阅读说明：本技术 一种速溶胶原蛋白微针及其制备方法 (Instant collagen microneedle and preparation method thereof ) 是由 肖建喜 李文华 于 2021-03-25 设计创作，主要内容包括：本发明属于护肤品领域,具体涉及一种速溶胶原蛋白微针及其制备方法。所述胶原蛋白微针包括重组胶原蛋白/动物胶原蛋白和透明质酸,所述重组胶原蛋白/动物胶原蛋白和透明质酸的混合物溶液制备获得的胶原蛋白微针成型性和溶解性良好；所述胶原蛋白微针还具有良好的微针形貌,针体力学性能优良,能够刺穿皮肤角质层；针体在插入皮肤后能够快速溶解,实现胶原蛋白的高效递送,促进胶原蛋白的吸收,帮助修复受损肌肤；所述胶原蛋白微针制备方法简单,制备过程中很好的保持了胶原蛋白的生物活性,制备工艺适于规模化生产。(The invention belongs to the field of skin care products, and particularly relates to an instant collagen microneedle and a preparation method thereof. The collagen microneedle comprises recombinant collagen/animal collagen and hyaluronic acid, and the collagen microneedle prepared from the mixture solution of the recombinant collagen/animal collagen and the hyaluronic acid has good formability and solubility; the collagen microneedle also has good microneedle appearance, the needle body has excellent mechanical property, and can pierce the stratum corneum; the needle body can be quickly dissolved after being inserted into the skin, so that the high-efficiency delivery of collagen is realized, the absorption of the collagen is promoted, and the damaged skin is helped to be repaired; the preparation method of the collagen microneedle is simple, the bioactivity of the collagen is well kept in the preparation process, and the preparation process is suitable for large-scale production.)

1. The raw material liquid for preparing the collagen microneedle is characterized by comprising a collagen solution and a hyaluronic acid solution, wherein the collagen is recombinant collagen or animal collagen.

2. The feed solution of claim 1, comprising a concentration ratio of 0.01 to 30 mg/ml: 5-30mg/ml of recombinant collagen and hyaluronic acid.

3. The feed solution of claim 1, comprising a concentration ratio of 0.01 to 0.7 mg/ml: 5-30mg/ml animal collagen and hyaluronic acid.

4. The feed solution of claim 3, wherein said animal collagen is yak collagen.

5. The stock solution of claim 1, further comprising a cosmetically acceptable functional ingredient, and/or a cosmetically acceptable auxiliary material.

6. A collagen microneedle prepared from the raw material liquid according to any one of claims 1 to 5.

7. A method of preparing a collagen microneedle according to claim 6, comprising:

(1) preparation of microneedle stock solution: heating and dissolving hyaluronic acid at 65-85 ℃, and then vacuum drying and defoaming; cooling to below 25 ℃, adding a collagen solution, and performing vacuum drying and defoaming to obtain a microneedle raw material solution;

(2) preparation of collagen microneedles: and (2) adding the microneedle raw material liquid obtained in the step (1) into a microneedle mould, vacuum drying and defoaming at the temperature of below 25 ℃, curing, and demoulding to obtain the collagen microneedle.

8. The method according to claim 7, wherein the collagen solution is an animal collagen solution, and the step (1) is: heating and dissolving hyaluronic acid at 65-85 ℃, and then vacuum drying and defoaming; cooling to below 25 ℃, adding the animal collagen solution, homogenizing, and carrying out vacuum drying and defoaming to obtain the microneedle raw material solution.

9. A collagen microneedle is characterized by comprising the following components in percentage by weight: 0.03-86% wt of recombinant collagen and 14% -99.97% wt of hyaluronic acid.

10. A collagen microneedle is characterized by comprising the following components in percentage by weight: 0.03-12% of animal collagen and 88% -99.97% of hyaluronic acid.

11. The collagen microneedle of claim 10, wherein the animal collagen is yak collagen.

12. A collagen microneedle according to any one of claims 9 to 11, further comprising a cosmetically acceptable functional ingredient, and/or a cosmetically acceptable auxiliary raw material.

13. Use of the collagen microneedle according to claim 6 or any one of claims 9 to 12 for the preparation of a cosmetic, cosmeceutical product.

Technical Field

The invention belongs to the field of skin care products, and particularly relates to an instant collagen microneedle and a preparation method thereof.

Background

Collagen is the most abundant protein in the human body, and accounts for about 30% of the total amount of human protein. Collagen, as the major structural protein, forms a molecular scaffold that provides mechanical strength and structural integrity to the human body, supporting and protecting soft tissues and internal organs. The collagen has high biological activity, participates in the interaction of a large amount of extracellular matrixes, and has a very close relation with cell regeneration, wound healing and the like. Collagen has the advantages of biocompatibility, biodegradability, bioabsorbability and the like, so that the collagen is widely applied to the fields of cosmetics, medical instruments and the like.

Collagen is the main structural component of skin, and the degradation, breakage, collapse and loss of collagen fibers in the skin can cause skin problems such as wrinkles, acne, dark yellow and the like; meanwhile, the collagen fiber network provides a good growth environment for fibroblasts, and excessive degradation of the collagen fiber network can influence the fibroblasts to express collagen, so that the collagen is further lost, and the skin aging is accelerated. Therefore, collagen is one of the most commonly used additives in skin care products, however, how collagen is taken as a protein with larger molecular weight to promote the absorption of collagen by skin is always a key problem in the collagen skin care products.

The micro-needle is a transdermal drug delivery device, is a micro-needle point structure which is manufactured by a micro-nano processing technology and has a micron-sized size, and consists of a square, round or eye mask-shaped patch base with a certain thickness and a conical needle point structure. Soluble microneedles are a physical permeation-promoting technique that uses arrays of micron-sized tips to pierce the stratum corneum of the skin to deliver drugs into the body. Because the microneedle only pierces the stratum corneum of the skin and does not touch subcutaneous tissues and nerves, the epidermal administration is realized, the damage to the subcutaneous tissues and the nerves of the skin can be reduced, and the microneedle has the advantages of painlessness, accuracy, high efficiency, convenience and the like. The preparation requirement of the soluble microneedle is very strict, the microneedle is required to have the fine appearance, the needle point of the microneedle is required to have good mechanical property to support the microneedle to puncture the epidermal layer, and the needle point can be quickly dissolved after penetrating into the skin. Due to the defects of poor formability, poor solubility and the like of the recombinant collagen and the animal collagen, the collagen microneedle cannot be successfully prepared by using the recombinant collagen or the animal collagen at present.

The invention unexpectedly discovers that the recombinant collagen microneedle with good formability can be prepared by adding hyaluronic acid in a certain proportion into the recombinant collagen, and the recombinant collagen microneedle has good microneedle appearance and mechanical property; meanwhile, the invention discovers that the animal collagen microneedle prepared by adding hyaluronic acid in a certain proportion into animal collagen not only has good microneedle appearance and needle body mechanical property, but also can be quickly dissolved after the needle body of the microneedle is inserted into the skin. The technical scheme of the invention solves the problem that the microneedle is difficult to prepare by the recombinant collagen/animal collagen, and the prepared recombinant collagen/animal collagen microneedle has good microneedle appearance and needle body mechanical property, can be quickly dissolved after being inserted into the skin, realizes the high-efficiency delivery of the collagen, promotes the absorption of the collagen, and repairs the damaged skin; meanwhile, the preparation method of the collagen microneedle is simple, the bioactivity of the collagen is well kept in the preparation process, and the preparation process is suitable for large-scale production.

Disclosure of Invention

Firstly, aiming at the defects of poor formability of recombinant collagen, poor solubility of animal collagen and the like in the prior art and the problem that a soluble collagen microneedle is difficult to prepare, the invention mixes the recombinant collagen/the animal collagen and hyaluronic acid according to a certain proportion to successfully prepare the collagen microneedle which has good formability and can be quickly dissolved. The method specifically comprises the following steps:

in a first aspect, the present invention provides a feedstock solution for preparing a collagen microneedle, the feedstock solution comprising a reconstituted collagen/animal collagen solution and a hyaluronic acid solution.

Preferably, the raw material liquid comprises a mixture of the following components in a concentration ratio of 0.01-30 mg/ml: 5-30mg/ml recombinant collagen and hyaluronic acid.

Preferably, the raw material liquid comprises a concentration ratio of 0.01-0.7 mg/ml: 5-30mg/ml animal collagen and hyaluronic acid.

Preferably, the animal collagen is yak collagen.

Preferably, the raw material liquid further comprises a cosmetically acceptable efficacy ingredient, and/or a cosmetically acceptable auxiliary raw material.

Preferably, the cosmetically acceptable functional components comprise one or more of nicotinamide, centella asiatica extract, wild chrysanthemum flower extract, peony extract, aloe extract, purslane extract, rose hydrosol, tea hydrosol, allantoin and salicylic acid.

Preferably, the cosmetically acceptable auxiliary raw material comprises any one or more of glucose, soluble starch, lactose, chondroitin sulfate, carboxymethyl cellulose IV, sodium carboxymethyl cellulose, chitosan, polyethylene glycol, polyvinyl alcohol, PVP, maltose, sodium alginate, polylactic acid and polyglycolic acid.

In a second aspect, the present invention provides a collagen microneedle prepared from the raw material liquid according to the first aspect.

In a third aspect, the present invention provides an application of the collagen microneedle according to the second aspect in preparing cosmetics and medical beauty products.

In a fourth aspect, the present invention provides a method for preparing a collagen microneedle according to the third aspect, the method comprising:

(1) preparation of microneedle stock solution: heating and dissolving hyaluronic acid at 65-85 ℃, and then vacuum drying and defoaming; cooling to below 25 ℃, adding a collagen solution, and performing vacuum drying and defoaming to obtain a microneedle raw material solution;

(2) preparation of collagen microneedles: and (2) adding the microneedle raw material liquid obtained in the step (1) into a microneedle mould, vacuum drying and defoaming at the temperature of below 25 ℃, curing, and demoulding to obtain the collagen microneedle.

Preferably, the collagen solution is an animal collagen solution, and the step (1) is: heating and dissolving hyaluronic acid at 65-85 ℃, and then vacuum drying and defoaming; cooling to below 25 ℃, adding the animal collagen solution, homogenizing, and carrying out vacuum drying and defoaming to obtain the microneedle raw material solution.

Preferably, the animal collagen is yak collagen.

In a fifth aspect, the present invention provides a collagen microneedle, which comprises the following components by weight: 0.04-86% wt of recombinant collagen and 14% -99.96% wt of hyaluronic acid.

Preferably, the collagen microneedle further comprises a cosmetically acceptable functional ingredient, and/or a cosmetically acceptable auxiliary material.

Preferably, the cosmetically acceptable functional components comprise one or more of nicotinamide, centella asiatica extract, wild chrysanthemum flower extract, peony extract, aloe extract, purslane extract, rose hydrosol, tea hydrosol, allantoin and salicylic acid.

Preferably, the cosmetically acceptable auxiliary raw material comprises any one or more of glucose, soluble starch, lactose, chondroitin sulfate, carboxymethyl cellulose IV, sodium carboxymethyl cellulose, chitosan, polyethylene glycol, polyvinyl alcohol, PVP, maltose, sodium alginate, polylactic acid and polyglycolic acid.

In a sixth aspect, the present invention provides an application of the collagen microneedle according to the fifth aspect in preparing cosmetics and medical cosmetic products.

In a seventh aspect, the present invention provides a collagen microneedle, which comprises the following components by weight: 0.03-12% wt of animal collagen and 88% -99.96% wt of hyaluronic acid.

Preferably, the animal collagen is yak collagen.

Preferably, the collagen microneedle further comprises a cosmetically acceptable functional ingredient, and/or a cosmetically acceptable auxiliary material.

Preferably, the cosmetically acceptable functional components comprise one or more of nicotinamide, centella asiatica extract, wild chrysanthemum flower extract, peony extract, aloe extract, purslane extract, rose hydrosol, tea hydrosol, allantoin and salicylic acid.

Preferably, the cosmetically acceptable auxiliary raw material comprises any one or more of glucose, soluble starch, lactose, chondroitin sulfate, carboxymethyl cellulose IV, sodium carboxymethyl cellulose, chitosan, polyethylene glycol, polyvinyl alcohol, PVP, maltose, sodium alginate, polylactic acid and polyglycolic acid.

In an eighth aspect, the present invention provides an application of the collagen microneedle according to the seventh aspect in preparing cosmetics and medical cosmetic products.

The invention has the beneficial effects that:

(1) the invention discovers that the recombinant collagen microneedle prepared by adding hyaluronic acid in a certain proportion into the recombinant collagen has good formability, and solves the problem that the soluble microneedle is difficult to prepare due to poor formability of the recombinant collagen; the prepared recombinant collagen microneedle has good microneedle appearance, and the needle body has excellent mechanical property and can pierce the stratum corneum of skin; the needle body of the microneedle can be quickly dissolved after being inserted into the skin, so that the high-efficiency delivery of collagen is realized, the absorption of the collagen is promoted, and the damaged skin is repaired;

(2) according to the invention, hyaluronic acid is added into animal collagen in a certain proportion, so that the prepared animal collagen microneedle can be quickly dissolved, and the problem that the dissoluble microneedle is difficult to prepare due to poor solubility of the animal collagen is solved; the prepared animal collagen microneedle has good microneedle appearance, and the needle body has excellent mechanical property and can pierce the stratum corneum; the needle body of the microneedle can be quickly dissolved after being inserted into the skin, so that the high-efficiency delivery of collagen is realized, the absorption of the collagen is promoted, and the damaged skin is repaired;

(3) the method is simple, the biological activity of the collagen is well maintained in the preparation process, and the preparation process is suitable for large-scale production;

(4) hyaluronic acid is a beneficial ingredient in skin, can quickly fill skin tiny defects and help an epidermal structure to keep moisture; meanwhile, the needle body can be added with functional components acceptable for cosmetics, and the functional components are delivered to the dermis layer through the rapid dissolution of the needle body and are rapidly absorbed, so that the effects of supplementing skin nutrition, repairing damaged skin, promoting blood circulation and the like are achieved.

(5) Other cosmetically acceptable functional components can be added into the microneedle, so that the microneedle has various beautifying effects.

Drawings

FIG. 1 SEM image and microscopic observation image of recombinant collagen microneedle

FIG. 2 is a vertical fluorescence microscope bright field channel for observing the dissolving effect of the recombinant collagen microneedle;

FIG. 3 is a front view of agarose gel pad diffusion of recombinant collagen microneedles;

FIG. 4 agarose gel pad diffusion profile of recombinant collagen microneedles;

FIG. 5 is a H & E staining diagram of a pigskin tissue after a congo red-labeled recombinant collagen microneedle punctures the pigskin;

fig. 6 an SEM image of an animal collagen microneedle;

fig. 7 is a vertical fluorescence microscope bright field channel for observing the dissolving effect of the animal collagen micro-needle.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited to the following.

The recombinant collagen described in the following embodiments is collagen obtained in animal, plant or microorganism expression systems using transgenic technology and gene recombination technology. The recombinant collagen described in the following examples is obtained by fermentation of escherichia coli, but is not limited to the above method, and may also be prepared by other methods, including pichia pastoris fermented recombinant collagen, recombinant human collagen, and the like.

The recombinant collagen was prepared according to the literature references (He M, Zhang Y, Munyemana J C, et al. tuning the structural details of the mineral proteins via collagen-structured biochemical [ J ]. Journal of Materials Chemistry B,2017,5(7): 1423-. However, the present invention is not limited to the recombinant collagen prepared by the above method, and any other recombinant collagen prepared by any method is applicable to the technical solution for preparing the recombinant collagen microneedle according to the present invention, and therefore, any recombinant collagen prepared by any method is within the scope of the present invention.

The animal collagen described in the following examples is yak collagen, but is not limited to yak collagen; the yak collagen is extracted and purified from yak tendons by an enzymolysis method.

The micro-needle consists of a square, round or eye mask-shaped patch base with a certain thickness and a conical needle point structure, wherein the patch base and the needle point structure are integrally connected and are prepared by a mould method, the mould is a PDMS female mould, and the array characteristics of the mould are as follows: the needles were 800 μm long, the diameter of the bottom of the needles was 225 μm, the tip distance was 650 μm, and the number array was 20X 20.

Collagen microneedle formability criteria described in the following examples: whether the micro-needle can be completely taken down from the mold or not and whether the needle points are regularly arranged or not is observed by naked eyes after the micro-needle is taken down; collagen microneedle solubility criteria: whether the microneedle body is completely dissolved within 2 min.

Example 1

Weighing hyaluronic acid, adding quantitative water, and preparing into a mixture with the concentration: 1. 5, 10, 15, 20, 25, 30mg/ml hyaluronic acid solution; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 80 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; after cooling, recombinant collagen was added to a final concentration of: 0.5mg/ml, uniformly stirring, putting into a vacuum drying oven again, drying and defoaming at constant temperature of-50 kPa for 12h at 25 ℃ to obtain the microneedle raw material solution.

Adding a certain amount of microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying and defoaming at the constant temperature of-50 kPa and 25 ℃, recovering the atmospheric pressure every 4 hours, repeating the steps twice, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the recombinant collagen microneedles.

The microneedle effects of hyaluronic acid concentrations ranging from 1mg/ml to 35mg/ml and recombinant collagen concentrations ranging from 0.5mg/ml are shown in Table 1. The results show that when the concentration of hyaluronic acid is 5mg/ml-30mg/ml and the concentration of recombinant collagen is 0.5mg/ml, namely the concentration ratio of hyaluronic acid to recombinant collagen is 10-60:1, the recombinant collagen and hyaluronic acid can be mixed to form a solution, and the microneedle prepared from the raw material solution has good formability and can be quickly dissolved in water.

TABLE 1 microneedle Effect of hyaluronic acid and 0.5mg/ml recombinant collagen in different concentrations

Note: "instant" means that the microneedle is rapidly dissolved in water within 2min to form a solution

Example 2

Weighing hyaluronic acid, adding quantitative water to prepare a hyaluronic acid solution with the concentration of 15 mg/ml; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 65 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; after cooling, recombinant collagen was added to give a final concentration of recombinant collagen: 0.01, 0.1, 0.3, 0.5, 0.7, 1.0, 5.0, 10.0, 20.0 and 30.0mg/ml, uniformly stirring, putting into a vacuum drying oven again, drying and defoaming at the constant temperature of 20 ℃ under the vacuum degree of-50 KPa for 12 hours to obtain the microneedle raw material solution.

Adding a certain amount of microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at a constant temperature of 20 ℃ under a vacuum degree of-50 kPa, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the recombinant collagen microneedles.

The microneedle effects of the recombinant collagen of 0.01mg/ml to 30.0mg/ml and 15mg/ml in different concentrations are shown in table 2. The result shows that when the concentration of the recombinant collagen is 0.01mg/ml to 30.0mg/ml and the concentration of the hyaluronic acid is 15mg/ml, namely the concentration ratio of the hyaluronic acid to the recombinant collagen is 0.5 to 1500:1, the recombinant collagen and the hyaluronic acid can be mixed to form a solution, and the microneedle prepared from the raw material solution has good formability and can be quickly dissolved in water.

TABLE 2 microneedle effect of different concentrations of recombinant collagen with 15mg/ml hyaluronic acid

Note: "instant" means that the microneedle is rapidly dissolved in water within 2min to form a solution

Example 3

According to the experimental results of example 1 and example 2, the conditions of microneedle formation and dissolution under the conditions of the extreme hyaluronic acid concentration and the extreme recombinant collagen concentration were examined in this example. Weighing hyaluronic acid, adding quantitative water to prepare hyaluronic acid solutions with the concentrations of 5mg/ml and 30 mg/ml; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 65 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; and after cooling, adding the recombinant collagen into the hyaluronic acid group with the concentration of 30mg/ml until the concentration of the recombinant collagen is 30mg/ml, adding the recombinant collagen into the hyaluronic acid group with the concentration of 5mg/ml until the concentration of the recombinant collagen is 0.01mg/ml, uniformly stirring, putting into a vacuum drying oven again, and drying and defoaming at the constant temperature of 20 ℃ under the vacuum degree of 50kPa for 12 hours to obtain the microneedle raw material solution.

Adding a quantitative microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at a constant temperature of-50 kPa and 20 ℃, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the recombinant collagen microneedles.

The results of the extreme concentration points of the hyaluronic acid-recombinant collagen are shown in table 3. The results showed that when the concentration of the recombinant collagen was 30mg/ml at the upper limit and that of the hyaluronic acid was 30mg/ml at the upper limit, the recombinant collagen and the hyaluronic acid were mixed to form a solution, and that the microneedle prepared from the raw material solution had good moldability and was rapidly dissolved in water, and that when the concentration of the collagen was 0.01mg/ml at the lower limit and that of the hyaluronic acid was 5mg/ml at the lower limit, the same results were obtained.

TABLE 3 microneedle effect of boundary concentration recombinant collagen and hyaluronic acid

Note: "instant" means that the microneedle is rapidly dissolved in water within 2min to form a solution

Example 4

1. Preparation of recombinant collagen microneedle

Weighing hyaluronic acid, adding quantitative water to prepare hyaluronic acid solutions with the concentrations of 5mg/ml and 15 mg/ml; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 75 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 Kpa at 55 ℃, drying and defoaming for 12h, taking out, and cooling; two groups are arranged, one group is added with recombinant collagen marked by FITC to a hyaluronic acid solution group with 5mg/ml until the concentration of the recombinant collagen is 0.01mg/ml, the other group is added with recombinant collagen marked by FITC to a hyaluronic acid solution group with 15mg/ml until the concentration of the recombinant collagen is 0.5mg/ml, the mixture is uniformly stirred and then is put into a vacuum drying oven again, the vacuum degree of 50kPa is kept, and the microneedle raw material solution is obtained after constant-temperature drying and defoaming at the temperature of 20 ℃ for 12 hours.

Adding a quantitative microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at a constant temperature of-50 kPa and 20 ℃, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the recombinant collagen microneedles.

2. Recombinant collagen microneedle performance assay

(1) Recombinant collagen microneedle SEM detection

And (3) detection process: attaching 5mg/ml hyaluronic acid-0.01 mg/ml recombinant collagen microneedle to a sample table through conductive adhesive, uniformly distributing a conductive layer on the surface of the sample table through a vacuum gold-plating method for 30s, and obtaining SEM pictures under different magnifications in the same selected area by using a field emission scanning electron microscope.

As a result, as shown in fig. 1(a) and (B), a single microneedle tip has a tip shape, the size of the microneedle is the same as the designed size, and a plurality of tips are uniformly distributed in the lateral and longitudinal directions, have the same height and tip diameter, and are ideal microneedle shapes, indicating that they have the potential to form uniform channels in the skin.

(2) Recombinant collagen distribution observed by upright microscope fluorescence field of recombinant collagen microneedle

And (3) detection process: the microneedles were placed on a glass slide, the shape and distribution of the microneedles were observed under the bright field condition of a Nikon-ECLIPSE 80i/DS-Ri2/NIS-ElementsD imaging system, and then the microneedles were excited by switching to the fluorescence excitation condition with the excitation wavelength of 460nm to 550nm, and photographs were taken under the 4 Xcondition. A row of needle points in the micro-needle are taken to take pictures under 4X and 10X magnification under a bright field channel.

As shown in fig. 1(C), after recombinant collagen is labeled with FITC and FITC molecules not bound to the recombinant collagen are removed by a dialysis method, uniformly distributed green fluorescence (FITC autofluorescence) is observed in a fluorescence channel of the recombinant collagen microneedle prepared by compounding with hyaluronic acid, and the fluorescence intensity is uniform, indicating that the recombinant collagen is uniformly distributed in the microneedle. When observed in a bright field channel, the results are shown in fig. 1(D), and the single tips of the microneedles are all in a tapered structure.

(3) Dissolution result of recombinant collagen microneedle body

The experimental process comprises the following steps: the shape and distribution of the microneedles were observed under the bright field condition of a Nikon-ECLIPSE 80i/DS-Ri2/NIS-ElementsD imaging system by using a single-row tip array, and photographs were obtained under 4X magnification and 10X magnification. Taking pigskin with a proper size, attaching the pigskin to the surface of the pigskin through uniform external force, starting timing, taking down the pigskin after 3min to obtain a single-row needle tip array, and observing and comparing under a bright field channel of a Nikon-ECLIPSE 80i/DS-Ri2/NIS-ElementsD imaging system.

The dissolution conditions of the needle bodies before and after the use of the microneedle are observed and compared through a bright field stereomicroscope, the result is shown in figure 2, a Nikon-ECLIPSE 80i/DS-Ri2/NIS-ElementsD imaging system observes the needle body of the recombinant collagen microneedle from the side, the structure of the needle body is complete before the use, and the needle body is completely dissolved after the use for 3min, which indicates that the recombinant collagen microneedle prepared by the invention has good solubility and can be completely dissolved in the use process.

(4) Microneedle diffusion experiments on agarose gel pads

Marking the micro-needle with 0.1% Congo red, preparing an agarose gel pad in advance, enabling the micro-needle to be in contact with agarose through uniform external force from 0min, starting timing, recording the red diffusion conditions of the front side and the side by taking 5min as a time interval unit, observing for 30min totally, and representing the dissolving and diffusing capacity of the micro-needle through the diffusion conditions of the Congo red dye uniformly distributed in the micro-needle.

As a result, as shown in fig. 3 and 4, it can be seen from the side (fig. 3) that the congo red-labeled recombinant collagen microneedles were diffused on the agarose gel pad; from the front (fig. 4), it can be seen that the congo red labeled recombinant collagen microneedle is placed on the agarose gel pad for 30min, and after the microneedle is taken off, the congo red labeled recombinant collagen has diffused into the agarose gel pad. That is, the recombinant collagen in the recombinant collagen microneedle according to the present invention can effectively permeate into the skin.

(5) Congo red marked recombinant collagen microneedle puncture experiment

Attaching the 0.1% Congo red marked recombinant collagen microneedle to the surface of the pigskin, carrying out a transdermal experiment, taking down the recombinant collagen microneedle after 15min, processing the pigskin tissue into tissue slices with the thickness of 10 mu m and 15 mu m by a frozen slice method, and observing the tissue slices under the condition of a vertical fluorescent microscope bright field through H & E staining.

The result is shown in fig. 5, the recombinant collagen microneedle of the invention can puncture the surface layer of the pigskin, and has good mechanical property.

Example 5

Preparing animal collagen microneedles: weighing hyaluronic acid, adding quantitative water to prepare a hyaluronic acid solution with the concentration of 15 mg/ml; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 65 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; after cooling, respectively adding yak collagen solutions with different concentrations to obtain yak collagen with the final concentration: 0.01, 0.1, 0.3, 0.5, 0.7, 1.0 and 2.0mg/ml of stock solution, uniformly stirring, controlling the temperature to be lower than 25 ℃, homogenizing by a high-speed shearing homogenizing emulsifying machine, putting the mixed solution into a vacuum drying oven again after homogenizing, drying and defoaming at the constant temperature of-50 kPa vacuum degree and 20 ℃ for 12 hours to obtain the microneedle raw material solution.

Adding a quantitative microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at a constant temperature of-50 kPa and 20 ℃, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the animal collagen microneedles.

The microneedle effects of yak collagen with different concentrations of 0.01mg/ml-2.0mg/ml and 15mg/ml hyaluronic acid are shown in table 4. The result shows that when the concentration of yak collagen is 0.01mg/ml-0.7mg/ml and the concentration of hyaluronic acid is 15mg/ml, namely the concentration ratio of hyaluronic acid to animal collagen is 21.4-1500:1, the animal collagen and hyaluronic acid can be mixed to form a solution, and the microneedle prepared from the raw material solution has good formability and can be quickly dissolved in water.

TABLE 4 microneedle effect of different concentrations of animal collagen and 15mg/ml hyaluronic acid

Note: "insoluble" means that the microneedle does not dissolve rapidly in water into solution; "instant" means that the microneedle is rapidly dissolved in water within 2min to form a solution

Example 6

Preparing animal collagen microneedles: weighing hyaluronic acid, adding quantitative water, and preparing into a mixture with the concentration: 1. 5, 10, 15, 20, 25, 30, 35mg/ml hyaluronic acid solution; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 80 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; cooling, adding yak collagen to make the final concentration of the yak collagen be 0.5mg/ml, uniformly stirring, controlling the temperature to be lower than 25 ℃, homogenizing by a high-speed shearing homogenizing emulsifying machine, homogenizing, putting the mixed solution into a vacuum drying oven again, and drying and defoaming at the constant temperature of-50 kPa under 25 ℃ for 12 hours to obtain the microneedle raw material solution.

Adding a quantitative microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at a constant temperature of-50 kPa and 25 ℃, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the animal collagen microneedles.

The microneedle effects of hyaluronic acid of different concentrations, 1mg/ml-35mg/ml and yak collagen of 0.5mg/ml, are shown in table 5. The concentration of hyaluronic acid is 5mg/ml-30mg/ml, when the concentration of yak collagen is 0.5mg/ml, namely the concentration ratio of hyaluronic acid to animal collagen is 10-60:1, animal collagen and hyaluronic acid can be mixed to form a solution, and the microneedle prepared from the raw material solution has good formability and can be quickly dissolved in water.

TABLE 5 microneedle Effect of different concentrations of hyaluronic acid and 0.5mg/ml animal collagen

Note: "instant" means that the microneedle is rapidly dissolved in water within 2min to form a solution

Example 7

Preparing animal collagen microneedles: according to the experimental results of example 5 and example 6, the conditions of microneedle formation and dissolution under the conditions of the extreme hyaluronic acid concentration and the extreme animal collagen concentration were examined in this example. Weighing hyaluronic acid, adding quantitative water to prepare hyaluronic acid solutions with the concentrations of 5mg/ml and 30 mg/ml; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 65 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; adding yak collagen into 5mg/ml and 30mg/ml hyaluronic acid solutions respectively until the final concentration of the yak collagen is 0.01mg/ml and 0.7mg/ml, uniformly stirring, controlling the temperature to be lower than 25 ℃, homogenizing by a high-speed shearing homogenizing emulsifying machine, putting the mixed solution into a vacuum drying oven again after homogenizing, and drying and defoaming at the constant temperature of 25 ℃ under the vacuum degree of-50 kPa for 12 hours to obtain the microneedle raw material solution.

Adding a quantitative microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at a constant temperature of-50 kPa and 20 ℃, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the animal collagen microneedles.

The results of the hyaluronic acid complex yak collagen extremal concentration points are shown in table 6. The results show that when yak collagen is at the upper concentration limit of 0.7mg/ml and hyaluronic acid is at the upper concentration limit of 30mg/ml, animal collagen and hyaluronic acid can be mixed to form a solution, and the microneedle prepared from the raw material solution has good moldability and can be rapidly dissolved in water, and when animal collagen is at the lower concentration limit of 0.01mg/ml and hyaluronic acid is at the lower concentration limit of 5mg/ml, the same results can be obtained.

TABLE 6 microneedle Effect of boundary concentration animal collagen and hyaluronic acid

Note: "instant" means that the microneedle is rapidly dissolved in water within 2min to form a solution

Example 8

1. Preparation of animal collagen microneedle

Weighing hyaluronic acid, adding quantitative water to prepare hyaluronic acid solutions with the concentrations of 5mg/ml and 15 mg/ml; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 65 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; after cooling, adding yak collagen into a hyaluronic acid solution group with the concentration of 5mg/ml until the concentration of the yak collagen is 0.05mg/ml, adding yak collagen into a hyaluronic acid solution group with the concentration of 15mg/ml until the concentration of the yak collagen is 0.5mg/ml, homogenizing by a high-speed shearing homogenizing emulsifying machine, controlling the temperature to be lower than 25 ℃, after homogenizing, putting into a vacuum drying oven again, and drying and defoaming at the constant temperature of-50 kPa for 12 hours at the temperature of 20 ℃ to obtain the microneedle raw material solution.

Adding a quantitative microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at a constant temperature of-50 kPa and 20 ℃, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the animal collagen microneedles.

2. Animal collagen microneedle performance assay

(1) Animal collagen microneedle SEM detection

And (3) detection process: obliquely attaching animal collagen microneedles prepared from 5mg/ml hyaluronic acid and 0.01mg/ml yak collagen to a sample table through conductive adhesive, uniformly distributing a conductive layer on the surface of the animal collagen microneedles by a vacuum gold-plating method for 30s, and obtaining SEM images by using a field emission scanning electron microscope under different magnifications in the same selected area.

As a result, as shown in fig. 6(a), (B), (C), and (D), the single microneedle tip has a shape of a needle tip, the size of the microneedle is the same as the designed size, and the plurality of needle tips have uniform distribution in the lateral and longitudinal directions, have the same height and diameter of the needle tip, and are an ideal microneedle shape, indicating that it has a potential to form a uniform channel in the skin.

(2) Dissolution result of animal collagen microneedle body

The experimental process comprises the following steps: the shape and distribution of the microneedles were observed under bright field conditions of a Nikon-ECLIPSE 80i/DS-Ri2/NIS-ElementsD imaging system by using an array of individual tips, and photographs were obtained at 4X and 10X magnification. Taking pig skin with a proper size, attaching the micro-needle to the surface of the pig skin through a uniform external force, starting timing, taking down the micro-needle after 3min to obtain a row of single needle points, and observing and comparing under a bright field channel of a Nikon-ECLIPSE 80i/DS-Ri2/NIS-ElementsD imaging system.

The dissolution conditions of the needle bodies before and after the use of the animal collagen microneedle are observed by a bright field stereomicroscope, and the result is shown in fig. 7, the Nikon-ECLIPSE 80i/DS-Ri2/NIS-ElementsD imaging system observes the needle body of the animal collagen microneedle from the side, the structure of the needle body is complete before the use, and the needle body is completely dissolved after the use for 3min, which indicates that the animal collagen microneedle prepared by the invention has good solubility and can be completely dissolved in the use process.

Example 9

In this example, the effect comparison of the hyaluronic acid and other common replica agents and common thickening plastic components (glucose, soluble starch, lactose, chondroitin sulfate, chitosan, hyaluronic acid, carboxymethyl cellulose iv, sodium carboxymethyl cellulose, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone PVP) for preparing collagen microneedles is considered, specifically:

preparing the solution with the concentration of 5 mg/mL; stirring the solution until no powder caking substance is visible to naked eyes, putting the solution into a vacuum drying oven, heating to 75 ℃, and keeping the temperature for 30 min; taking out, stirring uniformly again, putting into a vacuum drying oven, keeping the vacuum degree of-50 kPa at 55 ℃, drying and defoaming for 12h, and taking out; cooling, adding 0.01mg/ml recombinant collagen, stirring, placing into a vacuum drying oven again, drying under-50 kPa vacuum degree at 15 deg.C for deaeration for 12h to obtain microneedle raw material solution.

Adding a quantitative microneedle raw material solution into a PDMS mold, placing the PDMS mold into a vacuum drying oven, drying at constant temperature of-50 kPa and 15 ℃, repeatedly recovering the atmospheric pressure every 4 hours, vacuumizing again, defoaming for 8 hours, adding the microneedle raw material solution with the same volume again, repeating the above operations, drying and defoaming for 3 times in vacuum until the microneedles are dried, and demolding to obtain the recombinant collagen microneedles.

The effect of microneedles formulated from different materials is shown in table 7. The result shows that the glucose, the chondroitin sulfate, the hyaluronic acid and the polyvinylpyrrolidone have dissolving capacity and can be uniformly mixed with the recombinant collagen; but only hyaluronic acid and recombinant collagen have microneedle forming capability, and the prepared microneedles can be quickly dissolved. The results show that only hyaluronic acid and recombinant collagen can be made into collagen microneedles with good formability and solubility.

TABLE 7 microneedle Effect prepared from different starting materials

Note: "instant" means that the microneedle is rapidly dissolved in water within 2min to form a solution

The above description is only for details of a specific exemplary embodiment of the present invention, and it is obvious to those skilled in the art that various modifications and changes may be made in the present invention in the practical application process according to specific preparation conditions, and the present invention is not limited thereto. All that comes within the spirit and principle of the invention is to be understood as being within the scope of the invention.