WO2022266841A1 - Produit de micro-aiguilles biologiques produisant de l'hydrogène et son procédé de préparation et son utilisation - Google Patents

Produit de micro-aiguilles biologiques produisant de l'hydrogène et son procédé de préparation et son utilisation Download PDF

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Publication number
WO2022266841A1
WO2022266841A1 PCT/CN2021/101532 CN2021101532W WO2022266841A1 WO 2022266841 A1 WO2022266841 A1 WO 2022266841A1 CN 2021101532 W CN2021101532 W CN 2021101532W WO 2022266841 A1 WO2022266841 A1 WO 2022266841A1
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Prior art keywords
hydrogen
microneedle
producing
bacteria
backing
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PCT/CN2021/101532
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English (en)
Chinese (zh)
Inventor
郑斌
王志云
郭庆陆
王涛
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天津大学
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Priority to PCT/CN2021/101532 priority Critical patent/WO2022266841A1/fr
Publication of WO2022266841A1 publication Critical patent/WO2022266841A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the invention relates to the field of biological microneedles, in particular to a biological microneedle product which contains biological microneedles and can generate hydrogen from the biological microneedles.
  • the present invention also relates to a method for preparing the biological microneedle product and an application of the biological microneedle product.
  • oxidative stress oxidative stress
  • numerous symptoms such as tiredness, general weakness, muscle and joint pain, indigestion, anxiety, depression, itchy skin, headaches , as well as difficulty concentrating and infections that are difficult to heal.
  • the most common diseases induced by elevated levels of oxidative stress are heart disease, cancer, osteoarthritis, rheumatoid arthritis, diabetes, tumors, spinal cord injury, aging And neurodegenerative problems, such as Alzheimer's disease, Parkinson's disease, etc.
  • Hydrogen is a signaling molecule in animals, especially in humans. It has anti-oxidative stress effects and is widely used in the treatment of oxidative stress diseases (especially rheumatoid arthritis). It inhibits the expression of MAPK, NF- ⁇ B and TGF- ⁇ 1 by activating the Nrf2 signaling pathway, and exerts antioxidant and anti-inflammatory effects. After hydrogen treatment, the joint activity score increased, and the levels of oxidation indicators and inflammatory factors decreased. In addition, biosafety and excellent tissue diffusibility are unique advantages of hydrogen.
  • Biohydrogen-producing microorganisms are capable of producing hydrogen gas. They can be broadly divided into four groups, including green algae, cyanobacteria (also known as blue-green algae), photosynthetic bacteria, and fermentative bacteria. These microorganisms have the advantages of green, energy saving, and environmental protection. Among them fermentative bacteria, especially Clostridium butyricum (Clostridium butyricum, C.B.) and Enterobacter aerogenes as facultative anaerobes aerogenes, E.A.), which widely exist in soil, water and other environments, have low requirements for growth conditions, can ferment a variety of sugars to produce hydrogen, and are excellent candidates for hydrogen-producing microorganisms.
  • green algae also known as blue-green algae
  • photosynthetic bacteria and fermentative bacteria.
  • fermentative bacteria especially Clostridium butyricum (Clostridium butyricum, C.B.) and Enterobacter aerogenes as facultative anaerobes aerogenes, E.A.),
  • Microneedle drug delivery is a new type of local transdermal drug delivery technology, which combines the convenience of plaster and the effectiveness of subcutaneous injection, avoids the shortcomings of other drug delivery methods, and has the advantages of not touching the nerve, safe and painless , Efficient penetration and many other advantages.
  • Microneedle products usually include a plurality of microneedles whose length generally does not exceed about 1 mm.
  • the microneedles can form micro-channels in the stratum corneum of the skin, break through the barrier of the stratum corneum of the skin, and promote the penetration of drugs, thereby reducing the penetration of drugs in the stratum corneum. Accumulation increases the dose of drugs reaching the epidermis, dermis and subcutaneous tissue. Therefore, microneedle products are widely used to promote the transdermal absorption of small molecules and macromolecules, and to treat obesity, diabetes, cancer and many other diseases, and have broad application prospects.
  • the use of microneedle products is extremely convenient, no professional training is required, patients can self-administer medication, the risk of accidental needle stick injuries is low, and it is easy to dispose of after use.
  • Chinese patent application CN112206202A discloses a microneedle loaded with live bacteria for the treatment of skin diseases.
  • the microneedles are loaded with live functional bacteria, and these bacteria include one or more of Bacillus subtilis, Bacillus licheniformis, yeast, Escherichia coli and Paenibacillus polymyxa.
  • the purpose of the present invention is to make full use of the advantages of the microneedle itself and the advantages of the biological hydrogen-producing microorganism itself, to provide a hydrogen-producing biological microneedle product, which generates hydrogen through the hydrogen-producing active bacteria and sugars loaded in the microneedle, After the microneedle product is applied to the skin, efficient transdermal absorption of hydrogen can be achieved, and it can fully relieve and treat common diseases caused by elevated levels of oxidative stress, and solve the problems of traditional drug resistance, side effects, and infiltration. Poor sex and other shortcomings.
  • the present invention provides the following technical solutions:
  • a hydrogen-producing biological microneedle product comprising a backing and a hydrogen-producing biological microneedle array attached to one side of the backing, the hydrogen-producing biological microneedle array comprising a plurality of microneedles, wherein each The root microneedle comprises a matrix and hydrogen-producing active bacteria and sugars loaded in the matrix, and the hydrogen-producing active bacteria can use the sugars to produce hydrogen, wherein the sugars are preferably selected from glucose, maltose, fructose, Lactose, sucrose or a mixture of two or more thereof, more preferably selected from glucose, the content of the sugar in the microneedle is about 2.5% by weight to about 10% by weight based on the weight of the matrix %, preferably about 5% by weight to about 7.5% by weight, more preferably about 6% by weight, the content of the hydrogen-producing active bacteria in the microneedles is about 1 ⁇ 10 7 cfu to about 1 ⁇ 10 per gram of matrix weight 9 cfu, preferably about 1 ⁇ 10 8 c
  • Scheme 2 The hydrogen-producing biological microneedle product according to scheme 1, wherein the hydrogen-producing active bacteria are selected from cyanobacteria, green algae, photosynthetic bacteria, fermenting bacteria or a mixture of two or more of these bacteria, preferably selected from The fermenting bacteria are more preferably selected from Clostridium butyricum and/or Enterobacter aerogenes, most preferably selected from Enterobacter aerogenes.
  • Scheme 3 The hydrogen-producing biological microneedle product according to scheme 1 or 2, wherein the matrix is formed by crosslinking and/or drying and curing an aqueous solution comprising one or more of the following substances: polyethylene glycol alcohol diacrylate, silk fibroin, methacrylate gelatin, carboxymethylcellulose, trehalose, hyaluronic acid, polylactic-co-glycolic acid, polylactic acid, galactose, polyvinylpyrrolidone, and polyvinyl alcohol, Preference is given to methacrylate gelatin and/or hyaluronic acid, more preferably methacrylate gelatin.
  • Scheme 4 The hydrogen-producing biological microneedle product according to any one of schemes 1 to 3, wherein the backing is formed by crosslinking and/or drying and curing an aqueous solution comprising one or more of the following substances Made of: polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin, carboxymethylcellulose, trehalose, hyaluronic acid, polylactic-co-glycolic acid, polylactic acid, galactose, polyvinylpyrrolidone and polyvinyl alcohol, preferably hyaluronic acid.
  • Scheme 6 A method for preparing a hydrogen-producing biological microneedle product according to any one of schemes 1 to 5, the method comprising the following steps:
  • the total content of the active hydrogen-producing bacteria is about 0.2 ⁇ 10 7 cfu/mL to about 0.4 ⁇ 10 9 cfu/mL, preferably about 0.3 ⁇ 10 8 cfu/mL, wherein
  • the hydrogen-producing active bacteria are preferably selected from cyanobacteria, green algae, photosynthetic bacteria, fermenting bacteria or a mixture of two or more of these bacteria, more preferably from fermenting bacteria, especially preferably from Clostridium butyricum and/or Enterobacter aerogenes, most
  • the concentration of the matrix material is about 20% by weight to about 40% by weight based on the total weight of the mixed solution, preferably about 25% by weight to about 35% by weight, more preferably about 30% by weight, and the concentration of the sugar From about 10 mg/mL to about 20 mg/mL, preferably about 15 mg/mL
  • the carbohydrate is preferably selected from glucose, maltose, fructose, lactose, sucrose or a mixture of two or more thereof, more preferably from glucose
  • the matrix material is preferably selected from polyethylene glycol Diacrylate, silk fibroin, methacrylate gelatin, carboxymethylcellulose, trehalose, hyaluronic acid, polylactic-co-glycolic acid, polylactic acid, galactose, polyvinylpyrrolidone, polyvinyl alcohol,
  • a microneedle mold comprising an upper surface and a shaped hole extending downward from the upper surface, wherein the shaped hole has a tip and a bottom end, the tip is far away from the upper surface, the The base plane is flush with the upper surface, the distance from the tip to the base is preferably from about 200 ⁇ m to about 1 mm, the base preferably has a base diameter from about 100 ⁇ m to about 500 ⁇ m, and adjacent The spacing between tips is preferably from about 300 ⁇ m to about 800 ⁇ m;
  • step (2) placing the mixed solution obtained in step (2) in the forming hole and filling at least a part of the volume of the forming hole, preferably filling up the forming hole;
  • the backing material is cross-linked to form a continuous backing layer, so that the hydrogen-producing biological microneedle array is attached to the backing solution layer or the backing layer, and the backing material is preferably selected from polyethylene glycol Diacrylate, silk fibroin, methacrylate gelatin, carboxymethylcellulose, trehalose, hyaluronic acid, polylactic-co-glycolic acid, polylactic acid, galactose, polyvinylpyrrolidone, polyvinyl alcohol, or their A mixture of two or more of, preferably hyaluronic acid; and
  • step (6) Drying and curing the backing solution layer or backing layer obtained in step (6) and the hydrogen-producing bio-microneedle array at the same time to form the hydrogen-producing bio-microneedle product.
  • Scheme 7 The hydrogen-producing biological microneedle product according to any one of schemes 1 to 5 or the hydrogen-producing biological microneedle product prepared according to the method of scheme 6 is used to alleviate and/or treat animal bodies, preferably human oxidative stress diseases (preferably heart disease, cancer, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, diabetes, tumor, spinal cord injury, aging, Alzheimer's disease and/or Parkinson's disease).
  • human oxidative stress diseases preferably heart disease, cancer, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, diabetes, tumor, spinal cord injury, aging, Alzheimer's disease and/or Parkinson's disease.
  • Scheme 8 The hydrogen-producing biological microneedle product according to any one of schemes 1 to 5 or the hydrogen-producing biological microneedle product prepared according to the method of scheme 6 is used to prepare and relieve and/or treat the oxidative stress of the animal body, preferably the human body Use of the medical device for disease, preferably heart disease, cancer, osteoarthritis, rheumatoid arthritis, diabetes, tumor, spinal cord injury, aging, Alzheimer's disease and/or Parkinson's disease.
  • disease preferably heart disease, cancer, osteoarthritis, rheumatoid arthritis
  • diabetes preferably tumor
  • spinal cord injury aging
  • Alzheimer's disease and/or Parkinson's disease aging of Alzheimer's disease and/or Parkinson's disease.
  • the present invention makes full use of the advantages of the microneedle itself and the advantages of the hydrogen-producing active bacteria itself, and provides a hydrogen-producing biological microneedle product, which is loaded on
  • the hydrogen-producing active bacteria and sugars in the microneedles produce hydrogen gas, which can achieve efficient transdermal absorption of hydrogen gas after the microneedle product is applied to the skin, and have sufficient relief for common diseases induced by increased levels of oxidative stress And therapeutic effect, solve the traditional drug resistance, many side effects, poor permeability and other shortcomings.
  • the advantages of the present invention are:
  • the hydrogen gas produced by the hydrogen-producing microneedle product of the present invention is a signal molecule in the human body and has an anti-oxidative stress effect. It is used in the treatment of oxidative stress diseases such as rheumatoid arthritis by activating the Nrf2 signaling pathway , Inhibit the expression of MAPK, NF- ⁇ B and TGF- ⁇ 1, and exert anti-oxidation and anti-inflammatory effects. After hydrogen treatment, the joint activity scores (including clinical scores, paw thickness, etc.) were improved, and the levels of inflammatory factors were reduced.
  • the hydrogen-producing microneedle product of the present invention can pierce the cuticle of the skin that limits drug absorption, and promote the diffusion of hydrogen molecules without causing pain.
  • biosafety and excellent tissue diffusibility are unique advantages of hydrogen.
  • the hydrogen-producing microneedle medical product of the present invention utilizes microneedles and live gas-producing bacteria to ferment and produce hydrogen, which has stability and high efficiency.
  • the present invention prepares microneedles by inverting the microneedle template, which is simple, easy to operate, low in price, reusable, does not require high technical requirements, and is easy to control the basic shape of the microneedle array, which is safe Strong, suitable for promotion.
  • Fig. 1 is a side view schematically illustrating a part of the biological microneedle product of the present invention.
  • Fig. 2 is a side view schematically illustrating a part of a microneedle mold used to prepare the biological microneedle product of the present invention.
  • FIG 3 is a photo illustrating the hydrogen production effect of the biological microneedle product of Preparation Example 2 of the present invention.
  • the present invention relates to a hydrogen-producing biological microneedle product 100 .
  • the hydrogen-producing biomicroneedle product 100 of the present invention includes a backing 120 and a hydrogen-producing biomicroneedle array attached to one side of the backing 120, the hydrogen-producing biomicroneedle array It includes a plurality of microneedles 110, wherein each microneedle 110 includes a matrix and active hydrogen-producing bacteria and sugars loaded in the matrix, and the active hydrogen-producing bacteria can use the sugars to generate hydrogen.
  • the sugar is not particularly limited, as long as it can be fermented by the hydrogen-producing active bacteria and stably produce enough hydrogen.
  • the sugars may include but not limited to glucose, maltose, fructose, lactose, sucrose or a mixture of two or more thereof, more preferably glucose.
  • the content of the sugar in the microneedles 110 can vary in a wide range depending on the hydrogen-producing active bacteria used.
  • the content of the sugar in the microneedle 110 is preferably about 2.5 wt% to about 10 wt%, preferably about 5 wt% to about 7.5 wt%, based on the weight of the matrix, More preferably about 6% by weight.
  • the sugar content is less than about 2.5% by weight, the nutrients contained in the microneedles 110 are not enough to satisfy the growth of active hydrogen-producing bacteria, which will lead to a decrease in hydrogen production efficiency.
  • the content of the sugar is higher than about 10% by weight, the mechanical strength of the microneedles 110 may decrease significantly.
  • the type of the hydrogen-producing active bacteria is not particularly limited, as long as it can ferment the sugar and stably produce sufficient hydrogen.
  • the hydrogen-producing active bacteria are preferably selected from cyanobacteria, green algae, photosynthetic bacteria, fermenting bacteria or a mixture of two or more of these bacteria, more preferably from fermenting bacteria, especially preferably from butyric acid Clostridium acid and/or Enterobacter aerogenes, most preferably selected from Enterobacter aerogenes.
  • the hydrogen-producing biological microneedle product 100 of the present invention requires cross-linking and/or drying and curing steps in the preparation process, and these cross-linking and/or drying and curing steps may require the use of ultraviolet, heat, electronic Bacteria, radiation and other means, and Clostridium butyricum and/or Enterobacter aerogenes, which belong to fermentative bacteria species, after going through processes such as ultraviolet crosslinking, the determination of bacterial viability shows that very short ultraviolet light will not significantly affect The activity of these bacteria, and because Enterobacter aerogenes and/or Clostridium butyricum are widely present in soil, water and other environments, the growth conditions are not high, and they can ferment a variety of sugars to produce hydrogen, which is an excellent hydrogen-producing microorganism.
  • the active hydrogen-producing bacteria used in the present invention are preferably Enterobacter aerogenes and/or Clostridium butyricum, most preferably selected from Enterobacter aerogenes.
  • the content of the hydrogen-producing active bacteria in the microneedle 110 is not particularly limited, as long as it can ferment the sugar and stably produce Just enough hydrogen.
  • the content of the hydrogen-producing bacteria in the microneedles 110 is preferably in the range of about 1 ⁇ 10 7 cfu to about 1 ⁇ 10 9 cfu per gram of substrate weight, preferably about 1 ⁇ 10 8 cfu to In the range of about 1.5 x 10 8 cfu, most preferably about 1.2 x 10 8 cfu.
  • the content of the hydrogen-producing bacteria in the microneedles 110 can be determined by plate counting.
  • the content of the hydrogen-producing active bacteria is lower than about 1 ⁇ 10 7 cfu/g, the concentration of the hydrogen-producing active bacteria is too low, resulting in insufficient hydrogen production efficiency; if the content of the hydrogen-producing active bacteria is higher than about If it is 1 ⁇ 10 9 cfu/g, the nutrients contained in the microneedles 110 will be insufficient to satisfy the sufficient growth of hydrogen-producing active bacteria, which will also lead to a decrease in hydrogen production efficiency.
  • the matrix material forming the matrix is not particularly limited, as long as the common matrix materials used in the field for preparing microneedle products can be used in the present invention.
  • the matrix is preferably formed by including the following substances The aqueous solution of one or more of them is cross-linked and/or dried and solidified: polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin, carboxymethyl cellulose, trehalose, hyaluronic acid acid, polylactic-co-glycolic acid, polylactic acid, galactose, polyvinylpyrrolidone and polyvinyl alcohol, preferably methacrylate gelatin and/or hyaluronic acid, more preferably methacrylate gelatin.
  • the matrix is preferably selected from methacrylate gelatin and/or hyaluronic acid.
  • Methacrylate gelatin (GelMA) is prepared from methacrylic anhydride (MA) and gelatin (Gelatin), in which they are cross-linked by ultraviolet light irradiation under the mediation of photosensitizers to form a porous structure with a certain strength .
  • GelMA has excellent biocompatibility.
  • Hyaluronic acid is a mucopolysaccharide that protects the skin and can be used to accelerate wound healing.
  • the backing material forming the backing 120 is not particularly limited, as long as the commonly used backing materials used in the field for preparing microneedles can be used in this invention. invention.
  • the backing 120 is preferably crosslinked and / or formed after drying: polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin, carboxymethylcellulose, trehalose, hyaluronic acid, polylactic-co-glycolic acid, polylactic acid, semi Lactose, polyvinylpyrrolidone and polyvinyl alcohol, preferably hyaluronic acid.
  • hyaluronic acid is a kind of mucopolysaccharide, which has the function of protecting the skin and can be applied to accelerate wound healing, so it is preferably used as the backing material in the present invention.
  • the thickness of the formed backing 120 is not limited, but considering that the backing 120 has a certain strength and flexibility, the thickness of the backing 120 is preferably about 0.1 to about 15 mm, more preferably about 1 to about 10 mm, most preferably about 2 to about 3 mm.
  • the materials forming the base body and the backing 120 are the same. In these cases, the combination of the microneedle 110 and the backing 120 is more stable, and in the method for preparing the hydrogen-producing biomicroneedle product 100 as described below, the microneedle array and the backing 120 can be made One-piece molding simplifies the preparation process.
  • the materials of the matrix and the backing 120 are preferably different, for example, the matrix material is methacrylate gelatin, and the backing material is hyaluronic acid .
  • the microneedle 110 has a certain strength and has a porous structure and excellent biocompatibility, and at the same time, the backing 120 has a better protective effect on the skin and can be used to accelerate wound healing.
  • the size and shape of the microneedle 110 are not particularly limited, which can be determined according to the site where the hydrogen-producing biological microneedle product 100 of the present invention is applied and the disease to be treated. And vary in a wide range.
  • each microneedle 110 has a tip and a bottom end, the tip is far away from the backing 120, and the microneedle
  • the needle 110 is attached to the backing 120 via the bottom end, and the height h from the tip to the bottom end is not particularly limited, but is preferably about 200 ⁇ m to about 1 mm.
  • the height h is preferably not less than about 200 ⁇ m, otherwise the microneedles 110 are not easy to pierce the stratum corneum of some parts of animal body, preferably human body.
  • the height h is also preferably not higher than about 1 mm, otherwise it will pierce the stratum corneum of some parts of the animal body, preferably the human body, to reach the nerve layer, thereby causing pain.
  • the bottom end of the microneedle 110 preferably has a bottom diameter w of about 100 ⁇ m to about 500 ⁇ m.
  • the diameter w of the bottom end is preferably not less than about 100 ⁇ m, otherwise the mechanical strength of the microneedles 110 will be insufficient and easily broken.
  • the diameter w of the bottom end is also preferably not greater than about 500 ⁇ m, otherwise, after the hydrogen-producing biomicroneedle product 100 of the present invention is applied to some animal bodies, preferably certain parts of the human body, relatively small Large pores, causing problems with skin aesthetics and healing.
  • the three-dimensional shape of the microneedle 110 is not particularly limited, it can be cylindrical, conical, truncated conical, etc. or a combination thereof, preferably regular or irregular.
  • Regular cones, quasi-cones, triangular pyramids, quadrangular pyramids or higher-order pyramids, etc., and these cones, quasi-conical pyramids, triangular pyramids, quadrangular pyramids or higher-order pyramids can be regular pyramids or oblique pyramids.
  • the distance d between the tips of adjacent microneedles 110 is preferably about 300 ⁇ m to about 800 ⁇ m.
  • the distance within this range can make the hydrogen-producing biological microneedle product 100 of the present invention achieve optimal effects in many aspects, such as the depth of penetration into the stratum corneum and the efficiency of hydrogen delivery.
  • the multiple microneedles 110 in the hydrogen-producing biological microneedle product 100 of the present invention may also be the same or different from each other in terms of size, shape and composition.
  • the differences between the plurality of microneedles 110 can be in many aspects, including but not limited to shape, height h, bottom surface diameter w, tip distance d, matrix material contained, hydrogen-producing active bacteria species contained, and sugars, etc.
  • a part of the microneedles 110 may have a higher content of hydrogen-producing bacteria and sugars, while another part of the micro-needles 110 may have a lower content of hydrogen-producing bacteria and carbohydrate.
  • microneedles and microneedles 110 may have volumes that vary regularly and periodically at intervals. Under these circumstances, the applicability of the hydrogen-producing biological microneedle product 100 of the present invention to alleviate and/or treat various diseases can be broadened.
  • the microneedles 110 and/or the backing 120 in the hydrogen-producing biomicroneedle product 100 of the present invention can also contain technical fields as needed.
  • Other well-known additives including but not limited to molding agents, preservatives and photoinitiators (photosensitive mediators), such as 2,4-dihydroxybenzophenone, diphenyl ethyl ketone, etc.
  • the amount of the other auxiliary agents is preferably no more than about 10 wt%, more preferably about 5 wt%, most preferably no more than about 1 wt%, based on the weight of the matrix of the microneedle 110 and/or the backing 120.
  • the present invention also relates to a method for preparing the hydrogen-producing biological microneedle product 100 according to the present invention as described above, the method comprising the following steps 1-7.
  • Step 1 providing a liquid medium, autoclaving the liquid medium, inoculating the active hydrogen-producing bacteria, and culturing the active hydrogen-producing bacteria to obtain a liquid medium containing the active hydrogen-producing bacteria.
  • the liquid medium may be a nutrient broth liquid medium known in the art. Those skilled in the art are capable of conveniently determining the type of the liquid culture medium according to the selected hydrogen-producing active bacteria.
  • the culture process is also a common culture process in the art.
  • the hydrogen-producing active bacteria can be cultured at about 30°C to about 40°C, preferably at about 37°C for about 5 hours to about 20 hours, preferably about 12 hours to obtain the Liquid culture medium for active bacteria.
  • the content of the hydrogen-producing active bacteria is not particularly limited, as long as it matches the amount of the matrix material and sugars used in the following step 2 so that it can be used in the final microneedle
  • the content in the product 100 only needs to meet the range defined above.
  • the specific value of the content of the hydrogen-producing active bacteria can be determined according to the desired content of the hydrogen-producing active bacteria in the microneedle 110 and the concentration of the matrix material used in the following step 2 in the mixed solution. Sure.
  • the content of the hydrogen-producing active bacteria in the microneedle 110 is set to about 1 ⁇ 10 8 cfu/g, and the concentration of the matrix material used in the following step 2 in the mixed solution is set to When it is set at about 30% by weight, the final total content of the hydrogen-producing bacteria in the liquid medium should be set at about 0.3 ⁇ 10 8 cfu/mL.
  • the content of the hydrogen-producing active bacteria in the liquid medium is about 0.2 ⁇ 10 7 cfu/mL to about 0.4 ⁇ 10 9 cfu/mL, preferably about 0.3 ⁇ 10 8 cfu/mL, and the content may be Determined by methods known in the art, such as plate counting.
  • the hydrogen-producing active bacteria are preferably selected from cyanobacteria, green algae, photosynthetic bacteria, fermenting bacteria, or two or more of these bacteria A mixture of species, more preferably selected from fermenting bacteria, especially preferably selected from Clostridium butyricum and/or Enterobacter aerogenes, most preferably selected from Enterobacter aerogenes.
  • Step 2 Adding the matrix material capable of forming the matrix and the sugars to the liquid culture medium containing the subcultured hydrogen-producing active bacteria obtained in step 1 to form a mixed solution.
  • the concentration of the matrix material is not particularly limited, as long as the amount can ensure the formation of the microneedles 110 in the final biological microneedle product 100 of the present invention.
  • the content of the matrix material in the liquid medium is about 20% to about 40% by weight, preferably about 25% to about 35% by weight, more preferably about 30% by weight.
  • the microneedle 110 formed of the matrix material has both sufficient mechanical strength and certain porosity, so that the efficiency of hydrogen production can be optimized.
  • the concentration of the sugar is not particularly limited, as long as the amount can ensure that its content in the final microneedle product 100 meets the range defined above.
  • the carbohydrate is present at a concentration of about 10 mg/mL to about 20 mg/mL, more preferably about 15 mg/mL. mg/mL.
  • the base material is preferably selected from polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin, carboxymethyl fiber trehalose, hyaluronic acid, polylactic acid-glycolic acid copolymer, polylactic acid, galactose, polyvinylpyrrolidone, polyvinyl alcohol or a mixture of two or more thereof, more preferably methacrylate gelatin and/or hyaluronic acid, most preferably methacrylate gelatin; and said sugar is preferably selected from glucose, maltose, fructose, lactose, sucrose, more preferably selected from glucose.
  • Step 3 Provide a microneedle mold 200, as shown in Figure 2 of the specification, the microneedle mold 200 includes an upper surface 202 and includes a forming hole 201 extending downward from the upper surface 202, wherein the forming hole 201 It has a tip and a bottom, the tip is away from the upper surface 202 , and the bottom plane is flush with the upper surface 202 .
  • the three-dimensional shape of the forming hole 201 should match the shape of the desired microneedle 110 to be formed.
  • it can be cylindrical, conical, conical, etc. or a combination thereof, preferably regular or irregular conical, quasi-conical, triangular, quadrangular, or higher-order pyramids, and these cones , Class cones, triangular pyramids, quadrangular pyramids or higher pyramids can be regular pyramids or oblique pyramids.
  • the forming hole 201 should have a height h, a bottom width w, and a tip spacing d of the forming hole 201 corresponding to the height h, bottom width w, and tip spacing d of the microneedle 110 .
  • the shaped hole 201 may also have a height h and a bottom width w of the shaped hole 201 that are greater than the height h and bottom width w of the microneedle 110 . In the latter case, the formed microneedles 110 will not fill the forming holes 201 .
  • the upper surface 202 of the microneedle mold 200 (which includes the inner surface of the forming hole 201 ) may be coated with an anti-sticking layer.
  • the microneedle mold 200 is commercially available, for example, it can be a custom-made PDMS mold purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd., and the mold parameters can be determined according to the above-mentioned needle size needs to be customized.
  • the height h of all the forming holes 201 is about 600 ⁇ m
  • the width w of the bottom surface is about 320 ⁇ m
  • the tip distance d is about 500 ⁇ m.
  • the overall length of the mold 200 ⁇ width about 15 mm ⁇ about 15 mm.
  • Step 4 Put the mixed solution obtained in step 2 into the forming hole 201 and fill at least a part of the volume of the forming hole 201 .
  • the volume of the mixed solution filling the forming hole 201 is not particularly limited, but preferably fills at least about 1/4, at least about 1/3, or at least about 1/3 of the volume of the forming hole 201. 2. At least about 2/3, at least about 3/4, most preferably filling the forming hole 201 . If the forming hole 201 is not filled, the part of the volume of the forming hole 201 that is not filled by the microneedle 110 should be filled with the backing material solution in step 6 below.
  • Step 5 Cross-link and/or dry and solidify the mixed solution containing the matrix material, the sugar and the active hydrogen-producing bacteria in the forming hole 201, so as to form in the forming hole 201
  • the microneedles 110, a plurality of the microneedles 110 constitute the hydrogen-producing biological microneedle array.
  • the crosslinking can be realized by various means such as ultraviolet rays, heat, electron beams, and radiation.
  • the drying and curing can be achieved, for example, by natural drying or drying at a certain temperature in an oven.
  • those skilled in the art can determine that such crosslinking can be achieved by ultraviolet crosslinking in the presence of photosensitizing mediators known in the art.
  • the UV crosslinking time is preferably less than about 20 seconds, preferably about 5 seconds to about 15 seconds, more preferably about 10 seconds; the oven drying is preferably less than about 4 hours at a temperature lower than about 50°C, Preferably from about 1 hour to about 3 hours. Exceeding said upper limit may cause significant inactivation of hydrogen-producing bacteria.
  • Step 6 Apply a solution containing a backing material on the bottom surface of the microneedle 110 and the upper surface 202 of the microneedle mold 200 not covered by the microneedle 110 to form a backing solution layer, so that the The hydrogen-producing biological microneedle array is attached to the backing solution layer.
  • the backing material is preferably selected from polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin , carboxymethylcellulose, trehalose, hyaluronic acid, polylactic acid-glycolic acid copolymer, polylactic acid, galactose, polyvinylpyrrolidone, polyvinyl alcohol or a mixture of two or more of them, preferably transparent uric acid.
  • the backing material is optionally cross-linked to form a continuous backing layer, so that the hydrogen-producing biological microneedle array is attached to the backing layer.
  • the crosslinking can be achieved by various means such as ultraviolet rays, heat, electron beams, and radiation.
  • those skilled in the art can determine that such crosslinking can be achieved by ultraviolet crosslinking in the presence of photosensitizing mediators known in the art.
  • the UV crosslinking time is preferably less than about 20 seconds, preferably about 5 seconds to about 15 seconds, more preferably about 10 seconds. Exceeding the upper limit mentioned here can lead to a considerable inactivation of the hydrogen-producing bacteria.
  • Step 7 Dry and solidify the backing solution layer obtained in step 6 and optionally the microneedle array attached to the backing solution layer at the same time to form the hydrogen-producing biomicroneedle product 100 .
  • the drying can be achieved, for example, by natural drying or preferably drying in an oven at a certain temperature.
  • the oven drying is preferably at a temperature below about 50°C, for example at about 37°C for less than about 4 hours, preferably about 1 hour to about 3 hours. Exceeding said upper limit may cause significant inactivation of hydrogen-producing bacteria.
  • the drying step is preferably such that the water content in the microneedles 110 and the backing 120 is less than about 20% by weight, preferably less than about 10% by weight, most preferably less than About 5% by weight.
  • the present invention relates to the hydrogen-producing bio-microneedle product 100 in the first aspect above or the hydrogen-producing bio-microneedle product prepared according to the method for preparing the hydrogen-producing bio-microneedle product 100 in the above second aspect 100 Use for relieving and/or treating oxidative stress diseases in animals.
  • the present invention relates to the hydrogen-producing bio-microneedle product 100 in the first aspect above or the hydrogen-producing bio-microneedle product prepared according to the method for preparing the hydrogen-producing bio-microneedle product 100 in the above second aspect 100 Use for preparing a medical device for alleviating and/or treating oxidative stress diseases in animals.
  • the animal body is preferably a human body.
  • the oxidative stress disease preferably comprises heart disease, cancer, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, diabetes, tumor, spinal cord injury, aging, Alzheimer's disease and/or Parkinson's disease.
  • Liquid medium commercially purchased from the nutrient broth medium of Qingdao Rishui Biotechnology Co., Ltd.;
  • Clostridium butyricum (C.B.): ATCC19398, obtained from School of Food and Biological Engineering, Jiangsu University;
  • Enterobacter aerogenes E.A.: ATCC13048, obtained from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.;
  • Gelatin methacrylate (GelMA): self-made, 94% methacrylate commercially purchased from Shanghai Macklin Biochemical Technology Co., Ltd. and gelatin with a glue strength of 250 g commercially purchased from Shanghai Dibo Biotechnology Co., Ltd. Obtained by cross-linking by conventional methods in the art;
  • Hyaluronic acid (HA) 30% aqueous solution, molecular weight 800,000-1,000,000, purchased from Tianjin Sino Biochemical Technology Co., Ltd.;
  • 2,4-dihydroxybenzophenone commercially purchased from Tianjin Xiensiopude Technology Co., Ltd.;
  • UV curing instrument commercially purchased from Shanghai Luyang Instrument Co., Ltd.;
  • UV spectrophotometer evolution 220, commercially available from Thermo Scientific.
  • the hydrogen-producing biomicroneedle products 1 to 6 of the present invention were prepared by the following general preparation process.
  • the general preparation method comprises the following steps:
  • step (2) placing the mixed solution obtained in step (2) into the forming hole of the microneedle mold and filling the forming hole;
  • a solution containing a backing material as shown in Table 1 was applied to form a backing solution layer , and thermally curing the backing material to form a continuous backing, so that the hydrogen-producing biological microneedle array is attached to one side of the backing;
  • step (6) The backing obtained in step (6) and the microneedle array attached to one side of the backing were simultaneously heat-dried under the conditions shown in Table 1 so that the water content was less than 5% by weight, forming The hydrogen-producing biological microneedle product.
  • control sample was prepared using the same process as the general preparation process described above, except that the cultivation of hydrogen-producing bacteria was not carried out in step 1.
  • Table 2 Hydrogen production levels of the control sample and the samples of the hydrogen-producing biomicroneedle products prepared in Examples 1 to 6
  • Table 3 Effect data of the control sample and the samples of the hydrogen-producing biomicroneedle products prepared in Examples 1 to 6 in treating arthritis in mice
  • attachment and other terms should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral body; it can be directly connected, or An indirect connection through an intermediary may be an internal communication between two elements or an interaction relationship between two elements.
  • attachment and other terms should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral body; it can be directly connected, or An indirect connection through an intermediary may be an internal communication between two elements or an interaction relationship between two elements.
  • the term “about” used in the present invention has meanings known to those skilled in the art, preferably referring to the value modified by the term within ⁇ 50%, ⁇ 40%, ⁇ 30%, ⁇ 20%, ⁇ 10%, ⁇ 5% % or ⁇ 1% range.

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Abstract

L'invention concerne un produit de micro-aiguilles biologiques produisant de l'hydrogène. Le produit de micro-aiguilles biologiques produisant de l'hydrogène comprend un support et un réseau de micro-aiguilles biologiques produisant de l'hydrogène fixé à une surface latérale du support; le réseau de micro-aiguilles biologiques produisant de l'hydrogène comprend une pluralité de micro-aiguilles, dont chacune comprend un corps de base et une bactérie active produisant de l'hydrogène et un saccharide supporté dans le corps de base; et la bactérie active produisant de l'hydrogène peut produire de l'hydrogène à l'aide du saccharide. L'application du produit de micro-aiguilles sur un corps d'animal, en particulier la peau d'un corps humain, permet une absorption transdermique efficace de l'hydrogène, présente un soulagement et des effets thérapeutiques suffisants sur des maladies communes induites par des niveaux élevés de stress oxydatif, et surmonte les inconvénients de la résistance aux médicaments classiques, de nombreuses réactions secondaires, de mauvaise perméabilité, etc.
PCT/CN2021/101532 2021-06-22 2021-06-22 Produit de micro-aiguilles biologiques produisant de l'hydrogène et son procédé de préparation et son utilisation WO2022266841A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115025037A (zh) * 2022-06-10 2022-09-09 河北大学 一种负载活性小球藻的可分离微针及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109414575A (zh) * 2016-01-22 2019-03-01 特兰斯德姆公司 用微针阵列递送肉毒杆菌
WO2019048281A1 (fr) * 2017-09-06 2019-03-14 Stefano Scoglio Utilisation transdermique d'algues cyanobactériennes et/ou d'extraits de celles-ci, par l'intermédiaire de dispositifs de passage trans-pore de faible ou haute intensité, à des fins esthétiques et d'amélioration de la santé
CN111544758A (zh) * 2019-03-26 2020-08-18 华中科技大学同济医学院附属协和医院 一种载光敏剂的可溶性微针和微针阵列及制备方法
CN112206202A (zh) * 2020-11-20 2021-01-12 南京鼓楼医院 一种载活菌的微针的制备方法及应用
WO2021072313A1 (fr) * 2019-10-09 2021-04-15 Vaxess Technologies, Inc. Micro-aiguilles à base de fibroïne de soie et leurs utilisations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109414575A (zh) * 2016-01-22 2019-03-01 特兰斯德姆公司 用微针阵列递送肉毒杆菌
WO2019048281A1 (fr) * 2017-09-06 2019-03-14 Stefano Scoglio Utilisation transdermique d'algues cyanobactériennes et/ou d'extraits de celles-ci, par l'intermédiaire de dispositifs de passage trans-pore de faible ou haute intensité, à des fins esthétiques et d'amélioration de la santé
CN111544758A (zh) * 2019-03-26 2020-08-18 华中科技大学同济医学院附属协和医院 一种载光敏剂的可溶性微针和微针阵列及制备方法
WO2021072313A1 (fr) * 2019-10-09 2021-04-15 Vaxess Technologies, Inc. Micro-aiguilles à base de fibroïne de soie et leurs utilisations
CN112206202A (zh) * 2020-11-20 2021-01-12 南京鼓楼医院 一种载活菌的微针的制备方法及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDERSON ASHLEIGH, HEGARTY CATHERINE, CASIMERO CHARNETE, DAVIS JAMES: "Electrochemically Controlled Dissolution of Nanocarbon–Cellulose Acetate Phthalate Microneedle Arrays", APPLIED MATERIALS & INTERFACES, AMERICAN CHEMICAL SOCIETY, US, vol. 11, no. 39, 2 October 2019 (2019-10-02), US , pages 35540 - 35547, XP093017499, ISSN: 1944-8244, DOI: 10.1021/acsami.9b09674 *
LUO YAOJIA, LI YUAN-HONG: "Update of Melasma Treatment in 2020", CHINESE JOURNAL OF LASER MEDICINE & SURGERY, vol. 29, no. 5, 31 October 2020 (2020-10-31), pages 274 - 280, XP093017498, ISSN: 1003-9430, DOI: 10.13480/j.issn1003-9430.2020.0274 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115025037A (zh) * 2022-06-10 2022-09-09 河北大学 一种负载活性小球藻的可分离微针及其制备方法

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