CN113599530B - No-load phase-conversion hydrogel microneedle, chinese cobra neurotoxin phase-conversion hydrogel microneedle, and preparation methods and application thereof - Google Patents
No-load phase-conversion hydrogel microneedle, chinese cobra neurotoxin phase-conversion hydrogel microneedle, and preparation methods and application thereof Download PDFInfo
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- CN113599530B CN113599530B CN202110876432.9A CN202110876432A CN113599530B CN 113599530 B CN113599530 B CN 113599530B CN 202110876432 A CN202110876432 A CN 202110876432A CN 113599530 B CN113599530 B CN 113599530B
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- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
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Abstract
The invention discloses an empty-load phase-conversion hydrogel microneedle, a Chinese cobra neurotoxin phase-conversion hydrogel microneedle, a preparation method and application thereof, wherein the empty-load phase-conversion hydrogel microneedle takes a polyvinyl alcohol/glucan mixed solution (84/16, w/w) as a needle raw material, the Chinese cobra neurotoxin phase-conversion hydrogel microneedle takes a medicine carrying solution (a Chinese cobra neurotoxin medicine stock solution and a polyvinyl alcohol/glucan mixed solution with the mass ratio concentration of 84/16) as a needle raw material, a microneedle needle body part is prepared by centrifugation, a polyvinyl alcohol aqueous solution (18 percent, w/w) is taken as an adhesive layer solution, a polyvinyl alcohol aqueous solution (25 percent, w/w) is taken as a backing layer, and corresponding microneedles are prepared by repeated freezing-thawing and drying; the Chinese cobra neurotoxin phase-inversion hydrogel microneedle prepared by the method can obviously increase the skin permeability of neurotoxin, realize slow release and high-efficiency transdermal administration, and can be used for preparing transdermal administration analgesic preparations.
Description
Technical Field
The invention belongs to the technical field of microneedles, and particularly relates to an empty-load phase-conversion hydrogel microneedle, a Chinese cobra neurotoxin phase-conversion hydrogel microneedle, and a preparation method and application thereof.
Background
Pain is a physiological and pathological process accompanied by emotional response, which occurs when the body is subjected to noxious stimulus from the internal and external environments, and also a defensive response after the body is subjected to noxious stimulus. Commonly used analgesics are classified into peripheral analgesics and central analgesics according to their action mechanisms. Clinically, peripheral analgesics are mainly used for mild pain, including non-steroidal anti-inflammatory drugs such as aspirin, acetaminophen, ibuprofen, etc.; the central analgesic is mainly applied to moderately severe pain, has remarkable analgesic effect, and mainly comprises synthetic analgesic such as tramadol and opioid alkaloids such as morphine, pethidine and the like. Although the peripheral analgesic and the central analgesic have different degrees of analgesic efficacy, there are many adverse reactions in use, for example, nonsteroidal anti-inflammatory drugs have adverse reactions in addition to antipyretic, analgesic and anti-inflammatory effects, among which the most common adverse reactions include: gastrointestinal reactions (abdominal pain, nausea, vomiting, dyspepsia, gastric ulcer, etc.), liver damage, kidney damage, cardiovascular adverse reactions, etc., can also cause adverse reactions in the central nervous system, common tinnitus and dizziness; opioid central analgesics exert a strong analgesic effect and bring about addiction, wherein morphine has serious adverse reactions such as addiction and respiratory depression, and acute poisoning is caused, thereby causing death.
In recent years, with the deep research of analgesic drugs, natural analgesic drugs are the hot spot of current research due to high selectivity, good safety and small side effects. The Chinese cobra neurotoxin (Naja atra neurotoxin, NANT) is a low-dose and high-effect macromolecular protein polypeptide analgesic drug, has an analgesic effect different from that of opium, is slow in acting, high in acting titer, long in maintenance time, free of tolerance and addiction, has a good analgesic effect in clinical application, is suitable for treating chronic, intractable and persistent pain, is a novel potential analgesic drug, and has clinical application prospect.
At present, the domestic cobra peptide injection has the main component of cobra venom neurotoxin and is used for treating trigeminal neuralgia, advanced cancer pain, arthralgia and the like. However, the injection administration method is easy to cause pain and dislike of patients, and can cause too fast in-vivo drug distribution and have side effects of respiratory depression.
The Micro Needles (MNs) can puncture human skin to form self-repairable micro channels, the stratum corneum disorder is broken, meanwhile, the dermis layer is not touched, and the micro needle transdermal administration system has the dual advantages of injection administration and transdermal administration and has the advantages of rapidness, convenience, no pain and the like. Research shows that the transdermal drug delivery of the microneedle can obviously improve the transdermal rate and the absorption capacity of the drug, and particularly has good effect and application prospect in the field of transdermal preparation research of macromolecular substances such as protein, polypeptide, DNA, RNA and the like.
In 2012, the drug research team taught by Shanghai university of transportation Jin Ta first proposed Phase-inversion hydrogel microneedles (Phase-transition hydrogel-forming microneedles, PTH-MNs) whose matrix was designed from the drug adjuvant polyvinyl alcohol (Polyvinyl alcohol, PVA), and the PVA-formed Phase-inversion microneedles physically crosslinked to form microcrystalline domains after repeated freeze-thaw cycles, so that the microneedles could remain in a tenacious hydrated state and no longer dissolve in body fluids, thereby being able to remain on the skin to ensure sustained release of drug within the microneedles, and then be completely removed from the skin. The insulin phase inversion hydrogel microneedles were prepared by Yang et al (Yang SX, wu F, liu JG, et al phase-Transition Microneedle Patches for Efficient and Accurate Transdermal Delivery of Insulin [ J ]. Advanced Fuctional Materials,2015,25,4633-4641.), and experimental results indicate that the water-swelling but water-insoluble nature of the phase inversion hydrogel microneedles ensures efficient transdermal delivery of insulin and sufficient mechanical strength to be removed from the skin completely in a hydrated state without depositing unwanted substances.
Based on the research, in order to reduce the deposition of the microneedle in the skin, increase the drug loading rate of the drug and the drug compliance of a patient, the research of selecting the phase-inversion hydrogel microneedle as a cobra neurotoxin transdermal administration preparation provides a theoretical basis for the research and development of transdermal delivery of protein polypeptides and macromolecular drugs.
Disclosure of Invention
The technical aim of the invention is to provide an empty-load phase-conversion hydrogel microneedle, a Chinese cobra neurotoxin phase-conversion hydrogel microneedle, and a preparation method and application thereof.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the no-load phase inversion hydrogel microneedle comprises an no-load needle body, an adhesion layer and a backing layer, wherein the adhesion layer and the backing layer are sequentially attached to the top of the no-load needle body;
the empty needle body is prepared from a mixed solution of polyvinyl alcohol and dextran;
the adhesive layer and the backing layer are both made of aqueous polyvinyl alcohol solution.
Further, the empty needle body is prepared from a polyvinyl alcohol and dextran mixed solution with the mass ratio concentration of 84/16;
the solution of the adhesive layer is a polyvinyl alcohol aqueous solution with the mass ratio concentration of 18%;
the backing layer is a thin layer prepared from a polyvinyl alcohol aqueous solution with a mass ratio concentration of 25%.
The invention also provides a China cobra neurotoxin phase inversion hydrogel microneedle which comprises a medicine carrying needle body, an adhesion layer and a backing layer, wherein the adhesion layer and the backing layer are sequentially attached to the top of the medicine carrying needle body;
the drug-carrying needle body is prepared from a drug-carrying solution, and the drug-carrying solution consists of a Chinese cobra neurotoxin solution and auxiliary materials; the auxiliary material is a mixed solution of polyvinyl alcohol and glucan;
The solution of the adhesive layer is a polyvinyl alcohol aqueous solution with the mass ratio concentration of 18%;
the backing layer is a thin layer prepared from a polyvinyl alcohol aqueous solution with a mass ratio concentration of 25%.
Further, the auxiliary material is a mixed solution of polyvinyl alcohol and dextran with the mass ratio concentration of 84/16;
the invention also provides a preparation method of the Chinese cobra neurotoxin phase inversion hydrogel microneedle, which is prepared from a needle body drug-carrying solution, an adhesive layer solution and a backing layer through centrifugation, repeated freezing-thawing and finally drying.
Further, the backing layer is prepared by the steps of: spreading 25% polyvinyl alcohol aqueous solution on square glass plate, uniformly pressing with another glass plate, supporting the two glass plates with a flat pad with thickness of about 1mm, freezing at-20deg.C for 8 hr, thawing at 4deg.C for 4 hr, and freezing-thawing for 2 times to obtain backing layer.
Further, the specific preparation steps of the Chinese cobra neurotoxin phase inversion hydrogel microneedle are as follows:
preparing a needle body drug carrying solution;
placing the needle body medicine carrying solution into a microneedle mould, injecting the needle body medicine carrying solution into holes of the microneedle mould by adopting a centrifugal method, scraping off redundant needle body medicine carrying solution, adding an adhesive layer solution, sticking a backing layer, and removing bubbles and redundant adhesive layer solution to prepare the microneedle patch;
And (3) after the microneedle patch is subjected to freeze-thawing cycle, removing the microneedle patch from the microneedle mould, and finally drying to obtain the Chinese cobra neurotoxin phase-converted hydrogel microneedle.
Further, the preparation method of the needle drug-carrying solution comprises the following steps:
mixing the powder of the Chinese cobra neurotoxin with ultrapure water to obtain a medicinal stock solution;
according to the required microneedle drug loading concentration, a proper amount of drug stock solution is taken and added into a mixed solution of polyvinyl alcohol and dextran with the mass ratio concentration of 84/16, and the mixed solution is uniformly mixed and stirred to obtain the needle drug loading solution which is uniform and stable and has no phase separation.
Further, the freezing-thawing conditions of the microneedle patch were: freezing at-20deg.C for 8 hr, and thawing at 4deg.C for 4 hr.
The invention also provides the application of the preparation method of the empty-load phase-inversion hydrogel microneedle or the Chinese cobra neurotoxin phase-inversion hydrogel microneedle in the preparation of transdermal administration analgesic preparations.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides an empty-load phase-conversion hydrogel microneedle, a Chinese cobra neurotoxin phase-conversion hydrogel microneedle (NANT-PTH-MNs), a preparation method and application thereof, wherein the empty-load phase-conversion hydrogel microneedle takes a polyvinyl alcohol/dextran mixed solution (84/16, w/w) as a needle raw material of the empty-load phase-conversion hydrogel microneedle, the Chinese cobra neurotoxin phase-conversion hydrogel microneedle takes a drug-carrying solution (a Chinese cobra neurotoxin drug stock solution and a polyvinyl alcohol/dextran mixed solution with the mass ratio concentration of 84/16) as a needle raw material, a microneedle needle body part is prepared by a centrifugal method, and further takes a polyvinyl alcohol aqueous solution (18%, w/w) as an adhesive layer solution, and a polyvinyl alcohol aqueous solution (25%, w/w) as a backing layer, and the empty-carrying or drug-carrying (Chinese cobra neurotoxin) phase-conversion hydrogel microneedle is prepared through repeated freezing-thawing and drying, wherein after repeated freezing-thawing circulation processes, a microcrystalline state can be kept in a tough state, the microneedle can be insoluble in the body fluid, so that the microneedle can be left in skin, and can be kept in a continuous and unnecessary drug can be completely removed from the skin;
(2) The micropin type vertical integrity of the hydrogel phase-converted by the Chinese cobra neurotoxin prepared by the preparation method provided by the invention has the advantages of ordered arrangement, good mechanical property, in-vitro swelling rate of more than 210%, and sufficient space channel for drug delivery; in addition, the drug loading rate of the microneedle is 23.4%, so that an effective analgesic therapeutic amount can be achieved; the cumulative release rate of the microneedle in vitro release is approximately 85%; the cumulative transdermal rate of the microneedle in vitro transdermal can reach 80%; the NANT-PTH-MNs have good stability and are more suitable for being stored in a low-temperature environment; the micro needle can not cause adverse reactions such as skin allergy after being removed after application, and has no side effect or small side effect;
(3) The method has the advantages that the content of the loaded Chinese cobra neurotoxin in the micropin has little influence on the release trend and the accumulated release rate, the content of the neurotoxin in the micropin can be adjusted according to the dosage requirement of the neurotoxin in actual animal administration in the later period, and the product flexibility is high;
(4) The prepared hydrogel microneedle for phase inversion of the Chinese cobra neurotoxin can obviously increase the skin permeability of the neurotoxin, and realize slow release and high-efficiency transdermal administration;
(5) According to the invention, through the analgesic effect research of the Chinese cobra neurotoxin phase-inversion hydrogel microneedle, the high-dose Chinese cobra neurotoxin phase-inversion hydrogel microneedle is found to be capable of remarkably reducing the twisting times of mice and improving the twisting inhibition rate, and is capable of remarkably prolonging the pain threshold of the mice, and has a good analgesic effect.
Drawings
FIG. 1 is a morphology of PTH-MNs prepared with different excipients in example 1 of the present invention, wherein A1-A2 are PVA/HA constituent pins, B1-B2 are PVA/CMC constituent pins, and C1-C2 are PVA/Dex constituent pins;
FIG. 2 shows the results of skin penetration by PTH-MNs mice prepared with different PVA/Dex contents in example 1 of the present invention, wherein A is PVA/Dex 90/10 (w/w), B is PVA/Dex 88/12 (w/w), C is PVA/Dex 84/16 (w/w), and D is PVA/Dex 82/18 (w/w);
FIG. 3 is a flow chart of the preparation of the neurotoxin phase inversion hydrogel microneedle of the present invention;
FIG. 4 is a NANT-PTH-MNs topography of example 2 of the present invention, wherein 4A is an optical micrograph of a microneedle and 4B is a scanning electron microscope of the microneedle;
FIG. 5 shows the mechanical properties of NANT-PTH-MNs of example 2 of the present invention, wherein A is a rat skin penetration map ex vivo, B is a rat penetration test in vivo, and C is a rat skin HE staining map;
FIG. 6 shows the swelling results of NANT-PTH-MNs in example 2 of the present invention, n=3;
Fig. 7 shows the effect of different drug loading on the release of the drug from the nan-PTH-MNs in example 2 of the present invention, n=3;
FIG. 8 shows the results of the irritation test of NANT-PTH-MNs on rat skin in example 2 of the present invention;
fig. 9 shows the cumulative permeation curve of the nan-PTH-MNs in example 2 of the present invention, n=6.
Detailed Description
The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. The experimental methods used in the examples below, unless otherwise indicated, are conventional methods, and the reagents, methods and apparatus used, unless otherwise indicated, are conventional in the art.
Example 1: optimal needle auxiliary material prescription screening of China cobra neurotoxin phase-converted hydrogel microneedle
In practical application, the patch part of the microneedle is required to be closely adhered to the skin, so that the patch needs to have certain flexibility, and the needle part of the hydrogel microneedle only penetrates through the stratum corneum of the skin to release the drug into the skin to play a role, so that the microneedle needs to have enough mechanical strength, and therefore, prescription screening is required for auxiliary materials for constructing the patch part and auxiliary materials for the needle part.
The main auxiliary material suitable for preparing the phase-inversion hydrogel microneedle is polyvinyl alcohol, and due to practical application limitations, a composite material formed by mixing two or more polymer materials often has the properties of the two polymer materials, is better than any polymer, and can show a special composite property. Polyvinyl alcohol has good toughness, and is generally used with brittle polymer materials such as polysaccharide, but the proportion of polysaccharide in the prescription is controlled, otherwise, the network structure of the hydrogel is damaged. Therefore, the invention takes polyvinyl alcohol (PVA), hyaluronic Acid (HA), carboxymethyl cellulose (CMC) and Dextran (DEX) as auxiliary materials, adopts a centrifugal method to prepare the phase-inversion hydrogel microneedle by two steps, and screens out the optimal prescription by microscopic observation and preliminary evaluation of mechanical strength of the needle type of the prepared blank phase-inversion hydrogel microneedle.
1.1 raw materials
The raw materials used in this example are as follows:
the above raw materials are not limited to those from the above manufacturers, but may be obtained by other manufacturers or by self-configuration.
1.2 preparation of blank phase-inverted hydrogel microneedles (PTH-MNs)
Numerous methods of manufacturing microneedles are available, such as laser cutting techniques, reactive ion etching, and micro-molding. The preparation method of the different types of microneedles is different, and the hydrogel microneedles are suitable for a micro-molding method. At present, the micro-molding method is used for preparing the micro-needle in two ways, namely a negative pressure method and a centrifugal method, and the phase-inversion hydrogel micro-needle is prepared by adopting the centrifugal method.
1.2.1 preparation of PVA solution
16g of PVA is weighed and placed in a wide-mouth bottle, 84g of ultrapure water is added, the mixture is heated to 95 ℃ under stirring, the temperature is kept for 3 to 4 hours to enable the PVA to be completely dissolved, then the mixture is naturally cooled at room temperature until the PVA solution is free of bubbles, and the solution is clear and transparent, namely, the swelling is complete, so that the PVA aqueous solution with the concentration of 16% (w/w) is obtained.
1.2.2 preparation of PVA/HA solution
Firstly absorbing a certain amount of the prepared PVA solution, then adding a certain amount of HA powder into the PVA solution, stirring the mixture to uniformly mix the PVA solution and the HA powder, and preparing mixed solutions of which the PVA/HA ratio is 98/2, 96/4, 94/6, 92/8, 90/10, 88/12, 86/14, 84/16, 82/18 and 80/20 (w/w) respectively.
1.2.3 preparation of PVA/CMC solution
Firstly absorbing a certain amount of the prepared PVA solution, then adding a certain amount of CMC powder into the PVA solution, stirring the mixture to uniformly mix the PVA solution and the CMC powder, and preparing mixed solutions of which the PVA/CMC ratio is 98/2, 96/4, 94/6, 92/8, 90/10, 88/12, 86/14, 84/16, 82/18 and 80/20 (w/w) respectively.
1.2.4 preparation of PVA/Dex solution
Firstly sucking a certain amount of the prepared PVA solution, then adding a certain amount of Dex powder into the PVA solution, stirring the mixture to uniformly mix the PVA solution and the Dex powder, and preparing mixed solutions of which the PVA/Dex ratio is 98/2, 96/4, 94/6, 92/8, 90/10, 88/12, 86/14, 84/16, 82/18 and 80/20 (w/w) respectively.
1.2.5 preparation of adhesive layer solution
18g of PVA is weighed and placed in a wide-mouth bottle, 82g of ultrapure water is added, the mixture is heated to 95 ℃ under stirring, the temperature is kept for 3 to 4 hours to enable the PVA to be completely dissolved, then the mixture is naturally cooled at room temperature until the PVA solution is free of bubbles, the solution is clear and transparent, and the PVA aqueous solution with the concentration of 18% (w/w) is obtained, namely the adhesive layer solution.
1.2.6 preparation of backing layer
Weighing 25g of PVA, placing in a wide-mouth bottle, adding 75g of ultrapure water, heating to 95 ℃ under stirring, and preserving heat for 3-4 hours to completely dissolve the PVA, so as to obtain a PVA aqueous solution with the concentration of 25% (w/w); spreading proper amount of solution on square glass plate, pressing with another identical glass plate (with a flat pad with thickness of about 1mm between two glass plates), freezing at-20deg.C for 8 hr, thawing at 4deg.C for 4 hr, and freezing-thawing for 2 times to obtain backing layer with certain toughness and hardness.
1.2.7 preparation of drug-free microneedles
Taking 300 mu L of PVA/HA, PVA/CMC and PVA/Dex solutions prepared in the above 1.2.2, 1.2.3 and 1.2.4 in different proportions, respectively placing the solutions into a microneedle mould (with the specifications of needles, cones, 800 mu m long, 300 mu m bottom width, 900 mu m needle spacing and 15X 15 array), injecting the mixed solution into holes of the microneedle mould by adopting a centrifugal method (3000 rpm,10 min), scraping off redundant mixed solution, adding a small amount of adhesive layer solution, sticking a backing layer, and removing bubbles; freezing the prepared microneedle at-20 ℃ for 8 hours, thawing at 4 ℃ for 4 hours, performing freeze-thawing cycle for 2 times, removing the microneedle from the mold, and drying for 48 hours at 25 ℃ in an oven to obtain a blank phase-inversion hydrogel microneedle (PTH-MNs), namely an empty phase-inversion hydrogel microneedle, wherein the phase-inversion hydrogel microneedle does not contain the Chinese cobra neurotoxin.
1.3 characterization of blank phase inversion hydrogel microneedles
1.3.1 morphology of microneedles prepared from different excipients
And (3) placing the PTH-MNs prepared in the step (1.2) under an optical microscope (such as an SZ780 continuous variable magnification stereomicroscope), and observing the formability of the micro-needle, such as whether the micro-needle is broken, bent needle and the like.
Microscopic observation of typical graphs of microneedles prepared by taking three auxiliary materials of PVA/HA, PVA/CMC and PVA/Dex in different proportions as microneedle needle materials, as shown in FIG. 1, graphs A1 and A2 are PTH-MNs prepared by PVA/HA of 90/10 (w/w) and 84/16 (w/w) respectively; FIGS. B1 and B2 are PTH-MNs prepared with PVA/CMC of 90/10 (w/w) and 84/16 (w/w), respectively; FIGS. C1 and C2 are PTH-MNs prepared with PVA/Dex of 90/10 (w/w) and 84/16 (w/w), respectively;
microscopic observation of the micro-needles prepared from three auxiliary materials in different proportions can show that the needle body of the PTH-MNs prepared from the PVA/HA and the PVA/CMC in different proportions is not vertical or bent. The typical microneedle micrograph of FIG. 1 intuitively shows that the microneedles prepared from PVA/HA (FIGS. A1-A2) and PVA/CMC (FIGS. B1-B2) cannot be subjected to subsequent experiments due to poor needle shape; the needle material is a microneedle (figures C1-C2) prepared from PVA/Dex, and the needle is vertical and complete and is arranged in a regular rectangle.
1.3.2 mechanical Properties of microneedles prepared with different excipients
According to the shape detection results of the micro-needles prepared by the 1.3.1 different auxiliary materials, the needle type of PTH-MNs prepared by mixing the PVA and the Dex auxiliary materials is complete and clear in matrix, so that PVA/Dex is selected as a needle preparation material of the PTH-MNs. PTH-MNs with different PVA/Dex content ratios are subjected to rat in vitro skin penetration experiments to detect the mechanical properties of PTH-MNs with different PVA/Dex content ratios.
Rat ex vivo skin penetration experiment: taking SD rat, after anesthesia with aconite, killing the rat by breaking neck, using pet shaver and depilatory cream to depilation the back of rat, taking down skin of the back, and removing muscle and fat on the skin; cleaning the removed skin with normal saline, wrapping in preservative film and tinfoil paper, and storing in a refrigerator at-20deg.C; taking out the mouse skin, thawing in physiological saline for 30min, then sucking water on the skin with filter paper, and placing one side of the horny layer of the mouse skin upwards; the microneedle was placed on the skin of a rat, the microneedle was pressed by a finger to puncture the skin of a healthy intact rat ex vivo, the microneedle was left in the skin for 3min, the microneedle was taken out, immediately dyed with trypan blue solution, and after 10min, the trypan blue dye remaining on the skin was cleaned with a paper towel, and the mechanical strength of the microneedle was determined by observing the blue hole formed on the skin, and the result is shown in fig. 2.
As can be seen from fig. 2, the phase-inversion hydrogel microneedles with different contents of PVA/Dex formed different numbers of blue holes on the skin of rats, indicating that the mechanical strength of the microneedles made with different contents of PVA/Dex also varies; when the ratio of PVA/Dex in the needle auxiliary material solution is 84/16 (figure 2C), most of blue holes are clear to naked eyes, and the mechanical strength is reduced along with the increase of the PVA content in the needle auxiliary material solution and the PVA.
In conclusion, the appearance form of PTH-MNs prepared by uniformly mixing PVA, HA, CMC and Dex in different proportions is inspected, and the microneedle prepared by taking PVA/Dex as a needle material is complete in needle shape and HAs no defect; further, on the premise of ensuring good needle type of the micro needle, mechanical strength of different contents of PVA/Dex is inspected by performing an in vitro skin penetration experiment of a rat, and the mechanical strength is found to be better when the content ratio of PVA/Dex is 84/16. Therefore, the PVA/Dex solution content ratio of 84/16 is determined as the preferable auxiliary material for constructing the microneedle body, and subsequent experiments are carried out.
Example 2: preparation and performance verification of China cobra neurotoxin phase-conversion hydrogel microneedle
2.1 preparation of NANT-PTH-MNs
2.1.1 preparation of PVA solution
An aqueous PVA solution having a concentration of 16% (w/w) was prepared as described in step 1.2.1.
2.1.2 preparation of PVA/Dex solution
Firstly sucking a certain amount of the prepared PVA solution, then adding a certain amount of Dex powder into the PVA solution, stirring the mixture to uniformly mix the powder, and preparing a mixed solution with the PVA/Dex ratio of 84/16 (w/w).
2.1.3 preparation of PVA/Dex needle drug-carrying solution
When preparing the needle body drug-carrying solution, the different adding modes of the drug can lead to different distribution states of the drug in the mixed solution, and experiments show that the direct addition of the Chinese cobra neurotoxin powder into the polyvinyl alcohol/glucan mixed solution can lead to uneven distribution of the drug in the solution, and the phenomenon of light yellow caking can occur, probably because the viscosity of the mixed solution influences the dissolution of the drug in the solution. Therefore, the invention selects to prepare the Chinese cobra neurotoxin drug solution firstly, and then adds the drug-containing solution into the mixed solution of polyvinyl alcohol and dextran, and the specific steps are as follows:
weighing a proper amount of NANT powder (Chinese cobra neurotoxin crude drug with purity more than 99%, wherein the embodiment is selected from Zhejiang Zheshan cobra), fully dissolving in a small amount of ultrapure water to prepare NANT drug stock solution, adding a proper amount of drug-containing stock solution into a proper amount of prepared PVA/Dex solution (84/16, w/w), stirring for at least 2h to fully and uniformly mix the drug with the auxiliary material solution, and preparing into a uniform and stable PVA/Dex needle drug-carrying solution without phase separation.
2.1.4 preparation of adhesive layer solution
The adhesive layer solution is a PVA aqueous solution with the concentration of 18% (w/w) and is prepared by adopting the method described in the step 1.2.5.
2.1.5 preparation of backing layer
The backing layer was an aqueous PVA solution having a concentration of 25% (w/w) and was prepared as described in reference to step 1.2.6.
2.1.6 preparation of microneedles
Referring to FIG. 3, 300. Mu.L of the PVA/Dex needle drug-carrying solution prepared in step 2.1.2 is placed in a microneedle mould (specification: needle, cone, length 800 μm, bottom width 300 μm, needle spacing 900 μm, 15X 15 array), the mixed solution is injected into the holes of the microneedle mould by centrifugation (3000 rpm,10 min), the redundant mixed solution is scraped off, a small amount of adhesive layer solution is added, and a backing layer is attached to remove bubbles; freezing the prepared microneedle at-20deg.C for 8h, thawing at 4deg.C for 4h, freezing-thawing for 2 times, removing the microneedle from the mold, and oven drying at 25deg.C for 48h to obtain the final product.
2.2 in vitro analysis of NANT-PTH-MNs
2.2.1 microneedle morphology
And (3) placing the NANT-PTH-MNs prepared according to 2.1 under an optical microscope and a scanning electron microscope, and observing the formability of the micro needle, such as whether the micro needle is broken, bent needle and the like.
As a result, it was found that NANT-PTH-MNs prepared according to 2.1 contained 225 microneedles distributed in a 15X 15 array. The single needle type of the micro needle is cone, the needle body is 800 μm, and the diameter of the bottom end of the needle body is 300 μm. The shape of the micro needle is observed under an optical microscope and a scanning electron microscope, as shown in fig. 4A and 4B, the shape of NANT-PTH-MNs is a cone array, the needle tip is sharp, the needle body surface is smooth, the microscopic shape is good, and the effect of adding the medicine on the needle shape of the micro needle can be obtained.
2.2.2 mechanical Properties
2.2.2.1 murine skin puncture experiments
SD rats were anesthetized with uliose, sacrificed by neck breakage, the backs of the rats were dehaired with a pet shaver and depilatory cream, the skin on the backs was removed, and the muscles and fat on the skin were removed. Cleaning the removed skin with normal saline, wrapping in preservative film and tinfoil paper, and storing in refrigerator at-20deg.C. The mouse skin was removed, thawed in physiological saline for 30min, then the skin was blotted with filter paper to remove moisture, and the stratum corneum of the mouse skin was placed with one side facing upwards. NANT-PTH-MNs were placed on the rat skin, finger pressed to puncture healthy intact rat skin ex vivo, left in the skin for 3min, NANT-PTH-MNs were removed, immediately stained with trypan blue solution, after 10min residual trypan blue dye on the skin was removed with paper towel, and mechanical strength of NANT-PTH-MNs was determined by observing blue holes formed on the skin.
The results are shown in FIG. 5A. The NANT-PTH-MNs prepared according to 2.1 formed blue holes clearly visible to the naked eye on rat skin, indicating that NANT-PTH-MNs could successfully penetrate the skin and deliver the drug.
2.2.2.2 histological analysis of mouse skin
To confirm the puncture performance of NANT-PTH-MNs on living rats, SD rats were anesthetized with pullulan, hair on the backs thereof was removed with a pet shaver and depilatory cream, the microneedles were removed after pressing the prepared NANT-PTH-MNs on the back skin of the rats for 5min, the microneedle administration area was visually observed, and photographs were taken, and the results are shown in FIG. 5B; the rats were then sacrificed and the skin immediately after microneedle penetration was peeled off and fixed by immersing the skin in 4% paraformaldehyde, leaving histological sections aside. The skin was cut to a thickness of 5 μm using a cryostat and stained with hematoxylin and eosin (HE stain), and then loaded into a slide glass, and the penetration of the skin ex-vivo was observed by photographing with an inverted microscope, further illustrating the penetration effect of the skin in-vivo with a microneedle, and the result is shown in fig. 5C.
As can be seen in fig. 5B, the microneedles form a clearly visible array of microneedles on the skin.
As can be seen from fig. 5C, the nan-PTH-MNs formed cone-like pinhole morphology in the skin, indicating that the prepared microneedles can form macroscopic pinhole arrays after being administered to the back of rats, and skin tissue sections also demonstrate that the microneedles can form clear microneedle apertures on the skin surface, providing a channel for transdermal delivery of drugs. Through a series of identification such as visual inspection of the skin surface and tissue sections of the skin, the NANT-PTH-MNs prepared by the invention can be proved to have good mechanical properties, and can provide a certain experiment and theoretical basis for the drug in the subsequent experimental microneedle to enter the skin for playing a role.
2.2.3 in vitro swelling experiments
The mechanism of releasing the medicine by the phase-inversion hydrogel microneedle is that the microneedle can absorb interstitial fluid when penetrating into the skin, and the needle body can swell, so that a micro-pore channel is provided for conveying the medicine, and therefore the swelling condition of the phase-inversion hydrogel microneedle is examined.
Taking NANT-PTH-MNs prepared according to 2.1, recording initial mass W L The method comprises the steps of carrying out a first treatment on the surface of the Wrapping the microneedle with polytetrafluoroethylene film and tinfoil paper to expose the needle body; placing in the hole of a 24-hole plate, adding 1ml of ultrapure water, inverting the wrapped microneedle into the hole, and immersing the exposed needle body in water; the microneedles were taken out of the 24-well plate at 1, 2, 4, 8, 12, 18, 24, 32, 40, 50, 60, 90, 120, 150, 180min, and the surface excess water was wiped off, and then the mass at 0 was designated as W 0 At t is marked as W t In (W) t -W 0 )/W L Representing the swelling ratio of the microneedle at t, the weight of each sample in the above experimentAnd repeating for 3 times. The swelling condition of the microneedle was examined by plotting t on the abscissa and the swelling ratio on the ordinate.
As shown in FIG. 6, the result shows that the NANT-PTH-MNs has a swelling rate of 180% at 60min, and then the swelling rate gradually decreases to be gentle, and the swelling rate at 180min is about 210%. The volume of the micro needle after swelling is increased due to the fact that the needle body of the micro needle is swelled after absorbing water, the micro needle is changed from a glassy state before swelling into a hydrogel state, and a large enough conveying channel can be provided for the medicine to enter the skin to be absorbed.
2.2.4 investigation of the needle drug-loading
NANT-PTH-MNs prepared according to 2.1 were taken and placed in 2mL of PBS (pH 7.4) solution, so that the microneedles were completely immersed and fully swelled for 24 hours, and the neurotoxin content of all needle bodies in the microneedles was measured by HPLC.
Preparing NANT-PTH-MNs with different drug concentrations by the method described in the section 2.1, and detecting the content of NANT in the microneedle by High Performance Liquid Chromatography (HPLC);
the chromatographic conditions were as follows:
chromatographic column: dikma Plastil ODS column (4.6 mm. Times.150 mm,5 um)
Mobile phase a:0.1% TFA in water
Mobile phase B:0.1% TFA-acetonitrile solution
Wavelength: 277nm
Flow rate: 1.0mL/min
Temperature: 35 DEG C
Sample injection amount: 20uL
Elution was performed using a binary high pressure gradient, and the elution procedure is as shown in table 1.
TABLE 1 binary high pressure gradient elution time program
The drug loading of NANT in the three formulations with different concentrations is shown in Table 2, and the drug loading rates of the three different drug concentrations are not different, about 23.4%.
TABLE 2 drug loading of NANT-PTH-MNs at different drug concentrations (n=6)
2.2.5 in vitro Release Studies
Taking the prepared NANT-PTH-MNs, wrapping the microneedle with polytetrafluoroethylene film and tinfoil paper to expose the needle body; placing in the hole of a 24-hole plate, adding 1.5mL of PBS (pH 7.4), inverting the wrapped microneedle into the hole, and immersing the exposed needle point into water; placing a 24-hole plate in a constant-temperature shaking incubator, setting the temperature to 37 ℃ and the rotating speed to 100r/min, and carrying out an in-vitro release test; samples were taken at 0.5, 1, 2, 3, 4, 5, 6, 8, 10 and 12h for HPLC analysis, respectively, and the in vitro release profile of the microneedles was plotted. Note that each sampling was completed and the liquid in each well of the 24-well plate was replaced with 1.5mL of fresh PBS, and each set of samples was repeated 3 times.
The invention examines the in vitro release condition of neurotoxin in NANT-PTH-MNs prepared by the prescription of two different drug concentrations (the drug concentration in the prescription is 1.5mg/ml,100 ug/tablet, and the drug concentration in the prescription is 3mg/ml,200 ug/tablet). As shown in fig. 7, the results show that the in vitro release trend of the neurotoxin in the microneedles is basically consistent at different drug loading rates, and the cumulative release rate at 12 hours is close to 85%, which indicates that the content of the neurotoxin in the microneedles has little influence on the release trend and the cumulative release rate, and the content of the neurotoxin in the microneedles can be adjusted according to the dosage requirement of the neurotoxin in actual animal administration in the later period.
2.2.6 skin irritation
The skin of the back of the rat was administrated for 30min, the test sites were observed at 0min,10min,1h,2h,8h,12h time periods after the microneedle was removed, and the irritation of the micro-needle to the skin was evaluated according to the degree of the skin of the back of the rat that was slightly red or edematous.
The skin condition of the rats after administration is shown in fig. 8. The trace of the skin of the back of the rat was strongly pressed to subside much within 10min, and the trace of the pressing was hardly seen after 2 h; with the extension of time, the back skin is quickly recovered to be intact after 8 hours of administration, and the result shows that the skin of the rat cannot be stimulated when the microneedle is administered, so that the skin cannot be red, swollen and allergic.
2.2.7 in vitro transdermal Studies
Microneedle dosing group: taking the in-vitro mouse skin which is prepared previously and stored at the temperature of minus 20 ℃, shearing the in-vitro mouse skin into a proper size by scissors, thawing the skin at room temperature before starting an experiment, and stabilizing the skin at 37 ℃ for 3 hours; fixing mouse skin between diffusion chamber and receiving chamber of permeation diffusion device, placing NANT-PTH-MNs on treated isolated mouse skin with thumb and applying force with certain force, fixing with medical adhesive tape after 3min, and ensuring skin integrity during treatment; the stratum corneum faces the diffusion chamber and the dermis faces the receiving chamber; adding 12mL (PBS, pH 7.4) of receiving solution into the receiving chamber, discharging bubbles, enabling the liquid level to be in full contact with the skin, setting the magnetic stirring speed to 600r/min to simulate the internal circulation of blood and tissue fluid under the skin of a human body, and circulating in a constant-temperature water bath at 32+/-0.5 ℃; starting timing after administration, taking 0.5mL of receiving solution respectively at 10min,20min,30min,1h,2h,3h,4h,6h,8h and 12h, and simultaneously supplementing an equal volume of fresh receiving solution; the obtained sample is filtered by a 0.45 mu m filter membrane, the content of neurotoxin in the sample liquid is measured according to the chromatographic condition under the item of 2.2.4, and the accumulated permeation curve of the medicine is drawn by SPSS 23.0 data processing.
Control group: selecting the same dose of NANT aqueous solution, and the other steps are the same as those of the microneedle administration group;
during the in vitro transdermal experiments, the cumulative permeation curve of NANT-PTH-MNs over 12h is shown in FIG. 9. As can be seen from the figure, the cumulative neurotoxin permeability in NANT-PTH-MNs was almost zero in the first 20min, perhaps because the neurotoxin permeability was too low in the first 20min, which was not detected by HPLC, but after 30min it was evident that some neurotoxin penetrated the skin, and within 3h the cumulative neurotoxin permeability had reached 50% and after 6h approximately 80%. The aqueous NANT solution of the control group had a permeability of about 10% within 12 hours. The micro-needle can be used for achieving the purpose of promoting the transdermal absorption of the medicine by penetrating the stratum corneum, and in-vitro permeation test results show that the skin permeability of neurotoxin can be obviously increased by using the micro-needle, and the mechanism of promoting permeation by the physics of the micro-needle is further described, so that slow release and high-efficiency transdermal administration can be realized.
2.2.8 microneedle stability detection
Respectively storing the prepared NANT-PTH-MNs at normal temperature, 4 ℃ and-20 ℃ for 1, 2 and 3 months, then taking out the microneedles in different time periods, respectively placing the microneedles in 1.5mL PBS (pH 7.4) buffer solution, wrapping the microneedles with polytetrafluoroethylene films and tinfoil paper, exposing the needle body, fully immersing the microneedles, and fully swelling for 24 hours; HPLC was used to determine the NANT content of all needles in NANT-PTH-MNs.
The NANT-PTH-MNs prepared by the invention is carried out in a freezing (-20 ℃) to thawing (4 ℃) circulating environment, so that the drug stability of the microneedle under different storage conditions is examined, and the result is shown in a table 3, the longer the preservation time is, the lower the neurotoxin content in the NANT-PTH-MNs is under the same storage condition; and when NANT-PTH-MNs are stored under different conditions for the same time, the neurotoxin content in the microneedle is the highest and the change degree of the neurotoxin content is the smallest when NANT-PTH-MNs are stored under the condition of minus 20 ℃. From this, NANT-PTH-MNs have good stability and are more suitable for storage in low temperature environments.
TABLE 3 drug content ratio (%)
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In conclusion, the NANT-PTH-MNs prepared by taking PVA/Dex (84/16, w/w) as an auxiliary material has complete needle shape, certain mechanical property and stable drug loading, can effectively carry NANT to penetrate the skin, gradually recover the skin after about 8 hours of drug administration, does not generate red swelling, inflammation and other reactions after drug administration, and can be used as a drug administration carrier in the aspect of transdermal drug administration treatment of pain.
Example 3: analgesic property research of Chinese cobra neurotoxin phase-converted hydrogel microneedle
Has research on the Chinese cobra neurotoxin with excellent pain relieving effect and excellent application foreground in treating arthralgia and cancer pain. The NANT-PTH-MNs prepared in example 2 are taken as test groups, and pain threshold values of the mice in each test group on thermal stimulation and pain caused by chemical stimulation acetic acid after administration are observed to investigate whether the NANT-PTH-MNs can produce good analgesic effect on the mice, so that a reference basis is provided for clinical application of the NANT-PTH-MNs.
3.1 animal Material
The male and female half of Kunming mice are 18-22g in weight, and are provided by the laboratory animal center of the university of Anhui medical science, and the qualification number is as follows: SCXK (Anhui) 2017-001. No water was forbidden during the 12h before the experiment.
3.2, experimental methods
3.2.1 acetic acid torsion experiment
Test grouping: the mice were divided into 6 groups at random, namely, a blank group (physiological saline), a pethidine group (20 mg/kg), a PTH-MNs group, a NANT-PTH-MNs high dose group (120 mug/kg), a NANT-PTH-MNs low dose group (60 mug/kg) and a NANT aqueous solution group (60 mug/kg);
test procedure: the experiment was performed in a room temperature environment at 25 ℃): the pethidine group and the NANT aqueous solution group are administrated by intraperitoneal injection of 0.1mL/10g, the PTH-MNs group and the NANT-PTH-MNs group are administrated by back administration, the mice in the blank control group are administrated by intraperitoneal injection of physiological saline with the same volume as the NANT aqueous solution group, and each group of mice is administrated continuously for 3d according to the dosage. Each group of mice was intraperitoneally injected with 1% HAc solution (0.1 mL/10 g) 1h after the last dose, and the number of twists of each group of mice was recorded over 15 min.
The torque inhibition ratio (%) = (average number of twists of control group-average number of twists of administration group)/average number of twists of control group×100% ] was calculated as follows.
Data results using SPSS 23.0 statistical softwareThe data from each group was compared to the data from the control group using the Dunnett-t test. P (P)<0.05 as a significance-related level.
The mice were given a torsion reaction within 15min after intraperitoneal injection of 1% HAc solution, and the number of torsion reactions was reduced for each group of mice after further injection of HAc solution 1h after the last administration, and the results are shown in table 4.
Compared with a blank control group, the pethidine group, the NANT-PTH-MNs high dose group, the NANT-PTH-MNs low dose group and the NANT aqueous solution group can obviously reduce the torsion reaction times of mice caused by injecting HAc solution, and the difference has statistical significance (P < 0.05). Compared with the NANT aqueous solution group, the NANT-PTH-MNs high dose group reduces the number of times of twisting mice, and the NANT-PTH-MNs low dose group has fewer times of twisting mice than the NANT aqueous solution group, which indicates that the NANT-PTH-MNs low dose group has good analgesic effect without the NANT aqueous solution group, and the difference has statistical significance (P < 0.05). Compared with the meperidine group, the analgesic effect of the NANT-PTH-MNs high dose group is not significantly different from that of the meperidine; the NANT-PTH-MNs low dose group is not as good as the pethidine analgesic effect, and the difference has statistical significance (P < 0.05).
TABLE 4 influence of neurotoxin phase inversion hydrogel micro on acetic acid induced mouse torsion reactionn＝8)
*P<0.05, compared with the normal group, ▲ P<0.05 compared with NANT aqueous solution group
3.2.2 experiments with hotplate method
Female mice are taken, a hot plate instrument is used for measuring the pain threshold of the mice before formal experiments, the temperature is set to 55.0+/-0.5 ℃, the time(s) required for the mice to start to enter the hot plate instrument until the feet are licked is recorded, and the average value of the time(s) is measured for 2 times and taken as a pain threshold index. Mice with stable response and no jump in the range of 15-25s were selected for the experiment and used as pain threshold before dosing.
Qualified female mice were randomly divided into 6 groups, a blank group (physiological saline), a pethidine group (20 mg/kg), a PTH-MNs group, a NANT-PTH-MNs high dose group (120. Mu.g/kg), a NANT-PTH-MNs low dose group (60. Mu.g/kg) and a NANT aqueous solution group (60. Mu.g/kg). The pethidine group and the NANT aqueous solution group are administrated by intraperitoneal injection of 0.1mL/10g, the PTH-MNs group and the NANT-PTH-MNs group are administrated by back administration, the mice in the control group are administrated by intraperitoneal injection of physiological saline with the same volume as the NANT aqueous solution group, and each group of mice is administrated continuously for 3d according to the dosage. The response time(s) from the mice to the occurrence of licking of the hind feet was recorded as pain threshold 1.5 hours after the last dose of each group of mice, the pain threshold extension time and the pain threshold improvement percentage were calculated, and the analgesic effect of the NANT-PTH-MNs high and low dose groups was compared. Percentage improvement in pain threshold (TB%) = (post-dose pain threshold-pre-dose pain threshold)/pre-dose pain threshold x 100%.
The results are shown in Table 5, the pain threshold of mice to pain response latency is obviously prolonged in the pethidine group, the NANT-PTH-MNs high dose group, the NANT-PTH-MNs low dose group and the NANT aqueous solution group compared with the blank control group, the pain threshold difference has statistical significance (P < 0.05), the pain threshold of the NANT-PTH-MNs high dose group is prolonged for a longer time, and the pain relieving effect is better (P < 0.05) compared with the NANT aqueous solution group; the pain relieving effect of the NANT-PTH-MNs low dose group is slightly poorer than that of the NANT aqueous solution group, and the pain threshold difference has statistical significance (P is less than 0.05); compared with the pethidine group, the analgesic effect of the NANT-PTH-MNs high-dose group is not significantly different from that of the pethidine group; the NANT-PTH-MNs low dose group is not as good as the pethidine analgesic effect, and the difference has statistical significance (P < 0.05).
TABLE 5 influence of NANT-PTH-MNs on the pain response of mice caused by thermal stimulusn＝8)/>
*P<0.05, compared with the normal group, ▲ P<0.05 compared with NANT aqueous solution group
Compared with the NANT solution group, the NANT-PTH-MNs high dose group has obvious analgesic effect, while the NANT-PTH-MNs low dose group has less good analgesic effect than the NANT aqueous solution. NANT-PTH-MNs are used in applications where drug loss occurs, perhaps due to transdermal and release rates, resulting in reduced amounts of drug entering the body. The loss of the drug in the NANT-PTH-MNs high dose group is relatively less than that in the NANT-PTH-MNs low dose group, which also results in better analgesic effect in the NANT-PTH-MNs high dose group than in the NANT aqueous solution group, and poorer analgesic effect in the NANT-PTH-MNs low dose group than in the NANT aqueous solution group.
In conclusion, the PTH-MNs and NANT-PTH-MNs prepared by the invention have complete needle type, regular matrix arrangement and good mechanical properties. The drug-loading rate of NANT-PTH-MNs can reach effective therapeutic dose, in-vitro release, in-vitro transdermal and in-vivo analgesic effects, and NANT-PTH-MNs can exert good analgesic effects through transdermal administration, thereby providing new ideas and strategies for development and treatment of novel analgesic therapeutic dosage forms.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the invention without departing from the principles thereof are intended to be within the scope of the invention as set forth in the following claims.
Claims (8)
1. The no-load phase inversion hydrogel microneedle is characterized by comprising an no-load needle body, an adhesion layer and a backing layer, wherein the adhesion layer and the backing layer are sequentially attached to the top of the no-load needle body;
the empty needle body is prepared from a mixed solution of polyvinyl alcohol and dextran; the empty needle body is prepared from a polyvinyl alcohol and dextran mixed solution with the mass ratio concentration of 84/16;
The adhesive layer and the backing layer are both made of polyvinyl alcohol aqueous solution; the solution of the adhesive layer is a polyvinyl alcohol aqueous solution with the mass ratio concentration of 18%; the backing layer is a thin layer prepared from a polyvinyl alcohol aqueous solution with a mass ratio concentration of 25%.
2. A Chinese cobra neurotoxin phase-inversion hydrogel microneedle is characterized in that,
the drug-loaded needle comprises a drug-loaded needle body, an adhesive layer and a backing layer, wherein the adhesive layer and the backing layer are sequentially attached to the top of the drug-loaded needle body;
the drug-carrying needle body is prepared from a drug-carrying solution, and the drug-carrying solution consists of a Chinese cobra neurotoxin solution and auxiliary materials;
the auxiliary material is a mixed solution of polyvinyl alcohol and dextran with the mass ratio concentration of 84/16;
the solution of the adhesive layer is a polyvinyl alcohol aqueous solution with the mass ratio concentration of 18%;
the backing layer is a thin layer prepared from a polyvinyl alcohol aqueous solution with a mass ratio concentration of 25%.
3. The method for preparing the Chinese cobra neurotoxin phase inversion hydrogel microneedle according to claim 2, which is characterized in that the preparation method comprises the steps of centrifugating a needle body drug carrying solution, an adhesive layer solution and a backing layer, repeatedly freezing and thawing, and finally drying.
4. The method for preparing the phase-converted hydrogel microneedle of the Chinese cobra neurotoxin according to claim 2, which is characterized in that: the backing layer is prepared by the steps of: spreading 25% polyvinyl alcohol aqueous solution on square glass plate, uniformly pressing with another glass plate, supporting the two glass plates with a flat pad with thickness of about 1mm, freezing at-20deg.C for 8 hr, thawing at 4deg.C for 4 hr, and freezing-thawing for 2 times to obtain backing layer.
5. The method for preparing the phase-converted hydrogel microneedle of the Chinese cobra neurotoxin according to claim 3, which is characterized in that: the specific preparation steps of the Chinese cobra neurotoxin phase inversion hydrogel microneedle are as follows:
preparing a needle body drug carrying solution;
placing the needle body medicine carrying solution into a microneedle mould, injecting the needle body medicine carrying solution into holes of the microneedle mould by adopting a centrifugal method, scraping off redundant needle body medicine carrying solution, adding an adhesive layer solution, sticking a backing layer, and removing bubbles and redundant adhesive layer solution to prepare the microneedle patch;
and (3) after the microneedle patch is subjected to freeze-thawing cycle, removing the microneedle patch from the microneedle mould, and finally drying to obtain the Chinese cobra neurotoxin phase-converted hydrogel microneedle.
6. The method for preparing the phase-converted hydrogel microneedle of the Chinese cobra neurotoxin according to claim 5, which is characterized in that:
the preparation method of the needle drug-carrying solution comprises the following steps:
mixing the powder of the Chinese cobra neurotoxin with ultrapure water to obtain a medicinal stock solution;
according to the required microneedle drug loading concentration, a proper amount of drug stock solution is taken and added into a mixed solution of polyvinyl alcohol and dextran with the mass ratio concentration of 84/16, and the mixed solution is uniformly mixed and stirred to obtain the needle drug loading solution which is uniform and stable and has no phase separation.
7. The method for preparing the phase-converted hydrogel microneedle of the Chinese cobra neurotoxin according to claim 5, which is characterized in that: the freezing-thawing conditions of the microneedle patch were: freezing at-20deg.C for 8 hr, and thawing at 4deg.C for 4 hr.
8. Use of the empty phase inversion hydrogel microneedle of claim 1 or the chinese cobra neurotoxin phase inversion hydrogel microneedle of claim 3 or 4 or the method of preparation of any one of claims 2-7 for the preparation of a transdermal administration analgesic formulation.
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