Preparation method and application of soluble microneedle array patch capable of adjusting oral mucosa flora

Abstract

The invention discloses a preparation method and application of a soluble microneedle array patch capable of adjusting oral mucosa flora, and belongs to the technical field of biological medicines.The soluble microneedle array patch is composed of a microneedle substrate and a soluble microneedle body, and the microneedle substrate and the soluble microneedle body are made of biocompatible materials; the composition contains isomaltooligosacharide, chitosan, hyaluronic acid, pvp k-90, an EcN coated preparation and mint powder. The microneedle is prepared by dissolving the auxiliary materials chitosan and hyaluronic acid at a specific pH value, adding the prebiotics and the EcN coating preparation, feeding into a mold, and drying. According to the effects of promoting probiotic proliferation and enhancing wound repair of prebiotics and the effect of realizing physical isolation of the auxiliary material chitosan and hyaluronic acid, the wound flora is regulated, so that the wound healing effect of treating difficult-to-heal oral ulcer caused by radiotherapy and chemotherapy is achieved; a new thought is provided for treating diseases such as difficult-to-heal oral ulceration caused by radiotherapy and chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing a microneedle array patch that can be used to repair wounds of oral diseases such as refractory oral ulcers caused by radiotherapy and chemotherapy, and its application in treating oral ulcers. Background Art

[0002] Escherichia coli Nissle 1917 (EcN) is a widely studied probiotic that plays the following roles in the oral cavity: First, it promotes the growth of healthy oral flora by producing beneficial metabolites, such as short-chain fatty acids, thereby helping to maintain the balance of the oral microecology. Second, EcN reduces the colonization and growth of pathogens in the oral cavity through a competitive inhibition mechanism, reducing the risk of oral diseases. Furthermore, the metabolic activity of EcN promotes the release of nutrients in the oral cavity, providing necessary nutrients for beneficial microorganisms and promoting their proliferation. Finally, EcN can also produce antibacterial substances such as bacteriocins, which can effectively inhibit the growth activity of pathogens in the oral cavity and help maintain oral health. Difficult-to-heal oral ulcers caused by radiotherapy and chemotherapy, medically known as oral mucositis, are one of the common side effects of cancer treatment. This type of oral ulcer is mainly caused by direct or indirect damage to oral mucosal cells by radiotherapy or chemotherapy drugs. It is characterized by persistent, difficult-to-heal ulcers in the oral mucosa, often accompanied by pain, infection and dysfunction. In order to treat oral diseases such as difficult-to-heal oral ulcers and repair wounds, and regulate the oral mucosal flora, it is necessary to find a substance that can effectively regulate the flora. Prebiotics are a class of substances that can be used by beneficial bacteria. They play an important role in regulating and promoting the balance and health of the flora. They mainly include some soluble fiber substances, among which functional oligosaccharides are more common. The role of prebiotics in regulating the flora is mainly reflected in the following aspects: 1. Promoting the growth of beneficial bacteria. Prebiotics can provide substrates for nutrition and growth of beneficial bacteria, especially lactic acid bacteria such as Bifidobacterium and Lactobacillus. These beneficial bacteria help maintain the balance and stability of the oral mucosal flora. 2. Enhance immune function. Appropriate intake of prebiotics can regulate the function of the immune system, promote the activity of immune cells and anti-inflammatory response, thereby enhancing the body's immune defense ability. 3. Reduce the attachment of pathogens. Prebiotics can compete with the mucosa for nutrition and attachment sites, reduce the attachment and growth of pathogens, and thus inhibit the reproduction and infection of harmful bacteria.

[0003] As one of the defense barriers, the mucosa blocks the entry of external microorganisms. In order to maximize the therapeutic effect of prebiotics, the wound surface should be kept as small as possible to prevent infection by other bacteria or fungi during drug delivery. Microneedle arrays are a new way to promote drug delivery developed in recent years. They are composed of many tiny needles and are used to enhance mucosal adhesion and deliver drugs or perform other treatments. These microneedles are usually nano or submillimeter in size and can penetrate the surface layer of the mucosa, creating tiny channels that allow drugs to penetrate into the deep tissues of the mucosa. Microneedle arrays have a series of advantages such as improving drug permeability, reducing dosage and side effects, providing personalized treatment, and improving patient compliance. They are the most suitable way to deliver drugs with EcN-encapsulated preparations and regulate oral mucosal flora.

[0004] Based on this, the present invention designs and prepares an EcN coating preparation made of oligofructose by using an electrostatic physical adsorption method, thereby increasing the in vivo biocompatibility and replication ability of EcN and improving the colonization and accumulation level of EcN in the oral cavity. Summary of the invention

[0005] In order to treat the healing of difficult-to-heal oral ulcer wounds caused by radiotherapy and chemotherapy and regulate the oral mucosal flora, the present invention designs and prepares a soluble microneedle array patch carrying an EcN coating preparation, which enhances oral mucosal adhesion through microneedles and maximizes the flora regulation effect.

[0006] The technical solution adopted by the present invention is:

[0007] A soluble microneedle array patch carrying an EcN coating preparation that can regulate oral mucosal flora is composed of a microneedle substrate and a soluble microneedle body, wherein the microneedle substrate and the soluble microneedle body are made of biocompatible materials, including oligomaltolsaccharide, chitosan, hyaluronic acid, pvp k-90, an EcN coating preparation and mint powder. The mass percentage of chitosan is 5%-10%, and the mass percentage of hyaluronic acid is less than 5%. The EcN coating preparation is prepared from EcN and oligofructose.

[0008] The microneedles of the present invention are made of biocompatible materials to ensure that the materials can be integrated with the oral mucosa. Under the premise of ensuring a small wound surface, the defect of non-soluble microneedles causing secondary trauma is further overcome. The prebiotic components contained in the microneedles remain in an active but unrecovered state. When the microneedle array is inserted into the oral mucosa, the microneedle needles will be dissolved by the interstitial fluid of the epidermis and the upper dermis, releasing active prebiotics to the epidermis and dermis tissues and diffusing. The active prebiotics begin to slowly recover and diffuse under the skin, and exert a therapeutic effect by regulating the flora environment of the mucosal wound site.

[0009] The soluble microneedle array patch for regulating oral mucosal flora of the present invention is as follows Figure 1 , prepared by the following method:

[0010] (1) Add glacial acetic acid to deionized water to adjust the pH value to 3-4;

[0011] (2) weighing isomaltooligosaccharide, chitosan, hyaluronic acid, pvp k-90, EcN coating preparation and mint powder according to proportion, and dissolving them in deionized water adjusted to a pH value;

[0012] (3) After stirring for 3-4 hours, ultrasonicate and let stand for 2-3 hours;

[0013] (4) Pour the dissolved material into a mold and centrifuge at 3000 rpm for 15-20 min;

[0014] (5) removing the mold and adding dissolved material to the surface again to form a bulge;

[0015] (6) Dry at 37°C for 36-48 hours and remove the microneedle array patch.

[0016] The specifications of the polydimethylsiloxane (PDMS) microneedle mold are pyramid or cone-shaped with a height of 1-2 mm and a diameter of 0.3-0.6 mm.

[0017] Prebiotics refer to organic substances that are not digested and absorbed by the host but can selectively promote the growth and reproduction of beneficial bacteria such as bifidobacteria in the body, thereby improving the health of the host. Functional oligosaccharides are the most common. Prebiotics can regulate the flora and promote the proliferation of probiotics, thereby enhancing wound repair and playing a major therapeutic role in microneedles.

[0018] Chitosan and hyaluronic acid mainly play the role of physical isolation, among which hyaluronic acid can play a small role in reducing cytotoxicity.

[0019] PVP K-90 mainly increases the viscosity of the mixed solution, making it easier to shape.

[0020] The EcN coated preparation mainly serves to increase the biocompatibility and replication ability of EcN in vivo, and improve the colonization and accumulation level of EcN in the oral cavity.

[0021] Mint powder mainly plays a flavor-correcting role, covering up the original strong bitter taste and improving patient compliance.

[0022] The invention discloses an application of the soluble microneedle array patch capable of regulating oral mucosal flora in the preparation of a wound healing device for treating oral refractory ulcers caused by radiotherapy and chemotherapy.

[0023] The soluble microneedle array patch capable of regulating oral mucosal flora of the present invention is used in the preparation of local drug delivery systems such as oral mucosal drug delivery.

[0024] The soluble microneedle array patch for regulating oral mucosal flora of the present invention is simple to prepare and has a clear mechanism.

[0025] Beneficial effects of the present invention:

[0026] The present invention limits the size of the microneedle body to ensure that the microneedle penetrates the oral mucosa when used. Since the microneedle tip does not reach the deep layer of the oral mucosa where nerve endings are richly distributed, no pain is generated. In addition, the microneedle body is made of biocompatible materials and can be integrated with the mucosa without causing trauma and inflammation, thereby overcoming the defect of non-soluble microneedles causing trauma, and can break through the barrier function of the oral mucosa without pain or bleeding for the user, achieving the effect of effectively delivering the EcN coated preparation to the deep layer of the mucosa ( Figure 2 ).

[0027] The technology of delivering EcN coated preparations by soluble microneedle array of the present invention has no obvious mucosal irritation and well solves the problem that EcN is difficult to penetrate into the mucosa to exert its effect. The soluble microneedle array represents a new technology for delivering EcN into the mucosa.

[0028] In summary, the present invention is based on the role of prebiotics in promoting the proliferation of probiotics and enhancing wound repair, as well as the role of the auxiliary material chitosan and hyaluronic acid in achieving physical isolation, to achieve the effect of regulating the wound flora, thereby treating the wound healing of difficult-to-heal oral ulcers caused by radiotherapy and chemotherapy. The present invention provides a new idea for the treatment of diseases such as difficult-to-heal oral ulcers caused by chemotherapy / radiotherapy, and is expected to become a blessing for alleviating the pain of patients with difficult-to-heal oral ulcers. The present invention will provide a new theory and advanced solution for oral diseases such as difficult-to-heal oral ulcers caused by radiotherapy and chemotherapy, and also provide promising strategies and exploration ideas for the clinical application of EcN, which has important scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The pictures are real objects. From top to bottom, they are the mixture state of active ingredients dissolved in deionized water, microneedle mold, and soluble microneedle array patch.

[0030] Figure 2 A diagram showing the effect of the needle tips dissolving after the soluble microneedle array patch of the present invention is applied to the skin;

[0031] Figure 3 A is a top view of a microneedle loaded with oligofructose preparation, B is an inverted microscope image of a side view of a microneedle loaded with EcN preparation, and C is a laser confocal microscope image of a side view of a microneedle loaded with EcN@OFT;

[0032] Figure 4 A0min, B 1min, C 5min, D 15min after administration of microneedle patch, the recovery of the back skin of mice; E 0min and F15min after administration of microneedle patch, the enlarged picture of the recovery of the back skin of mice;

[0033] Figure 5 Side views of the microneedle penetrating the dorsal skin of mice. A 0min, B 1min, C 5min, D 15min dissolution.

[0034] Figure 6 Image of 24h colony plate spread culture of EcN coating preparation prepared for oligofructose;

[0035] Figure 7 Image of 48h colony plate spread culture of EcN coating preparation prepared with oligofructose. DETAILED DESCRIPTION

[0036] The above contents of the present invention are further described in detail through the following specific examples, but the examples do not limit the present invention.

[0037] Example 1: LB liquid culture medium preparation

[0038] Take 1g of peptone, 0.5g of yeast powder, and 1g of NaCl respectively, stir and dissolve them in a 250ml conical flask with appropriate amount of deionized water, make the volume to 100ml, seal it with kraft paper and string, sterilize it at 121℃ in a high-pressure steam sterilizer for 30min, then prepare LB liquid culture medium, and place it in a UV clean bench for use.

[0039] Example 2: Cultivation of EcN

[0040] Take out the frozen bacteria from the -80℃ refrigerator, quickly take out 100μl, add it to a culture tube containing 4ml LB liquid culture medium, and place it in a shaker (37℃, 220r / min) for 12h. Take 1ml of the cultured bacterial solution and add it to 100ml LB liquid culture medium that has been sterilized, and then place it in a shaker (37℃, 220r / min) for 4h.

[0041] Example 3: Preparation of EcN Coated Preparation

[0042] Weigh 10 mg of oligofructose and dissolve it in 1 ml of phosphate buffered saline (PBS) solution (pH = 7.4) to obtain a PBS solution of water-soluble materials. Take 1 ml of newly passaged EcN bacterial solution for 1 day and place it in two 15 ml centrifuge tubes, centrifuge at 3000 r / min for 10 min, discard the supernatant, resuspend the obtained precipitate in PBS, and measure the absorbance value at optical density (OD) = 600 with a multifunctional microplate reader. Dilute to the same OD value of each group by adding deionized water, and control the concentration of the two obtained bacterial solutions to be 5×10 7 The mixture was centrifuged at 3000 r / min for 10 min, the supernatant was discarded, one portion was resuspended in 1 ml PBS solution (pH = 7.4), and the other portion was resuspended in the PBS solution of the water-soluble material obtained above.

[0043] The two bacterial solutions were placed on a shaker (37°C, 220 r / min) for 45 min, then ultrafiltrated and centrifuged at 3000 r / min for 5 min. The supernatant was discarded, and the resulting precipitate was washed three times with distilled water and resuspended in PBS to obtain EcN solution and EcN@OFT preparation solution.

[0044] Figure 3 A is a top view of the microneedle loaded with oligofructose preparation, B is a side view inverted microscope image of the microneedle loaded with EcN preparation, and C is a side view laser confocal microscope image of the microneedle loaded with EcN@OFT.

[0045] Example 4: Preparation of soluble microneedle array patch

[0046] Prebiotics refer to some organic substances that are not digested and absorbed by the host but can selectively promote the growth and reproduction of beneficial bacteria such as bifidobacteria in the body, thereby improving the health of the host, including functional oligosaccharides (such as fructooligosaccharides, xylooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, etc.), polysaccharides (microalgae discovered at this stage such as spirulina, arthrospira, etc.), extracts of some natural plants (such as vegetables, Chinese herbal medicines, wild plants, etc.), protein hydrolysates, polyols, etc. Among them, functional oligosaccharides are the most common prebiotics.

[0047] Oligosaccharide is a common prebiotic. Its viscosity is higher than that of sucrose solution of the same concentration and lower than that of maltose. It is easier to operate than maltose during the experiment and has no adverse effects on the physical and chemical properties of other auxiliary materials. The viscosity is higher than that of sucrose, and it is easier to maintain a stable structure. Oligosaccharide has excellent heat resistance and acid resistance. The 50% concentration syrup will not decompose when heated for a long time at pH = 3 and 120°C, which is suitable for various treatment environments. Oligosaccharide is added to drinking water, and mice are free to take it within 1 year. The daily intake is 2.7-5.0g / kg body weight of isomaltose. After long-term feeding tests, there are no abnormalities in the anatomical results and blood tests. There is no mutogenicity in the bacterial regression mutation test and the chromosome abnormality test of cultured cells. Therefore, the safety of oligosaccharide is extremely high. Therefore, the present invention selects oligosaccharide as the main drug to regulate the balance of the flora of the oral mucosa, promote the growth of probiotics, and treat oral diseases such as oral ulcers.

[0048] In order to better play the role of oligosaccharides in regulating the balance of bacterial flora, we chose chitosan as the substrate of the microneedle array. Chitosan is the product of the natural polysaccharide chitin with some acetyl groups removed. It has the characteristics of biodegradability, biocompatibility, and non-toxicity. Due to its guaranteed safety, it is widely used in the field of pharmacy and is a common excipient in pharmacy.

[0049] The original prescription adopted by the present invention is:

[0050] Isomaltooligosaccharide 5mg

[0051] Chitosan 30mg

[0052] Deionized water 1.5ml

[0053] The preparation method adopted by the present invention is as follows: adding an appropriate amount of deionized water into a beaker, and adjusting the pH value to 3-4 with glacial acetic acid; weighing isomaltooligosaccharide and chitosan according to the mass ratio in a vial, and dissolving them with deionized water with the adjusted pH value; adding a stirring bar, stirring on the instrument for 3-4 hours, ultrasonicating and standing for 2 hours; putting the dissolved substance into a mold, and centrifuging it at 3000r / min for 15 minutes; taking out the mold, and adding the dissolved substance again until a small bulge is formed; drying at 37°C for 36 hours, and taking out the microneedle array patch.

[0054] Embodiment 5:

[0055] The soluble microneedle array patch prepared according to the prescription of Example 4 can play a certain therapeutic role. In order to further improve the therapeutic effect of the microneedles, it was decided to select a substance that can not only cooperate with oligosaccharides to regulate the balance of the flora but also achieve physical isolation, and add it to the original prescription as an auxiliary material.

[0056] Hyaluronic acid is an acidic mucopolysaccharide. It was first isolated from the vitreous of bovine eyes by Meyer and other professors of ophthalmology at Columbia University in the United States in 1934. With its unique molecular structure and physical and chemical properties, hyaluronic acid has many important physiological functions in the body, such as lubricating joints, regulating the permeability of blood vessel walls, regulating the diffusion and movement of proteins, water and electrolytes, and promoting wound healing.

[0057] A certain proportion of hyaluronic acid is added to the original prescription to give full play to the physical isolation effect of hyaluronic acid, and to maximize the cooperation with prebiotics to regulate the oral mucosal flora, promote the reproduction of probiotics, and achieve better therapeutic effects.

[0058] The adjusted prescription according to Example 4 is as follows:

[0059]

[0060] The preparation method adopted in this prescription is as follows: add an appropriate amount of deionized water to a beaker, and select glacial acetic acid to adjust the pH value to 3-4; add isomaltooligosaccharide, chitosan and hyaluronic acid in a small bottle according to the mass ratio, and dissolve them with deionized water with adjusted pH value; add a stirring bar, stir on the instrument for 3-4 hours, ultrasonicate and let stand for 2 hours; put the dissolved substance into a mold, and centrifuge it at 3000r / min for 15 minutes; take out the mold, and add the dissolved substance again to form a small bulge; dry it at 37°C for 36 hours, and take out the microneedle array patch.

[0061] Embodiment 6:

[0062] To further improve the therapeutic effect of the soluble microneedle array patch prepared in Example 5, the concentration of hyaluronic acid was increased by combining prebiotics to better regulate the oral mucosal flora.

[0063] A series of hyaluronic acid solutions with gradient concentrations were prepared and added to the microneedle mold, and the concentrations of hyaluronic acid were 0, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, and 25 mg / ml, respectively. A series of blank microneedles with gradient concentrations of hyaluronic acid were obtained by drying under the same conditions.

[0064] Through research, it was found that within a certain concentration range, as the concentration of hyaluronic acid increases, the therapeutic effect improves, and the two show a positive correlation. However, when hyaluronic acid exceeds a certain range, it will affect the dissolution of oligosaccharides, resulting in insufficient content of the main active ingredients. Experiments have shown that 10mg / ml is the most suitable hyaluronic acid concentration.

[0065] The adjusted prescription according to Example 5 is as follows:

[0066]

[0067] The preparation method adopted in this prescription is as follows: add an appropriate amount of deionized water to a beaker, and select glacial acetic acid to adjust the pH value to 3-4; add isomaltooligosaccharide, chitosan and hyaluronic acid in a small bottle according to the mass ratio, and dissolve them with deionized water with adjusted pH value; add a stirring bar, stir on the instrument for 3-4 hours, ultrasonicate and let stand for 2 hours; put the dissolved substance into a mold, and centrifuge it at 3000r / min for 15 minutes; take out the mold, and add the dissolved substance again to form a small bulge; dry it at 37°C for 36 hours, and take out the microneedle array patch.

[0068] Embodiment 7:

[0069] The soluble microneedle array patch prepared according to Example 6 has insufficient hardness and is relatively brittle as a whole, making it difficult to directly penetrate the oral mucosa during use. We chose to add a suitable adhesive to the microneedles to solve this problem.

[0070] PVP K-90 is a widely used adhesive in pharmaceuticals and is commonly used in solid preparations. Its powder can also be directly added to other powders in a dry state and mixed, and then granulated by adding water, alcohol or hydroalcoholic solution. In oral or parenteral administration, PVP K-90 can also be used as a solubilizer to accelerate the dissolution of poorly soluble drugs from solid preparations. In addition, PVP K-90 solution can also be used as a coating material.

[0071] The adjusted prescription according to Example 6 is as follows:

[0072]

[0073] The preparation method adopted in this prescription is as follows: add an appropriate amount of deionized water to a beaker, and select glacial acetic acid to adjust the pH value to 3-4; add isomaltooligosaccharide, chitosan, hyaluronic acid and pvp k-90 in a small bottle according to the mass ratio, and dissolve them with deionized water with adjusted pH value; add a stirring bar, stir on the instrument for 3-4 hours, ultrasonicate and let stand for 2 hours; put the dissolved substance into a mold, and centrifuge it at 3000r / min for 15 minutes; take out the mold, and add the dissolved substance again to form a small bulge; dry it at 37°C for 36 hours, and take out the microneedle array patch.

[0074] Embodiment 8:

[0075] In order to further improve the hardness of the soluble microneedle array patch prepared in Example 7 and avoid the needle tip breaking due to insufficient hardness during use, which would prevent the needle tip from piercing the mucosal barrier and exerting its efficacy, the concentration of PVPK-90 was increased.

[0076] 0, 3, 5, 7, 9 and 11 mg of PVP K-90 were dissolved in 1.5 ml of deionized water to prepare a series of PVP K-90 solutions with gradient concentrations and added to the microneedle mold. A series of blank microneedles with gradient concentrations of PVP K-90 were obtained by drying under the same conditions. By applying the same force, we found that within a certain range, the hardness of the microneedle increased with the increase of PVP K-90 concentration. Beyond a certain range, the viscosity of the mixed solution is too high and the fluidity is very poor, making it difficult to form. The study found that the most suitable concentration of PVP K-90 is 9 mg / 1.5 ml.

[0077] The adjusted prescription according to Example 7 is as follows:

[0078]

[0079]

[0080] The preparation method adopted in this prescription is as follows: add an appropriate amount of deionized water to a beaker, and select glacial acetic acid to adjust the pH value to 3-4; add isomaltooligosaccharide, chitosan, hyaluronic acid and pvp k-90 in a small bottle according to the mass ratio, and dissolve them with deionized water with adjusted pH value; add a stirring bar, stir on the instrument for 3-4 hours, ultrasonicate and let stand for 2 hours; put the dissolved substance into a mold, and centrifuge it at 3000r / min for 15 minutes; take out the mold, and add the dissolved substance again to form a small bulge; dry it at 37°C for 36 hours, and take out the microneedle array patch.

[0081] Example 9: Preparation of EcN-loaded microneedle delivery system

[0082] In order to increase the in vivo biocompatibility and replication ability of EcN and improve the colonization and accumulation level of EcN in the oral mucosa, the present invention decides to add the EcN coating preparation as an excipient in the preferred prescription.

[0083] The adjusted prescription according to Example 8 is as follows:

[0084]

[0085] The preparation method adopted in this prescription is as follows: add an appropriate amount of deionized water to a beaker, and select glacial acetic acid to adjust the pH value to 3-4; add isomaltooligosaccharide, chitosan, hyaluronic acid, pvp k-90 and EcN solution / EcN@OFT preparation solution in a small bottle according to the mass ratio, and dissolve them with deionized water with adjusted pH value; add a stirring bar, stir on the instrument for 3-4 hours, ultrasonicate and let stand for 2 hours; put the dissolved substance into a mold, and centrifuge it at 3000r / min for 15 minutes; take out the mold, and add the dissolved substance again to form a small bulge; dry it at 37°C for 36 hours, and take out the microneedle array patch.

[0086] Figure 4 A0min, B 1min, C 5min, D 15min after administration of the microneedle patch of this prescription. Enlarged view of the recovery of the dorsal skin of the mouse at E 0min and F15min after administration of the microneedle patch.

[0087] Figure 5 Side views of the dissolution of the microneedle of this prescription after penetrating into the dorsal skin of mice. A 0min, B 1min, C 5min, D 15min.

[0088] Embodiment 10:

[0089] Flavor correctors refer to pharmaceutical excipients used in medicines to improve or mask the bitter taste of medicines, making it difficult for patients to detect the strong bitter taste (or other peculiar smells such as spicy, irritating, etc.) of medicines. So far, flavor correctors that have been used or are being used in medicine processing include: various taste-screening agents, lipid substances (including lecithin, surfactants), hydrophilic drug embedding materials (such as polyacrylic resin pharmaceutical coating agents, modified starch carbohydrates, cyclodextrin glucose oligomers, etc.), as well as ion exchange resins, proteins, zeolites and other various new pharmaceutical excipients.

[0090] In the actual application process of the microneedles prepared according to Example 8, patients generally reported that the microneedle array patch prepared according to the original prescription had a strong bitter taste when treating oral ulcers, which was not conducive to the treatment of the disease. In order to change the taste of the original microneedle patch and improve the compliance of patients, we need to select a suitable flavoring agent to cover up the original bitter taste.

[0091] Mint powder is a commonly used flavoring agent in the field of pharmacy. Its cooling and analgesic effects are often used to mask the bitterness of other drugs, relieve the stinging of local oral mucosa, and improve patient compliance. Since mint powder is widely used in the field of food processing, its safety is greatly guaranteed. Further research has found that mint powder does not interact with other ingredients in the soluble microneedle array patch prepared in the present invention, and has the characteristics of acid and high temperature resistance, ensuring that its physical and chemical properties will not change during the entire production process, and no other products will be produced.

[0092] The adjusted prescription according to Example 8 is as follows:

[0093]

[0094] The preparation method adopted in this prescription is as follows: add an appropriate amount of deionized water to a beaker, and select glacial acetic acid to adjust the pH value to 3-4; add isomaltooligosaccharide, chitosan, hyaluronic acid, pvp k-90 and mint powder in a small bottle according to the weight ratio, and dissolve them with deionized water with adjusted pH value; add a stirring bar, stir on the instrument for 3-4 hours, ultrasonicate and let stand for 2 hours; put the dissolved substance into a mold, and centrifuge it at 3000r / min for 15 minutes; take out the mold, and add the dissolved substance again to form a small bulge; dry it at 37°C for 36 hours, and take out the microneedle array patch.

[0095] Combined with Examples 9-10, the optimal prescription of the present invention is obtained, which is as follows:

[0096]

[0097] Example 11: After successfully designing and preparing the EcN coated preparation coated with oligofructose, we will verify whether this material has a promoting effect on the replication ability of EcN. At the same time, we will conduct an in-depth study on the feasibility of loading the coated preparation into the microneedle, focusing on whether the EcN has replication activity after being released from the microneedle, and whether it retains the enhanced replication ability of the original preparation.

[0098] 11.1 Preparation of liquid LB medium

[0099] Take 1g of peptone, 0.5g of yeast powder, and 1g of NaCl respectively, make up to 100ml with deionized water in a 250ml conical flask, stir thoroughly to dissolve, seal with kraft paper and string, sterilize at 121℃ in a high-pressure steam autoclave for 30min to prepare LB culture medium, and place it in a UV clean bench for use.

[0100] 11.2 Preparation of solid LB medium

[0101] Take 1g of peptone, 0.5g of yeast powder, and 1g of NaCl respectively, make up to 100ml with deionized water in a 250ml conical flask, stir thoroughly to dissolve, add 1.5g of agar powder, seal with kraft paper and string, sterilize at 121℃ in a high-pressure steam autoclave for 30min, pour evenly onto 10 sterile culture dishes under a microbial clean bench while hot, and obtain LB solid culture medium after cooling to room temperature.

[0102] 11.3 Colony plate spreading culture

[0103] Centrifuge the EcN solution and the EcN coating preparation solution in an ultracentrifuge (3300r / min) for 10 minutes, obtain the precipitate and resuspend it in the centrifuge tube containing the LB liquid culture medium, and take 2 kinds of 20μl bacterial solution and dilute it to 1ml with liquid LB culture medium for testing. Under the microbiological clean bench, take 5 kinds of 50μl diluted bacterial solution to be tested, drop it on the surface of the newly prepared LB solid culture medium, and use a newly sterilized triangular rake to spin-coat the bacterial solution until it is evenly distributed on the surface of the culture medium. Repeat the above steps once to obtain 2 colony coating culture plates, a total of two batches, and place them in a microbiological incubator (37°C, 5% CO 2 ) for 24 h, and observe and take photos after 48 h to record the results. Figure 6-7 .

[0104] From the above experimental results, it can be seen that: (1) the amount of EcN replication increased significantly with time; (2) the replication amount of the EcN@OFT group was significantly higher than that of the blank group EcN group, indicating that the EcN@OFT group has a promoting effect on the replication ability of EcN; the control group material OFT to be used in the present invention has a certain degree of promoting effect on the replication ability of EcN, which preliminarily verifies the effect of the preparation on the replication ability of EcN after being loaded into the soluble microneedles.

Claims

1. A soluble microneedle array patch that can regulate oral mucosal flora, It is characterized in that It consists of a microneedle substrate and a soluble microneedle body, wherein the microneedle substrate and the soluble microneedle body are made of biocompatible materials, including oligomaltose, chitosan, hyaluronic acid, pvp k-90, EcN coating preparation and mint powder; wherein the EcN coating preparation is prepared from EcN and oligofructose.

2. A soluble microneedle array patch capable of regulating oral mucosal flora according to claim 1, It is characterized in that The preparation method of the EcN coating preparation specifically comprises the following steps: Preparation of LB liquid culture medium: Take 1g of peptone, 0.5g of yeast powder, and 1g of NaCl respectively, stir and dissolve them with deionized water, and after constant volume, seal them with kraft paper and string, sterilize them in a high-pressure steam sterilizer at 121℃ for 30min to prepare LB liquid culture medium, and place it in a UV clean bench for use; Culture of EcN: Take out the frozen bacteria from the -80℃ freezer, quickly take out 100μl, add it to a culture tube containing 4ml LB liquid culture medium, put it in a shaker at 37℃, 220r / min, culture for 12h, take out 1ml of the cultured bacterial solution and add it to 100ml LB liquid culture medium, then put it in a shaker at 37℃, 220r / min, culture for 4h; Preparation of EcN coating preparation: Weigh 10 mg oligofructose and dissolve it in 1 ml pH = 7.4 phosphate buffer solution to obtain a PBS solution of water-soluble materials. Take 1 ml of EcN bacterial solution that has been newly subcultured for 1 day and place it in two 15 ml centrifuge tubes. Centrifuge at 3000 r / min for 10 min, discard the supernatant, and resuspend the obtained precipitate in PBS. Use a multifunctional microplate reader to measure the absorbance value at the optical density OD = 600. Dilute it to the same OD value of each group by adding deionized water, and control the concentration of the two obtained bacterial solutions to be 5×10 7 / ml, centrifuged at 3000r / min for 10min, discarded the supernatant, resuspended one portion in 1ml PBS solution, and resuspended the other portion in the PBS solution of the obtained water-soluble material; The two bacterial solutions were placed in a shaker at 37°C, 220 r / min, shaken for 45 min, and then ultrafiltered and centrifuged at 3000 r / min for 5 min. The supernatant was discarded, and the resulting precipitate was washed three times with distilled water and resuspended in PBS to obtain EcN solution and EcN envelope preparation solution.

3. A soluble microneedle array patch capable of regulating oral mucosal flora according to claim 2, It is characterized in that The ratio of oligosaccharide isomaltose, chitosan, hyaluronic acid, pvp k-90 and EcN coating preparation is:

4. A method for preparing a soluble microneedle array patch capable of regulating oral mucosal flora according to any one of claims 1 to 3, It is characterized in that The following steps are involved: (1) Add glacial acetic acid to deionized water to adjust the pH value to 3-4; (2) weighing isomaltooligosaccharide, chitosan, hyaluronic acid, pvp k-90, and EcN coating preparation according to proportion and dissolving them in deionized water adjusted to a pH value; (3) After stirring for 3-4 hours, ultrasonicate and let stand for 2-3 hours; (4) Pour the dissolved material into a mold and centrifuge; (5) removing the mold and adding dissolved material to the surface again to form a bulge; (6) Dry at 37°C for 36-48 hours and remove the microneedle array patch.

5. A method for preparing a soluble microneedle array patch capable of regulating oral mucosal flora according to claim 4, It is characterized in that In the step (2), mint powder is also added.

6. A method for preparing a soluble microneedle array patch capable of regulating oral mucosal flora according to claim 4, It is characterized in that In the step (2), the ratio of oligomaltodextrin, chitosan, hyaluronic acid, pvp k-90 and EcN coating preparation is:

7. A method for preparing a soluble microneedle array patch capable of regulating oral mucosal flora according to claim 4, It is characterized in that In the step (4), the dissolved substance is poured into a mold and centrifuged at 3000 r / min for 15-20 min.

8. Use of a soluble microneedle array patch capable of regulating oral mucosal flora according to any one of claims 1 to 3 in the preparation of a wound healing device for treating oral refractory ulcers caused by radiotherapy and chemotherapy.

9. Use of a soluble microneedle array patch capable of regulating oral mucosal flora according to any one of claims 1 to 3 in the preparation of oral mucosal drug delivery and local mucosal drug delivery systems.

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