WO2023098158A1 - Needle-medicine integrated hydrogel microneedle - Google Patents
Needle-medicine integrated hydrogel microneedle Download PDFInfo
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- WO2023098158A1 WO2023098158A1 PCT/CN2022/114243 CN2022114243W WO2023098158A1 WO 2023098158 A1 WO2023098158 A1 WO 2023098158A1 CN 2022114243 W CN2022114243 W CN 2022114243W WO 2023098158 A1 WO2023098158 A1 WO 2023098158A1
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- microneedle
- cinnamon oil
- acupuncture
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- medicine
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the technical field of medicine, and in particular relates to a hydrogel microneedle integrating acupuncture and medicine.
- acupoints are special parts where Qi and blood flow into and out of the viscera and meridians, and acupoints are closely connected with deep tissues and organs and communicate with each other. Stimulating the acupoints of the human body through acupuncture, massage, drug administration, etc. can dredge the meridians, reconcile yin and yang, promote qi and blood circulation, stimulate the effect of acupoints, and achieve the purpose of strengthening the body, eliminating pathogens, and preventing and curing diseases.
- Umbilical drug administration is one of the commonly used umbilical therapy methods in traditional Chinese medicine.
- acupoint administration is acupoint injection and acupoint application, but the compliance of patients with acupoint injection is poor. Due to the barrier effect of the skin and intradermal microblood Rapid transport, it is difficult to concentrate the drug in the subcutaneous deep acupoint tissue.
- Microneedles is a new transdermal drug penetration enhancing technology integrating injection and transdermal, which is painless and minimally invasive.
- Microneedles can be made of metal, ceramics, silicon or polymer materials, with a length of 100-1000 ⁇ m.
- the treatment method combining microneedle and drug can be given by acupuncture first, or the drug can be attached and loaded in the needle body and then directly transported into the body. Since acupuncture can form drug delivery channels on the skin surface, it can effectively promote the percutaneous absorption of drugs and enhance the therapeutic effect.
- Microneedling is painless and minimally invasive, and patients are highly compliant.
- Cinnamon oil has the effect of warming the middle and dispelling cold.
- acupoint therapy if it can be delivered to the deep layer of the skin at the acupoints, it can fully exert its efficacy and achieve the purpose of treatment; especially the administration of umbilical acupoints.
- cinnamon oil is attached to the needle body of the microneedle for acupuncture treatment, the drug loading may be insufficient; if it is entrapped in the needle body, the release problem in the body has not been solved.
- the present invention aims to overcome the defects of the prior art, combines acupuncture in the umbilical area with acupoint administration, develops a microneedle loaded with medicine, and realizes an integrated acupoint treatment strategy of "needle” and "medicine", which is easy to operate, and enhance the therapeutic effect.
- the invention provides a hydrogel microneedle with integrated acupuncture and medicine (HFMNs-CIO@NCs), which comprises a microneedle matrix and a drug entrapped in the microneedle matrix, and the microneedle matrix is gelatin and tannic acid Hydrogels formed by co-products.
- HFMNs-CIO@NCs integrated acupuncture and medicine
- the medicine contained in the microneedle matrix is a nanocapsule, more preferably a cinnamon oil nanocapsule, that is, the described acupuncture-medicine integrated hydrogel microneedle is a cinnamon oil nanocapsule hydrogel microneedle (HFMNs- CIO@NCs).
- cinnamon oil nanocapsules are cinnamon oil encapsulated in the shell-core structure formed by chitosan and sodium alginate; the particle diameter of the cinnamon oil nanocapsules is 50-500nm, and the The drug loading amount of cinnamon aldehyde in the cinnamon oil nanocapsules is 26wt%-36wt%.
- the mass ratio of the microneedle matrix to the cinnamon oil nanocapsules is 0.2-0.5:1.
- the microneedle matrix is gelatin and tannic acid cross-linked product (Gel-Tan), which is obtained by the cross-linking reaction between the amino group and carboxyl group in gelatin and the hydroxyl group in tannic acid.
- the preparation method of needle matrix comprises the following steps:
- the mass ratio of the tannic acid, glycerin and gelatin is 0.02 ⁇ 0.05:0.05 ⁇ 0.2:1, preferably 0.04:0.1:1; the concentration of the gelatin solution is 100 ⁇ 200mg/mL, the tannic acid The solution concentration is 40-80 mg/mL.
- step (1) the pH is adjusted to 5.5, and the reaction is carried out at 50-60° C. for 2-16 hours.
- the cinnamon oil nanocapsules are prepared by the controllable gelation of alginate induced by cations, and the preparation method of the cinnamon oil nanocapsules comprises the following steps:
- step (b) Add the calcium chloride solution dropwise to the O/W emulsion in step (a), and stir for 15 to 60 minutes;
- step (c) Add chitosan solution dropwise in the solution of step (b), continue to stir after dropping, leave standstill;
- the phospholipid is egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine;
- the phospholipids have a purity greater than 95%, more preferably greater than 98%.
- the alcohol is ethanol.
- the weight ratio of described cinnamon oil to phospholipid, calcium chloride and chitosan is 4 ⁇ 6:1:0.4 ⁇ 0.6:0.2 ⁇ 0.4
- the sodium alginate solution concentration described in step (a) 0.3 ⁇ 1mg/mL
- the consumption ratio of cinnamon oil and alcohol is 30 ⁇ 60mg/mL
- the calcium chloride solution concentration described in step (b) is 0.3 ⁇ 1mg/mL
- the chitosan described in step (c) The solution concentration is 0.3-1mg/mL.
- the cinnamon oil is prepared according to the provisions of the Pharmacopoeia, and is the volatile oil of cinnamon, wherein the content of cinnamon aldehyde complies with the quality standards for cinnamon oil (Cinnamon Oil, CIO) in the 2020 edition of "Chinese Pharmacopoeia”.
- step (a) sodium alginate is stirred in pure water at 200-600rmp until dissolved to obtain a sodium alginate solution; in step (a), the alcohol solution of cinnamon oil and phospholipids is added to the seaweed while ultrasonically After mixing, continue to sonicate for 10 to 20 minutes, and then sonicate the probe for 10 to 20 minutes in an ice bath.
- step (b) calcium chloride is added dropwise to the O/W emulsion in step (a) at a rate of 6-10 mL/h, and after the dropwise addition is completed, stirring is continued at 200-500 rpm for 20-40 min.
- step (c) chitosan is dissolved with 0.5% ⁇ 2% acetic acid aqueous solution, and adjusts pH to 4.5-5.5, preferred pH value is 5; And chitosan solution is stirred while stirring at the speed of 8 ⁇ 15mL/h Add to the solution in step (b), after the dropwise addition, continue stirring at 200-500 rpm for 2-6 hours, and let stand for 8-16 hours.
- step (d) centrifuge at 100,000 to 150,000 ⁇ g for 45 to 120 min, and take the precipitate.
- the invention provides a method for preparing the hydrogel microneedle integrating acupuncture and medicine.
- the steps include: mixing the drug solution and the microneedle matrix solution, forming and drying.
- the preparation method of cinnamon oil nanocapsule hydrogel microneedles comprises: mixing the cinnamon oil nanocapsule solution and the microneedle matrix solution, forming and drying. Specifically, the cinnamon oil nanocapsule solution and the microneedle matrix solution are evenly mixed, and then cast in a microneedle mold, put into the mold under reduced pressure, and then vacuum-dried, and then demoulded.
- the invention adopts the microneedle matrix obtained by cross-linking gelatin and tannic acid, which is easy to form and demould, has good mechanical strength and high swelling property.
- the loading of nanocapsules can enhance the strength of microneedles.
- the above-mentioned cinnamon oil nanocapsule hydrogel microneedle can be used to prepare a medicine for treating dysmenorrhea of cold coagulation and blood stasis type. Using this kind of microneedle can realize the integrated treatment strategy of acupuncture and medicine.
- the microneedle of the present invention can deliver the cinnamon oil nanocapsules to the deep part of the acupoint area.
- the hydrogel microneedle absorbs water and swells, continuously stimulates the tissue of the acupoint area, stimulates the effect of the acupoint area, and achieves acupuncture to dredge the meridian, promote qi and dissipate blood stasis, Therapeutic effects such as pain relief.
- Nanocapsules (Nanocapsules, NCs) technology uses polymer films to embed and encapsulate trace amounts of drug molecules.
- the embedded substance is called the core material, and the polymer covering the core material is called the wall material.
- the particle size of nanocapsules is at the nanometer level, and its nanosize effect makes it have good biocompatibility, targeting and sustained release.
- Chitosan Choitson, CS
- the N-deacetylation product of chitin is a linear polysaccharide composed of glucosamine and N-acetylglucosamine units linked by ⁇ -(1 ⁇ 4) glycosidic bonds.
- Chitosan can be dissolved in acidic medium after deacetylation treatment, and becomes the only polysaccharide with high positive charge density due to the protonation of amino groups on its main chain. Chitosan also has characteristics such as non-toxicity, biocompatibility and biodegradability.
- Sodium Alginate Sodium Alginate, SA
- SA sodium Alginate
- the sodium alginate and calcium ion cross-linked product is used as the shell of the nanocapsule
- the cinnamon oil is used as the core
- the cinnamon oil nanocapsule is prepared by an ion gel method and modified with chitosan.
- the gelatin and tannic acid cross-linked product was used as the hydrogel microneedle matrix, and the cinnamon oil nanocapsules were mixed evenly with the microneedle matrix, and the hydrogel microneedles were prepared by casting method, which could better encapsulate cinnamon oil and be used for umbilical cord.
- the cinnamon oil nanocapsules can be delivered to the deep part of the acupoint area for better drug efficacy.
- Hydrogel forming microneedles consist of swelling materials and drug depots. After the hydrogel microneedles are pierced into the skin, the swelling material and the drug reservoir in the microneedle array can absorb the interstitial fluid so that the needle body swells and releases the drug. Therefore, by using microneedles to carry drugs, it is possible to combine acupuncture in the umbilicus region with acupoint administration, and realize the integrated treatment of "needle” and "medicine”.
- the invention provides a new type of integrated acupuncture-medicine microneedle, which can be used for acupoint therapy.
- the microneedle is used as a carrier, and the extract of traditional Chinese medicine and its preparation, especially the nanocapsule of the extract of traditional Chinese medicine, are used for acupuncture and drug delivery at the acupoints at the same time. Medicine, exerting the comprehensive effect of acupuncture and drug therapy.
- the cinnamon oil nanocapsule hydrogel microneedle (HFMNs-CIO@NCs) prepared by the present invention is used for administration in the umbilical area (Shenque point) to treat dysmenorrhea of cold coagulation and blood stasis type.
- the cinnamon oil nanocapsules are delivered to the deep part of the acupoint area.
- the hydrogel microneedle absorbs water and swells, continuously stimulating the acupoint area tissue, stimulating the acupoint area effect, and achieving the therapeutic effects of acupuncture to dredge the meridians, promote qi and dissipate blood stasis, and relieve pain.
- the invention uses the cross-linked product of gelatin and tannic acid as the hydrogel microneedle matrix, which is easy to form and demould, has good mechanical strength and high swelling property. And the loading of nanocapsules can enhance the strength of microneedles.
- the shell-core structure formed by chitosan and sodium alginate is used to entrap cinnamon oil to form cinnamon oil nanocapsules; the cross-linked product of gelatin and tannic acid is used as the microneedle matrix to encapsulate cinnamon oil.
- the prepared cinnamon oil nanocapsule hydrogel microneedles can not only deliver cinnamon oil nanocapsules to the deep layer of the skin in the acupoint area, but also give full play to the effect of cinnamon oil in warming and dispelling cold, and at the same time, the microneedle itself absorbs water and swells, which can stimulate the acupoints Area tissue, stimulating the effect of point area, to achieve the purpose of treatment.
- Administration of cinnamon oil nanocapsules, hydrogel, and microneedles to the umbilicus can improve the symptoms of cold coagulation syndrome in rats with dysmenorrhea of cold coagulation and blood stasis type, prolong the writhing latency period, reduce the number of writhing times and writhing scores, and significantly increase the PGE2 of uterine tissue content, reduce PGF2 ⁇ content and PGF2 ⁇ /PGE2 ratio, significantly increase the number of mast cells and mast cell degranulation rate in Shenque acupoint area, thereby significantly improving the therapeutic effect on rats with cold coagulation and blood stasis type dysmenorrhea.
- microneedle of the present invention can realize the integrated treatment strategy of acupuncture and medicine, and it is expected that the microneedle can be used to carry free medicine and its preparation, and to carry a variety of medicines and their preparations, which can be further used in the clinical treatment of acupoint therapy, which is worthy of clinical promotion Use;
- the microneedle preparation method of the present invention is simple, does not require special equipment and harsh conditions, is easy to realize large-scale production, and has strong practical value.
- Fig. 1 is the transmission electron micrograph of the cinnamon oil nanocapsule prepared by the embodiment of the present invention 2;
- Fig. 2 is the scanning electron micrograph of the cinnamon oil nanocapsule hydrogel microneedle prepared in Example 3 of the present invention, wherein: the magnification of A is 100 times, and the magnification of B is 350 times;
- Fig. 3 is the hydrogel microneedle (GB-Tan-MNs-CIO@NCs) loaded with cinnamon oil nanocapsules prepared in Example 3 of the present invention and the blank hydrogel microneedle (GB-Tan- MNs) mechanical strength test pattern;
- FIG. 4 shows the hydrogel microneedles (GB-Tan-MNs-CIO@NCs) loaded with cinnamon oil nanocapsules prepared in Example 3 of the present invention and the blank hydrogel microneedles (GB-Tan- MNs) swelling test collection;
- Fig. 5 is the optical photo of the rhodamine B-labeled blank microneedle patch pierced into rat isolated skin prepared in Example 3 of the present invention
- Fig. 6 is the toluidine blue stained light microscope photo of mast cells in rat Shenque acupoint area obtained in Example 4 of the present invention, wherein: (A, B) is a blank group, C is a model group, D is an acupuncture group, and E is a blank Microneedle group, F is drug-loaded microneedle group.
- Embodiment 1 Preparation of cinnamon oil nanocapsules
- cinnamon medicinal material stipulated in the 2020 edition of "Chinese Pharmacopoeia”
- weigh 200.2g of cinnamon medicinal material crush it into a 20-mesh coarse powder, add 8 times the amount of water, soak for 1h, extract by steam distillation at 200°C for 6h, and collect volatile oil to obtain 3mL Cinnamon essential oil.
- the cinnamon aldehyde content in cinnamon volatile oil is 93.1 ⁇ 4.45%, which meets the quality standards for cinnamon oil (Cinnamon Oil, CIO) in the 2020 edition of "Chinese Pharmacopoeia”.
- the chitosan solution was dripped into the above-mentioned sodium alginate solution while stirring (1000rpm) at a speed of 10mL ⁇ h -1 , continued to stir at 300rpm for 3h after the dropwise addition, left to stand overnight, centrifuged at 140,000 ⁇ g for 60min, and removed the above Then add 20 mL of purified water to the precipitate to disperse to obtain cinnamon oil nanocapsules (CIO@NCs), which are stored at 4°C for later use.
- CIO@NCs cinnamon oil nanocapsules
- the dried HFMNs-CIO@NCs were taken, and the surface was evenly sprayed with gold powder, and placed under a scanning electron microscope under vacuum conditions for morphological observation.
- Fig. 2 is a scanning electron micrograph of the hydrogel microneedle of the cinnamon oil-loaded nanocapsule prepared in Example 3 of the present invention, wherein, the magnification of A is 100 times, and the magnification of B is 350 times. It can be seen from Figure 2 that the demolding rate of HFMNs-CIO@NCs is 100%. After demoulding, the needle tip is intact and undamaged.
- the needle body is pyramid-shaped and consists of four-sided cone-shaped needle bodies. The base thickness is uniform and the array is arranged neatly.
- the microneedle patch is a square with a side length of 1.4 cm. The microneedle array is 10 ⁇ 10.
- the length of the microneedle base is about 600 ⁇ m, the width is about 600 ⁇ m, and the height of the microneedles is about 1450 ⁇ m.
- the distance between the base of the microneedles is 600 ⁇ m, and the distance between the needle tips is 900 ⁇ m.
- the needle body is placed upwards on the test plate of the texture analyzer, and a cylindrical probe with a diameter of 5 mm is set to compress the microneedle at a speed of 30 mm/min until the extrusion deformation is 40%.
- the initial trigger force was 0.05N, and the force-displacement curve of the microneedle was drawn.
- Fig. 3 is the hydrogel microneedle mechanical strength test spectrum of the cinnamon oil nanocapsules prepared in Example 3 of the present invention, as can be seen from Fig. 3, the mechanical strength of the BSP composite microneedle before and after drug loading can reach more than 18N. Since the microneedle mold is an array of 11 ⁇ 11, the calculated mechanical strengths of HFMNs and HFMNs-CIO@NCs are 0.15 N/needle and 0.18 N/needle, respectively, that is, the loading of nanocapsules slightly increases the strength of microneedles.
- microneedle patches Take blank microneedle patches (HFMNs) and drug-loaded microneedle patches (HFMNs-CIO@NCs), and record the initial mass W L .
- the microneedle sheets with different prescriptions were wrapped with polytetrafluoroethylene film and tin foil in turn, so that the needle tip was exposed.
- Add 0.7 mL of PBS to each well of the 24-well plate place the wrapped microneedle patch upside down in the well, and immerse the exposed needle tip in water. At 5, 15, 30, 60, and 90 minutes, the microneedle patch was taken out from the 24-well plate, and the excess water on the surface was wiped off, and then weighed.
- the mass at 0 was recorded as W 0
- the mass at t was recorded as W t
- (W t -W 0 )/W L represents the swelling rate of the microneedle patch at time t
- the above test was repeated 3 times for each group of samples. Take t as the abscissa and the swelling rate as the ordinate respectively to investigate the effect of drug loading on the swelling rate of microneedles.
- Fig. 4 is the hydrogel microneedle swelling property test spectrum of the loaded cinnamon oil nanocapsule prepared by the embodiment of the present invention 3, as seen from Fig. 4, the swelling rate of blank microneedle reaches 350% in 30min, after that, the swelling rate gradually decreases, It tends to be gentle, and the swelling rate is about 430% at 90 minutes. However, the swelling rate of the microneedles increased slightly after loading the drug, reaching 390% at 30 minutes, and 470% at 90 minutes. The swelling degree and swelling speed were better than those of blank microneedles.
- Rhodamine-labeled microneedles were prepared by labeling blank microneedle matrix with water-soluble dye rhodamine B. Press the surface of the isolated skin of the rat with your hands for 5 minutes, remove the microneedle patch, and observe the ability of the microneedle to penetrate the skin.
- Fig. 5 is an optical photograph of the rhodamine B-labeled blank microneedle patch prepared in Example 3 of the present invention piercing the isolated rat skin. It can be seen from Fig. 5 that the microneedle can successfully penetrate the isolated rat skin.
- Example 4 Study on the analgesic effect of hydrogel microneedles loaded with cinnamon oil nanocapsules (HFMNs-CIO@NCs) on model rats with cold coagulation and blood stasis type dysmenorrhea
- Female SD rats with a body weight of 180-220 g, were divided into the following four groups: (1) blank control group, (2) model group, (3) umbilicus acupuncture group, (4) blank microneedle umbilicus treatment group, (5 ) containing cinnamon oil nanocapsule microneedle umbilical acupuncture treatment group. Except for the blank control group, the model group and each treatment group were divided into groups after successful modeling.
- Rats in model group and treatment group were subcutaneously injected with estradiol benzoate, once a day, for 10 consecutive days; the dose was 2.5 mg ⁇ kg -1 on the first and 10th day, and 1 mg ⁇ kg -1 in the rest of the time; Put the rats in an ice-water bath for 10 minutes.
- epinephrine the animals were placed in ice water for 5 minutes, and after 5 minutes, the rats were taken out and put back into the cages for feeding.
- estradiol benzoate On the 12th day, 1 hour after the subcutaneous injection of estradiol benzoate, the rats were immediately intraperitoneally injected with oxytocin (dose 10U ⁇ kg -1 ) to prepare a rat model of dysmenorrhea in combination with estrogen and oxytocin in ice water bath. Observe the writhing reaction behavior of rats to judge the success of modeling.
- the blank control group was given normal diet, free drinking water, and no treatment.
- subcutaneous injection of normal saline was given to the thigh every day. , 0.25mL/monkey on the 10th day, subcutaneous injection twice on the 11th day, 0.1mL/bird each time, with an interval of 2h.
- 0.25 mL of normal saline was injected subcutaneously, and 1 hour later, oxytocin (dose 10 U ⁇ kg -1 ) was injected intraperitoneally immediately, and the writhing response behavior of the rats was observed.
- Rats were anesthetized by inhalation of isoflurane. After the rats were anesthetized, the coarse hairs around the Shenque acupoint were removed with an electric shaver, and then the fine hairs around the Shenque acupoint were removed with a depilatory cream. Rats were given treatment on the 6th day after modeling, once a day for a total of 7 treatments.
- the Shenque acupoint was regarded as the dial, and the intradermal needle was used to take the 6 o’clock position and stab horizontally towards the corresponding acupoint area.
- the needle was fixed with medical adhesive tape, and the needle was retained for 30 minutes at a depth of 4-5 mm.
- the rats were fixed in a supine position, and the blank or drug-loaded microneedles were placed in the Shenque acupoint area of the rats, and pressed with hands for 1 min, so that the microneedles penetrated into the Shenque acupoints of the rats, fixed with medical adhesive tape, and the needles were left for 30 min.
- the blank group and the model group were grasped and fixed synchronously with the above-mentioned groups, and no treatment was given.
- the symptoms improved, the weight remained stable or gradually increased, the spirit improved, the hair became brighter, and the skin color of the ears, limbs, tail and other parts appeared pink.
- the stool is slightly moist, and the fecal matter is slightly soft.
- Table 1 The results are shown in Table 1.
- the writhing reaction latency of rats in the model group was significantly shortened, and the number of writhing times and writhing scores were significantly increased, indicating that the model of dysmenorrhea rats with cold coagulation and blood stasis was successfully established.
- the number of writhing times in the umbilical acupuncture treatment group was significantly reduced (P ⁇ 0.05), and the number of writhing times and writhing scores in the blank microneedle umbilicus treatment group were significantly reduced (P ⁇ 0.05).
- the writhing latency period of the microneedle umbilicus treatment group was significantly prolonged (P ⁇ 0.05), and the number of writhing times and writhing ratings were significantly reduced (P ⁇ 0.01), indicating that each treatment group had a certain effect on the dysmenorrhea model rats of cold coagulation and blood stasis type. Among them, the treatment effect of the microneedle umbilicus treatment group loaded with cinnamon oil nanocapsules was the most significant.
- the rat was anesthetized by intraperitoneal injection of 3% pentobarbital sodium 1.0mL (150mg/kg), and the uterus was quickly removed on the ice tray of the sterile workbench, weighed, and the sample was placed in a -80°C refrigerator Save for later use.
- the content of PGF2 ⁇ in the model group was significantly increased (P ⁇ 0.01), and the ratio of PGF2 ⁇ /PGE2 was significantly increased (P ⁇ 0.05), indicating that the model of dysmenorrhea rats with cold coagulation and blood stasis was successfully established.
- the PGF2 ⁇ content in the umbilical acupuncture treatment group and the blank microneedle umbilicus treatment group significantly decreased (P ⁇ 0.01), and the ratio of PGF2 ⁇ /PGE2 decreased significantly (P ⁇ 0.05).
- the content of PGE2 in the microneedling umbilicus treatment group was significantly increased (P ⁇ 0.01), and the content of PGF2 ⁇ and the ratio of PGF2 ⁇ /PGE2 were significantly reduced (P ⁇ 0.05), indicating that each treatment group had a significant effect on the treatment of dysmenorrhea model rats with low cold blood stasis type. All have a certain therapeutic effect, and the treatment effect of the microneedle umbilicus treatment group loaded with cinnamon oil nanocapsules is the most significant.
- the rats were anesthetized by intraperitoneal injection of 1.0 mL (150 mg/kg) of 3% pentobarbital sodium, and the skin of the Shenque acupoint area of the rat and the subcutaneous muscle tissue (3mm ⁇ 3mm ⁇ 3mm), washed with normal saline, fixed with 4% paraformaldehyde, used for toluidine blue staining, and photographed under a 10 ⁇ 40 light microscope, the results are shown in Figure 6.
- Figure 6 is a light microscope photograph of mast cells stained with toluidine blue in the rat Shenque acupoint area obtained in Example 4 of the present invention, wherein: A, B. Blank group C. Model group, D. Acupuncture group E. Blank microneedle group F. Drug-loaded microneedle group; as can be seen from Figure 6 and Table 3, compared with the blank group, the number of mast cells and the mast cell degranulation rate in the Shenque acupoint area of the rats in the model group were significantly increased, with a statistical difference (P ⁇ 0.05) . Compared with the model group, the number of mast cells and the degranulation rate of mast cells in the Shenque acupoint area in each treatment group were significantly increased (P ⁇ 0.01).
- the acupuncture-medicine integrated treatment strategy provided by the present invention can deliver the drug to the deep layer of the skin in the acupoint area, exert its therapeutic effect in the acupoint area, and at the same time, the microneedle body absorbs water and swells, which can stimulate the tissue in the acupoint area and stimulate Acupoint effect to achieve the purpose of treatment.
- Administration of cinnamon oil nanocapsules, hydrogel, and microneedles to the umbilicus can improve the symptoms of dysmenorrhea model rats of cold coagulation and blood stasis type, prolong the writhing latency period, reduce the number of writhing times and writhing scores, significantly increase the content of PGE2 in uterine tissue, and reduce PGF2 ⁇
- the content and the ratio of PGF2 ⁇ /PGE2 significantly increased the number of mast cells and the degranulation rate of mast cells in the Shenque acupoint area, indicating that the administration of cinnamon oil nanocapsules hydrogel microneedles to the umbilical region can exert the effect of acupuncture at acupoints and improve the effect of cold coagulation.
- the symptoms of blood stasis type dysmenorrhea play an important role.
- the integrated treatment strategy of acupuncture and medicine is expected to use microneedles to carry free medicine and its preparations, which can be further used in the clinical treatment of acupoint therapy, and it is worthy of clinical promotion and use.
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Abstract
The present invention relates to the technical field of medicines. Disclosed is a needle-medicine integrated hydrogel microneedle. A microneedle matrix is hydrogel formed by a gelatin and tannic acid cross-linked product, a medicine is entrapped in the matrix, and the medicine is cinnamon oil nanocapsules (CIO@NCs). The cinnamon oil nanocapsule hydrogel microneedle (HFMNs-CIO@NCs) can better entrap the CIO@NCs and deliver the CIO@NCs to the deep layer of the skin in an acupoint area to fully exert the effect of warming spleen and stomach for dispelling cold of cinnamon oil; and moreover, the microneedle itself absorbs water to swell, and can stimulate the tissue in the Shenque acupoint area to excite an acupoint area effect, thereby improving symptoms of cold coagulation of a cold coagulation and blood stasis type dysmenorrhea model rat. The needle-medicine integrated hydrogel microneedle can be used for various entrapment medicines and preparations thereof, is further used for clinical treatment of acupoint therapy, and has clinical popularization and use values.
Description
本申请要求于2021年12月03日提交中国专利局、申请号为202111466714.8、发明名称为“一种针药一体化水凝胶微针”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application with the application number 202111466714.8 and the title of the invention "a hydrogel microneedle integrating acupuncture and medicine" submitted to the China Patent Office on December 03, 2021, the entire contents of which are incorporated by reference in this application.
本发明属于医药技术领域，具体涉及一种针药一体化的水凝胶微针。The invention belongs to the technical field of medicine, and in particular relates to a hydrogel microneedle integrating acupuncture and medicine.
传统中医理论认为穴位(腧穴)是气血输注出入脏腑经络的特殊部位，穴位与深部组织器官密切联系、互相输通。通过针灸、推拿、给药等方式刺激人体的穴位，可以起到疏通经络、调和阴阳、行气活血的作用，激发穴区效应，达到扶正祛邪、防治疾病的目的。脐部给药为中医常用的脐疗方法之一。中医认为在脐部给药后，药物经皮进入穴区组织，可刺激穴区激发特殊调节作用，同时发挥药物的治疗作用。近年来，腹针、揿针、脐针等用于痛经治疗的临床报道日益增多，通过细针、浅刺，刺激神阙穴区，结合艾灸、药物敷贴等，起到调节脏腑经络、行气、散瘀、止痛的效果。当前临床应用中，针灸需要专业医技人员，穴位给药的主要途径为穴位注射和穴区敷药，而穴位注射患者依从性较差，穴区敷药由于皮肤的屏障作用及皮内微血的快速转运，难以将药物浓集于皮下深层的穴区组织。The theory of traditional Chinese medicine believes that acupoints (acupoints) are special parts where Qi and blood flow into and out of the viscera and meridians, and acupoints are closely connected with deep tissues and organs and communicate with each other. Stimulating the acupoints of the human body through acupuncture, massage, drug administration, etc. can dredge the meridians, reconcile yin and yang, promote qi and blood circulation, stimulate the effect of acupoints, and achieve the purpose of strengthening the body, eliminating pathogens, and preventing and curing diseases. Umbilical drug administration is one of the commonly used umbilical therapy methods in traditional Chinese medicine. Traditional Chinese medicine believes that after the drug is administered at the umbilicus, the drug enters the acupoint tissue through the skin, which can stimulate the acupoint area to stimulate special regulation, and at the same time exert the therapeutic effect of the drug. In recent years, there have been more and more clinical reports on the use of abdominal acupuncture, pressing needles, and umbilical acupuncture for the treatment of dysmenorrhea. Through fine needles and shallow needling, stimulating the Shenque acupoint area, combined with moxibustion and drug application, it can regulate viscera, meridians, Promoting qi, dissipating blood stasis, and relieving pain. In the current clinical application, acupuncture and moxibustion require professional medical technicians. The main ways of acupoint administration are acupoint injection and acupoint application, but the compliance of patients with acupoint injection is poor. Due to the barrier effect of the skin and intradermal microblood Rapid transport, it is difficult to concentrate the drug in the subcutaneous deep acupoint tissue.
微针(Microneedles,MNs)是一种集注射和透皮为一体的新兴的经皮给药促透技术，具有无痛、微创的特点。微针可用金属、陶瓷、硅或高分子聚合物材料制备，长度为100～1000μm，有单根微针和多枚集成的微针阵列，可根据需要制成笔式、滚轮、贴片等不同形式。微针与药物相结合的治疗方法，可先行针刺再给药，也可将药物附着、包载于针体后直接输送到体内。由于针刺后可在皮肤表面形成药物输送孔道，能有效促进药物经皮吸收，增强治疗效果。微针具有无痛、微创的特点，患者依从性高。Microneedles (MNs) is a new transdermal drug penetration enhancing technology integrating injection and transdermal, which is painless and minimally invasive. Microneedles can be made of metal, ceramics, silicon or polymer materials, with a length of 100-1000 μm. There are single microneedles and multiple integrated microneedle arrays, which can be made into pen type, roller, patch, etc. according to needs. form. The treatment method combining microneedle and drug can be given by acupuncture first, or the drug can be attached and loaded in the needle body and then directly transported into the body. Since acupuncture can form drug delivery channels on the skin surface, it can effectively promote the percutaneous absorption of drugs and enhance the therapeutic effect. Microneedling is painless and minimally invasive, and patients are highly compliant.
肉桂油具有温中散寒的效果，在穴位疗法的临床中，如果能够递送到穴位皮肤深层，能充分发挥功效，达到治疗的目的；尤其是脐部穴区给药。Cinnamon oil has the effect of warming the middle and dispelling cold. In the clinical practice of acupoint therapy, if it can be delivered to the deep layer of the skin at the acupoints, it can fully exert its efficacy and achieve the purpose of treatment; especially the administration of umbilical acupoints.
如果将肉桂油附着在微针针体上，进行针刺治疗，载药量可能不足；如果包载于针体，在体内的释放问题并未解决。If cinnamon oil is attached to the needle body of the microneedle for acupuncture treatment, the drug loading may be insufficient; if it is entrapped in the needle body, the release problem in the body has not been solved.
发明内容Contents of the invention
本发明旨在克服现有技术缺陷，将脐区针刺与穴位给药相结合，开发了一种包载药物的微针，实现“针”“药”的一体化穴位治疗策略，易于操作，并增强治疗效果。The present invention aims to overcome the defects of the prior art, combines acupuncture in the umbilical area with acupoint administration, develops a microneedle loaded with medicine, and realizes an integrated acupoint treatment strategy of "needle" and "medicine", which is easy to operate, and enhance the therapeutic effect.
本发明提供了一种针药一体化水凝胶微针(HFMNs-CIO@NCs)，包括微针基质和包载于微针基质内的药物，所述微针基质为明胶与单宁酸交联产物形成的水凝胶。The invention provides a hydrogel microneedle with integrated acupuncture and medicine (HFMNs-CIO@NCs), which comprises a microneedle matrix and a drug entrapped in the microneedle matrix, and the microneedle matrix is gelatin and tannic acid Hydrogels formed by co-products.
优选的，包载于微针基质内的药物为纳米胶囊，更优选为肉桂油纳米胶囊，即所述的针药一体化水凝胶微针为肉桂油纳米胶囊水凝胶微针(HFMNs-CIO@NCs)。Preferably, the medicine contained in the microneedle matrix is a nanocapsule, more preferably a cinnamon oil nanocapsule, that is, the described acupuncture-medicine integrated hydrogel microneedle is a cinnamon oil nanocapsule hydrogel microneedle (HFMNs- CIO@NCs).
所述的肉桂油纳米胶囊(CIO@NCs)，是将肉桂油包载于壳聚糖与海藻酸钠形成的壳-核结构中；所述肉桂油纳米胶囊的粒径为50～500nm，所述肉桂油纳米胶囊中桂皮醛的载药量为26wt％～36wt％。The cinnamon oil nanocapsules (CIO@NCs) are cinnamon oil encapsulated in the shell-core structure formed by chitosan and sodium alginate; the particle diameter of the cinnamon oil nanocapsules is 50-500nm, and the The drug loading amount of cinnamon aldehyde in the cinnamon oil nanocapsules is 26wt%-36wt%.
在本发明中，分别以明胶和肉桂油用量计，微针基质与肉桂油纳米胶囊的质量比为0.2～0.5:1。In the present invention, based on the amount of gelatin and cinnamon oil respectively, the mass ratio of the microneedle matrix to the cinnamon oil nanocapsules is 0.2-0.5:1.
在本发明中，所述的微针基质为明胶与单宁酸交联产物(Gel-Tan)，由明胶中的氨基与羧基与单宁酸中的羟基发生交联反应而得，所述微针基质的制备方法包括以下步骤：In the present invention, the microneedle matrix is gelatin and tannic acid cross-linked product (Gel-Tan), which is obtained by the cross-linking reaction between the amino group and carboxyl group in gelatin and the hydroxyl group in tannic acid. The preparation method of needle matrix comprises the following steps:
(1)将单宁酸溶液缓慢滴加到明胶溶液中，调节pH至5～6，45～65℃反应2～16h；(1) Slowly add the tannic acid solution dropwise to the gelatin solution, adjust the pH to 5-6, and react at 45-65°C for 2-16 hours;
(2)反应液中加入甘油并搅拌均匀。(2) Glycerin was added to the reaction liquid and stirred evenly.
所述单宁酸、甘油和明胶的质量比为0.02～0.05:0.05～0.2:1，优选为0.04:0.1:1；所述的明胶溶液浓度为100～200mg/mL，所述的单宁酸溶液浓度为40～80mg/mL。The mass ratio of the tannic acid, glycerin and gelatin is 0.02～0.05:0.05～0.2:1, preferably 0.04:0.1:1; the concentration of the gelatin solution is 100～200mg/mL, the tannic acid The solution concentration is 40-80 mg/mL.
优选的，步骤(1)中，pH调节至5.5，50～60℃反应2～16h。Preferably, in step (1), the pH is adjusted to 5.5, and the reaction is carried out at 50-60° C. for 2-16 hours.
在本发明中，所述肉桂油纳米胶囊为利用阳离子诱导的海藻酸盐的可控凝胶作用制备而得，所述肉桂油纳米胶囊的制备方法包括以下步骤：In the present invention, the cinnamon oil nanocapsules are prepared by the controllable gelation of alginate induced by cations, and the preparation method of the cinnamon oil nanocapsules comprises the following steps:
(a)将肉桂油与磷脂溶解于醇，加入到海藻酸钠溶液中，超声分散，得到O/W乳液；(a) dissolving cinnamon oil and phospholipids in alcohol, adding them to sodium alginate solution, and ultrasonically dispersing to obtain an O/W emulsion;
(b)氯化钙溶液滴加到步骤(a)的O/W乳液中，搅拌15～60min；(b) Add the calcium chloride solution dropwise to the O/W emulsion in step (a), and stir for 15 to 60 minutes;
(c)步骤(b)的溶液中滴加壳聚糖溶液，滴加后继续搅拌，静置；(c) Add chitosan solution dropwise in the solution of step (b), continue to stir after dropping, leave standstill;
(d)分离取沉淀即为肉桂油纳米胶囊。(d) Separation and precipitation are cinnamon oil nanocapsules.
在本发明中，所述的磷脂为蛋黄卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷脂酰胆碱或二棕榈酰磷脂酰胆碱；所述磷脂的纯度大于95％，更优选为大于98％。In the present invention, the phospholipid is egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine; The phospholipids have a purity greater than 95%, more preferably greater than 98%.
优选的，所述的醇为乙醇。Preferably, the alcohol is ethanol.
在本发明中，所述的肉桂油与磷脂、氯化钙和壳聚糖的重量比为4～6:1:0.4～0.6:0.2～0.4，步骤(a)所述的海藻酸钠溶液浓度为0.3～1mg/mL，肉桂油与醇的用量比为30～60mg/mL；步骤(b)所述的氯化钙溶液浓度为0.3～1mg/mL；步骤(c)所述的壳聚糖溶液浓度为0.3～1mg/mL。In the present invention, the weight ratio of described cinnamon oil to phospholipid, calcium chloride and chitosan is 4～6:1:0.4～0.6:0.2～0.4, the sodium alginate solution concentration described in step (a) 0.3～1mg/mL, the consumption ratio of cinnamon oil and alcohol is 30～60mg/mL; the calcium chloride solution concentration described in step (b) is 0.3～1mg/mL; the chitosan described in step (c) The solution concentration is 0.3-1mg/mL.
在本发明中，所述的肉桂油按照药典之规定制取，是肉桂的挥发油，其中桂皮醛的含量符合2020年版《中国药典》中关于肉桂油(Cinnamon Oil，CIO)的质量标准规定。In the present invention, the cinnamon oil is prepared according to the provisions of the Pharmacopoeia, and is the volatile oil of cinnamon, wherein the content of cinnamon aldehyde complies with the quality standards for cinnamon oil (Cinnamon Oil, CIO) in the 2020 edition of "Chinese Pharmacopoeia".
优选的，步骤(a)中，海藻酸钠在纯水中以200～600rmp搅拌至溶解，制得海藻酸钠溶液；步骤(a)中，肉桂油与磷脂的醇溶液边超声边加入到海藻酸钠溶液中，混合后继续超声10～20min，再于冰浴条件下探头超声10～20min。Preferably, in step (a), sodium alginate is stirred in pure water at 200-600rmp until dissolved to obtain a sodium alginate solution; in step (a), the alcohol solution of cinnamon oil and phospholipids is added to the seaweed while ultrasonically After mixing, continue to sonicate for 10 to 20 minutes, and then sonicate the probe for 10 to 20 minutes in an ice bath.
步骤(b)中，氯化钙以6～10mL/h的速度滴加到步骤(a)的O/W乳液中，滴加完毕后，200～500rpm继续搅拌20～40min。In step (b), calcium chloride is added dropwise to the O/W emulsion in step (a) at a rate of 6-10 mL/h, and after the dropwise addition is completed, stirring is continued at 200-500 rpm for 20-40 min.
步骤(c)中，壳聚糖用0.5％～2％醋酸水溶液溶解，并调节pH至4.5-5.5，优选的pH值为5；并且壳聚糖溶液以8～15mL/h的速度边搅拌边加入到步骤(b)的溶液中，滴加完毕后，200～500rpm继续搅拌2～6h，静置8～16h。In step (c), chitosan is dissolved with 0.5%～2% acetic acid aqueous solution, and adjusts pH to 4.5-5.5, preferred pH value is 5; And chitosan solution is stirred while stirring at the speed of 8～15mL/h Add to the solution in step (b), after the dropwise addition, continue stirring at 200-500 rpm for 2-6 hours, and let stand for 8-16 hours.
步骤(d)中，100000～150000×g离心45～120min，取沉淀。In step (d), centrifuge at 100,000 to 150,000×g for 45 to 120 min, and take the precipitate.
本发明提供了上述针药一体化水凝胶微针的制备方法，步骤包括：将药物溶液与微针基质溶液混匀后成型并干燥。The invention provides a method for preparing the hydrogel microneedle integrating acupuncture and medicine. The steps include: mixing the drug solution and the microneedle matrix solution, forming and drying.
在本发明中，当药物为肉桂油纳米胶囊时，肉桂油纳米胶囊水凝胶微针的制备方法步骤包括：将肉桂油纳米胶囊溶液与微针基质溶液混匀后成型并干燥。具体为：肉桂油纳米胶囊溶液与微针基质溶液混合均匀，再浇铸于微针模具内，经减压入模、真空干燥后脱模制成。In the present invention, when the medicine is cinnamon oil nanocapsules, the preparation method of cinnamon oil nanocapsule hydrogel microneedles comprises: mixing the cinnamon oil nanocapsule solution and the microneedle matrix solution, forming and drying. Specifically, the cinnamon oil nanocapsule solution and the microneedle matrix solution are evenly mixed, and then cast in a microneedle mold, put into the mold under reduced pressure, and then vacuum-dried, and then demoulded.
本发明采用明胶与单宁酸交联得到的微针基质，易于成型和脱模，机 械强度好，溶胀性高。纳米胶囊的载入可以加强微针的强度。The invention adopts the microneedle matrix obtained by cross-linking gelatin and tannic acid, which is easy to form and demould, has good mechanical strength and high swelling property. The loading of nanocapsules can enhance the strength of microneedles.
在本发明中，上述肉桂油纳米胶囊水凝胶微针可用于制备治疗寒凝血瘀型痛经的药物。利用这种微针能够实现针药一体化治疗策略。本发明的微针可将肉桂油纳米胶囊递送至穴区深部，同时，水凝胶微针吸水溶胀，不断地刺激穴区组织，激发穴区效应，达到针刺疏通经络、行气散瘀、止痛等的治疗效果。In the present invention, the above-mentioned cinnamon oil nanocapsule hydrogel microneedle can be used to prepare a medicine for treating dysmenorrhea of cold coagulation and blood stasis type. Using this kind of microneedle can realize the integrated treatment strategy of acupuncture and medicine. The microneedle of the present invention can deliver the cinnamon oil nanocapsules to the deep part of the acupoint area. At the same time, the hydrogel microneedle absorbs water and swells, continuously stimulates the tissue of the acupoint area, stimulates the effect of the acupoint area, and achieves acupuncture to dredge the meridian, promote qi and dissipate blood stasis, Therapeutic effects such as pain relief.
纳米胶囊(Nanocapsules,NCs)技术利用高分子聚合物薄膜将微量的药物分子包埋封装，其中被包埋的物质被称为芯材，包覆芯材的高分子聚合物被称为壁材。一般纳米胶囊粒径为纳米级别，其纳米尺寸效应使其具有良好的生物相容性、靶向性以及缓释性。壳聚糖(Chitson,CS)是甲壳素的N-脱乙酰化产物，是一种线性多糖，由氨基葡萄糖和N-乙酰氨基葡萄糖单元通过β-(1→4)糖苷键连接。壳聚糖经脱乙酰处理后，能在酸性介质中溶解，由于其主链上的氨基质子化而成为唯一一种具有高正电荷密度的多糖。壳聚糖还具有如无毒性、生物相容性和生物降解性等特点。海藻酸钠(Sodium Alginate,SA)是从褐藻和海藻中提取的一种天然多糖，它是一种水溶性线性多糖，羧酸基团使海藻酸盐带负电荷，因此能够与带正电荷的分子静电作用。本发明中，以海藻酸钠与钙离子交联产物作为纳米胶囊外壳，以肉桂油作为内核，通过离子凝胶法制备肉桂油纳米胶囊，并以壳聚糖修饰。以明胶与单宁酸交联产物作为水凝胶微针基质，将肉桂油纳米胶囊与微针基质混合均匀，浇铸法制备水凝胶微针，可以较好地包载肉桂油，用于脐区给药，可将肉桂油纳米胶囊递送至穴区深部更好地发挥药效。Nanocapsules (Nanocapsules, NCs) technology uses polymer films to embed and encapsulate trace amounts of drug molecules. The embedded substance is called the core material, and the polymer covering the core material is called the wall material. Generally, the particle size of nanocapsules is at the nanometer level, and its nanosize effect makes it have good biocompatibility, targeting and sustained release. Chitosan (Chitson, CS), the N-deacetylation product of chitin, is a linear polysaccharide composed of glucosamine and N-acetylglucosamine units linked by β-(1→4) glycosidic bonds. Chitosan can be dissolved in acidic medium after deacetylation treatment, and becomes the only polysaccharide with high positive charge density due to the protonation of amino groups on its main chain. Chitosan also has characteristics such as non-toxicity, biocompatibility and biodegradability. Sodium Alginate (Sodium Alginate, SA) is a natural polysaccharide extracted from brown algae and seaweed. It is a water-soluble linear polysaccharide. Molecular electrostatic interaction. In the present invention, the sodium alginate and calcium ion cross-linked product is used as the shell of the nanocapsule, and the cinnamon oil is used as the core, and the cinnamon oil nanocapsule is prepared by an ion gel method and modified with chitosan. The gelatin and tannic acid cross-linked product was used as the hydrogel microneedle matrix, and the cinnamon oil nanocapsules were mixed evenly with the microneedle matrix, and the hydrogel microneedles were prepared by casting method, which could better encapsulate cinnamon oil and be used for umbilical cord. The cinnamon oil nanocapsules can be delivered to the deep part of the acupoint area for better drug efficacy.
水凝胶微针(Hydrogel forming microneedles,HFMNs)由溶胀材料和药物贮库组成。水凝胶微针刺入皮肤后，微针阵列中的溶胀材料和药物贮库可吸收细胞间隙液从而使针体溶胀，释放药物。因此，以微针包载药物，可以将脐区针刺与穴位给药相结合，实现“针”“药”的一体化治疗。Hydrogel forming microneedles (HFMNs) consist of swelling materials and drug depots. After the hydrogel microneedles are pierced into the skin, the swelling material and the drug reservoir in the microneedle array can absorb the interstitial fluid so that the needle body swells and releases the drug. Therefore, by using microneedles to carry drugs, it is possible to combine acupuncture in the umbilicus region with acupoint administration, and realize the integrated treatment of "needle" and "medicine".
本发明提供一种新型针药一体化微针，可用于穴位治疗，以微针作为载体，包载中药提取物及其制剂，尤其是中药提取物的纳米胶囊，同时给予穴区针刺和给药，发挥针刺和药物治疗的综合效应。The invention provides a new type of integrated acupuncture-medicine microneedle, which can be used for acupoint therapy. The microneedle is used as a carrier, and the extract of traditional Chinese medicine and its preparation, especially the nanocapsule of the extract of traditional Chinese medicine, are used for acupuncture and drug delivery at the acupoints at the same time. Medicine, exerting the comprehensive effect of acupuncture and drug therapy.
本发明所制备的肉桂油纳米胶囊水凝胶微针(HFMNs-CIO@NCs)用于脐区(神阙穴)给药治疗寒凝血瘀型痛经，经脐部穴区(神阙穴)可将肉桂 油纳米胶囊递送至穴区深部，同时，水凝胶微针吸水溶胀，不断地刺激穴区组织，激发穴区效应，达到针刺疏通经络、行气散瘀、止痛等的治疗效果。The cinnamon oil nanocapsule hydrogel microneedle (HFMNs-CIO@NCs) prepared by the present invention is used for administration in the umbilical area (Shenque point) to treat dysmenorrhea of cold coagulation and blood stasis type. The cinnamon oil nanocapsules are delivered to the deep part of the acupoint area. At the same time, the hydrogel microneedle absorbs water and swells, continuously stimulating the acupoint area tissue, stimulating the acupoint area effect, and achieving the therapeutic effects of acupuncture to dredge the meridians, promote qi and dissipate blood stasis, and relieve pain.
与现有技术相比，本发明有益效果为：Compared with the prior art, the beneficial effects of the present invention are:
本发明以明胶与单宁酸交联产物作为水凝胶微针基质，易于成型和脱模，机械强度好，溶胀性高。而且纳米胶囊的载入可以加强微针的强度。载肉桂油纳米胶囊时，以壳聚糖与海藻酸钠形成的壳-核结构包载肉桂油，形成肉桂油纳米胶囊；采用明胶与单宁酸交联产物作为微针基质，包载肉桂油纳米胶囊，制备成的肉桂油纳米胶囊水凝胶微针不但可将肉桂油纳米胶囊递送到穴区皮肤深层，充分发挥肉桂油温中散寒的作用，同时微针本身吸水溶胀，能够刺激穴区组织，激发穴区效应，达到治疗目的。肉桂油纳米胶囊水凝胶微针脐部给药可以改善寒凝血瘀型痛经模型大鼠的寒凝症的症状，延长扭体潜伏期，减少扭体次数及扭体评分，显著升高子宫组织PGE2含量，降低PGF2α含量及PGF2α/PGE2比值，显著增加神阙穴区肥大细胞个数与肥大细胞脱颗粒率，从而显著提高对大鼠寒凝血瘀型痛经的治疗作用。The invention uses the cross-linked product of gelatin and tannic acid as the hydrogel microneedle matrix, which is easy to form and demould, has good mechanical strength and high swelling property. And the loading of nanocapsules can enhance the strength of microneedles. When loading cinnamon oil nanocapsules, the shell-core structure formed by chitosan and sodium alginate is used to entrap cinnamon oil to form cinnamon oil nanocapsules; the cross-linked product of gelatin and tannic acid is used as the microneedle matrix to encapsulate cinnamon oil. Nanocapsules, the prepared cinnamon oil nanocapsule hydrogel microneedles can not only deliver cinnamon oil nanocapsules to the deep layer of the skin in the acupoint area, but also give full play to the effect of cinnamon oil in warming and dispelling cold, and at the same time, the microneedle itself absorbs water and swells, which can stimulate the acupoints Area tissue, stimulating the effect of point area, to achieve the purpose of treatment. Administration of cinnamon oil nanocapsules, hydrogel, and microneedles to the umbilicus can improve the symptoms of cold coagulation syndrome in rats with dysmenorrhea of cold coagulation and blood stasis type, prolong the writhing latency period, reduce the number of writhing times and writhing scores, and significantly increase the PGE2 of uterine tissue content, reduce PGF2α content and PGF2α/PGE2 ratio, significantly increase the number of mast cells and mast cell degranulation rate in Shenque acupoint area, thereby significantly improving the therapeutic effect on rats with cold coagulation and blood stasis type dysmenorrhea.
利用本发明的微针可实现针药一体化治疗策略，可望利用微针担载游离药及其制剂，包载多种药物及其制剂，进一步地用于穴位疗法的临床治疗，值得临床推广使用；另外，本发明的微针制备方法简单，无需特殊设备和苛刻条件，易于实现规模化生产，具有极强的实用价值。Using the microneedle of the present invention can realize the integrated treatment strategy of acupuncture and medicine, and it is expected that the microneedle can be used to carry free medicine and its preparation, and to carry a variety of medicines and their preparations, which can be further used in the clinical treatment of acupoint therapy, which is worthy of clinical promotion Use; In addition, the microneedle preparation method of the present invention is simple, does not require special equipment and harsh conditions, is easy to realize large-scale production, and has strong practical value.
图1是本发明实施例2制备的肉桂油纳米胶囊的透射电镜图；Fig. 1 is the transmission electron micrograph of the cinnamon oil nanocapsule prepared by the embodiment of the present invention 2;
图2是本发明实施例3制备的肉桂油纳米胶囊水凝胶微针扫描电镜图，其中：A放大倍数为100倍，B放大倍数为350倍；Fig. 2 is the scanning electron micrograph of the cinnamon oil nanocapsule hydrogel microneedle prepared in Example 3 of the present invention, wherein: the magnification of A is 100 times, and the magnification of B is 350 times;
图3是本发明实施例3制备的载肉桂油纳米胶囊的水凝胶微针(GB-Tan-MNs-CIO@NCs)以及对照的未载药的空白水凝胶微针(GB-Tan-MNs)机械强度测试图谱；Fig. 3 is the hydrogel microneedle (GB-Tan-MNs-CIO@NCs) loaded with cinnamon oil nanocapsules prepared in Example 3 of the present invention and the blank hydrogel microneedle (GB-Tan- MNs) mechanical strength test pattern;
图4是本发明实施例3制备的载肉桂油纳米胶囊的水凝胶微针(GB-Tan-MNs-CIO@NCs)以及对照的未载药的空白水凝胶微针(GB-Tan-MNs)溶胀性测试图谱；Figure 4 shows the hydrogel microneedles (GB-Tan-MNs-CIO@NCs) loaded with cinnamon oil nanocapsules prepared in Example 3 of the present invention and the blank hydrogel microneedles (GB-Tan- MNs) swelling test collection;
图5是本发明实施例3制备的罗丹明B标记的空白微针贴片刺入大鼠离体皮肤光学照片；Fig. 5 is the optical photo of the rhodamine B-labeled blank microneedle patch pierced into rat isolated skin prepared in Example 3 of the present invention;
图6是本发明实施例4获得的大鼠神阙穴区肥大细胞甲苯胺蓝染色光镜照片，其中：(A，B)为空白组，C为模型组，D为针灸组，E为空白微针组，F为载药微针组。Fig. 6 is the toluidine blue stained light microscope photo of mast cells in rat Shenque acupoint area obtained in Example 4 of the present invention, wherein: (A, B) is a blank group, C is a model group, D is an acupuncture group, and E is a blank Microneedle group, F is drug-loaded microneedle group.
以下将结合具体实施例对本发明提供的技术方案进行详细说明，应理解下述具体实施方式仅用于说明本发明而不用于限制本发明的范围。The technical solutions provided by the present invention will be described in detail below in conjunction with specific examples. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
实施例1 肉桂油纳米胶囊的制备 Embodiment 1 Preparation of cinnamon oil nanocapsules
1.1肉桂油的提取1.1 Extraction of cinnamon oil
参照2020年版《中国药典》规定之肉桂药材，称取200.2g肉桂药材，粉碎成20目的粗粉，加8倍量的水，浸泡1h，200℃水蒸气蒸馏法提取6h，收集挥发油，得3mL肉桂挥发油。采用HPLC检测，肉桂挥发油中桂皮醛含量为93.1±4.45％，符合2020年版《中国药典》中关于肉桂油(Cinnamon Oil，CIO)的质量标准规定。Referring to the cinnamon medicinal material stipulated in the 2020 edition of "Chinese Pharmacopoeia", weigh 200.2g of cinnamon medicinal material, crush it into a 20-mesh coarse powder, add 8 times the amount of water, soak for 1h, extract by steam distillation at 200°C for 6h, and collect volatile oil to obtain 3mL Cinnamon essential oil. Using HPLC detection, the cinnamon aldehyde content in cinnamon volatile oil is 93.1±4.45%, which meets the quality standards for cinnamon oil (Cinnamon Oil, CIO) in the 2020 edition of "Chinese Pharmacopoeia".
1.2肉桂油纳米胶囊的制备1.2 Preparation of cinnamon oil nanocapsules
称取60mg海藻酸钠加纯化水至100mL配制成浓度为0.6mg·mL
-1的溶液，60℃加热并，300rpm搅拌30min至溶解，加1％醋酸水溶液调节pH至5.5，备用。
Weigh 60 mg of sodium alginate and add purified water to 100 mL to prepare a solution with a concentration of 0.6 mg mL -1 , heat and mix at 60 ° C, stir at 300 rpm for 30 min until dissolved, add 1% acetic acid aqueous solution to adjust the pH to 5.5, and set aside.
称取120mg肉桂油与24mg大豆卵磷脂(纯度98.6％)，加入2mL乙醇搅拌均匀。上述溶液边超声分散边加入上述海藻酸钠溶液中，继续超声15min，然后探头超声15min(冰浴)，得到蓝色乳光的O/W乳液。Weigh 120mg of cinnamon oil and 24mg of soybean lecithin (98.6% purity), add 2mL of ethanol and stir evenly. Add the above solution into the above sodium alginate solution while ultrasonically dispersing, continue ultrasonication for 15 minutes, and then probe ultrasonically for 15 minutes (ice bath), to obtain a blue opalescent O/W emulsion.
称取12mg氯化钙，加纯化水20mL配制成0.6mg·mL
-1的溶液。将氯化钙溶液以8.5ml·h
-1的速度边搅拌(1000rpm)边滴入上述O/W乳液中，滴加完毕以300rpm继续搅拌30min。
Weigh 12 mg of calcium chloride and add 20 mL of purified water to prepare a solution of 0.6 mg·mL -1 . The calcium chloride solution was added dropwise into the above O/W emulsion while stirring (1000rpm) at a speed of 8.5ml·h -1 , and stirring was continued at 300rpm for 30min after the dropwise addition.
称取6mg壳聚糖以10mL1％醋酸水溶液溶解，配制成浓度为0.6mg·mL
-1的溶液，以4％NaOH溶液调节pH至5.0。然后将壳聚糖溶液以10mL·h
-1的速度边搅拌(1000rpm)边滴入上述海藻酸钠溶液中，滴加完毕以300rpm继续搅拌3h，静置过夜，140,000×g离心60min，除去上清，沉淀加20mL纯化水分散，即得肉桂油纳米胶囊(CIO@NCs)，4℃贮存备 用。
Weigh 6 mg of chitosan and dissolve it in 10 mL of 1% acetic acid aqueous solution to prepare a solution with a concentration of 0.6 mg·mL -1 , and adjust the pH to 5.0 with 4% NaOH solution. Then the chitosan solution was dripped into the above-mentioned sodium alginate solution while stirring (1000rpm) at a speed of 10mL·h -1 , continued to stir at 300rpm for 3h after the dropwise addition, left to stand overnight, centrifuged at 140,000×g for 60min, and removed the above Then add 20 mL of purified water to the precipitate to disperse to obtain cinnamon oil nanocapsules (CIO@NCs), which are stored at 4°C for later use.
实施例2 载肉桂油纳米胶囊的水凝胶微针的制备Example 2 Preparation of hydrogel microneedles loaded with cinnamon oil nanocapsules
1.3载肉桂油纳米胶囊的水凝胶微针的制备1.3 Preparation of hydrogel microneedles loaded with cinnamon oil nanocapsules
称取4g明胶，加入15mL纯化水，于55℃加热，搅拌(300rpm)1h至全溶。称取160mg单宁酸，加入5mL纯化水，300rpm搅拌溶解。将单宁酸溶液缓慢滴加入明胶溶液中，以1％氢氧化钠溶液调节pH至5.5，于55℃加热反应4h。向明胶-单宁酸溶液中加入400mg甘油，搅拌均匀，得水凝胶微针基质。Weigh 4g of gelatin, add 15mL of purified water, heat at 55°C, and stir (300rpm) for 1h until completely dissolved. Weigh 160 mg of tannic acid, add 5 mL of purified water, and stir at 300 rpm to dissolve. The tannic acid solution was slowly added dropwise to the gelatin solution, the pH was adjusted to 5.5 with 1% sodium hydroxide solution, and the reaction was heated at 55°C for 4h. Add 400 mg of glycerin to the gelatin-tannic acid solution and stir evenly to obtain a hydrogel microneedle matrix.
取20mL肉桂油纳米胶囊溶液，与2mL水凝胶微针基质搅拌混匀，取1mL浇铸于洁净的微针模具中，-0.08mPa真空环境下放置15min，刮去上层未入模基质，同时补充新的基质溶液。于30℃干燥箱中干燥完全，脱模即得载肉桂油纳米胶囊的水凝胶微针(HFMNs-CIO@NCs)。Take 20mL of cinnamon oil nanocapsule solution, stir and mix with 2mL hydrogel microneedle matrix, take 1mL and cast it in a clean microneedle mold, place it in a vacuum environment of -0.08mPa for 15min, scrape off the upper layer of matrix that has not been molded, and add New matrix solution. After drying in a drying oven at 30°C, the hydrogel microneedles loaded with cinnamon oil nanocapsules (HFMNs-CIO@NCs) were obtained after demoulding.
实施例3 载肉桂油纳米胶囊的水凝胶微针(HFMNs-CIO@NCs)的评价与表征Example 3 Evaluation and characterization of hydrogel microneedles loaded with cinnamon oil nanocapsules (HFMNs-CIO@NCs)
3.1 HFMNs-CIO@NCs的形态表征3.1 Morphological characterization of HFMNs-CIO@NCs
取干燥后的HFMNs-CIO@NCs，表面均匀喷洒金粉，真空条件置于扫描电子显微镜下对其进行形态学观察。The dried HFMNs-CIO@NCs were taken, and the surface was evenly sprayed with gold powder, and placed under a scanning electron microscope under vacuum conditions for morphological observation.
图2是本发明实施例3制备的载肉桂油纳米胶囊的水凝胶微针扫描电镜图，其中，A放大倍数为100倍，B放大倍数为350倍。由图2可见，HFMNs-CIO@NCs脱模率达100％，脱模后针尖完整无破损，针体呈金字塔型，由四面锥型针体组成，基底厚度均一，阵列排列整齐。微针贴片为正方形，边长1.4cm，其中微针阵列为10×10，微针基底长度约为600μm，宽度约为600μm，微针高度约1450μm。微针基底间距为600μm，针尖间距为900μm。Fig. 2 is a scanning electron micrograph of the hydrogel microneedle of the cinnamon oil-loaded nanocapsule prepared in Example 3 of the present invention, wherein, the magnification of A is 100 times, and the magnification of B is 350 times. It can be seen from Figure 2 that the demolding rate of HFMNs-CIO@NCs is 100%. After demoulding, the needle tip is intact and undamaged. The needle body is pyramid-shaped and consists of four-sided cone-shaped needle bodies. The base thickness is uniform and the array is arranged neatly. The microneedle patch is a square with a side length of 1.4 cm. The microneedle array is 10×10. The length of the microneedle base is about 600 μm, the width is about 600 μm, and the height of the microneedles is about 1450 μm. The distance between the base of the microneedles is 600 μm, and the distance between the needle tips is 900 μm.
3.2 HFMNs-CIO@NCs的机械强度3.2 Mechanical strength of HFMNs-CIO@NCs
分别取干燥后的HFMNs-CIO@NCs，确保针体形态完好、基底平整均一。针体向上置于质构仪的测试板，设置直径为5mm的圆柱探头以30mm/min的速度压缩微针，直到挤压形变量为40％。初始触发力为0.05N，绘制微针的力—位移曲线。Take the dried HFMNs-CIO@NCs separately to ensure that the shape of the needle body is intact and the base is even and uniform. The needle body is placed upwards on the test plate of the texture analyzer, and a cylindrical probe with a diameter of 5 mm is set to compress the microneedle at a speed of 30 mm/min until the extrusion deformation is 40%. The initial trigger force was 0.05N, and the force-displacement curve of the microneedle was drawn.
图3是本发明实施例3制备的载肉桂油纳米胶囊的水凝胶微针机械强 度测试图谱，由图3可见，载药前后BSP复合微针的机械强度均可达18N以上。由于微针模具为11×11阵列，计算HFMNs和HFMNs-CIO@NCs的机械强度分别为0.15N/针、0.18N/针，即纳米胶囊的载入使微针强度略有增加。Fig. 3 is the hydrogel microneedle mechanical strength test spectrum of the cinnamon oil nanocapsules prepared in Example 3 of the present invention, as can be seen from Fig. 3, the mechanical strength of the BSP composite microneedle before and after drug loading can reach more than 18N. Since the microneedle mold is an array of 11×11, the calculated mechanical strengths of HFMNs and HFMNs-CIO@NCs are 0.15 N/needle and 0.18 N/needle, respectively, that is, the loading of nanocapsules slightly increases the strength of microneedles.
3.3 HFMNs-CIO@NCs的溶胀性3.3 Swellability of HFMNs-CIO@NCs
取空白微针贴片(HFMNs)与载药微针贴片(HFMNs-CIO@NCs)，记录初始质量W
L。依次用聚四氟乙烯膜和锡箔纸包裹不同处方的微针片，使针尖露出。在24孔板的每个孔内加入PBS 0.7mL，将包裹好的微针贴片倒置于孔内，使露出的针尖浸没在水中。于5、15、30、60、90min时将微针贴片从24孔板内取出，擦干表面多余水分后称定，将0时的质量记为W
0，t时记为W
t，以(W
t-W
0)/W
L表示微针贴片在t时的溶胀率，上述试验每组样品重复3次。分别以t为横坐标、溶胀率为纵坐标作图，考察载药对微针溶胀率的影响。
Take blank microneedle patches (HFMNs) and drug-loaded microneedle patches (HFMNs-CIO@NCs), and record the initial mass W L . The microneedle sheets with different prescriptions were wrapped with polytetrafluoroethylene film and tin foil in turn, so that the needle tip was exposed. Add 0.7 mL of PBS to each well of the 24-well plate, place the wrapped microneedle patch upside down in the well, and immerse the exposed needle tip in water. At 5, 15, 30, 60, and 90 minutes, the microneedle patch was taken out from the 24-well plate, and the excess water on the surface was wiped off, and then weighed. The mass at 0 was recorded as W 0 , and the mass at t was recorded as W t . (W t -W 0 )/W L represents the swelling rate of the microneedle patch at time t, and the above test was repeated 3 times for each group of samples. Take t as the abscissa and the swelling rate as the ordinate respectively to investigate the effect of drug loading on the swelling rate of microneedles.
图4是本发明实施例3制备的载肉桂油纳米胶囊的水凝胶微针溶胀性测试图谱，由图4可见，空白微针在30min时溶胀率达350％，之后溶胀速度逐渐减小，趋于平缓，在90min时溶胀率约430％。而微针载药后溶胀率略有升高，在30min时溶胀率即达390％，且在90min时达到470％，其溶胀程度以及溶胀速度均优于空白微针。Fig. 4 is the hydrogel microneedle swelling property test spectrum of the loaded cinnamon oil nanocapsule prepared by the embodiment of the present invention 3, as seen from Fig. 4, the swelling rate of blank microneedle reaches 350% in 30min, after that, the swelling rate gradually decreases, It tends to be gentle, and the swelling rate is about 430% at 90 minutes. However, the swelling rate of the microneedles increased slightly after loading the drug, reaching 390% at 30 minutes, and 470% at 90 minutes. The swelling degree and swelling speed were better than those of blank microneedles.
3.4微针皮肤刺入性3.4 Microneedle skin penetration
取SD大鼠，10％水合氯醛麻醉，用剃毛刀腹部脱毛，生理盐水擦洗，手术刀剥离腹部皮肤，剔除皮下组织和脂肪，生理盐水洗净，-4℃冰箱中保存，一周内使用。Take SD rats, anesthetized with 10% chloral hydrate, depilate the abdomen with a razor, scrub with normal saline, peel off the abdominal skin with a scalpel, remove subcutaneous tissue and fat, wash with normal saline, store in a -4°C refrigerator, and use within one week .
采用水溶性染料罗丹明B标记空白微针基质，制备罗丹明标记的微针。以手按压于大鼠离体皮肤表面，维持5min，取下微针贴片，观察微针刺入皮肤的能力。Rhodamine-labeled microneedles were prepared by labeling blank microneedle matrix with water-soluble dye rhodamine B. Press the surface of the isolated skin of the rat with your hands for 5 minutes, remove the microneedle patch, and observe the ability of the microneedle to penetrate the skin.
图5是本发明实施例3制备的罗丹明B标记的空白微针贴片刺入大鼠离体皮肤光学照片，由图5可见，微针可以成功刺入大鼠离体皮肤。Fig. 5 is an optical photograph of the rhodamine B-labeled blank microneedle patch prepared in Example 3 of the present invention piercing the isolated rat skin. It can be seen from Fig. 5 that the microneedle can successfully penetrate the isolated rat skin.
实施例4 载肉桂油纳米胶囊的水凝胶微针(HFMNs-CIO@NCs)对寒凝血瘀型痛经模型大鼠的镇痛效应研究Example 4 Study on the analgesic effect of hydrogel microneedles loaded with cinnamon oil nanocapsules (HFMNs-CIO@NCs) on model rats with cold coagulation and blood stasis type dysmenorrhea
4.1动物分组4.1 Animal grouping
雌性SD大鼠，体质量180～220g，为以下四组：(1)空白对照组，(2)模型组，(3)脐部针灸组，(4)空白微针脐部治疗组，(5)载肉桂油纳米胶囊微针脐部刺治疗组。除空白对照组外，模型组与各治疗组在造模成功后再分组。Female SD rats, with a body weight of 180-220 g, were divided into the following four groups: (1) blank control group, (2) model group, (3) umbilicus acupuncture group, (4) blank microneedle umbilicus treatment group, (5 ) containing cinnamon oil nanocapsule microneedle umbilical acupuncture treatment group. Except for the blank control group, the model group and each treatment group were divided into groups after successful modeling.
4.2寒凝血瘀型痛经模型的制备4.2 Preparation of dysmenorrhea model of cold coagulation and blood stasis type
按照文献方法(中华中医药学会.痛经动物模型制备规范(草案)[J].中国实验方剂学杂志，2018)建立寒凝血瘀型痛经大鼠模型。连续给予动物苯甲酸雌二醇，提高子宫敏感性；再注射缩宫素，产生类似痛经反应。冰水浴可致动物出现体温降低。According to the literature method (China Association of Traditional Chinese Medicine. Dysmenorrhea Animal Model Preparation Specification (Draft) [J]. Chinese Journal of Experimental Formulas, 2018), a rat model of dysmenorrhea of cold coagulation and blood stasis type was established. Continuous administration of estradiol benzoate to animals increased uterine sensitivity; re-injection of oxytocin produced a similar dysmenorrhea response. Ice-water baths can cause hypothermia in animals.
模型组及治疗组大鼠皮下注射苯甲酸雌二醇，每天1次，连续10d；第1，10天剂量为2.5mg·kg
-1，其余时间的剂量均为1mg·kg
-1；同时每天将大鼠置于冰水浴中10min，为促进血瘀模型的形成，可在第11天给大鼠皮下注射2次盐酸肾上腺素(8μg·kg
-1)，间隔2h，在第2次注射盐酸肾上腺素后，将动物置于冰水中5min，5min后将大鼠拿出放回笼子中饲养。第12天大鼠皮下注射苯甲酸雌二醇1h后，立即腹腔注射缩宫素(剂量10U·kg
-1)，制备成雌激素与缩宫素联用冰水浴大鼠痛经模型。观察大鼠扭体反应行为，判断造模成功。
Rats in model group and treatment group were subcutaneously injected with estradiol benzoate, once a day, for 10 consecutive days; the dose was 2.5 mg·kg -1 on the first and 10th day, and 1 mg·kg -1 in the rest of the time; Put the rats in an ice-water bath for 10 minutes. In order to promote the formation of the blood stasis model, on the 11th day, subcutaneously inject epinephrine hydrochloride (8 μg kg -1 ) into the rats twice, with an interval of 2 hours, and inject hydrochloric acid for the second time After epinephrine, the animals were placed in ice water for 5 minutes, and after 5 minutes, the rats were taken out and put back into the cages for feeding. On the 12th day, 1 hour after the subcutaneous injection of estradiol benzoate, the rats were immediately intraperitoneally injected with oxytocin (dose 10U·kg -1 ) to prepare a rat model of dysmenorrhea in combination with estrogen and oxytocin in ice water bath. Observe the writhing reaction behavior of rats to judge the success of modeling.
空白对照组正常饲食，自由饮水，不做任何治疗，于造模第1～10天，每天给予股部皮下注射生理盐水，第1天0.25mL/只，第2～9天0.1mL/只，第10天0.25mL/只，第11天皮下注射2次，每次0.1mL/只，间隔2h。第12天皮下注射生理盐水0.25mL/只，1h后，立即腹腔注射缩宫素(剂量10U·kg
-1)，观察大鼠扭体反应行为。
The blank control group was given normal diet, free drinking water, and no treatment. On the 1st to 10th day of modeling, subcutaneous injection of normal saline was given to the thigh every day. , 0.25mL/monkey on the 10th day, subcutaneous injection twice on the 11th day, 0.1mL/bird each time, with an interval of 2h. On the 12th day, 0.25 mL of normal saline was injected subcutaneously, and 1 hour later, oxytocin (dose 10 U·kg -1 ) was injected intraperitoneally immediately, and the writhing response behavior of the rats was observed.
4.3取穴方法4.3 Point selection method
按照文献方法(纪峰.成年雌性大鼠“神阙”穴定位方法探析[C].第十七届针灸对机体功能的调节机制及针灸临床独特经验研讨会会议论文集,2014)采用“比较解剖取穴法”取大鼠神阙穴：下肢自然松弛和部分拉伸状态下，大鼠“神阙”穴位于第4,5对乳头中点连线约上1/3处。下肢完全拉伸的情况，“神阙”穴基本上与第4对乳头平行。According to the literature method (Ji Feng. Analysis of the location method of "Shenque" point in adult female rats [C]. Proceedings of the 17th Symposium on the Regulation Mechanism of Acupuncture and Moxibustion on Body Function and the Unique Experience of Acupuncture and Moxibustion, 2014) the method of "comparison" was adopted. "Anatomical acupoint selection method" select rat Shenque acupoint: in the state of natural relaxation and partial stretching of the lower limbs, the rat "Shenque" acupoint is located at about the upper 1/3 of the line connecting the midpoints of the 4th and 5th pair of nipples. When the lower limbs are fully stretched, the "Shenque" point is basically parallel to the fourth pair of nipples.
4.4治疗方法4.4 Treatment methods
于造模第5天给予大鼠吸入异氟烷麻醉，大鼠麻醉后用电动剃毛刀剔 除大鼠神阙穴周围粗毛，然后以脱毛膏除去神阙穴周围细毛。于造模第6天给予大鼠治疗，每日治疗1次，共治疗7次。On the 5th day of modeling, the rats were anesthetized by inhalation of isoflurane. After the rats were anesthetized, the coarse hairs around the Shenque acupoint were removed with an electric shaver, and then the fine hairs around the Shenque acupoint were removed with a depilatory cream. Rats were given treatment on the 6th day after modeling, once a day for a total of 7 treatments.
4.4.1皮内法4.4.1 Intradermal method
大鼠仰卧固定后，把神阙穴看做表盘，采用皮内针，取6点位，向相应的穴区方位横刺，用医用胶布固定，留针30分钟，进针深度4～5mm。After the rats were fixed in a supine position, the Shenque acupoint was regarded as the dial, and the intradermal needle was used to take the 6 o’clock position and stab horizontally towards the corresponding acupoint area. The needle was fixed with medical adhesive tape, and the needle was retained for 30 minutes at a depth of 4-5 mm.
4.4.2微针法4.4.2 Microneedling
将大鼠仰卧固定，将空白或载药微针置于大鼠神阙穴穴区，以手按压1min，使微针刺入大鼠神阙穴，以医用胶布固定，留针30min。The rats were fixed in a supine position, and the blank or drug-loaded microneedles were placed in the Shenque acupoint area of the rats, and pressed with hands for 1 min, so that the microneedles penetrated into the Shenque acupoints of the rats, fixed with medical adhesive tape, and the needles were left for 30 min.
空白组与模型组与上述各组做同步抓取、固定，不做治疗。The blank group and the model group were grasped and fixed synchronously with the above-mentioned groups, and no treatment was given.
4.5指标检测4.5 Index detection
4.5.1大鼠状态变化4.5.1 Rat state changes
记录大鼠饮食、体重，观察大鼠毛发色泽，动作行为，精神状态。Record the diet and body weight of the rats, observe the hair color, behavior and mental state of the rats.
造模过程中，模型组体重维持稳定或逐渐减轻，后期出现蜷缩少动，喜扎堆，偶有寒战，朦胧欲睡，两眼无神，反应迟钝，被毛蓬松竖立无光泽，耳部、四肢、尾部等部位肤色呈现粉紫红，大便湿润，粪质变软。根据杨佳敏(寒凝血瘀证动物模型制备方法的评价与选择[J].中医学报，2014)，造模大鼠寒症表征评分为中度。各治疗组大鼠前期造模过程中状态与模型组相同，后期治疗过程中症状有所改善，体重维持稳定或逐渐增长，精神好转，毛发转亮，耳部、四肢、尾部等部位肤色呈现粉红，大便稍湿润，粪质稍变软。During the modeling process, the weight of the model group remained stable or gradually decreased. In the later stage, they curled up and moved less, liked to get together, occasionally shivered, drowsy, dull eyes, unresponsive, fluffy and dull hair, ears and limbs The color of the body, tail and other parts is pink and purple, the stool is moist, and the feces become soft. According to Yang Jiamin (Evaluation and Selection of Animal Model Preparation Methods for Cold Condensation and Blood Stasis Syndrome [J]. Chinese Medical Journal, 2014), the cold symptoms of model rats were scored as moderate. The state of the rats in each treatment group was the same as that of the model group in the early stage of modeling. During the later stage of treatment, the symptoms improved, the weight remained stable or gradually increased, the spirit improved, the hair became brighter, and the skin color of the ears, limbs, tail and other parts appeared pink. , the stool is slightly moist, and the fecal matter is slightly soft.
4.5.2扭体反应4.5.2 Writhing Response
观察空白组、模型组腹腔注射缩宫素20min内大鼠出现扭体反应的潜伏期、扭体次数及扭体评分。针灸组、空白微针脐部治疗组及载肉桂油纳米胶囊微针脐部治疗组最后一次治疗后，腹腔注射缩宫素，观察20min内每组大鼠出现扭体反应的潜伏期、扭体次数及扭体评分。Observe the latency, times of writhing and writhing scores of the rats in the blank group and model group within 20 minutes of intraperitoneal injection of oxytocin. After the last treatment in the acupuncture group, the blank microneedle treatment group and the cinnamon oil nanocapsule microneedle treatment group, oxytocin was injected intraperitoneally, and the latency and the number of writhing reactions in rats in each group were observed within 20 minutes and writhing scores.
根据Schmauss(In vivo studies on spinal opiate receptor systemsmediating antinociceptionⅡpharmacological profiles suggesting a differentialassociation of mu，delta and kappa receptors with visceral chemical andcutaneous thermal stimuli inthe rat[J].J Pharmacol Exp Ther，l984)提出的行为学评分标准，将扭体行为分为0～3四个等级。0级：正常姿势(手爪 平放盒底或正常探查行为)；1级：身体斜向一边；2级：后肢伸展，后爪背屈，躯体伸展伴频繁地盆骨侧向旋转；3级：腹部肌肉收缩，后肢后伸。扭体潜伏期为注射缩宫素后至出现扭体反应的时间；扭体评分＝0级(次数)×0分+1级(次数)×1分+2级(次数)×2分+3级(次数)×3分。结果如表1所示。According to Schmauss (In vivo studies on spinal opiate receptor systems mediating antinociceptionⅡpharmacological profiles suggesting a differential association of mu, delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat[J ].J Pharmacol Exp Ther, l984) proposed behavioral scoring standard, will Writhing behavior is divided into four grades from 0 to 3. Grade 0: normal posture (paw flat on bottom of box or normal exploratory behavior); Grade 1: body tilted to one side; Grade 2: hindlimb extension, hind paw dorsiflexion, body extension with frequent lateral pelvic rotation; grade 3 : contraction of abdominal muscles, extension of hind limbs. The writhing latency period is the time from the injection of oxytocin to the writhing reaction; writhing score = grade 0 (times) x 0 points + grade 1 (times) x 1 point + grade 2 (times) x 2 points + grade 3 (number of times) × 3 points. The results are shown in Table 1.
与空白组相比，模型组大鼠扭体反应潜伏期明显缩短，扭体次数及扭体评分显著性增加，表明寒凝血瘀型大鼠痛经模型造模成功。与模型组相比，脐部针灸治疗组扭体次数显著减少(P﹤0.05)，空白微针脐部治疗组扭体次数及扭体评分显著降低(P﹤0.05)，载肉桂油纳米胶囊的微针脐部治疗组扭体潜伏期显著延长(P﹤0.05)，扭体次数及扭体评极显著性降低(P﹤0.01)，表明各治疗组对寒凝血瘀型痛经模型大鼠均具有一定的治疗作用，其中，载肉桂油纳米胶囊的微针脐部治疗组治疗效果最显著。Compared with the blank group, the writhing reaction latency of rats in the model group was significantly shortened, and the number of writhing times and writhing scores were significantly increased, indicating that the model of dysmenorrhea rats with cold coagulation and blood stasis was successfully established. Compared with the model group, the number of writhing times in the umbilical acupuncture treatment group was significantly reduced (P﹤0.05), and the number of writhing times and writhing scores in the blank microneedle umbilicus treatment group were significantly reduced (P﹤0.05). The writhing latency period of the microneedle umbilicus treatment group was significantly prolonged (P﹤0.05), and the number of writhing times and writhing ratings were significantly reduced (P﹤0.01), indicating that each treatment group had a certain effect on the dysmenorrhea model rats of cold coagulation and blood stasis type. Among them, the treatment effect of the microneedle umbilicus treatment group loaded with cinnamon oil nanocapsules was the most significant.
表1各组大鼠扭体反应比较(n＝6)Table 1 Comparison of writhing responses of rats in each group (n=6)
注：与空白组比较，#P<0.05，##P<0.01；与模型组比较，*P﹤0.05，**P﹤0.01。Note: Compared with blank group, #P<0.05, ##P<0.01; compared with model group, *P﹤0.05, **P﹤0.01.
4.5.3 ELISA法测定大鼠子宫组织内前列腺素E2(PGE2)、前列腺素F2α(PGF2α)含量4.5.3 Determination of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) content in rat uterine tissue by ELISA
注射缩宫素1h后，大鼠腹腔注射3％戊巴比妥钠1.0mL(150mg/kg)麻醉，于无菌工作台冰盘上迅速摘取子宫，称重，样品置于-80℃冰箱保存待用。One hour after the injection of oxytocin, the rat was anesthetized by intraperitoneal injection of 3% pentobarbital sodium 1.0mL (150mg/kg), and the uterus was quickly removed on the ice tray of the sterile workbench, weighed, and the sample was placed in a -80°C refrigerator Save for later use.
临用前将子宫样品取出，室温下解冻。取适量样品加生理盐水制成10％的子宫组织匀浆，以3000r/min低温离心15min，取上清液，按照ELISA试剂盒说明书方法测定PGE2、PGF2α含量。结果如表2所示。Uterine samples were taken out before use and thawed at room temperature. Take an appropriate amount of sample and add physiological saline to make 10% uterine tissue homogenate, centrifuge at 3000r/min for 15min at low temperature, take the supernatant, and measure the content of PGE2 and PGF2α according to the ELISA kit instructions. The results are shown in Table 2.
与空白组相比，模型组PGF2α含量极显著性升高(P﹤0.01)， PGF2α/PGE2比值显著性增大(P﹤0.05)，表明寒凝血瘀型大鼠痛经模型造模成功。与模型组相比，脐部针灸治疗组与空白微针脐部治疗组PGF2α含量极显著性减少(P﹤0.01)，PGF2α/PGE2比值显著性减小(P﹤0.05)，载肉桂油纳米胶囊的微针脐部治疗组PGE2含量极显著性增加(P﹤0.01)，PGF2α含量及PGF2α/PGE2比值显著性减小(P﹤0.05)，表明各治疗组对低寒凝血瘀型痛经模型大鼠均有一定的治疗作用，其中载肉桂油纳米胶囊的微针脐部治疗组治疗效果最显著。Compared with the blank group, the content of PGF2α in the model group was significantly increased (P﹤0.01), and the ratio of PGF2α/PGE2 was significantly increased (P﹤0.05), indicating that the model of dysmenorrhea rats with cold coagulation and blood stasis was successfully established. Compared with the model group, the PGF2α content in the umbilical acupuncture treatment group and the blank microneedle umbilicus treatment group significantly decreased (P﹤0.01), and the ratio of PGF2α/PGE2 decreased significantly (P﹤0.05). The content of PGE2 in the microneedling umbilicus treatment group was significantly increased (P﹤0.01), and the content of PGF2α and the ratio of PGF2α/PGE2 were significantly reduced (P﹤0.05), indicating that each treatment group had a significant effect on the treatment of dysmenorrhea model rats with low cold blood stasis type. All have a certain therapeutic effect, and the treatment effect of the microneedle umbilicus treatment group loaded with cinnamon oil nanocapsules is the most significant.
表2各组大鼠子宫PGE2、PGF2α含量及PGF2α/PGE2比值比较(n＝6)Table 2 Comparison of PGE2, PGF2α content and PGF2α/PGE2 ratio in the uterus of rats in each group (n=6)
注：与对照组比较，#P<0.05，##P<0.01；与模型组比较，*P﹤0.05，**P﹤0.01。Note: Compared with the control group, #P<0.05, ##P<0.01; compared with the model group, *P﹤0.05, **P﹤0.01.
4.5.4穴区肥大细胞数目、脱颗粒率4.5.4 Number of mast cells and degranulation rate in acupoint area
注射缩宫素1h后，大鼠腹腔注射3％戊巴比妥钠1.0mL(150mg/kg)麻醉，于无菌工作台冰盘上迅速取大鼠神阙穴区皮肤连同皮下肌肉组织(3mm×3mm×3mm)，生理盐水冲洗，以4％多聚甲醛固定，用于甲苯胺蓝染色，在10×40倍光镜下拍摄照片，结果如图6所示。每只大鼠穴区取3张组织切片，随机选取3个视野，肌层1个视野、筋膜层2个视野，于10×20倍光镜下观察切片肥大细胞形态并计数(包括完整的和脱颗粒的)，计数结果以“个数/高倍视野(个/HPF)”表示，结果如表3所示。One hour after the injection of oxytocin, the rats were anesthetized by intraperitoneal injection of 1.0 mL (150 mg/kg) of 3% pentobarbital sodium, and the skin of the Shenque acupoint area of the rat and the subcutaneous muscle tissue (3mm ×3mm×3mm), washed with normal saline, fixed with 4% paraformaldehyde, used for toluidine blue staining, and photographed under a 10×40 light microscope, the results are shown in Figure 6. Three tissue slices were taken from the acupoint area of each rat, and three visual fields were randomly selected, one visual field in the muscular layer and two visual fields in the fascia layer, and the morphology of mast cells in the slices was observed and counted under a light microscope of 10×20 times (including intact and degranulated), the counting results are expressed in "number/high power field (number/HPF)", and the results are shown in Table 3.
表3大鼠神阙穴区肥大细胞计数、脱颗粒率比较(n＝6)Table 3 Comparison of mast cell count and degranulation rate in Shenque acupoint area of rats (n=6)
注：与对照组比较，#P<0.05，##P<0.01；与模型组比较，*P﹤0.05，**P﹤0.01。Note: Compared with the control group, #P<0.05, ##P<0.01; compared with the model group, *P﹤0.05, **P﹤0.01.
图6为本发明实施例4所得大鼠神阙穴区肥大细胞甲苯胺蓝染色光镜照片，其中：A、B.空白组C.模型组，D.针灸组E.空白微针组F.载药微针组；由图6与表3可知，与空白组相比，模型组大鼠神阙穴区肥大细胞个数与肥大细胞脱颗粒率明显增多，具有统计学差异(P<0.05)。与模型组比较，各治疗组神阙穴区肥大细胞个数与肥大细胞脱颗粒率极显著性增多(P<0.01)。Figure 6 is a light microscope photograph of mast cells stained with toluidine blue in the rat Shenque acupoint area obtained in Example 4 of the present invention, wherein: A, B. Blank group C. Model group, D. Acupuncture group E. Blank microneedle group F. Drug-loaded microneedle group; as can be seen from Figure 6 and Table 3, compared with the blank group, the number of mast cells and the mast cell degranulation rate in the Shenque acupoint area of the rats in the model group were significantly increased, with a statistical difference (P<0.05) . Compared with the model group, the number of mast cells and the degranulation rate of mast cells in the Shenque acupoint area in each treatment group were significantly increased (P<0.01).
由上述可见，本发明提供的一种针药一体化治疗策略可将药物递送至穴区皮肤深层，发挥其在穴区的治疗作用，同时微针针体吸水溶胀，能够刺激穴区组织，激发穴区效应，达到治疗目的。肉桂油纳米胶囊水凝胶微针脐部给药可以改善寒凝血瘀型痛经模型大鼠的症状，延长扭体潜伏期，减少扭体次数及扭体评分，显著升高子宫组织PGE2含量，降低PGF2α含量及PGF2α/PGE2比值，显著增加神阙穴区肥大细胞个数与肥大细胞脱颗粒率，说明肉桂油纳米胶囊水凝胶微针脐部给药可以发挥经穴针刺效应，对改善寒凝血瘀型痛经的症状具有重要作用。该针药一体化治疗策略可望利用微针包载游离药及其制剂，进一步地用于穴位疗法的临床治疗，值得临床推广使用。It can be seen from the above that the acupuncture-medicine integrated treatment strategy provided by the present invention can deliver the drug to the deep layer of the skin in the acupoint area, exert its therapeutic effect in the acupoint area, and at the same time, the microneedle body absorbs water and swells, which can stimulate the tissue in the acupoint area and stimulate Acupoint effect to achieve the purpose of treatment. Administration of cinnamon oil nanocapsules, hydrogel, and microneedles to the umbilicus can improve the symptoms of dysmenorrhea model rats of cold coagulation and blood stasis type, prolong the writhing latency period, reduce the number of writhing times and writhing scores, significantly increase the content of PGE2 in uterine tissue, and reduce PGF2α The content and the ratio of PGF2α/PGE2 significantly increased the number of mast cells and the degranulation rate of mast cells in the Shenque acupoint area, indicating that the administration of cinnamon oil nanocapsules hydrogel microneedles to the umbilical region can exert the effect of acupuncture at acupoints and improve the effect of cold coagulation. The symptoms of blood stasis type dysmenorrhea play an important role. The integrated treatment strategy of acupuncture and medicine is expected to use microneedles to carry free medicine and its preparations, which can be further used in the clinical treatment of acupoint therapy, and it is worthy of clinical promotion and use.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.
Claims (20)
- 一种针药一体化水凝胶微针，其特征在于，包括微针基质和包载于所述微针基质内的药物，所述微针基质为明胶与单宁酸交联产物形成的水凝胶。An acupuncture-medicine integrated hydrogel microneedle, characterized in that it includes a microneedle matrix and a drug contained in the microneedle matrix, and the microneedle matrix is a water gel formed by a cross-linked product of gelatin and tannic acid. gel.
- 根据权利要求1所述的针药一体化水凝胶微针，其特征在于，所述药物为纳米胶囊。The acupuncture-medicine integrated hydrogel microneedle according to claim 1, wherein the medicine is a nanocapsule.
- 根据权利要求2所述的针药一体化水凝胶微针，其特征在于，所述的药物为肉桂油纳米胶囊，所述肉桂油纳米胶囊的粒径为50～500nm，所述肉桂油纳米胶囊中桂皮醛的载药量为26wt％～36wt％。The acupuncture-medicine integrated hydrogel microneedle according to claim 2, wherein the drug is cinnamon oil nanocapsules, the particle diameter of the cinnamon oil nanocapsules is 50-500nm, and the cinnamon oil nanocapsules The drug loading amount of cinnamaldehyde in the capsule is 26wt%-36wt%.
- 根据权利要求3所述的针药一体化水凝胶微针，其特征在于，所述肉桂油纳米胶囊中，肉桂油包载于壳聚糖与海藻酸钠形成的壳-核结构中。The acupuncture-medicine integrated hydrogel microneedle according to claim 3, characterized in that, in the cinnamon oil nanocapsules, cinnamon oil is contained in a shell-core structure formed of chitosan and sodium alginate.
- 根据权利要求3所述的针药一体化水凝胶微针，其特征在于，所述微针基质与肉桂油纳米胶囊的质量比为0.2～0.5:1。The acupuncture-medicine integrated hydrogel microneedle according to claim 3, characterized in that the mass ratio of the microneedle matrix to the cinnamon oil nanocapsule is 0.2-0.5:1.
- 根据权利要求1、2或5所述的针药一体化水凝胶微针，其特征在于，所述的微针基质的制备方法包括以下步骤：The acupuncture-medicine integrated hydrogel microneedle according to claim 1, 2 or 5, wherein the preparation method of the microneedle matrix comprises the following steps:(1)将单宁酸溶液缓慢滴加到明胶溶液中，调节pH至5～6，45～65℃反应2～16h；(1) Slowly add the tannic acid solution dropwise to the gelatin solution, adjust the pH to 5-6, and react at 45-65°C for 2-16 hours;(2)反应液中加入甘油并搅拌均匀。(2) Glycerin was added to the reaction liquid and stirred evenly.
- 根据权利要求6所述的针药一体化水凝胶微针，其特征在于，所述单宁酸、甘油和明胶的质量比为0.02～0.05:0.05～0.2:1；所述的明胶溶液浓度为100～200mg/mL，所述的单宁酸溶液浓度为40～80mg/mL。The acupuncture-medicine integrated hydrogel microneedle according to claim 6, characterized in that the mass ratio of the tannic acid, glycerin and gelatin is 0.02-0.05:0.05-0.2:1; the concentration of the gelatin solution 100-200 mg/mL, and the concentration of the tannic acid solution is 40-80 mg/mL.
- 根据权利要求3所述的针药一体化水凝胶微针，其特征在于，所述肉桂油纳米胶囊为利用阳离子诱导的海藻酸盐的可控凝胶作用制备而得。The acupuncture-medicine integrated hydrogel microneedle according to claim 3, characterized in that the cinnamon oil nanocapsules are prepared by utilizing the controllable gelation of alginate induced by cations.
- 根据权利要求3所述的针药一体化水凝胶微针，其特征在于，所述肉桂油纳米胶囊以海藻酸钠与钙离子交联产物作为纳米胶囊外壳，以肉桂油作为内核，通过离子凝胶法制备，并以壳聚糖修饰。The acupuncture-medicine integrated hydrogel microneedle according to claim 3, wherein the cinnamon oil nanocapsule uses sodium alginate and calcium ion cross-linked product as the nanocapsule shell, uses cinnamon oil as the core, and passes through the cinnamon oil nanocapsule. Prepared by gel method and modified with chitosan.
- 根据权利要求3或9所述的针药一体化水凝胶微针，其特征在于，所述肉桂油纳米胶囊的制备方法包括以下步骤：The acupuncture-medicine integrated hydrogel microneedle according to claim 3 or 9, wherein the preparation method of the cinnamon oil nanocapsules comprises the following steps:(a)将肉桂油与磷脂溶解于醇，加入到海藻酸钠溶液中，超声分散，得到O/W乳液；(a) dissolving cinnamon oil and phospholipids in alcohol, adding them to sodium alginate solution, and ultrasonically dispersing to obtain an O/W emulsion;(b)氯化钙溶液滴加到步骤(a)的O/W乳液中，搅拌15～60min；(b) Add the calcium chloride solution dropwise to the O/W emulsion in step (a), and stir for 15 to 60 minutes;(c)步骤(b)的溶液中滴加壳聚糖溶液，滴加后继续搅拌，静置；(c) Add chitosan solution dropwise in the solution of step (b), continue to stir after dropping, leave standstill;(d)分离取沉淀即为肉桂油纳米胶囊。(d) Separation and precipitation are cinnamon oil nanocapsules.
- 根据权利要求10所述的针药一体化水凝胶微针，其特征在于，所述的磷脂为蛋黄卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷脂酰胆碱或二棕榈酰磷脂酰胆碱。The acupuncture-medicine integrated hydrogel microneedle according to claim 10, wherein the phospholipids are egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine, dimeat Myristoylphosphatidylcholine or dipalmitoylphosphatidylcholine.
- 根据权利要求10所述的针药一体化水凝胶微针，其特征在于，所述的醇为乙醇。The acupuncture-medicine integrated hydrogel microneedle according to claim 10, wherein the alcohol is ethanol.
- 根据权利要求10所述的针药一体化水凝胶微针，其特征在于，所述的肉桂油与磷脂、氯化钙和壳聚糖的重量比为4～6:1:0.4～0.6:0.2～0.4，步骤(a)所述的海藻酸钠溶液浓度为0.4～0.8mg/mL，肉桂油与醇的用量比为40～100mg/mL；步骤(b)所述的氯化钙溶液浓度为0.4～1mg/mL；步骤(c)所述的壳聚糖溶液浓度为0.4～1mg/mL。The acupuncture-medicine integrated hydrogel microneedle according to claim 10, wherein the weight ratio of the cinnamon oil to the phospholipid, calcium chloride and chitosan is 4～6:1:0.4～0.6: 0.2～0.4, the sodium alginate solution concentration described in step (a) is 0.4～0.8mg/mL, and the consumption ratio of cinnamon oil and alcohol is 40～100mg/mL; The calcium chloride solution concentration described in step (b) 0.4～1mg/mL; the chitosan solution concentration described in step (c) is 0.4～1mg/mL.
- 根据权利要求10所述的针药一体化水凝胶微针，其特征在于，所述步骤(a)为：所述海藻酸钠在纯水中以200～600rmp搅拌至溶解，制得海藻酸钠溶液；所述肉桂油与磷脂的醇溶液边超声边加入到海藻酸钠溶液中，混合后继续超声10～20min，再于冰浴条件下探头超声10～20min，得到O/W乳液。The acupuncture-medicine integrated hydrogel microneedle according to claim 10, characterized in that the step (a) is: stirring the sodium alginate in pure water at 200-600 rpm until dissolved to obtain alginic acid Sodium solution: the alcohol solution of cinnamon oil and phospholipids is added to the sodium alginate solution while ultrasonicating, after mixing, ultrasonication is continued for 10-20 minutes, and the probe is ultrasonicated for 10-20 minutes in an ice bath to obtain an O/W emulsion.
- 根据权利要求10所述的针药一体化水凝胶微针，其特征在于，所述步骤(b)为：将所述氯化钙溶液以6～10mL/h的速度滴加到步骤(a)的O/W乳液中，滴加完毕后，200～500rpm继续搅拌20～40min。The acupuncture-medicine integrated hydrogel microneedle according to claim 10, characterized in that the step (b) is: adding the calcium chloride solution dropwise to the step (a) at a rate of 6-10 mL/h. ) O/W emulsion, after the dropwise addition, continue to stir at 200-500rpm for 20-40min.
- 根据权利要求10所述的针药一体化水凝胶微针，其特征在于，所述步骤(c)为：将壳聚糖用0.5％～2％醋酸水溶液溶解，并调节pH至4.5～5.5，得到壳聚糖溶液；所述壳聚糖溶液以8～15mL/h的速度边搅拌边加入到步骤(b)的溶液中，滴加完毕后，200～500rpm继续搅拌2～6h，静置8～16h。The acupuncture-medicine integrated hydrogel microneedle according to claim 10, wherein the step (c) is: dissolving chitosan with 0.5%-2% acetic acid aqueous solution, and adjusting the pH to 4.5-5.5 , to obtain a chitosan solution; the chitosan solution is added to the solution of step (b) while stirring at a speed of 8 to 15 mL/h, after the dropwise addition, continue to stir at 200 to 500 rpm for 2 to 6 hours, and let it stand 8～16h.
- 根据权利要求10所述的针药一体化水凝胶微针，其特征在于，所述步骤(d)为：100000～150000×g离心45～120min，取沉淀。The acupuncture-medicine integrated hydrogel microneedle according to claim 10, characterized in that the step (d) is: centrifuge at 100,000-150,000×g for 45-120 min, and collect the precipitate.
- 权利要求1～17任一项所述针药一体化水凝胶微针的制备方法，其特征在于，步骤包括：将药物溶液与微针基质溶液混匀后成型并干燥。The preparation method of acupuncture-medicine integrated hydrogel microneedles according to any one of claims 1 to 17, characterized in that the steps include: mixing the drug solution and the microneedle matrix solution, forming and drying.
- 根据权利要求18所述的制备方法，其特征在于，所述制备方法为： 肉桂油纳米胶囊溶液与微针基质溶液混合均匀，再浇铸于微针模具内，经减压入模、真空干燥后脱模制成。The preparation method according to claim 18, characterized in that, the preparation method is: mixing the cinnamon oil nanocapsule solution and the microneedle matrix solution evenly, and then casting it in a microneedle mold, putting it into the mold under reduced pressure, and vacuum drying Released from the mold.
- 权利要求3～17任一项所述的针药一体化水凝胶微针在制备治疗寒凝血瘀型痛经的药物方面的应用。The application of the acupuncture-medicine integrated hydrogel microneedle according to any one of claims 3 to 17 in the preparation of medicine for treating dysmenorrhea of cold coagulation and blood stasis type.
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- 2022-08-23 WO PCT/CN2022/114243 patent/WO2023098158A1/en unknown
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| CN108245481A (en) * | 2018-01-12 | 2018-07-06 | 南京工业大学 | Micropin and microneedle patch |
| US20210322743A1 (en) * | 2020-04-20 | 2021-10-21 | Indian Institute Of Technology Bombay | Microneedle array patch for drug delivery and production method thereof |
| CN114146048A (en) * | 2021-12-03 | 2022-03-08 | 上海中医药大学 | Needle and medicine integrated hydrogel microneedle |
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| ZHANG, YONGTAI: "A Therapeutic Strategy of Combining Acupuncture and Drug Delivery at the Same Acupuncture Point-Taking Umbilical Therapy as an Example", CHINA'S NATUROPATHY, vol. 28, no. 5, 31 March 2020 (2020-03-31), XP009546872 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116650477A (en) * | 2023-06-14 | 2023-08-29 | 黑龙江迪龙制药有限公司 | Pharmaceutical composition containing ozagrel sodium for resisting platelet aggregation and preparation method thereof |
| CN116650477B (en) * | 2023-06-14 | 2023-11-10 | 黑龙江迪龙制药有限公司 | Pharmaceutical composition containing ozagrel sodium for resisting platelet aggregation and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114146048B (en) | 2023-03-14 |
| CN114146048A (en) | 2022-03-08 |
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