WO2022266841A1 - Hydrogen-producing biological microneedle product and preparation method therefor and use thereof - Google Patents
Hydrogen-producing biological microneedle product and preparation method therefor and use thereof Download PDFInfo
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- 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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- hydrogen
- microneedle
- producing
- bacteria
- backing
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- 239000001257 hydrogen Substances 0.000 title claims abstract description 212
- 229910052739 hydrogen Inorganic materials 0.000 title claims abstract description 212
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 title claims abstract description 211
- 238000002360 preparation method Methods 0.000 title description 14
- 238000004519 manufacturing process Methods 0.000 title description 8
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 150000002431 hydrogen Chemical class 0.000 claims abstract description 7
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 30
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- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
A hydrogen-producing biological microneedle product. The hydrogen-producing biological microneedle product comprises a backing and a hydrogen-producing biological microneedle array attached to a side surface of the backing; the hydrogen-producing biological microneedle array comprises a plurality of microneedles, each of which comprises a base body and a hydrogen-producing active bacterium and a saccharide supported in the base body; and the hydrogen-producing active bacterium can produce hydrogen by using the saccharide. The application of the microneedle product to an animal body, especially the skin of a human body, achieves efficient transdermal absorption of hydrogen, has sufficient relief and therapeutic effects on common diseases induced by elevated oxidative stress levels, and overcomes the shortcomings of conventional drug resistance, many side reactions, poor permeability, etc.
Description
本发明涉及生物微针领域，具体涉及其中包含生物微针并且能够由该生物微针产生氢气的生物微针制品。本发明还涉及制备所述生物微针制品的方法和所述生物微针制品的用途。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.
动物体，尤其是人体的几乎所有器官都很容易受到氧化应激带来的伤害，症状表现不计其数，如疲倦、全身无力、肌肉和关节痛、消化不良、焦虑、抑郁、皮肤瘙痒、头痛，以及注意力难以集中和感染难以痊愈等。在动物体，尤其是人体内，由氧化应激水平升高诱发的最常见疾病有心脏病、癌症、骨关节炎、风湿性关节炎、类风湿性关节炎、糖尿病、肿瘤、脊髓损伤、衰老以及神经退化性问题，例如阿尔兹海默症、帕金森病等。Almost all organs of the animal body, especially the human body, are susceptible to damage from oxidative stress, with 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. In animals, especially humans, 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.
氢气是动物体，尤其是人体内的信号分子，具有抗氧化应激作用，被广泛应用于氧化应激疾病（尤其例如类风湿关节炎）的治疗中。其通过激活Nrf2信号通路，抑制MAPK、NF-κB和TGF-β1的表达，发挥抗氧化与抗炎作用。氢气治疗后关节活动评分表现为升高，氧化指标与炎症因子水平均降低。此外，生物安全性和优异的组织扩散性是氢气独有的优点。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-κB and TGF-β1 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.
生物制氢微生物能够产生氢气。它们大体可分为四类，包括绿藻、蓝细菌（也称为蓝藻）、光合细菌和发酵细菌。这些微生物具有绿色、节能、环保等优点。其中特别是发酵细菌，尤其是丁酸梭菌（Clostridium
butyricum，C.B.）和作为兼性厌氧菌的产气肠杆菌（Enterobacter
aerogenes，E.A.），广泛存在于土壤、水等环境中，生长条件要求不高，能够发酵多种糖类产生氢气，是产氢微生物的优异候选者。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.
然而，高效利用生物制氢微生物产生的氢气治疗或缓解氧化应激疾病是困难的。However, it is difficult to efficiently utilize hydrogen produced by biohydrogen-producing microorganisms to treat or alleviate oxidative stress diseases.
微针给药是一种新型的局部经皮给药技术，结合了贴膏剂的便捷性和皮下注射给药的有效性，规避了其它给药方式的不足，具有不触及神经、安全、无痛、高效渗透等多种优点。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.
微针制品通常包括多根长度一般不超过约1 mm的微针，所述微针能够在皮肤角质层形成微型通道，突破皮肤角质层的阻隔，促进药物的渗透，从而减少药物在角质层的蓄积量，增加到达表皮、真皮及皮下组织的药物剂量。因此微针制品被广泛应用于促进小分子、大分子药物的透皮吸收，用于肥胖症、糖尿病、癌症等诸多疾病的治疗，具有广泛的应用前景。另外，微针制品的使用极为方便，不需要专业培训，患者可以自行给药，意外***风险低，且使用后易于处理。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. In addition, 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.
现有技术中已经有人将活细菌和微针进行结合实现某些医疗目的，例如中国专利申请CN112206202A公开了一种载活菌的微针用于皮肤病的治疗。所述微针载有活的功能性细菌，这些细菌包括枯草芽孢杆菌、地衣芽孢杆菌、酵母菌、大肠杆菌、多黏类芽孢杆菌中的一种或多种。In the prior art, some people have combined live bacteria and microneedles to achieve certain medical purposes. For example, 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.
然而，现有技术中尚没有文献公开将生物制氢微生物与微针技术相结合，实现对由氧化应激水平升高诱发的常见疾病的高效缓解和治疗。However, there is no literature in the prior art that discloses the combination of biohydrogen-producing microorganisms and microneedle technology to achieve efficient relief and treatment of common diseases induced by elevated levels of oxidative stress.
本发明的目的就是要充分利用微针自身的优点和生物制氢微生物自身的优点，提供一种产氢生物微针制品，其通过负载在微针中的产氢活性菌和糖类产生氢气，能够在将该微针制品施用到皮肤后实现氢气的高效透皮吸收，对由氧化应激水平升高诱发的常见疾病有充分的缓解和治疗作用，解决传统药物耐药、副反应多、渗透性差等缺点。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.
为了解决所述技术问题，本发明提供以下技术方案：In order to solve the technical problem, the present invention provides the following technical solutions:
方案1. 一种产氢生物微针制品，其包含背衬和附着于所述背衬的一个侧面的产氢生物微针阵列，所述产氢生物微针阵列包含多根微针，其中每根微针包含基体以及负载在所述基体中的产氢活性菌和糖类，所述产氢活性菌能够利用所述糖类产生氢气，其中所述糖类优选选自葡萄糖、麦芽糖、果糖、乳糖、蔗糖或它们中的两种或更多种的混合物，更优选选自葡萄糖，所述糖类在所述微针中的含量为基于所述基体重量计的约2.5重量%至约10重量%，优选约5重量%至约7.5重量%，更优选约6重量%，所述产氢活性菌在所述微针中的含量为每克基体重量约1×10
7 cfu至约1×10
9 cfu，优选约1×10
8
cfu至约1.5×10
8
cfu，最优选约1.2×10
8 cfu。
Scheme 1. 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 cfu to about 1.5×10 8 cfu, most preferably about 1.2×10 8 cfu.
方案2. 根据方案1的产氢生物微针制品，其中所述产氢活性菌选自蓝藻、绿藻、光合细菌、发酵细菌或这些细菌中的两种或更多种的混合物，优选选自发酵细菌，更优选选自丁酸梭菌和/或产气肠杆菌，最优选选自产气肠杆菌。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.
方案3. 根据方案1或2的产氢生物微针制品，其中所述基体是通过将包含以下物质中的一种或多种的水溶液进行交联和/或干燥固化后形成的：聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮和聚乙烯醇，优选甲基丙烯酸酯明胶和/或透明质酸，更优选甲基丙烯酸酯明胶。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.
方案4. 根据方案1至3中任一项的产氢生物微针制品，其中所述背衬是通过将包含以下物质中的一种或多种的水溶液进行交联和/或干燥固化后形成的：聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮和聚乙烯醇，优选透明质酸。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.
方案5. 根据方案1至4中任一项的产氢生物微针制品，其中所述每根微针具有尖端和底端，所述尖端远离所述背衬，所述微针经由所述底端附着于所述背衬，所述尖端至底端的距离为约200 μm至约1 mm，所述底端具有约100 μm至约500 μm的底端直径，和相邻的微针尖端之间的间距为约300 μm至约800 μm。Scheme 5. The hydrogen-producing biological microneedle product according to any one of schemes 1 to 4, wherein each microneedle has a tip and a bottom end, the tip is far away from the backing, and the microneedle passes through the bottom The end is attached to the backing, the distance from the tip to the base is about 200 μm to about 1 mm, the base has a base diameter of about 100 μm to about 500 μm, and between adjacent microneedle tips The pitch is about 300 μm to about 800 μm.
方案6. 制备根据方案1至5中任一项的产氢生物微针制品的方法，该方法包括以下步骤：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:
（1）提供液体培养基，将该液体培养基高压灭菌后接种所述产氢活性菌，并培养所述产氢活性菌以获得含有传代产氢活性菌的液体培养基，优选在约30℃至约40℃下，优选约37℃下培养所述产氢活性菌约5小时至约20小时，优选约12小时以获得含有传代产氢活性菌的液体培养基，其中在所述含有传代产氢活性菌的液体培养基中，所述产氢活性菌的总含量为约0.2×10
7
cfu/mL至约0.4×10
9
cfu/mL，优选约0.3×10
8 cfu/mL，其中所述产氢活性菌优选选自蓝藻、绿藻、光合细菌、发酵细菌或这些细菌中的两种或更多种的混合物，更优选选自发酵细菌，尤其优选选自丁酸梭菌和/或产气肠杆菌，最优选选自产气肠杆菌；
(1) Provide a liquid medium, inoculate the hydrogen-producing active bacteria after autoclaving the liquid medium, and cultivate the hydrogen-producing active bacteria to obtain a liquid medium containing the hydrogen-producing active bacteria, preferably at about 30 °C to about 40°C, preferably at about 37°C, culturing the hydrogen-producing active bacteria for about 5 hours to about 20 hours, preferably about 12 hours to obtain a liquid medium containing passaged hydrogen-producing active bacteria, wherein In the liquid medium of active hydrogen-producing bacteria, 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 preferably selected from Enterobacter aerogenes;
（2）向由步骤（1）获得的含有传代产氢活性菌的液体培养基中添加能够形成所述基体的基体材料和所述糖类以形成混合溶液，其中在所述混合溶液中，所述基体材料的浓度为基于所述混合溶液总重量计的约20重量%至约40重量%，优选约25重量%至约35重量%，更优选约30重量%，和所述糖类的浓度为约10 mg/mL至约20 mg/mL，优选约15
mg/mL，所述糖类优选选自葡萄糖、麦芽糖、果糖、乳糖、蔗糖或它们中的两种或更多种的混合物，更优选选自葡萄糖，所述基体材料优选选自聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮、聚乙烯醇或它们中的两种或更多种的混合物，更优选甲基丙烯酸酯明胶和/或透明质酸，最优选甲基丙烯酸酯明胶；(2) Adding the matrix material capable of forming the matrix and the sugar to the liquid culture medium containing the passaged hydrogen-producing active bacteria obtained in step (1) to form a mixed solution, wherein in the mixed solution, the 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, and 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, or their A mixture of two or more of, more preferably methacrylate gelatin and/or hyaluronic acid, most preferably methacrylate gelatin;
（3）提供微针模具，该微针模具包含上表面和从所述上表面向下延伸的成型孔，其中所述成型孔具有尖端和底端，所述尖端远离所述上表面，所述底端平面与所述上表面齐平，所述尖端至底端的距离优选为约200 μm至约1 mm，所述底端优选具有约100 μm至约500 μm的底端直径，和相邻的尖端之间的间距优选为约300 μm至约800 μm；(3) providing 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 μm to about 1 mm, the base preferably has a base diameter from about 100 μm to about 500 μm, and adjacent The spacing between tips is preferably from about 300 μm to about 800 μm;
（4）将由步骤（2）获得的混合溶液置于所述成型孔中并填充所述成型孔容积的至少一部分，优选填满所述成型孔；(4) 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;
（5）使在所述成型孔中的包含所述基体材料、所述糖类和所述产氢活性菌的混合溶液进行交联，优选紫外交联约5秒至约15秒，优选约10秒，和/或进行干燥固化，优选热固化，从而在所述成型孔内形成所述微针，多个所述微针构成所述产氢生物微针阵列，每根微针具有尖端和底端，所述微针的尖端相对于所述微针的底端远离所述上表面；(5) Cross-linking the mixed solution containing the matrix material, the sugar and the hydrogen-producing active bacteria in the forming hole, preferably ultraviolet cross-linking for about 5 seconds to about 15 seconds, preferably about 10 seconds second, and/or carry out drying and curing, preferably thermal curing, so as to form the microneedles in the forming hole, a plurality of the microneedles constitute the hydrogen-producing biomicroneedle array, and each microneedle has a tip and a bottom end, the tip of the microneedle is far away from the upper surface relative to the bottom end of the microneedle;
（6）在所述微针的底端表面和所述微针模具的未被所述微针遮盖的上表面上施加包含背衬材料的溶液以形成背衬溶液层，并任选将所述背衬材料进行交联形成连续的背衬层，从而使所述产氢生物微针阵列附着于所述背衬溶液层或所述背衬层，所述背衬材料优选选自聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮、聚乙烯醇或它们中的两种或更多种的混合物，优选透明质酸；和(6) applying a solution containing a backing material on the bottom end surface of the microneedle and the upper surface of the microneedle mold not covered by the microneedle to form a backing solution layer, and optionally 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
（7）将由步骤（6）获得的背衬溶液层或背衬层并任选同时将所述产氢生物微针阵列进行干燥固化形成所述产氢生物微针制品。(7) 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.
方案7. 根据方案1至5中任一项的产氢生物微针制品或根据方案6的方法制备的产氢生物微针制品用于缓解和/或治疗动物体，优选人体的氧化应激疾病（优选心脏病、癌症、骨关节炎、风湿性关节炎、类风湿性关节炎、糖尿病、肿瘤、脊髓损伤、衰老、阿尔兹海默症和/或帕金森病）的用途。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).
方案8. 根据方案1至5中任一项的产氢生物微针制品或根据方案6的方法制备的产氢生物微针制品用于制备缓解和/或治疗动物体，优选人体的氧化应激疾病（优选心脏病、癌症、骨关节炎、风湿性关节炎、类风湿性关节炎、糖尿病、肿瘤、脊髓损伤、衰老、阿尔兹海默症和/或帕金森病）的医疗装置的用途。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.
通过以上本发明的各个技术方案实现了本发明的目的，即本发明充分利用了微针自身的优点和产氢活性菌自身的优点，提供了一种产氢生物微针制品，其通过负载在微针中的产氢活性菌和糖类产生氢气，能够在将所述微针制品施用到皮肤后实现氢气的高效透皮吸收，对由氧化应激水平升高诱发的常见疾病有充分的缓解和治疗作用，解决了传统药物耐药、副反应多、渗透性差等缺点。The purpose of the present invention is achieved through the various technical solutions of the present invention above, that is, 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.
具体地，本发明的优势在于：Specifically, the advantages of the present invention are:
（1）本发明的产氢微针制品所产生的氢气是人体内的信号分子，具有抗氧化应激作用，被应用于氧化应激疾病如类风湿关节炎的治疗中，通过激活Nrf2信号通路，抑制MAPK、NF-κB和TGF-β1的表达，发挥抗氧化与抗炎作用，氢气治疗后关节活动评分（包括临床评分、足爪部厚度等）表现为改进，炎症因子水平降低。(1) 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-κB and TGF-β1, 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.
（2）本发明的产氢微针制品可刺破限制药物吸收的皮肤角质层，促进氢气分子的扩散，但不至于引起疼痛。此外，生物安全性和优异的组织扩散性是氢气独有的优点。(2) 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. In addition, biosafety and excellent tissue diffusibility are unique advantages of hydrogen.
（3）本发明的产氢微针医疗制品利用微针与产气活菌结合发酵产氢的方式具有稳定性与高效性。(3) 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.
（4）本发明以微针模板倒模制备微针，方法简单，操作方便，价格低廉，可重复利用，不需要很高的技术要求，且易对微针阵列的基本形貌进行控制，安全性强，适宜推广。(4) 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.
为了更清楚地说明本发明，下面将对本发明的说明书附图进行描述和说明。In order to illustrate the present invention more clearly, the accompanying drawings of the present invention will be described and illustrated below.
显而易见地，下面描述中的附图仅仅说明了本发明的一些示例性实施方案的某些方面，对于本领域普通技术人员来说，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。需要说明的是，本发明的说明书附图仅是示意性的，其中描绘的部件尺寸及尺寸比例并不代表产品真实的尺寸及比例，而仅是为了示意性地呈现各部件之间的位置关系或连接关系。为了方便绘图与理解，部件的尺寸可能作出了不同比例的缩放。此外，相同或类似的附图标记表示相同或类似的构件。Apparently, the accompanying drawings in the following description only illustrate certain aspects of some exemplary embodiments of the present invention, and those skilled in the art can obtain Additional drawings. It should be noted that the accompanying drawings in the description of the present invention are only schematic, and the dimensions and proportions of the components depicted therein do not represent the real dimensions and proportions of the product, but are only for schematically presenting the positional relationship between the components or connection relationship. For the convenience of drawing and understanding, the dimensions of components may be scaled in different proportions. In addition, the same or similar reference numerals denote the same or similar members.
图1是示意性说明本发明的生物微针制品一部分的侧视图。Fig. 1 is a side view schematically illustrating a part of the biological microneedle product of the present invention.
图2是示意性说明用于制备本发明的生物微针制品的微针模具一部分的侧视图。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.
图3是说明本发明制备实施例2的生物微针制品的产氢效果的照片。3 is a photo illustrating the hydrogen production effect of the biological microneedle product of Preparation Example 2 of the present invention.
附图标记说明Explanation of reference signs
100
生物微针制品100
Biological Microneedle Products
110
微针110
Microneedles
120
背衬120
Backing
200
微针模具200
Micro needle mold
201
成型孔201
Forming hole
202
上表面。202
upper surface.
下文中，将参照附图详细描述本发明。Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
在第一方面，本发明涉及一种产氢生物微针制品100。如说明书附图1所示的，本发明的产氢生物微针制品100包含背衬120和附着于所述背衬120的一个侧面的产氢生物微针阵列，所述产氢生物微针阵列包含多根微针110，其中每根微针110包含基体以及负载在所述基体中的产氢活性菌和糖类，所述产氢活性菌能够利用所述糖类产生氢气。In a first aspect, the present invention relates to a hydrogen-producing biological microneedle product 100 . As shown in Figure 1 of the specification, 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.
在如上所述的本发明的产氢生物微针制品100中，所述糖类没有特别限制，只要其能够被所述产氢活性菌发酵并稳定地产生足够的氢气即可。在本发明中，所述糖类可以包括但不限于葡萄糖、麦芽糖、果糖、乳糖、蔗糖或它们中的两种或更多种的混合物，更优选葡萄糖。In the hydrogen-producing biological microneedle product 100 of the present invention as described above, the sugar is not particularly limited, as long as it can be fermented by the hydrogen-producing active bacteria and stably produce enough hydrogen. In the present invention, the sugars may include but not limited to glucose, maltose, fructose, lactose, sucrose or a mixture of two or more thereof, more preferably glucose.
所述糖类在所述微针110中的含量可以根据所采用的产氢活性菌不同而在宽范围内变化。例如，在本发明中，所述糖类在所述微针110中的含量优选为基于所述基体重量计的约2.5重量%至约10重量%，优选约5重量%至约7.5重量%，更优选约6重量%。在此，所述糖类的含量若低于约2.5重量%，则所述微针110中含有的营养成分不足以满足产氢活性菌的良好生长，从而会导致产氢效率下降。所述糖类的含量若高于约10重量%，则可能会导致所述微针110的机械强度显著下降。The content of the sugar in the microneedles 110 can vary in a wide range depending on the hydrogen-producing active bacteria used. For example, in the present invention, 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. Here, if 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. If the content of the sugar is higher than about 10% by weight, the mechanical strength of the microneedles 110 may decrease significantly.
在如上所述的本发明的产氢生物微针制品100中，所述产氢活性菌的种类没有特别限制，只要其能够发酵所述糖类并稳定地产生足够的氢气即可。在本发明中，所述产氢活性菌优选选自蓝藻、绿藻、光合细菌、发酵细菌或这些细菌中的两种或更多种的混合物，更优选选自发酵细菌，尤其优选选自丁酸梭菌和/或产气肠杆菌，最优选选自产气肠杆菌。In the hydrogen-producing biological microneedle product 100 of the present invention as described above, 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. In the present invention, 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.
在本发明的情况下，由于本发明的产氢生物微针制品100在制备过程中需要交联和/或干燥固化步骤，并且这些交联和/或干燥固化步骤可能需要采用紫外、热、电子束、辐射等多种手段，而属于发酵细菌种类的丁酸梭菌和/或产气肠杆菌在经由诸如紫外交联的过程后，通过对菌活力的测定表明极短紫外光照不会显著影响这些菌的活力，并且还由于产气肠杆菌和/或丁梭酸菌广泛存在于土壤、水等环境中，生长条件要求不高，能够发酵多种糖类产生氢气，是产氢微生物的优异候选者，因此，本发明所采用的产氢活性菌优选产气肠杆菌和/或丁梭酸菌，其中最优选选自产气肠杆菌。In the case of the present invention, since 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. Candidates, therefore, the active hydrogen-producing bacteria used in the present invention are preferably Enterobacter aerogenes and/or Clostridium butyricum, most preferably selected from Enterobacter aerogenes.
另外，在如上所述的本发明的产氢生物微针制品100中，所述产氢活性菌在所述微针110中的含量没有特别限制，只要其能够发酵所述糖类并稳定地产生足够的氢气即可。在本发明中，所述产氢活性菌在所述微针110中的含量优选在每克基体重量约1×10
7
cfu至约1×10
9
cfu范围内，优选约1×10
8 cfu至约1.5×10
8 cfu范围内，最优选约1.2×10
8
cfu。如本领域技术人员公知的，所述产氢活性菌在所述微针110中的含量可通过平板菌落计数法确定。在此，所述产氢活性菌的含量若低于约1×10
7 cfu/g，则产氢活性菌浓度太低，导致产氢效率不够，所述产氢活性菌的含量若高于约1×10
9
cfu/g，则会导致微针110中含有的营养成分不足以满足产氢活性菌的充分生长，从而也会导致产氢效率下降。
In addition, in the hydrogen-producing biological microneedle product 100 of the present invention as described above, 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. In the present invention, 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. As known to those skilled in the art, the content of the hydrogen-producing bacteria in the microneedles 110 can be determined by plate counting. Here, if 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.
在如上所述的本发明的产氢生物微针制品100中，其中形成所述基体的基体材料没有特别限制，只要本领域中采用的制备微针制品的常用基体材料都可用于本发明。然而，考虑到在本发明的情况下所形成的微针110需要具有一定的机械强度，并且固化后所述微针110还优选需要具有一定的孔隙度，所述基体优选是通过将包含以下物质中的一种或多种的水溶液进行交联和/或干燥固化后形成的：聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮和聚乙烯醇，优选甲基丙烯酸酯明胶和/或透明质酸，更优选甲基丙烯酸酯明胶。In the hydrogen-producing biological microneedle product 100 of the present invention as described above, 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. However, considering that the formed microneedle 110 needs to have a certain mechanical strength in the case of the present invention, and the microneedle 110 preferably needs to have a certain porosity after curing, 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.
在此，所述基体优选选自甲基丙烯酸酯明胶和/或透明质酸。甲基丙烯酸酯明胶（GelMA）是由甲基丙烯酸酐（MA）与明胶（Gelatin）制备获得，其中使它们在光敏剂介导下经紫外光照射发生交联，形成具有一定强度的孔状结构。GelMA具有优异的生物相容性。透明质酸是一种粘多糖，具有保护皮肤的作用，可应用于加速伤口的愈合。Here, 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.
在如上所述的本发明的产氢生物微针制品100中，其中形成所述背衬120的背衬材料没有特别限制，只要本领域中采用的制备微针的常用背衬材料都可用于本发明。然而，在本发明中，考虑到所形成的背衬120需要具有一定的机械强度和柔韧性，所述背衬120优选是通过将包含以下物质中的一种或多种的水溶液进行交联和/或干燥后形成的：聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮和聚乙烯醇，优选透明质酸。In the hydrogen-producing biological microneedle product 100 of the present invention as described above, 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. However, in the present invention, considering that the formed backing 120 needs to have certain mechanical strength and flexibility, 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.
如上所述的，透明质酸是一种粘多糖，具有保护皮肤的作用，可应用于加速伤口的愈合，因此优选在本发明中用作所述背衬材料。As mentioned above, 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.
此处，所形成的背衬120的厚度没有限制，但考虑到所述背衬120要具有一定的强度和柔韧性，优选所述背衬120的厚度为约0.1至约15 mm，更优选约1至约10 mm，最优选约2至约3 mm。Here, 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.
另外，优选在一些情况下，在本发明的产氢生物微针制品100中，形成所述基体和所述背衬120的材料是相同的。在这些情况下，微针110和背衬120的结合更稳固，并且在如下所述的制备所述产氢生物微针制品100的方法中，可以使所述微针阵列和所述背衬120一体成型，简化了制备过程。可另选地，在另一些情况下，所述基体和所述背衬120的材料优选是不同的，例如，所述基体材料是甲基丙烯酸酯明胶，和所述背衬材料是透明质酸。在这种情况下，所述微针110具有一定强度并具有孔状结构和优异的生物相容性，同时所述背衬120对皮肤有更好的保护作用，可用于加速伤口的愈合。In addition, preferably in some cases, in the hydrogen-producing biological microneedle product 100 of the present invention, 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. Alternatively, in other cases, 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 . In this case, 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.
需要说明的是，本领域技术人员完全有能力根据希望应用的情况对所述基体材料和背衬材料做出适当选择。It should be noted that those skilled in the art are fully capable of making appropriate choices for the base material and backing material according to the desired application.
另外，在本发明的产氢生物微针制品100中，所述微针110的尺寸和形状没有特别限制，其可根据本发明的产氢生物微针制品100所施用的部位和要治疗的疾病而在宽范围内变化。In addition, in the hydrogen-producing biological microneedle product 100 of the present invention, 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.
例如，如说明书附图1所示的，在本发明的产氢生物微针制品100中，所述每根微针110具有尖端和底端，所述尖端远离所述背衬120，所述微针110经由所述底端附着于所述背衬120，所述尖端至底端的高度h没有特别限制，但优选为约200 μm至约1 mm。所述高度h优选不低于约200 μm，否则所述微针110不易刺穿一些动物体，优选人体的某些部位的皮肤角质层。然而，所述高度h也优选不高于约1 mm，否则会刺穿一些动物体，优选人体的某些部位的皮肤角质层达到神经层，从而引起疼痛。For example, as shown in Figure 1 of the specification, in the hydrogen-producing biological microneedle product 100 of the present invention, 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 μm to about 1 mm. The height h is preferably not less than about 200 μm, otherwise the microneedles 110 are not easy to pierce the stratum corneum of some parts of animal body, preferably human body. However, 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.
另外，如说明书附图1所示的，在本发明的产氢生物微针制品100中，所述微针110的底端优选具有约100 μm至约500 μm的底端直径w。所述底端直径w优选不小于约100 μm，否则会使得微针110的机械强度不足，容易折断。另外，所述底端直径w也优选不大于约500 μm，否则会在将本发明的产氢生物微针制品100施用到一些动物体，优选人体的某些部位后，在皮肤上留下较大的孔洞，导致关于皮肤美观和愈合方面的问题。In addition, as shown in Fig. 1 of the specification, in the hydrogen-producing biological microneedle product 100 of the present invention, the bottom end of the microneedle 110 preferably has a bottom diameter w of about 100 μm to about 500 μm. The diameter w of the bottom end is preferably not less than about 100 μm, otherwise the mechanical strength of the microneedles 110 will be insufficient and easily broken. In addition, the diameter w of the bottom end is also preferably not greater than about 500 μm, 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.
另外，在本发明的产氢生物微针制品100中，所述微针110的立体形状没有特别限制，其可以是圆柱形、锥形、圆台形等或它们的结合，优选是规则的或不规则的圆锥、类圆锥、三棱锥、四棱锥或更高级棱锥等形状，并且这些圆锥、类圆锥、三棱锥、四棱锥或更高级棱锥可以是正锥体或斜锥体。In addition, in the hydrogen-producing biological microneedle product 100 of the present invention, 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.
另外，如附图1所示的，在本发明的产氢生物微针制品100中，相邻的微针110尖端之间的间距d优选为约300 μm至约800 μm。间距在此范围内可使得本发明的产氢生物微针制品100在刺入角质层深度和氢气给药效率等多方面均实现最优化效果。In addition, as shown in FIG. 1 , in the hydrogen-producing biological microneedle product 100 of the present invention, the distance d between the tips of adjacent microneedles 110 is preferably about 300 μm to about 800 μm. 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.
需要说明的是，本领域技术人员有能力根据实际应用情况对本发明的产氢生物微针制品100的微针110的形状和尺寸等做出适当选择。It should be noted that those skilled in the art are capable of making appropriate choices for the shape and size of the microneedles 110 of the hydrogen-producing biomicroneedle product 100 of the present invention according to actual application conditions.
另外，在实际应用中，本发明的产氢生物微针制品100中的多根微针110在尺寸、形状和组成等方面彼此之间也可以是相同或不同的。在此，多根微针110彼此之间的差异可以是多方面的，包括但不限于形状、高度h、底面直径w、尖端距离d、包含的基体材料、包含的产氢活性菌种类以及包含的糖类等。例如，在本发明的产氢生物微针制品100中，一部分微针110可具有较高含量的产氢活性菌以及糖类，而另一部分微针110可具有较低含量的产氢活性菌以及糖类。或者，所述微针微针110可间隔地具有规则周期变化的体积。在这些情况下，可以拓宽本发明的产氢生物微针制品100缓解和/或治疗多种疾病的适用性。In addition, in practical applications, 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. Here, 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. For example, in the hydrogen-producing biological microneedle product 100 of the present invention, 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. Alternatively, the 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.
另外，如本领域技术人员容易理解的，在本发明的产氢生物微针制品100中的微针110和/或背衬120中，除了上述明确说明的成分外，还可以根据需要包含本领域中公知的其它助剂，包括但不限于成型剂、防腐剂和光引发剂（光敏介导剂）等，例如2,4-二羟基二苯甲酮、二苯基乙酮等。所述其它助剂的量优选不超过所述微针110的基体和/或所述背衬120重量的约10重量%，更优选约5重量%，最优选不超过约1重量%。In addition, as those skilled in the art can easily understand, the microneedles 110 and/or the backing 120 in the hydrogen-producing biomicroneedle product 100 of the present invention, in addition to the above-mentioned clearly stated components, 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.
在本发明的第二方面，本发明还涉及制备如上所述的根据本发明的产氢生物微针制品100的方法，该方法包括以下步骤1-7。In the second aspect of the present invention, 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.
步骤1：提供液体培养基，将该液体培养基高压灭菌后接种所述产氢活性菌，并培养所述产氢活性菌以获得含有传代产氢活性菌的液体培养基。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.
在上述步骤1中，所述液体培养基可以是本领域中公知的营养肉汤液体培养基。本领域技术人员有能力根据所选用的产氢活性菌方便地确定所述液体培养基的种类。In the above step 1, 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.
另外，所述培养过程也是本领域中常用的培养过程。在本发明的情况下，优选可以将所述产氢活性菌在约30℃至约40℃下，优选约37℃下培养约5小时至约20小时，优选约12小时以获得含有传代产氢活性菌的液体培养基。In addition, the culture process is also a common culture process in the art. In the case of the present invention, preferably, 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.
在所述含有传代产氢活性菌的液体培养基中，所述产氢活性菌的含量没有特别限制，只要其与以下步骤2中使用的基体材料和糖类的用量匹配使得它在最终微针制品100中的含量符合上文限定的范围即可。例如，所述产氢活性菌的含量的具体数值可根据所述产氢活性菌在所述微针110中的希望的含量和在如下步骤2中所采用的基体材料在混合溶液中的浓度而确定。具体地，如果所述产氢活性菌在所述微针110中的含量被设定为约1×10
8 cfu/g，并且在如下步骤2中采用的基体材料在混合溶液中的浓度被设定为约30重量%的情况下，所述产氢活性菌在所述液体培养基中的最终总含量应被设定为约0.3×10
8 cfu/mL。
In the liquid culture medium containing subcultured hydrogen-producing 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. For example, 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. Specifically, if 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.
在此，优选所述产氢活性菌在液体培养基中的含量为约0.2×10
7 cfu/mL至约0.4×10
9 cfu/mL，优选约0.3×10
8
cfu/mL，所述含量可通过本领域中公知的方法测定，例如平板菌落计数法。
Here, it is preferable that 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.
另外，如上文关于本发明的产氢生物微针制品100的部分所述的，所述产氢活性菌优选选自蓝藻、绿藻、光合细菌、发酵细菌或这些细菌中的两种或更多种的混合物，更优选选自发酵细菌，尤其优选选自丁酸梭菌和/或产气肠杆菌，最优选选自产气肠杆菌。In addition, as described above in relation to the hydrogen-producing biological microneedle product 100 of the present invention, 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.
步骤2：向由步骤1获得的含有传代产氢活性菌的液体培养基中添加能够形成所述基体的基体材料和所述糖类以形成混合溶液。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.
在上述步骤2中，在所述混合溶液中，所述基体材料的浓度没有特别限制，只要其量能够确保形成本发明的最终生物微针制品100中的微针110即可。然而，优选所述基体材料在所述液体培养基中的含量为约20重量%至约40重量%，优选约25重量%至约35重量%，更优选约30重量%。在这个范围内，由所述基体材料形成的微针110既具有足够的机械强度，又具有一定的孔隙度，从而能够使产氢效率最佳。In the above step 2, in the 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. However, it is preferred that 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. Within this range, 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.
在上述步骤2中，在所述混合溶液中，所述糖类的浓度没有特别限制，只要其量能够确保它在最终微针制品100中的含量符合上文限定的范围即可。然而，优选所述糖类的浓度为约10 mg/mL至约20 mg/mL，更优选约15
mg/mL。In the above step 2, in the mixed solution, 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. However, it is preferred that 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.
另外，如上文关于本发明的产氢生物微针制品100的部分所述的，所述基体材料优选选自聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮、聚乙烯醇或它们中的两种或更多种的混合物，更优选甲基丙烯酸酯明胶和/或透明质酸，最优选甲基丙烯酸酯明胶；和所述糖类优选选自葡萄糖、麦芽糖、果糖、乳糖、蔗糖，更优选选自葡萄糖。In addition, as mentioned above in relation to the hydrogen-producing biological microneedle product 100 of the present invention, 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.
步骤3：提供微针模具200，如说明书附图2所示的，所述微针模具200包含上表面202并包含从所述上表面202向下延伸的成型孔201，其中所述成型孔201具有尖端和底端，所述尖端远离所述上表面202，所述底端平面与所述上表面202齐平。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 .
在上述步骤3中，在所述微针模具200中，所述成型孔201的立体形状要与希望形成的微针110的形状相匹配。如上所述的，其可以是圆柱形、锥形、圆台形等或它们的结合，优选是规则的或不规则的圆锥、类圆锥、三棱锥、四棱锥或更高级棱锥等形状，并且这些圆锥、类圆锥、三棱锥、四棱锥或更高级棱锥可以是正锥体或斜锥体。In the above step 3, in the microneedle mold 200, the three-dimensional shape of the forming hole 201 should match the shape of the desired microneedle 110 to be formed. As mentioned above, 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.
所述成型孔201应当具有与所述微针110的高度h、底面宽度w和尖端间距d相对应的成型孔201的高度h、底面宽度w和尖端间距d。然而，在一些情况下，所述成型孔201还可以具有大于所述微针110的高度h和底面宽度w的成型孔201的高度h和底面宽度w。在后者的情况下，所形成的微针110并不会充满所述成型孔201。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 . However, in some cases, 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 .
任选地，所述微针模具200的上表面202（其包括所述成型孔201的内表面）可涂覆有防粘层。Optionally, 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.
在本发明中，所述微针模具200是可商购获得的，例如其可以是商购自台州微芯医药科技公司的定制的PDMS模具，所述模具参数可根据如上所述的针体尺寸的需要进行定制。具体地，在本发明实施例中所采用的微针模具200中，所有成型孔201的高度h为约600 μm，底面宽度w为约320 μm，尖端间距d为约500 μm，所述微针模具200整体长×宽=约15 mm×约15 mm。In the present invention, 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. Specifically, in the microneedle mold 200 used in the embodiment of the present invention, the height h of all the forming holes 201 is about 600 μm, the width w of the bottom surface is about 320 μm, and the tip distance d is about 500 μm. The overall length of the mold 200×width=about 15 mm×about 15 mm.
步骤4：将由步骤2获得的混合溶液置于所述成型孔201中并填充所述成型孔201容积的至少一部分。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 .
在上述步骤4中，所述混合溶液填充所述成型孔201的容积量没有特别限制，但优选填充所述成型孔201容积量的至少约1/4，至少约1/3、至少约1/2、至少约2/3，至少约3/4，最优选填满所述成型孔201。在不填满所述成型孔201的情况下，成型孔201容积的未被微针110填充的部分应当在以下步骤6中被背衬材料溶液所填充。In the above step 4, 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.
步骤5：使在所述成型孔201中的包含所述基体材料、所述糖类和所述产氢活性菌的混合溶液进行交联和/或干燥固化，从而在所述成型孔201内形成所述微针110，多个所述微针110构成所述产氢生物微针阵列。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.
在上述步骤5中，所述交联可采用紫外、热、电子束、辐射等多种手段实现。所述干燥固化可例如通过自然干燥或烘箱中在一定温度下进行干燥实现。例如，本领域技术人员能够确定所述交联可通过在本领域中公知的光敏介导剂的存在下进行紫外交联实现。所述紫外交联的时间优选为小于约20秒，优选约5秒至约15秒，更优选约10秒；所述烘箱干燥优选是在低于约50℃的温度下干燥小于约4小时，优选约1小时至约3小时。超过所述上限可能会引起产氢活性菌的显著失活。In the above step 5, 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. For example, 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.
步骤6：在所述微针110的底端表面和所述微针模具200的未被所述微针110遮盖的上表面202上施加包含背衬材料的溶液以形成背衬溶液层，从而所述产氢生物微针阵列附着于所述背衬溶液层。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.
在上述步骤6中，如上文关于本发明的产氢生物微针制品100的部分所述的，所述背衬材料优选选自聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮、聚乙烯醇或它们中的两种或更多种的混合物，优选透明质酸。In the above step 6, as described above about the hydrogen-producing biological microneedle product 100 of the present invention, 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.
在上述步骤6中，任选将所述背衬材料进行交联形成连续的背衬层，从而所述产氢生物微针阵列附着于所述背衬层。在此，所述交联可采用紫外、热、电子束、辐射等多种手段实现。例如，本领域技术人员能够确定所述交联可通过在本领域中公知的光敏介导剂的存在下进行紫外交联实现。所述紫外交联的时间优选为小于约20秒，优选约5秒至约15秒，更优选约10秒。在此，超过所述上限可能会引起产氢活性菌的显著失活。In the above step 6, 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. Here, the crosslinking can be achieved by various means such as ultraviolet rays, heat, electron beams, and radiation. For example, 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.
步骤7：将由步骤6获得的背衬溶液层和任选同时将附着于所述背衬溶液层的微针阵列进行干燥固化形成所述产氢生物微针制品100。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 .
在上述步骤7中，所述干燥可例如通过自然干燥或或优选在烘箱中于一定温度下进行干燥实现。所述烘箱干燥优选是在低于约50℃的温度下，例如在约37℃下干燥小于约4小时，优选约1小时至约3小时。超过所述上限可能会引起产氢活性菌的显著失活。In the above step 7, 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.
在如上所述步骤6和7中，所述干燥步骤优选使所述微针110和所述背衬120中的水含量低于约20重量%，优选低于约10重量%，最优选低于约5重量%。In steps 6 and 7 as described above, 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.
在如上所述的制备本发明的产氢生物微针制品100的方法中，如上文所述的在本发明的产氢生物微针制品100方面描述的技术特征及其优选范围在此处仍然适用。In the method for preparing the hydrogen-producing bio-microneedle product 100 of the present invention as described above, the technical features described above in terms of the hydrogen-producing bio-microneedle product 100 of the present invention and their preferred ranges still apply here .
在本发明的第三方面，本发明涉及如上第一方面的产氢生物微针制品100或根据如上第二方面的制备所述产氢生物微针制品100的方法制备的产氢生物微针制品100用于缓解和/或治疗动物体的氧化应激疾病的用途。In the third aspect of the present invention, 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.
在本发明的第四方面，本发明涉及如上第一方面的产氢生物微针制品100或根据如上第二方面的制备所述产氢生物微针制品100的方法制备的产氢生物微针制品100用于制备缓解和/或治疗动物体的氧化应激疾病的医疗装置的用途。In the fourth aspect of the present invention, 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.
在上述第三和第四方面中，所述动物体优选是人体。所述氧化应激疾病优选包括心脏病、癌症、骨关节炎、风湿性关节炎、类风湿性关节炎、糖尿病、肿瘤、脊髓损伤、衰老、阿尔兹海默症和/或帕金森病。In the third and fourth aspects above, 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.
同样，在上述第三和第四方面中，如上文所述的在本发明的产氢生物微针制品100及其制备方法方面描述的技术特征及其优选范围在此处仍然适用。Likewise, in the third and fourth aspects above, the technical features and preferred ranges described above in terms of the hydrogen-producing biological microneedle product 100 and its preparation method of the present invention still apply here.
下文将通过示例性实施例来具体说明本发明及其优点。对示例性实施例的描述仅仅是说明性的，其决不作为对本公开及其应用或使用的任何限制。本公开可以以许多不同的形式实现，不限于这里所述的实施例。提供这些实施例是为了使本公开透彻且完整，并且向本领域技术人员充分表达本公开的范围。应注意到：除非另有说明，否则在这些实施例中阐述的部件和步骤的相对布置、材料的组分、数字表达式和数值等应被解释为仅仅是示例性的，而不是作为限制。The invention and its advantages will be described in detail below by means of exemplary embodiments. The description of the exemplary embodiments is illustrative only, and in no way serves as any limitation of the disclosure, its application or uses. The present disclosure can be implemented in many different forms and is not limited to the embodiments described here. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. It should be noted that unless otherwise stated, the relative arrangement of components and steps, material composition, numerical expressions and numerical values, etc. set forth in these embodiments should be interpreted as merely exemplary and not limiting.
使用的材料和仪器：Materials and instruments used:
液体培养基：商购自青岛日水生物技术有限公司的营养肉汤培养基；Liquid medium: commercially purchased from the nutrient broth medium of Qingdao Rishui Biotechnology Co., Ltd.;
丁酸梭菌（C.B.）：ATCC19398，得自江苏大学食品与生物工程学院；Clostridium butyricum (C.B.): ATCC19398, obtained from School of Food and Biological Engineering, Jiangsu University;
产气肠杆菌（E.A.）：ATCC13048，得自北京中科质检生物技术有限公司；Enterobacter aerogenes (E.A.): ATCC13048, obtained from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.;
甲基丙烯酸酯明胶（GelMA）：自制，由商购自上海麦克林生化科技有限公司的94%的甲基丙烯酸酯和商购自上海笛柏生物科技有限公司的胶强度为250 g的明胶根据本领域中常规方法交联获得；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;
透明质酸（HA）：30%水溶液，分子量80-100万，商购自天津希恩思生化科技有限公司；Hyaluronic acid (HA): 30% aqueous solution, molecular weight 800,000-1,000,000, purchased from Tianjin Sino Biochemical Technology Co., Ltd.;
微针模具：长×宽=15×15 mm，正圆锥成型孔的高度h：600 μm，底面宽度w：320 μm，尖端间距d：500 μm，商购自台州微芯医药科技公司；Microneedle mold: length × width = 15 × 15 mm, height h of positive conical forming hole: 600 μm, width w of bottom surface: 320 μm, distance between tips d: 500 μm, commercially purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd.;
2,4-二羟基二苯甲酮（光敏介导剂）：商购自天津希恩思奥普德科技有限公司；2,4-dihydroxybenzophenone (photosensitive mediator): commercially purchased from Tianjin Xiensiopude Technology Co., Ltd.;
紫外固化仪：商购自上海路阳仪器有限公司；UV curing instrument: commercially purchased from Shanghai Luyang Instrument Co., Ltd.;
紫外分光光度计：evolution 220，商购自Thermo
Scientific。UV spectrophotometer: evolution 220, commercially available from Thermo
Scientific.
the
制备实施例1至6：Preparation Examples 1 to 6:
以如下通用制备过程制备本发明的产氢生物微针制品1至6。所述通用制备方法包括以下步骤：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:
（1）提供所述液体培养基，将该液体培养基高压灭菌后接种所述产氢活性菌（C.B.或E.A.），并在37℃下培养12小时以获得含有如表1中所示含量的传代产氢活性菌的液体培养基；(1) Provide the liquid medium, inoculate the hydrogen-producing bacteria (C.B. or E.A.) after autoclaving the liquid medium, and culture it at 37°C for 12 hours to obtain the content as shown in Table 1 The liquid culture medium of the passaging hydrogen-producing active bacteria;
（2）向由步骤（1）获得的含有传代产氢活性菌的液体培养基中添加如表1中所示的量的基体材料（GelMA或HA）和糖类（葡萄糖或果糖）以形成混合溶液，并添加如表1中所示量的2,4-二羟基二苯甲酮；(2) Add matrix material (GelMA or HA) and sugar (glucose or fructose) in the amount shown in Table 1 to the liquid medium containing passaged hydrogen-producing active bacteria obtained in step (1) to form a mixed solution, and add 2,4-dihydroxybenzophenone in the amount shown in Table 1;
（3）提供所述微针模具；(3) Provide the microneedle mold;
（4）将由步骤（2）获得的混合溶液置于所述微针模具的成型孔中并填满所述成型孔；(4) placing the mixed solution obtained in step (2) into the forming hole of the microneedle mold and filling the forming hole;
（5）使在所述成型孔中的包含所述基体材料、所述糖类和所述产氢活性菌的混合溶液在如表1中所示的条件下进行交联，从而在所述成型孔内形成所述微针；(5) The mixed solution containing the matrix material, the sugar and the hydrogen-producing bacteria in the forming hole is cross-linked under the conditions shown in Table 1, so that forming said microneedles within the pores;
（6）在所述微针的底端表面和所述微针模具的未被所述微针遮盖的上表面上施加包含如表1中所示的背衬材料的溶液以形成背衬溶液层，并将所述背衬材料进行热固化形成连续的背衬，从而使所述产氢生物微针阵列附着于所述背衬的一个侧面；和(6) On the bottom end surface of the microneedle and the upper surface of the microneedle mold not covered by the microneedle, 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; and
（7）将由步骤（6）获得的背衬和附着于所述背衬的一个侧面的微针阵列在如表1中所示的条件下同时进行热干燥使水含量低于5重量%，形成所述产氢生物微针制品。(7) 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.
对照样品的制备：Preparation of control samples:
采用与如上所述的通用制备过程相同的过程制备对照样品，其中只是在步骤1中不进行产氢活性菌的培养。The 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.
the
表1：对照样品和制备实施例1至6的制备Table 1: Preparation of Control Samples and Preparation Examples 1 to 6
（1）基于液体培养基总重量计(1) Based on the total weight of the liquid medium
（2）基于基体重量计(2) Based on matrix weight
the
产氢效率评价实施例Example of Hydrogen Production Efficiency Evaluation
取对照样品和制备实施例1至6的产氢生物微针制品的样品浸没在4 ml蒸馏水中。浸没了所述产氢生物微针制品的浸泡液中能够观察到产生氢气，如说明书附图3中的照片所示的。Take the control sample and the samples of the hydrogen-producing biological microneedle products prepared in Examples 1 to 6 and immerse them in 4 ml of distilled water. Hydrogen gas generation can be observed in the immersion solution in which the hydrogen-producing biological microneedle product is submerged, as shown in the photo in Figure 3 of the specification.
如现有技术文献所公开的（参见Penghe Zhao等人，Local
generation of hydrogen for enhanced photothermal therapy，Nature communications，DOI:
10.1038/s41467-018-06630-2；和Cong Xu等人，Magnesium-Based Micromotors as Hydrogen Generators for
Precise Rheumatoid Arthritis Therapy，
Nano
letters，DOI: 10.1021/acs.nanolett.0c04438及该文献的支持信息（supporting information）），根据标准曲线定量法，通过MB吸光度法测定不同时间浸泡液中产生的氢气水平。
As disclosed in prior art literature (see Penghe Zhao et al., Local generation of hydrogen for enhanced photothermal therapy, Nature communications, DOI: 10.1038/s41467-018-06630-2; and Cong Xu et al., Magnesium-Based Micromotors as Hydrogen Generators for Precise Rheumatoid Arthritis Therapy, Nano letters , DOI: 10.1021/acs.nanolett.0c04438 and the supporting information (supporting information) of this document), according to the standard curve quantification method, the MB absorbance method was used to measure the hydrogen produced in the soaking solution at different times hydrogen level.
具体操作步骤为：制备亚甲基蓝（MB）探针溶液（300 μg/L）。然后将浸没过产氢生物微针制品的浸泡液样品200 μL加入到所述MB探针溶液200 μL中，通过紫外分光光度计测定664 nm处吸光度。MB在664 nm处有紫外特征吸收，被氢还原后吸光度降低，MB减少量与氢气的量线性相关。MB（蓝色）+ 2H+ + e-
=leucoMB(无色)。根据上述公式，可以使用氧化还原滴定体积分析法确定氢气在浸泡液中的浓度。The specific operation steps are as follows: prepare methylene blue (MB) probe solution (300 μg/L). Then, 200 μL of the soaking liquid sample immersed in the hydrogen-producing biomicroneedle product was added to 200 μL of the MB probe solution, and the absorbance at 664 nm was measured by an ultraviolet spectrophotometer. MB has a characteristic ultraviolet absorption at 664 nm, and its absorbance decreases after being reduced by hydrogen, and the amount of MB reduction is linearly related to the amount of hydrogen. MB (blue) + 2H+ + e-
= leucoMB (colorless). According to the above formula, the redox titration volumetric analysis method can be used to determine the concentration of hydrogen in the soaking solution.
测量结果如表2所示。The measurement results are shown in Table 2.
the
表2：对照样品和制备实施例1至6的产氢生物微针制品的样品的产氢水平Table 2: Hydrogen production levels of the control sample and the samples of the hydrogen-producing biomicroneedle products prepared in Examples 1 to 6
the
the
如现有技术文献Cong Xu等人，Magnesium-Based
Micromotors as Hydrogen Generators for Precise Rheumatoid Arthritis Therapy.
Nano
letters，DOI: 10.1021/acs.nanolett.0c04438及该文献的支持信息（supporting information）中说明的，用于关节炎治疗的氢气通过MB探针法进行定量，能够达到17 μM左右即有良好的治疗效果。
As described in the prior art document Cong Xu et al., Magnesium-Based Micromotors as Hydrogen Generators for Precise Rheumatoid Arthritis Therapy. Nano letters , DOI: 10.1021/acs.nanolett.0c04438 and the supporting information (supporting information) of this document, use The hydrogen gas used in the treatment of arthritis is quantified by the MB probe method, and it can reach about 17 μM to have a good therapeutic effect.
由表2中的数据可见，本发明制备实施例1至6的产氢生物微针制品均能在高达3小时的时间内持续释放氢气，并且最终能够达到良好的治疗效果。其中制备实施例2的产氢生物微针制品效果最佳。It can be seen from the data in Table 2 that the hydrogen-producing biological microneedle products prepared in Examples 1 to 6 of the present invention can continuously release hydrogen for up to 3 hours, and can finally achieve a good therapeutic effect. Among them, the hydrogen-producing biological microneedle product of Preparation Example 2 has the best effect.
the
治疗效果实施例Examples of therapeutic effects
建立小鼠关节炎模型，将对照样品和制备实施例1至6的产氢生物微针制品固定于小鼠关节处，通过临床评分，足爪部厚度和关节组织中炎症因子水平等评价微针缓解或治疗关节炎的有效性。Establish a mouse arthritis model, fix the control sample and the hydrogen-producing biological microneedle products prepared in Examples 1 to 6 on the joints of the mice, and evaluate the microneedles through clinical scores, paw thickness, and inflammatory factor levels in joint tissues Effectiveness in alleviating or treating arthritis.
具体操作步骤可参见现有技术文献Maimoona Qindeel等人，Surfactant-Free,
Self-Assembled Nanomicelles-Based Transdermal Hydrogel for Safe and Targeted
Delivery of Methotrexate against Rheumatoid Arthritis.
ACS NANO，2020年3月，DOI: 10.1021/acsnano.0c00364。
For specific operation steps, please refer to the prior art document Maimooona Qindeel et al., Surfactant-Free, Self-Assembled Nanocelles-Based Transdermal Hydrogel for Safe and Targeted Delivery of Methotrexate against Rheumatoid Arthritis. ACS NANO , March 2020, DOI: 10.1021/acsnano .0c00364.
试验结果如表3所示。The test results are shown in Table 3.
the
表3：对照样品和制备实施例1至6的产氢生物微针制品的样品治疗小鼠关节炎的效果数据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
the
由表3中的数据可见，本发明制备实施例1至6的产氢生物微针制品均对小鼠关节炎具有很好的治疗效果。其中制备实施例2的产氢生物微针制品效果最佳。It can be seen from the data in Table 3 that the hydrogen-producing biological microneedle products prepared in Examples 1 to 6 of the present invention all have good therapeutic effects on mouse arthritis. Among them, the hydrogen-producing biological microneedle product of Preparation Example 2 has the best effect.
the
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的主旨或基本特征的情况下，能够以其它的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内，不应将权利要求中的任何附图标记视为限制所涉及的权利要求。It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, but that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the invention, and any reference sign in a claim shall not be construed as limiting the claim concerned.
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其它实施方式。这些其它实施方式也涵盖在本发明的保护范围内。In addition, it should be understood that although this specification is described according to implementation modes, not each implementation mode only includes an independent technical solution, and this description in the specification is only for clarity, and those skilled in the art should take the specification as a whole , the technical solutions in the various embodiments can also be properly combined to form other implementations that can be understood by those skilled in the art. These other implementations are also covered within the protection scope of the present invention.
还应当理解，以上所述的具体实施例仅用于解释本发明，本发明的保护范围并不限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明/发明的保护范围之内。It should also be understood that the specific embodiments described above are only used to explain the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent replacement or change of the scheme and its inventive concepts shall fall within the protection scope of the present invention/invention.
本发明中使用的“包括”或者“包含”等类似的词语意指在该词前的要素涵盖在该词后列举的要素，并不排除也涵盖其它要素的可能。术语“内”、“外”、“上”、“下”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制，当被描述对象的绝对位置改变后，则该相对位置关系也可能相应地改变。在本发明中，除非另有明确的规定和限定，术语“附着”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。本发明中使用的术语“约”具有本领域技术人员公知的含义，优选指该术语所修饰的数值在其± 50%，± 40%，± 30%，± 20%，± 10%，± 5%或± 1%范围内。The words "comprising" or "comprising" and similar words used in the present invention mean that the element before the word covers the element listed after the word, and does not exclude the possibility of also covering other elements. The orientation or positional relationship indicated by the terms "inner", "outer", "upper", "lower" and the like are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or It is implied that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation of the present invention. When the absolute position of the described object changes, the relative positional relationship may also change accordingly . In the present invention, unless otherwise clearly specified and limited, the term "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. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention according to specific situations. 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.
本公开使用的所有术语（包括技术术语或者科学术语）与本公开所属领域的普通技术人员理解的含义相同，除非另外特别定义。还应当理解，在诸如通用词典中定义的术语应当被理解为具有与它们在相关技术的上下文中的含义相一致的含义，而不应用理想化或极度形式化的意义来解释，除非本文有明确地这样定义。All terms (including technical terms or scientific terms) used in the present disclosure have the same meaning as understood by one of ordinary skill in the art to which the present disclosure belongs, unless otherwise specifically defined. It should also be understood that terms defined in, for example, general-purpose dictionaries should be understood to have meanings consistent with their meanings in the context of the relevant technology, and should not be interpreted in idealized or extremely formalized meanings, unless explicitly stated herein defined in this way.
对于相关领域普通技术人员已知的技术、方法和设备可能不作为详细讨论，但在适当情况下，所述技术、方法和设备应当被视为说明书的一部分。Techniques, methods and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, such techniques, methods and devices should be considered part of the description.
本发明说明书中引用的现有技术文献所公开的内容整体均通过引用并入本发明中，并且因此是本发明公开内容的一部分。The disclosures of the prior art documents cited in the description of the present invention are incorporated by reference in their entirety into the present invention and are therefore part of the disclosure content of the present invention.
Claims (8)
- 一种产氢生物微针制品（100），其包含背衬（120）和附着于所述背衬（120）的一个侧面的产氢生物微针阵列，所述产氢生物微针阵列包含多根微针（110），其特征在于每根微针（110）包含基体以及负载在所述基体中的产氢活性菌和糖类，所述产氢活性菌能够利用所述糖类产生氢气，其中所述糖类优选选自葡萄糖、麦芽糖、果糖、乳糖、蔗糖或它们中的两种或更多种的混合物，更优选选自葡萄糖，所述糖类在所述微针（110）中的含量为基于所述基体重量计的约2.5重量%至约10重量%，优选约5重量%至约7.5重量%，更优选约6重量%，所述产氢活性菌在所述微针（110）中的含量为每克基体重量约1×10 7 cfu至约1×10 9 cfu，优选约1×10 8 cfu至约1.5×10 8 cfu，最优选约1.2×10 8 cfu。 A hydrogen-producing biomicroneedle product (100), comprising a backing (120) and a hydrogen-producing biomicroneedle array attached to one side of the backing (120), the hydrogen-producing biomicroneedle array comprising multiple A microneedle (110), characterized in that each microneedle (110) contains a matrix and hydrogen-producing active bacteria and sugars loaded in the matrix, and the hydrogen-producing active bacteria can use the sugars to generate hydrogen, Wherein the sugar is preferably selected from glucose, maltose, fructose, lactose, sucrose or a mixture of two or more thereof, more preferably selected from glucose, the sugar in the microneedle (110) The content 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, and the hydrogen-producing active bacteria are present in the microneedles (110 ) in an amount of about 1×10 7 cfu to about 1×10 9 cfu, preferably about 1×10 8 cfu to about 1.5×10 8 cfu, most preferably about 1.2×10 8 cfu per gram of matrix weight.
- 根据权利要求1的产氢生物微针制品（100），其中所述产氢活性菌选自蓝藻、绿藻、光合细菌、发酵细菌或这些细菌中的两种或更多种的混合物，优选选自发酵细菌，更优选选自丁酸梭菌和/或产气肠杆菌，最优选选自产气肠杆菌。The hydrogen-producing biological microneedle product (100) according to claim 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 Self-fermenting bacteria, more preferably selected from Clostridium butyricum and/or Enterobacter aerogenes, most preferably selected from Enterobacter aerogenes.
- 根据权利要求1或2的产氢生物微针制品（100），其中所述基体是通过将包含以下物质中的一种或多种的水溶液进行交联和/或干燥固化后形成的：聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮和聚乙烯醇，优选甲基丙烯酸酯明胶和/或透明质酸，更优选甲基丙烯酸酯明胶。The hydrogen-producing biomicroneedle product (100) according to claim 1 or 2, wherein the matrix is formed by crosslinking and/or drying and curing an aqueous solution containing one or more of the following substances: polyethylene Diol diacrylate, silk fibroin, methacrylate gelatin, carboxymethylcellulose, trehalose, hyaluronic acid, polylactic-co-glycolic acid, polylactic acid, galactose, polyvinylpyrrolidone, and polyvinyl alcohol , preferably methacrylate gelatin and/or hyaluronic acid, more preferably methacrylate gelatin.
- 根据权利要求1至3中任一项的产氢生物微针制品（100），其中所述背衬（120）是通过将包含以下物质中的一种或多种的水溶液进行交联和/或干燥固化后形成的：聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮和聚乙烯醇，优选透明质酸。The hydrogen-producing biological microneedle product (100) according to any one of claims 1 to 3, wherein the backing (120) is cross-linked and/or an aqueous solution containing one or more of the following substances Formed after drying and curing: Polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin, carboxymethyl cellulose, trehalose, hyaluronic acid, polylactic acid-glycolic acid copolymer, polylactic acid, galactose , polyvinylpyrrolidone and polyvinyl alcohol, preferably hyaluronic acid.
- 根据权利要求1至4中任一项的产氢生物微针制品（100），其中所述每根微针（110）具有尖端和底端，所述尖端远离所述背衬（120），所述微针（110）经由所述底端附着于所述背衬（120），所述尖端至底端的距离为约200 μm至约1 mm，所述底端具有约100 μm至约500 μm的底端直径，和相邻的微针（110）尖端之间的间距为约300 μm至约800 μm。The hydrogen-producing biological microneedle product (100) according to any one of claims 1 to 4, wherein each microneedle (110) has a tip and a bottom end, and the tip is far away from the backing (120), so The microneedle (110) is attached to the backing (120) via the bottom end, the distance from the tip to the bottom end is about 200 μm to about 1 mm, and the bottom end has a diameter of about 100 μm to about 500 μm The diameter of the bottom end, and the distance between the tips of adjacent microneedles (110) are about 300 μm to about 800 μm.
- 制备根据权利要求1至5中任一项的产氢生物微针制品（100）的方法，该方法包括以下步骤：The method for preparing the hydrogen-producing biological microneedle product (100) according to any one of claims 1 to 5, the method comprising the following steps:（1）提供液体培养基，将该液体培养基高压灭菌后接种所述产氢活性菌，并培养所述产氢活性菌以获得含有传代产氢活性菌的液体培养基，优选在约30℃至约40℃下，优选约37℃下培养所述产氢活性菌约5小时至约20小时，优选约12小时以获得含有传代产氢活性菌的液体培养基，其中在所述含有传代产氢活性菌的液体培养基中，所述产氢活性菌的总含量为约0.2×10 7 cfu/mL至约0.4×10 9 cfu/mL，优选约0.3×10 8 cfu/mL，其中所述产氢活性菌优选选自蓝藻、绿藻、光合细菌、发酵细菌或这些细菌中的两种或更多种的混合物，更优选选自发酵细菌，尤其优选选自丁酸梭菌和/或产气肠杆菌，最优选选自产气肠杆菌； (1) Provide a liquid medium, inoculate the hydrogen-producing active bacteria after autoclaving the liquid medium, and cultivate the hydrogen-producing active bacteria to obtain a liquid medium containing the hydrogen-producing active bacteria, preferably at about 30 °C to about 40°C, preferably at about 37°C, culturing the hydrogen-producing active bacteria for about 5 hours to about 20 hours, preferably about 12 hours to obtain a liquid medium containing passaged hydrogen-producing active bacteria, wherein In the liquid medium of active hydrogen-producing bacteria, 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 preferably selected from Enterobacter aerogenes;（2）向由步骤（1）获得的含有传代产氢活性菌的液体培养基中添加能够形成所述基体的基体材料和所述糖类以形成混合溶液，其中在所述混合溶液中，所述基体材料的浓度为基于所述混合溶液总重量计的约20重量%至约40重量%，优选约25重量%至约35重量%，更优选约30重量%，和所述糖类的浓度为约10 mg/mL至约20 mg/mL，优选约15 mg/mL，所述糖类优选选自葡萄糖、麦芽糖、果糖、乳糖、蔗糖或它们中的两种或更多种的混合物，更优选选自葡萄糖，所述基体材料优选选自聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮、聚乙烯醇或它们中的两种或更多种的混合物，更优选甲基丙烯酸酯明胶和/或透明质酸，最优选甲基丙烯酸酯明胶；(2) Adding the matrix material capable of forming the matrix and the sugar to the liquid culture medium containing the passaged hydrogen-producing active bacteria obtained in step (1) to form a mixed solution, wherein in the mixed solution, the 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 Preferably selected 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 or a mixture of two or more thereof, more preferably methacrylate gelatin and/or hyaluronic acid, most preferably methacrylate gelatin;（3）提供微针模具（200），该微针模具（200）包含上表面（202）和从所述上表面（202）向下延伸的成型孔（201），其中所述成型孔（201）优选尖端和底端，所述尖端远离所述上表面（202），所述底端平面与所述上表面（202）齐平，所述尖端至底端的距离优选为约200 μm至约1 mm，所述底端优选具有约100 μm至约500 μm的底端直径，和相邻的尖端之间的间距优选为约300 μm至约800 μm；(3) Provide a microneedle mold (200), the microneedle mold (200) includes an upper surface (202) and a forming hole (201) extending downward from the upper surface (202), wherein the forming hole (201) ) is preferably a tip and a bottom, the tip is far away from the upper surface (202), the bottom plane is flush with the upper surface (202), and the distance from the tip to the bottom is preferably about 200 μm to about 1 mm, the base preferably has a base diameter of about 100 μm to about 500 μm, and the spacing between adjacent tips is preferably about 300 μm to about 800 μm;（4）将由步骤（2）获得的混合溶液置于所述成型孔（201）中并填充所述成型孔（201）容积的至少一部分，优选填满所述成型孔（201）；(4) placing the mixed solution obtained in step (2) in the forming hole (201) and filling at least a part of the volume of the forming hole (201), preferably filling up the forming hole (201);（5）使在所述成型孔（201）中的包含所述基体材料、所述糖类和所述产氢活性菌的混合溶液进行交联，优选紫外交联约5秒至约15秒，优选约10秒，和/或进行干燥固化，优选热固化，从而在所述成型孔（201）内形成所述微针（110），多个所述微针（110）构成所述产氢生物微针阵列，每根微针（110）具有尖端和底端，所述微针（110）的尖端相对于所述微针（110）的底端远离所述上表面（202）；(5) cross-linking the mixed solution containing the matrix material, the sugar and the hydrogen-producing active bacteria in the forming hole (201), preferably ultraviolet cross-linking for about 5 seconds to about 15 seconds, Preferably about 10 seconds, and/or drying and curing, preferably thermal curing, so as to form the microneedles (110) in the forming hole (201), and a plurality of the microneedles (110) constitute the hydrogen-producing organism an array of microneedles, each microneedle (110) having a tip and a base, the tips of the microneedles (110) being farther away from the upper surface (202) than the bases of the microneedles (110);（6）在所述微针（110）的底端表面和所述微针模具（200）的未被所述微针（110）遮盖的上表面（202）上施加包含背衬材料的溶液以形成背衬溶液层，并任选将所述背衬材料进行交联形成连续的背衬层，从而使所述产氢生物微针阵列附着于所述背衬溶液层或所述背衬层，所述背衬材料优选选自聚乙二醇双丙烯酸酯、丝素蛋白、甲基丙烯酸酯明胶、羧甲基纤维素、海藻糖、透明质酸、聚乳酸-羟基乙酸共聚物、聚乳酸、半乳糖、聚乙烯吡咯烷酮、聚乙烯醇或它们中的两种或更多种的混合物，优选透明质酸；和(6) applying a solution containing a backing material on the bottom end surface of the microneedle (110) and the upper surface (202) of the microneedle mold (200) not covered by the microneedle (110) to forming a backing solution layer, and optionally crosslinking the backing material 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, 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 thereof, preferably hyaluronic acid; and（7）将由步骤（6）获得的背衬溶液层或背衬层并任选同时将所述产氢生物微针阵列进行干燥固化形成所述产氢生物微针制品（100）。(7) Drying and curing the backing solution layer or backing layer obtained in step (6) and the hydrogen-producing bio-microneedle array simultaneously to form the hydrogen-producing bio-microneedle product (100).
- 根据权利要求1至5中任一项的产氢生物微针制品（100）或根据权利要求6的方法制备的产氢生物微针制品（100）用于缓解和/或治疗动物体，优选人体的氧化应激疾病（优选心脏病、癌症、骨关节炎、风湿性关节炎、类风湿性关节炎、糖尿病、肿瘤、脊髓损伤、衰老、阿尔兹海默症和/或帕金森病）的用途。The hydrogen-producing biological microneedle product (100) according to any one of claims 1 to 5 or the hydrogen-producing biological microneedle product (100) prepared according to the method of claim 6 is used to alleviate and/or treat an animal body, preferably a human body Use in diseases of oxidative stress (preferably heart disease, cancer, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, diabetes, tumors, spinal cord injury, aging, Alzheimer's disease and/or Parkinson's disease) .
- 根据权利要求1至5中任一项的产氢生物微针制品（100）或根据权利要求6的方法制备的产氢生物微针制品（100）用于制备缓解和/或治疗动物体，优选人体的氧化应激疾病（优选心脏病、癌症、骨关节炎、风湿性关节炎、类风湿性关节炎、糖尿病、肿瘤、脊髓损伤、衰老、阿尔兹海默症和/或帕金森病）的医疗装置的用途。The hydrogen-producing biological microneedle product (100) according to any one of claims 1 to 5 or the hydrogen-producing biological microneedle product (100) prepared according to the method of claim 6 is used for preparing relief and/or treatment of animal bodies, preferably Diseases of oxidative stress in humans (preferably heart disease, cancer, osteoarthritis, rheumatoid arthritis, diabetes, tumors, spinal cord injury, aging, Alzheimer's disease and/or Parkinson's disease) Use of medical devices.
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