at 4°C for 15 min. This precipitate is the thylakoid.

[0052] (6) Add a buffer solution to the thylakoid, and process it with an ultrasonic cell disruptor for 0.1-2 h. Add 0.6 mL of 1 mg / mL PEG2000 aqueous solution to 3 mL of thylakoid PBS solution (containing 200 μg of thylakoid), and stir at 37°C for 2 h. Concentrate to 1 mL using a 10 kDa MWCO ultrafiltration tube at 3000 rpm to obtain PEGylated nanometer thylakoid.

[0053] Dilute the PEGylated nanometer thylakoid to the appropriate concentration, and use a Malvern particle size and potential analyzer to determine the particle size, as shown in Figure 1 .

[0054] (7) Finally, suspend 1 g of PEGylated nanometer thylakoid film in 0.4 M glucose, and freeze-dry it to obtain PEGylated nanometer thylakoid freeze-dried powder.

[0055] (8) The nano-capsule lyophilized powder is dissolved in water containing calcium ions (concentration of 50 mM) to prepare a nano-capsule solution (concentration of 25 mg / mL), and then sodium alginate powder containing a suitable penetration enhancer is dissolved in the above solution (concentration of 150 mg / mL) to prepare a sodium alginate solution containing nano-capsules.

[0056] (9) The nano-capsule lyophilized powder is dissolved in water to prepare a nano-capsule solution (concentration of 25 mg / mL), and then hyaluronic acid powder is dissolved in water (concentration of 150 mg / mL) to prepare a hyaluronic acid solution containing nano-capsules;

[0057] (10) A microneedle mold is prepared, and after ultrasonic cleaning of the microneedle mold, the sodium alginate solution containing nano-capsules is injected into the microneedle mold. After the nano-capsule solution fills the microneedle mold, centrifugation (10000 rpm, 20 min) is performed, and the microneedle mold is placed in a freeze vacuum drying box (0°C, 12 h). Vacuum is applied to remove air bubbles and moisture in the microneedle mold;

[0058] The microneedle mold is removed from the freeze vacuum drying box, and after removing the deionized water in the microneedle mold, the needle body is obtained. The hyaluronic acid solution containing nano-capsules is poured into the microneedle mold as a substrate, and centrifugation (10000 rpm, 20 min) is performed. The microneedle mold is again placed in a freeze vacuum drying box for drying treatment, and the microneedle mold is removed to obtain a soluble microneedle transdermal drug delivery system loaded with nano-capsules. The final transdermal drug delivery microneedle substrate has a thickness of 3 mm.

[0059] The soluble microneedle transdermal drug delivery system loaded with nano-capsules is packaged and sterilized by irradiation, and stored in a -20°C or -80°C refrigerator.

[0060] Example 2:

[0061] In this example, the PEGylated nano-capsules and the soluble microneedle transdermal drug delivery system prepared in Example 1 are tested.

[0062] 1. In vitro oxygen production capacity

[0063] 10 mL of non-PEGylated nano-capsule solution and 10 mL of PEGylated nano-capsule solution (both with a chlorophyll concentration of 40 μg / mL) are mixed with the same volume of 1 mM H2O2 solution, respectively. The change in dissolved oxygen value in the solution within 10 min is measured using a portable dissolved oxygen meter. As shown in Figure 2 PEGylated nano-capsules retain the oxygen production activity of nano-capsules and can catalyze the decomposition of hydrogen peroxide to produce a large amount of oxygen. The reaction gradually reaches equilibrium after 6 minutes.

[0064] 2. Characterization of the effect of calcium ions on the oxygen production capacity of hydrogen peroxidase and photosynthetic enzyme on nanoscale thylakoids in vitro

[0065] Two 10 mL aliquots of PEGylated nanoscale thylakoid solution (chlorophyll concentration of 40 μg / mL) were added with 100 μl of 0 mol / L and 1 mol / L calcium chloride solution, respectively, and then mixed with the same volume of 1 mM H2O2 solution. The change in the dissolved oxygen value in the solution was determined using a portable dissolved oxygen meter within 10 min. As shown in Figure 3 , calcium ions can improve the activity of hydrogen peroxidase and photosynthetic enzyme on nanoscale thylakoids, thereby improving the oxygen production activity of nanoscale thylakoids, and a large amount of oxygen is produced. The reaction gradually reaches equilibrium after 6 min.

[0066] 3. Quantitative measurement of NADPH production capacity

[0067] In a 3 mL reaction system (40 μg / mL PEGylated nanoscale thylakoids, 400 mM sucrose, 20 mM tricine NaOH pH = 7.8, 2.5 mM MgCl2, 30 mM NaCl, 1 mM glucose, 2 U / mL hexokinase, 500 μM ADP, 0.5 U / mL 6-phosphogluconate dehydrogenase), LED irradiation was performed.

[0068] Red light irradiation at ~630 nm was performed, and NADPH production was measured every 5 min by using an NADPH detection kit.

[0069] The detection results are shown in Figure 4 , which show that PEGylated nanoscale thylakoids significantly produce NADPH under light irradiation.

[0070] 4. The back of a mouse was treated by using a soluble microneedle transdermal drug delivery system for transdermal drug delivery, and then optical imaging of the mouse was obtained as shown in Figure 5 , it was found that the fluorescence intensity of the back of the mouse was obviously higher than that of other parts, indicating that the soluble microneedle transdermal drug delivery system prepared by using the application can make the drug enriched in the treated part after transdermal drug delivery, and can be applied to local treatment of diseases.

[0071] 5. After 7 days of tail vein injection of PEGylated nanoscale thylakoid membranes (10 mg / kg), as shown in Figure 6 , HE staining of important organs such as the heart, liver, spleen, lung and kidney of the mouse showed no obvious damage. It is indicated that the targeted nanoscale thylakoid membranes prepared by using the application have good biocompatibility.

[0072] The above examples are only illustrative of the application and do not constitute a limitation on the protection scope of the application. Any design identical or similar to the application falls within the protection scope of the application.

Claims

1. A method for preparing a soluble microneedle transdermal drug delivery system, characterized by, The method comprises the following steps: (1) storing the thylakoid membrane raw material in the dark, and consuming the starch stored in the chloroplast matrix; (2) mixing the stored raw material with water, crushing, filtering, centrifuging the filtrate, removing the supernatant, and collecting the precipitate; (3) adding a hypotonic lysis solution to the precipitate, homogenizing, storing in the dark, and centrifuging to obtain the thylakoids; (4) adding a buffer solution to the thylakoids, homogenizing, stirring, ultrafiltration, and concentration to obtain PEGylated nanometer thylakoids; (5) dissolving the PEGylated nanometer thylakoids in water containing calcium ions to obtain a nanometer thylakoid solution, dissolving sodium alginate in the nanometer thylakoid solution to obtain a sodium alginate solution containing nanometer thylakoids; (6) dissolving the PEGylated nanometer thylakoids in water, and then adding hyaluronic acid to obtain a hyaluronic acid solution containing nanometer thylakoids; (7) injecting the sodium alginate solution containing nanometer thylakoids into a microneedle mold, centrifuging, freezing and vacuum drying the microneedle mold to obtain a needle body, then adding the hyaluronic acid solution containing thylakoids as a substrate into the microneedle mold, centrifuging, freezing and vacuum drying again, taking out the microneedle mold, and demolding to obtain a soluble microneedle transdermal drug delivery system loaded with nanometer thylakoids; wherein: the thylakoid membrane raw material in step (1) comprises one or more of alfalfa, rape, sugar beet, dandelion, Arabidopsis thaliana, corn, tobacco, sunflower, lettuce, Chenopodium pumilum, Atriplex, spinach, and green algae; the mass ratio of the thylakoids to PEG2000 in step (4) is 1:(1-20); the concentration of nanometer thylakoids in the sodium alginate solution containing nanometer thylakoids in step (5) is 3-50 mg / mL, the concentration of calcium ions is 1-100 mM, and the concentration of sodium alginate is 10-300 mg / mL; the concentration of nanometer thylakoids in the hyaluronic acid solution containing nanometer thylakoids in step (6) is 3-50 mg / mL, and the concentration of hyaluronic acid is 10-300 mg / mL.

2. The preparation method of the soluble microneedle transdermal administration system according to claim 1, characterized in that: In step (3), the thylakoids are obtained by homogenizing, storing in the dark on ice for 0.5-2 h, centrifuging at 4-30℃ and 2000-20000 rpm for 5-30 min.

3. The preparation method of the soluble microneedle transdermal administration system according to claim 1, characterized in that: The sodium alginate solution containing nanometer thylakoids in step (5) further comprises a penetration enhancer.

4. The preparation method of the soluble microneedle transdermal administration system according to claim 1, characterized in that: The thickness of the substrate in step (7) is 1-5 mm.

5. The soluble microneedle transdermal drug delivery system prepared by the method of any one of claims 1-4.

6. The use of the soluble microneedle transdermal drug delivery system of claim 5 in the preparation of a pain treatment drug.

Images