Preparation method and application of bletilla striata polysaccharide modified nanocrystal-loaded microneedle

Abstract

The invention provides a preparation method and application of a bletilla striata polysaccharide modified nanocrystal-loaded microneedle, and the preparation method comprises the following steps: preparing andrographolide, first polyvinylpyrrolidone, polyvinyl alcohol and a first bearing liquid into a concentrated crystal suspension, and uniformly suspending andrographolide nanocrystals in the concentrated crystal suspension; mixing the concentrated and crystallized suspension and bletilla striata polysaccharide to obtain a needle solution; mixing second polyvinylpyrrolidone, glycerol and a second bearing solution to obtain a substrate solution; and putting the needle head solution and the matrix solution into a mold to form the bletilla striata polysaccharide modified nanocrystal-loaded microneedle. Experiments show that by adding the bletilla striata polysaccharide to form the needle solution, the healing rate of holes caused by puncture is effectively improved, and the risks of skin allergy and infection are avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly relates to a preparation method and application of bletilla striata polysaccharide-modified nanocrystalline-loaded microneedles. Background Art

[0002] Andrographolide (ADR) is a natural diterpenoid lactone isolated from the plant Andrographis paniculata, which has a wide range of biological activities, such as anti-cancer, anti-inflammatory, antibacterial, antiviral, and hepatoprotective effects. However, its poor solubility and low bioavailability seriously hinder its clinical application.

[0003] Nanocrystals (NCs) are submicron colloidal dispersions stabilized by surfactants or polymers. By reducing the particle size of drugs and increasing the specific surface area of drug particles, the solubility of hydrophobic drugs can be effectively improved. However, due to the multi-layer barrier effect of the skin, simply encapsulating drugs in conventional nanocarriers is not sufficient to effectively transdermally deliver poorly water-soluble drugs into the blood.

[0004] Further processing NCs into microneedles suitable for transdermal drug delivery can effectively solve the above problems. Microneedles (MN) are a new type of drug delivery system composed of needles. Their submillimeter-scale design can penetrate the stratum corneum of the skin to achieve painless and minimally invasive transdermal drug delivery of drug molecules. However, the skin holes created by the puncture of the microneedle needles damage the integrity of the skin and also increase the risk of skin allergy and infection. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method and application of bletilla striata polysaccharide-modified nanocrystalline-loaded microneedles, aiming to solve the technical problem that the microneedle drug delivery method in the prior art damages the integrity of the skin and also increases the risk of skin allergy and infection.

[0006] To achieve the above purpose, in the first aspect, the present invention provides a preparation method of bletilla striata polysaccharide-modified nanocrystalline-loaded microneedles, including the following steps:

[0007] Prepare andrographolide, the first polyvinylpyrrolidone, polyvinyl alcohol, and the first carrier liquid into a concentrated crystalline suspension, and andrographolide nanocrystals are uniformly suspended in the concentrated crystalline suspension;

[0008] Mix the concentrated crystalline suspension and bletilla striata polysaccharide to form a needle solution;

[0009] Mix the second polyvinylpyrrolidone, glycerol, and the second carrier liquid to form a base solution;

[0010] Place the needle solution and the matrix solution in a mold to form the nano-crystalline micro-needles modified with bletilla striata polysaccharide.

[0011] Furthermore, the mass ratio among andrographolide, polyvinylpyrrolidone and polyvinyl alcohol is 1:1:1, and the mass ratio of andrographolide to the first carrier liquid is 1:1000 - 1:50000.

[0012] Even further, the step of preparing andrographolide, the first polyvinylpyrrolidone, polyvinyl alcohol and the first carrier liquid into a concentrated crystalline suspension includes:

[0013] Place andrographolide, polyvinylpyrrolidone and polyvinyl alcohol in the first carrier liquid and stir to uniformly disperse andrographolide, polyvinylpyrrolidone and polyvinyl alcohol in the first carrier liquid and form a crude suspension;

[0014] Perform high-pressure homogenization treatment on the crude suspension through a high-shear homogenizing emulsifying dispersing machine to obtain a crude crystalline suspension;

[0015] Concentrate and dilute the crude crystalline suspension through a rotary evaporator to obtain a concentrated crystalline suspension.

[0016] Even further, the rotation speed of the high-pressure homogenization treatment is 10000 rpm - 20000 rpm, the time of the high-pressure homogenization treatment is 5 min - 20 min, the temperature of the high-pressure homogenization treatment is 41°C - 50°C, and the number of times of the high-pressure homogenization treatment is 10 - 30 times.

[0017] Even further, the step of concentrating and diluting the crude crystalline suspension through a rotary evaporator to obtain a concentrated crystalline suspension includes:

[0018] Place the crude crystalline suspension in an eggplant-shaped flask, perform vacuum pumping on the eggplant-shaped flask to make the pressure inside the eggplant-shaped flask reach a preset pressure;

[0019] Perform concentration treatment on the crude crystalline suspension in the eggplant-shaped flask after vacuum pumping to obtain a concentrated solution;

[0020] Perform dilution treatment on the concentrated solution to make the content of andrographolide nano-crystals in the concentrated solution reach a preset content and form a concentrated crystalline suspension.

[0021] Even further, the preset pressure is 0.09 Mpa, the concentration multiple of the concentration treatment is 50 times, and the preset content is 1 mg / ml - 20 mg / ml.

[0022] Even further, the particle size of the andrographolide nano-crystals is 200 nm - 800 nm.

[0023] Furthermore, the mass ratio of the concentrated crystallization suspension to the bletilla striata polysaccharide is 1:1.

[0024] Furthermore, the mass ratio of the second polyvinylpyrrolidone to the second carrier liquid is 1:1 to 1:10.

[0025] In a second aspect, the present invention provides an application of the bletilla striata polysaccharide-modified nano-crystalline micro-needles prepared by the method for preparing bletilla striata polysaccharide-modified nano-crystalline micro-needles described in the first aspect above in improving the transdermal absorption rate.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: Experiments show that by preparing the needle solution and the matrix solution into the micro-needle form, the penetration ability of the stratum corneum barrier can be improved, and the transdermal absorption rate can be increased to a certain extent; by preparing the concentrated crystallization suspension and then forming the needle solution, on the basis of the micro-needle form, the penetration of the poorly soluble traditional Chinese medicine active ingredient is further improved in the form of crystals, and the transdermal absorption rate is further increased; by adding the bletilla striata polysaccharide to form the needle solution, the healing rate of the holes caused by puncture is effectively improved, and the risks of skin allergy and infection are avoided. Description of the Drawings

[0027] Figure 1 It is a flow chart of the method for preparing bletilla striata polysaccharide-modified nano-crystalline micro-needles in the embodiment of the present invention;

[0028] Figure 2 It is a scanning electron micrograph of andrographolide in the embodiment of the present invention;

[0029] Figure 3 It is a transmission electron micrograph of andrographolide nano-crystals in the embodiment of the present invention;

[0030] Figure 4 It is an appearance diagram of the concentrated crystallization suspension in the embodiment of the present invention;

[0031] Figure 5 It is a scanning electron micrograph of the bletilla striata polysaccharide-modified nano-crystalline micro-needles in the embodiment of the present invention;

[0032] Figure 6 It is a graph of the in vitro release behavior of andrographolide (ADR) and its nano-crystals in the embodiment of the present invention;

[0033] Figure 7 It is a graph of the in vitro release behavior of different andrographolide micro-needle preparations in the embodiment of the present invention;

[0034] Figure 8 It is a graph of the transdermal results of applying ADR-MN, ADR-NCs-MN, and ADR-NCs-BPS-MN in the embodiment of the present invention;

[0035] Figure 9 These are the in vitro transdermal results graphs of ADR-MN, ADR-NCs-MN, and ADR-NCs-BPS-MN in the stratum corneum-stripped skin in the embodiments of the present invention;

[0036] Figure 10 These are the results graphs of the drug accumulation amounts in the skin at different depths after 2 hours of applying ADR-MN, ADR-NCs-MN, and ADR-NCs-BPS-MN in the embodiments of the present invention;

[0037] Figure 11 These are the graphs of the dissolution conditions of the microneedle tips at different times after applying different microneedles to the rat skin in the embodiments of the present invention. Among them, Figure 11 A is the result graph of ADR-MN, Figure 11 B is the result graph of ADR-NCs-MN, Figure 11 C is the result graph of ADR-NCs-BPS-MN;

[0038] Figure 12 These are the plasma concentration-time curves of ADR-MN, ADR-NCs-MN, and ADR-NCs-BPS-MN in the rat body in the embodiments of the present invention;

[0039] Figure 13 These are the graphs of the recovery conditions of the rat skin after applying different microneedles in the embodiments of the present invention. Among them, Figure 13 A is the recovery condition graph of ADR-MN, Figure 13 B is the recovery condition graph of ADR-NCs-MN, Figure 13 C is the recovery condition graph of ADR-NCs-BPS-MN;

[0040] Figure 14 These are the graphs of the hemostasis conditions of the rat tails after applying different microneedles in the embodiments of the present invention. Among them, 1 is the hemostasis condition graph of ADR-MN, 2 is the recovery condition graph of ADR-NCs-MN, and 3 is the recovery condition graph of ADR-NCs-BPS-MN;

[0041] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0042] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0043] It should be noted that when an element is referred to as being "fixed to" another element, it can be directly on the other element or there can also be an intermediate element. When an element is considered to be "connected to" another element, it can be directly connected to the other element or there may be an intermediate element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used in this article are only for the purpose of illustration.

[0044] Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of this invention in this article are only for the purpose of describing specific embodiments and are not intended to limit this invention. The term "and / or" used in this article includes any and all combinations of one or more of the related listed items.

[0045] Please refer to Figure 1 , in the first aspect, Embodiment 1 of the present invention provides a preparation method of berberine polysaccharide-modified nano-crystalline micro needles, including the following steps:

[0046] S10: Prepare andrographolide, the first polyvinylpyrrolidone, polyvinyl alcohol and the first carrier liquid into a concentrated crystal suspension, and andrographolide nano-crystals are uniformly suspended in the concentrated crystal suspension;

[0047] The mass ratio between andrographolide, the polyvinylpyrrolidone and the polyvinyl alcohol is 1:1:1. In this embodiment, the mass ratio between andrographolide and the first carrier liquid is 1:1000.

[0048] Specifically, the step S10 includes:

[0049] S110: Place andrographolide, polyvinylpyrrolidone and polyvinyl alcohol in the first carrier liquid and stir to make andrographolide, polyvinylpyrrolidone and polyvinyl alcohol uniformly disperse in the first carrier liquid and form a coarse suspension;

[0050] When stirring, it is necessary to ensure that andrographolide, polyvinylpyrrolidone and polyvinyl alcohol are fully swollen in the first carrier liquid. In this embodiment, the first carrier liquid is water.

[0051] Please refer to Figure 2 , characterize the morphology of andrographolide with a scanning electron microscope: stick andrographolide on the scanning electron microscope sample stage with conductive glue, place it in an ion sputtering instrument for gold spraying treatment to increase its conductivity. Open the sample chamber, put in the sample and then start pumping vacuum, set the photographing parameters, take pictures and save the photos. The shape of andrographolide is irregular and the particle size varies greatly.

[0052] S120: Subject the coarse suspension to high-pressure homogenization treatment using a high-shear homogenizing emulsifying disperser to obtain a crude crystalline suspension;

[0053] In this embodiment, the rotation speed of the high-pressure homogenization treatment is 10,000 rpm, the time of the high-pressure homogenization treatment is 5 min, the temperature of the high-pressure homogenization treatment is 41 °C, and the number of times of the high-pressure homogenization treatment is 10 times.

[0054] S130: Concentrate and dilute the crude crystalline suspension using a rotary evaporator to obtain a concentrated crystalline suspension;

[0055] Place the crude crystalline suspension in an eggplant-shaped flask, perform a vacuum pumping treatment on the eggplant-shaped flask to make the pressure inside the eggplant-shaped flask reach a preset pressure, and the preset pressure is 0.09 Mpa; perform a concentration treatment on the crude crystalline suspension inside the eggplant-shaped flask after the vacuum pumping treatment to obtain a concentrated solution, and the concentration multiple of the concentration treatment is 50 times. Dilute the concentrated solution to make the andrographolide nanocrystals in the concentrated solution reach a preset content and form a concentrated crystalline suspension. In this embodiment, HPLC is used to measure the drug content of the andrographolide nanocrystals in the concentrated solution, and the preset content is 1 mg / ml to 20 mg / ml.

[0056] It should be noted that in this embodiment, the particle size of the andrographolide nanocrystals is 800 nm. For the transdermal absorption rate, the larger the particle size of the crystals, the smaller the gain effect on improving the transdermal absorption rate. On the contrary, the smaller the particle size of the crystals, the greater the gain effect on improving the transdermal absorption rate. When the particle size exceeds 800 nm, it will lead to a significant decline in the transdermal absorption rate.

[0057] Please refer to Figure 3 and Figure 4 , and use a transmission electron microscope to observe the morphology of the andrographolide nanocrystals: Turn on the transmission electron microscope and pump vacuum; Pull the sample and the objective lamp bar to position 0; Apply high voltage. After pressing the HT key, press the 40 kv - 60 kv - 80 kv - 100 kv keys in sequence, adjust the filament phase, perform rough focusing, and adjust the centering of the condenser lens. The andrographolide nanocrystals have regular shapes and a narrow particle size distribution; The concentrated crystalline suspension appears milky white in appearance.

[0058] Please refer to Figure 6, a dialysis bag with a molecular weight of 3500 Da was used to investigate the in vitro release rate of the above-mentioned andrographolide nanocrystals and compare them with andrographolide. The release study was carried out in a constant temperature shaker, controlling the temperature within the range of 37.0 ± 0.5 and shaking at 120 rpm for 24 h. Two equal-concentration and equal-volume samples of andrographolide and andrographolide nanocrystals were prepared. The dialysis bag was cut into an appropriate size, tied tightly at one end with cotton thread, and an equal volume of andrographolide and andrographolide nanocrystals were pipetted from the other end of the dialysis bag and dropped into the dialysis bag. The dialysis bag containing the liquid medicine was placed into a centrifuge tube. The centrifuge tube was filled with PBS solution and placed in a constant temperature shaker. At 1, 2, 3, 4, 6, 8, 12, and 24 h, 200 μL of the sample solution was taken out from the receptor chamber, and fresh receptor solution with the same temperature and volume was added to ensure that the volume remained constant. The release amount of andrographolide within 24 h was about 32%, while the release amount of andrographolide nanocrystals within 24 h was about 64%, indicating that nanocrystals can significantly improve the release of the poorly soluble drug andrographolide.

[0059] S20: Mix the concentrated crystal suspension and bletilla striata polysaccharide to form a needle solution;

[0060] The mass ratio of the concentrated crystal suspension to the bletilla striata polysaccharide is 1:1.

[0061] S30: Mix the second polyvinylpyrrolidone, glycerol, and the second carrier solution to form a base solution;

[0062] Both the first polyvinylpyrrolidone and the second polyvinylpyrrolidone are essentially polyvinylpyrrolidone, and the names are only used to distinguish their addition in different steps. The mass ratio of the second polyvinylpyrrolidone to the second carrier solution is 1:1 to 1:10. When preparing the base solution, it is necessary to ensure that the second polyvinylpyrrolidone and the glycerol are fully swollen in the second carrier solution. In this embodiment, the second carrier solution is water.

[0063] S40: Place the needle solution and the matrix solution in a mold to form bletilla striata polysaccharide-modified nanocrystal-loaded microneedles;

[0064] It can be understood that the bletilla striata polysaccharide-modified nanocrystal-loaded microneedles are ADR-NCs-BSP-MIN.

[0065] The step S40 includes:

[0066] Pour the needle solution onto the mold, perform a first centrifugation treatment on the mold to enable the needle solution to fill the interior of the mold, remove the needle solution on the surface of the mold, and perform a first drying treatment to obtain a first mold; pour the base solution onto the first mold, perform a second centrifugation treatment on the first mold to enable the base solution to enter the interior of the first mold, remove the matrix solution on the surface of the first mold, and perform a second drying treatment to obtain a second mold.

[0067] In this embodiment, the centrifugation speeds of the first centrifugation treatment and the second centrifugation treatment are 4000 rpm to 5000 rpm, the temperatures of the first drying treatment and the second drying treatment are both 25 °C, and the times of the first drying treatment and the second drying treatment are both 12 h to 36 h.

[0068] In this embodiment, for the prepared Bletilla striata polysaccharide-modified nano-crystalline micro-needles, the needles are neatly distributed on the base. There are a total of 16×16 conical needles. The diameter of the bottom of each needle is 0.34 mm, the height of the needle is 0.8 mm, and the distance between the tips of two adjacent needles is 0.55 mm. By controlling the content of andrographolide nano-crystals in the concentrated crystal suspension at the preset content, controlling the mass ratio between the concentrated crystal suspension and Bletilla striata polysaccharide, controlling the mass ratio between the second polyvinylpyrrolidone and the second carrier liquid, controlling the rotation speed of the centrifugation treatment, the temperature and time of the drying treatment, the aims are to ensure the integrity of the needles, the integrity of the base, and the mechanical properties of the prepared Bletilla striata polysaccharide-modified nano-crystalline micro-needles, and to ensure that the micro-needles achieve the optimal quality effect.

[0069] Please refer to Figure 5 , and use a scanning electron microscope to observe the Bletilla striata polysaccharide-modified nano-crystalline micro-needles: Use tweezers to stick the Bletilla striata polysaccharide-modified nano-crystalline micro-needles on the scanning electron microscope sample stage, place them in an ion sputtering instrument for gold spraying treatment to increase their conductivity. Open the sample chamber, put in the sample, start pumping vacuum, fix the Bletilla striata polysaccharide-modified nano-crystalline micro-needles on the bracket, and place them under the scanning electron microscope for observation. The needles of the micro-needles are complete and the needle shape meets the requirements.

[0070] Embodiment 2 of the present invention provides a method for preparing Bletilla striata polysaccharide-modified nano-crystalline micro-needles, which is different from the method for preparing Bletilla striata polysaccharide-modified nano-crystalline micro-needles described in Embodiment 1 in that:

[0071] The mass ratio of andrographolide to the first carrier liquid is 1:50000.

[0072] Example 3 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles described in Example 1 in that:

[0073] The rotation speed of the high-pressure homogenization treatment is 20,000 rpm.

[0074] Example 4 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles described in Example 1 in that:

[0075] The time of the high-pressure homogenization treatment is 20 min.

[0076] Example 5 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles described in Example 1 in that:

[0077] The temperature of the high-pressure homogenization treatment is 50 °C.

[0078] Example 6 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles described in Example 1 in that:

[0079] The number of times of the high-pressure homogenization treatment is 30 times.

[0080] Example 7 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles described in Example 1 in that:

[0081] The mass ratio of andrographolide to the first carrier liquid is 1:40,000, the rotation speed of the high-pressure homogenization treatment is 15,000 rpm, the time of the high-pressure homogenization treatment is 10 min, the temperature of the high-pressure homogenization treatment is 45 °C, and the number of times of the high-pressure homogenization treatment is 20 times.

[0082] Comparative Example 1 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles described in Example 1 in that:

[0083] The mass ratio of andrographolide to the first carrier liquid is 1:500.

[0084] Comparative Example 2 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline micro-needles described in Example 1 in that:

[0085] The rotation speed of the high-pressure homogenization treatment is 5000 rpm.

[0086] Comparative Example 3 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline-loaded microneedles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline-loaded microneedles described in Example 1 in that:

[0087] The time of the high-pressure homogenization treatment is 2 min.

[0088] Comparative Example 4 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline-loaded microneedles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline-loaded microneedles described in Example 1 in that:

[0089] The temperature of the high-pressure homogenization treatment is 30 °C.

[0090] Comparative Example 5 of the present invention provides a method for preparing polysaccharide-bletilla striata-modified nano-crystalline-loaded microneedles, which is different from the method for preparing polysaccharide-bletilla striata-modified nano-crystalline-loaded microneedles described in Example 1 in that:

[0091] The number of times of the high-pressure homogenization treatment is 5 times.

[0092] The concentrated crystalline suspension is prepared according to the method for preparing polysaccharide-bletilla striata-modified nano-crystalline-loaded microneedles described in Examples 1 to 7 and Comparative Examples 1 to 5 of the present invention, and the particle size of andrographolide nano-crystals in the concentrated crystalline suspension is detected, and the detection results are shown in Table 1:

[0093] Table 1

[0094]

[0095] As can be seen from the above table, by controlling the mass ratio between andrographolide and the first carrier liquid and combining with the control of the parameters of the high-pressure homogenization treatment, it is possible to ensure that andrographolide nano-crystals with a particle size below 1000 nm are obtained, thereby ensuring the improvement effect on the transdermal absorption rate.

[0096] Comparative Example 6 of the present invention provides a method for preparing andrographolide microneedles, comprising the following steps:

[0097] Disperse andrographolide, the first polyvinylpyrrolidone and polyvinyl alcohol in the first carrier liquid, and obtain a needle solution by stirring;

[0098] Mix the second polyvinylpyrrolidone, glycerol and the second carrier liquid to form a base solution;

[0099] Place the needle solution and the matrix solution in a mold to form andrographolide microneedles. It can be understood that the andrographolide microneedles are ADR-MN.

[0100] Comparative Example 7 of the present invention provides a method for preparing andrographolide nanocrystalline microneedles, comprising the following steps:

[0101] Prepare a concentrated crystallization suspension by mixing andrographolide, the first polyvinylpyrrolidone, polyvinyl alcohol and the first carrier liquid, and andrographolide nanocrystals are uniformly suspended in the concentrated crystallization suspension;

[0102] Mix the second polyvinylpyrrolidone, glycerol and the second carrier liquid to form a base solution;

[0103] Place the concentrated crystallization suspension and the matrix solution in a mold to form andrographolide nanocrystalline microneedles. It can be understood that the andrographolide nanocrystalline microneedles are ADR-NCs-MN.

[0104] Prepare microneedles according to the methods described in Example 1, Comparative Example 6 and Comparative Example 7 of the present invention, namely, prepare the bletilla striata polysaccharide-modified nanocrystalline-loaded microneedles ADR-NCs-BSP-MN), the andrographolide microneedles (ADR-MN) and the andrographolide nanocrystalline microneedles (ADR-NCs-MN). And conduct effect detection on them respectively.

[0105] In vitro release investigation:

[0106] Please refer to Figure 7 , use a dialysis bag with a molecular weight cut-off of 3500 Da, cut it into a suitable size, tie one end of the dialysis bag tightly with a cotton thread, and add 1 mL of PBS solution. Put the prepared ADR-MN, ADR-NCs-MN, and ADR-NCs-BSP-MN into the dialysis bag and tie it tightly with a cotton thread to prevent the receiving liquid from entering the dialysis bag during the oscillation process. Place the dialysis bag containing the microneedles in a centrifuge tube. The centrifuge tube contains 20 mL of PBS and is placed in a constant temperature shaker. Control the temperature within the range of 37.0 ± 0.5 and shake at 120 rpm for 24 h. At 1, 2, 3, 4, 6, 8, 12, and 24 h, take out 200 μL of the sample solution from the receptor chamber and supplement fresh receptor solution with the same temperature and volume to ensure that the volume remains constant. After making the microneedles, the nanocrystals can still improve the in vitro release rate of andrographolide, and bletilla striata polysaccharide can further improve the release rate of the drug from the microneedles.

[0107] Transdermal absorption rate investigation:

[0108] Please refer to Figure 8, Take out the rat skin from a -20 °C refrigerator and soak it in PBS (pH = 7.4) buffer for 30 min. Take ADR-MN, ADR-NCs-MN, and ADR-NCs-BSP-MN and continuously insert them into the rat skin with the same force (30 N) for 10 s. Place the intact rat skin between the receiving chamber and the donor compartment of a Franz diffusion cell, add equal-concentration and equal-volume andrographolide suspension and andrographolide nanocrystals suspension, and place it between the receptor and donor compartments of the Franz diffusion cell. In a 37 ± 0.5 °C constant temperature water bath, PBS-T with a concentration of 0.01 mol·L -1 is used as the receiving medium, and the receiving chamber is stirred at 350 r·min -1 . Take out 200 μL of the sample solution from the receiving chamber at 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, and add an equal volume of fresh receptor solution at the same temperature to ensure that the volume remains constant. There is not much difference in the in vitro transdermal penetration of ADR prepared as NCs, indicating that after the drug particle size is reduced, it still cannot pass through the skin stratum corneum barrier; after ADR is prepared as MN, the transdermal rate of the drug increases by about 5 times, which shows that microneedles can penetrate the skin stratum corneum barrier and promote drug transdermal absorption; the transdermal rate of ADR-NCs-MN is higher than that of ADR-MN because when the microneedles contact the interstitial fluid in the skin, ADR-NCs have a smaller particle size and specific surface area, have a higher drug concentration gradient during the penetration process, and can more easily penetrate the skin structure. Therefore, more ADR-NCs molecules penetrate into the receiving chamber through the dermis layer; when ADR-NCs-BSP-MN contacts the interstitial fluid in the skin, due to the high solubility of BSP, it can dissolve the MN faster, release the drug from the MN, and promote the penetration of the drug into the deep layer of the skin. Therefore, the transdermal rate of ADR-NCs-BSP-MN is the highest.

[0109] Please refer to Figure 9 , Use skin without stratum corneum to investigate the permeability. The results show that the permeability of ADR-NCs-BSP is the best and that of ADR is the lowest. Compared with the transdermal absorption without removing the stratum corneum, the transdermal rate increases by about 4 times, indicating that the stratum corneum is the biggest barrier to drug absorption.

[0110] Investigation of skin accumulation:

[0111] Please refer to Figure 10After 2 h of applying each preparation to porcine skin, the skin accumulation of ADR-NCs-BSP-MN was the highest, followed by ADR-NCs-MN, and the lowest was ADR-MN. Compared with ADR-MN, the drug skin accumulation content of ADR-NCs-MN increased. These results indicate that ADR-NCs-MN dissolves in contact with skin interstitial fluid, and ADR-NCs has stronger skin penetration ability than ADR. ADR-NCs has a larger specific surface area and solubility, and has a higher drug concentration gradient during the penetration process, enabling the microneedles to dissolve rapidly when in contact with skin interstitial fluid and facilitating better drug penetration. BSP has good water solubility, and when the microneedles come into contact with skin interstitial fluid, it promotes the release of the drug from the needle solution.

[0112] Investigation of dissolution in skin:

[0113] Please refer to Figure 11 and use an optical microscope to record the dissolution of microneedles at different time points of 0, 1, 5, 10, and 20 min after application on the abdominal skin of live rats. The results showed that at 1 min, ADR-MN, ADR-NCs-MN, and ADR-NCs-BSP-MN had started to dissolve in the skin. This is because after the microneedles are applied to the skin and come into contact with the interstitial fluid in the skin, over time, the microneedle tips gradually dissolve. By 5 min, half of ADR-NCs-MN and ADR-NCs-BSP-MN had dissolved. By 20 min, ADR-NCs-MN and ADR-NCs-BSP-MN had completely dissolved, while ADR-MN had not completely dissolved. This indicates that preparing ADR into NCs can accelerate the dissolution of the microneedle tips in the skin in vivo, and the combination of BSP and NCs has a synergistic promoting effect on the dissolution of microneedles in the skin in vivo.

[0114] Pharmacokinetic investigation:

[0115] Please refer to Figure 12, 30 male SD rats weighing 190 - 210 g were selected. They were not fasted or water - restricted before and during the entire experiment. One day before drug administration, the rats were anesthetized with 5% chloral hydrate. The abdominal hair of the rats was removed using an electric clipper, and then depilatory cream was evenly applied to the abdominal area of the rats for 3 min. The depilatory cream was washed off with normal saline and dried to completely remove the abdominal hair of the rats. The 30 rats were divided into 5 groups: 1. Andrographolide gel group, 6 rats, 1 mL each; 2. Andrographolide gel containing laurocapram, 6 rats, 1 mL each; 3. ADR - MN, 6 rats, 4 tablets; 4. ADR - NCs - MN, 6 rats, 4 tablets; 5. ADR - NCs - BSP - MN, 6 rats, 4 tablets. For the 1st and 2nd gel groups, the drugs were applied on plastic wrap and then applied to the depilated abdominal area of the rats and fixed with medical tape for drug administration. For the 3rd, 4th, and 5th microneedle groups, the same operator inserted 4 microneedles into the depilated abdominal area of the rats with a constant force for 10 s, and then fixed it with medical tape for drug administration. Starting from the end of drug administration, blood was collected from the posterior orbital venous plexus of the rats at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h. Approximately 0.3 mL of blood was collected with a capillary tube and the blood sample was collected into a frozen 1.5 mL anticoagulant tube containing sodium heparin and stored at - 20 °C. After processing the plasma samples of each group, UPLC / MS was used for detection, and the analysis results were processed using DAS 3.3.0 pharmacokinetic software. The absolute bioavailability of ADRGel, ADRLaGel, ADR - MN, ADR - NCs - MN, and ADR - NCs - BSP - MN were 10.11%, 25.43%, 27.01%, 47.38%, and 54.54% respectively. Microneedles can improve the transdermal absorption of drugs; nanocrystals can further improve the transdermal absorption - promoting ability of microneedles; due to its fast - dissolving property, Bletilla striata polysaccharide can also further improve the transdermal absorption of drugs.

[0116] Investigation of pore healing:

[0117] (1) Skin recovery performance:

[0118] Please refer to Figure 13 , after inserting the microneedles into the abdominal skin of the rats for 5 minutes, the microneedles were removed and the recovery of the abdominal skin of the rats was observed: in the ADR - MN group, 60 min after removing the microneedles, although the pores formed by microneedle puncture were significantly reduced compared with 0 min, there were still a small number of pores that did not heal; the pore recovery rate of ADR - NCs - MN was slightly faster than that of ADR - MN; in the ADR - NCs - BSP - MN group, 30 min after removing the microneedles, the micropores left on the skin basically disappeared, and after 60 min, it almost recovered to the same as the original skin surface. The experiment shows that Bletilla striata polysaccharide has a promoting effect on the recovery of the pores left by microneedles on the skin.

[0119] (2) Hemostatic performance:

[0120] Please refer to Figure 14 , at a position 5 cm from the tail tip of the rat, cut it off with a scalpel. Then, put the cut tails into 1 mL of ADR, ADR-NCs, and ADR-NCs-BSP with a concentration of 1 mg·mL -1 for 30 s. After 30 s, take the tails out of the liquid medicine, gently wipe the residual liquid medicine on the tails with a tissue paper. After wiping, place the tails on the filter paper for 60 s, keeping the rat tails fixed on the filter paper. After 60 s, move the tails away from the filter paper. According to the same operation, put the rat tails into the liquid medicine for 30 s, take them out from the filter paper, wipe the liquid medicine with a tissue paper, and keep the points at different positions on the filter paper unmoved. Repeat this process in turn until the tails stop bleeding. Examine the hemostatic effect according to the size of the diffusion area of the surrounding blood on the filter paper, and examine the hemostatic time according to the number of blood spots on the filter paper. The results show that: the hemostatic effect of ADR is the worst, the hemostatic effect of ADR-NCs-BSP is the best, and the hemostatic effect of ADR-NCs is the second. For the rat tails applied with ADR-NCs, the degree of blood diffusion on the filter paper is not much different from that of ADR, but the hemostatic time is shorter than that of ADR. The above results indicate that BSP-modified ADR-NCs has a hemostatic effect. It is suggested that it has good wound healing performance.

[0121] Experiments show that by preparing the needle solution and the matrix solution into the micro-needle form, the penetration ability of the stratum corneum barrier can be improved, and the transdermal absorption rate can be increased to a certain extent; by preparing the concentrated crystalline suspension and then forming the needle solution, on the basis of the micro-needle form, the penetration of the poorly soluble traditional Chinese medicine active ingredients can be further improved in the form of crystals, further increasing the transdermal absorption rate; by adding the Bletilla striata polysaccharide to form the needle solution, the healing rate of the holes caused by puncture is effectively improved, avoiding the risks of skin allergy and infection.

[0122] In the second aspect, the present invention provides an application of the Bletilla striata polysaccharide-modified nano-crystalline-loaded micro-needles prepared by the preparation method of the Bletilla striata polysaccharide-modified nano-crystalline-loaded micro-needles as described in the above embodiments in improving the transdermal absorption rate.

[0123] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0124] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. A method for preparing nanocrystalline microneedles modified with Bletilla striata polysaccharide, characterized in that: The following steps are involved: preparing a concentrated crystal suspension from andrographolide, a first polyvinyl pyrrolidone, polyvinyl alcohol and a first carrier liquid, wherein andrographolide nanocrystals are uniformly suspended in the concentrated crystal suspension; Mixing the concentrated crystal suspension and Bletilla striata polysaccharide to form a needle solution; Mixing the second polyvinyl pyrrolidone, glycerol and the second carrier liquid to form a base solution; The needle solution and the matrix solution are placed in a mold to form the bletilla striata polysaccharide-modified nanocrystalline microneedles.

2. The method for preparing nanocrystalline microneedles modified with Bletilla striata polysaccharide according to claim 1, characterized in that: The mass ratio of the andrographolide, the polyvinyl pyrrolidone and the polyvinyl alcohol is 1:1:1, and the mass ratio of the andrographolide to the first carrier liquid is 1:1000 to 1:50000.

3. The method for preparing nanocrystalline microneedles modified with Bletilla striata polysaccharide according to claim 1, characterized in that: The step of preparing andrographolide, the first polyvinyl pyrrolidone, polyvinyl alcohol and the first carrier liquid into a concentrated crystal suspension comprises: Placing andrographolide, polyvinyl pyrrolidone and polyvinyl alcohol in a first carrier liquid and stirring the andrographolide, polyvinyl pyrrolidone and polyvinyl alcohol to uniformly disperse in the first carrier liquid to form a coarse suspension; The crude suspension is subjected to high-pressure homogenization treatment by a high-shear homogenizing emulsifier to obtain a crude crystalline suspension; The crude crystal suspension is concentrated and diluted by a rotary evaporator to obtain a concentrated crystal suspension.

4. The method for preparing nanocrystalline microneedles modified with Bletilla striata polysaccharide according to claim 3, characterized in that: The rotation speed of the high-pressure homogenization treatment is 10000rpm-20000rpm, the time of the high-pressure homogenization treatment is 5min-20min, the temperature of the high-pressure homogenization treatment is 41°C-50°C, and the number of high-pressure homogenization treatment is 10 times-30 times.

5. The method for preparing the Bletilla striata polysaccharide-modified nanocrystalline microneedles according to claim 3, characterized in that: The step of concentrating and diluting the crude crystalline suspension by a rotary evaporator to obtain a concentrated crystalline suspension comprises: placing the crude crystal suspension in an eggplant-shaped bottle, and performing a vacuum treatment on the eggplant-shaped bottle to make the eggplant-shaped bottle reach a preset pressure; Concentrating the crude crystal suspension in the eggplant-shaped bottle after vacuum treatment to obtain a concentrated solution; The concentrated solution is diluted so that the andrographolide nanocrystals in the concentrated solution reach a preset content and form a concentrated crystal suspension.

6. The method for preparing nanocrystalline microneedles modified with Bletilla striata polysaccharide according to claim 5, characterized in that: The preset pressure is 0.09 MPa, the multiple of the concentration treatment is 50 times, and the preset content is 1 mg / ml to 20 mg / ml.

7. The method for preparing the Bletilla striata polysaccharide-modified nanocrystalline microneedles according to claim 1, characterized in that: The particle size of the andrographolide nanocrystals is 200nm-800nm.

8. The method for preparing nanocrystalline microneedles modified with Bletilla striata polysaccharide according to claim 1, characterized in that: The mass ratio of the concentrated crystal suspension to the bletilla striata polysaccharide is 1:

1.

9. The method for preparing nanocrystalline microneedles modified with Bletilla striata polysaccharide according to claim 1, characterized in that: The mass ratio of the second polyvinyl pyrrolidone to the second carrier liquid is 1:1 to 1:

10.

10. Use of the bletilla striata polysaccharide-modified nanocrystalline microneedles prepared by the preparation method of bletilla striata polysaccharide-modified nanocrystalline microneedles as claimed in any one of claims 1 to 9 in improving transdermal absorption rate.

Images