Recombinant humanized collagen bionic microneedle for skin aging resistance and preparation method thereof

Abstract

The invention relates to a recombinant humanized collagen bionic microneedle for skin aging resistance, the microneedle comprises a needle tip, a base and a concave chamber, recombinant humanized collagen is loaded on the needle tip, the base and the concave chamber are made of hyaluronic acid, and the recombinant humanized collagen is uniformly distributed on the needle tip of the microneedle. Compared with a traditional smearing method and a conical microneedle, the bionic microneedle has a better anti-aging effect in a photoaging mouse model. The penetration depth of the micro-needle is obviously improved, and the solubility and the adhesiveness of the micro-needle are superior to those of the existing collagen micro-needle.

Description

Technical Field

[0001] The present invention belongs to the technical field of beauty products, and particularly relates to a recombinant humanized collagen biomimetic microneedle for skin anti-aging and a preparation method thereof. Background Art

[0002] With the aggravation of population aging, anti-aging research has increasingly become a global focus. As the largest organ of the human body, the aging of the skin is manifested as increased wrinkles, decreased elasticity, water loss, etc., which seriously affect people's appearance and self-confidence. Traditional anti-aging methods such as skin care products and laser treatment have deficiencies such as limited effects and large side effects, and are difficult to meet the growing needs of people. In recent years, microneedle technology, as a new type of transdermal drug delivery method, has shown great application potential in the anti-aging field due to its advantages of high efficiency, minimally invasive, and safety.

[0003] Recombinant humanized collagen is prepared by means of DNA recombinant technology, and encodes a full-length or partial amino acid sequence fragment by a specific type of human collagen gene, and is a combined form containing functional fragments of human collagen. Compared with traditional animal-derived collagen, recombinant humanized collagen has significant advantages: its biocompatibility is excellent, and it can effectively avoid the human immune rejection reaction; at the same time, it has good water solubility, and its biological activity is even 2.3 times that of human collagen. These characteristics have enabled it to be widely used in the field of anti-aging products. However, due to the large molecular weight of collagen, it is difficult to be effectively absorbed by the skin only through epidermal application, and collagen injection requires high professional skills for operators. Combining recombinant humanized collagen with microneedle technology can effectively solve the problem of low transdermal absorption rate of traditional collagen, and provide a new strategy for anti-aging treatment. However, currently existing collagen microneedles basically adopt traditional conical or quadrangular pyramid shapes. Due to reasons such as skin elasticity and deformation and the mechanical strength of microneedles, the microneedles cannot completely penetrate the skin, affecting the drug delivery efficiency. At the same time, the adhesion between the microneedles and the skin is weak, which may cause the microneedles to slide or fall off on the skin surface, affecting the stability of drug delivery.

[0004] Therefore, there is an urgent need for a new type of recombinant humanized collagen biomimetic microneedle for skin anti-aging and a preparation method thereof. Summary of the Invention

[0005] The object of the present invention is to address the problems existing in the above-mentioned prior art, and propose a recombinant humanized collagen biomimetic microneedle for skin anti-aging and a preparation method thereof.

[0006] A preparation method of a recombinant humanized collagen biomimetic microneedle for skin anti-aging according to the present invention includes the following steps:

[0007] Step 1: Design the shape of the microneedles using Abaqus software, optimize the geometric parameters through finite element analysis, and fabricate a polymethyl methacrylate (PMMA) punch and a polydimethylsiloxane (PDMS) mold.

[0008] Step 2: Mix recombinant humanized collagen and hyaluronic acid and inject them into the polydimethylsiloxane (PDMS) mold, and prepare recombinant humanized collagen biomimetic microneedles by the stepwise microforming method.

[0009] Further, the stepwise microforming method is specifically as follows:

[0010] Mix the prepolymer and the curing agent in a ratio of 10:1 (m / m), remove the bubbles under vacuum conditions, pour it into the mold, cure it under vacuum conditions, and finally peel it off from the PMMA mold to obtain a PDMS microneedle template.

[0011] Mix the recombinant humanized collagen solution and the hyaluronic acid solution in proportion. The concentration of recombinant humanized collagen is 0.1 mg / mL, and the concentration of hyaluronic acid is 15%. Pour the mixed solution into the tip part of the PDMS mold, and place the mold in a vacuum environment; after it dries, inject a 15% solution into the micro-mold to prepare the back lining layer, and the double-layer microneedles are formed after drying.

[0012] The present invention also relates to a recombinant humanized collagen biomimetic microneedle for skin anti-aging. The microneedle includes a needle tip, a base, and a recess. The base and the recess are composed of hyaluronic acid, and the needle tip is uniformly distributed with recombinant humanized collagen.

[0013] Further, the height of the microneedle is 500 - 1000 μm, the diameter is 250 - 500 μm, the needle tip spacing is 700 - 1200 μm, the width of the recess is 385 - 693 μm, the depth of the recess is 100 - 250 μm, and the height of the base is 100 - 250 μm.

[0014] Further, the height of the microneedle is 800 μm, the diameter is 250 μm, the needle tip spacing is 1200 μm, the width of the recess is 495 μm, the depth of the recess is 250 μm, and the height of the base is 120 μm.

[0015] Beneficial effects

[0016] The bionic microneedles of the present invention are jointly composed of a conical needle body, a base, and a recess. The tip part is loaded with recombinant humanized collagen, and the base and recess parts are prepared from hyaluronic acid. The microneedle patch has anti-aging effects in senescent cell models and photoaging mouse models. Compared with traditional application methods and conical microneedles, the bionic microneedles have better anti-aging effects in photoaging mouse models. It significantly increases the penetration depth of the microneedles, and its solubility and adhesiveness are superior to existing collagen microneedles. Description of the Drawings

[0017] Figure 1 It is a result diagram of the finite element analysis of the process of the microneedle piercing the skin in the present invention; among them, A) shows 5 different shapes of the microneedle; B) shows the comparison of the piercing forces of the 5 shapes of the microneedle; C) is an enlarged view of the comparison of the piercing forces of the 5 shapes of the microneedle; D) is the skin stress nephogram under the action of the microneedle (D represents the contact length between the microneedle and the dermis layer); E) is the penetration depth of the simulation models of different shapes of the microneedle; F) is the skin stress diagram of the simulation models of different shapes of the microneedle; G-J) show the influence of the tip width and height of the microneedle on the piercing force; K-N) show the influence of the groove width, depth, base width, and tip spacing of the microneedle on the penetration depth;

[0018] Figure 2 It is a schematic diagram of the preparation and characterization of the microneedle in the present invention; among them, A-B) show the morphology of the bionic microneedle; C-D) show the morphology of the ordinary conical microneedle; E-F) show the morphology of the octopus microneedle; G) is a comparison diagram of the solubility of the bionic microneedle, ordinary microneedle, and octopus microneedle; H) is a comparison diagram of the mechanical strength of the bionic microneedle, ordinary microneedle, and octopus microneedle; I-J) show the comparison of the adhesiveness of the bionic microneedle, ordinary microneedle, and octopus microneedle; K) shows the drug distribution at the tip of the bionic microneedle; L) shows the skin penetration performance of the bionic microneedle; M-N) show the transdermal drug delivery performance of the bionic microneedle; O) shows the stability of collagen in the bionic microneedle;

[0019] Figure 3 It is a schematic diagram of the in vitro anti-aging effect of the microneedle in the present invention; among them, A) shows the ROS scavenging ability of the microneedle; B-D) show the influence of the microneedle on the antioxidant enzyme activity of cells; E) shows the influence of the microneedle on the gene expression of P21, P16, P53, TNF-α, and MMP-9 in cells; F) shows the influence of the microneedle on the protein expression of P21, P16, and H3-K9 in cells;

[0020] Figure 4 It is a schematic diagram of the in vivo anti-aging effect and mechanism research of the microneedle in the present invention; among them, A) shows the HE staining and Masson staining of the mouse skin; B) shows the immunofluorescence staining of collagen and elastin in the mouse skin; C) shows the immunohistochemistry of MMP-9 / IL-6 / TNF-α in the mouse skin;

[0021] Figure 5Schematic diagram for quantitative analysis of the anti-aging effect of microneedles in vitro in the present invention; wherein A) is the quantitative analysis of the water content of mouse skin; B) is the quantitative analysis of the thickness of the dermis layer of mouse skin; C) is the quantitative analysis of the content of collagen fibers in mouse skin; D-E) are the quantitative analysis of the fluorescence intensity of collagen and elastin in mouse skin; F-H) are the quantitative analysis of the activity of antioxidant enzymes in mouse skin; I-K) are the quantitative analysis of the expression of MMP-9 / IL-6 / TNF-α in mouse skin. Detailed implementation manners

[0022] The following is combined with Figures 1 to 5 to specifically illustrate this implementation manner.

[0023] A recombinant humanized collagen biomimetic microneedle for skin anti-aging and its preparation method in the present invention, the preparation method includes the following steps:

[0024] Step 1: Construct a finite element skin model and a microneedle model through Abaqus software. The skin model consists of a stratum corneum, a dermis layer, and a subcutaneous tissue; a single microneedle model and a 3×3 microneedle array model are adopted. During the simulation process, the microneedle parameters are flexibly adjusted according to different simulation conditions;

[0025] The microneedle consists of a needle tip, a base, and a recess. The optimal parameters of the microneedle are: height 800μm, diameter 250μm, spacing 1200μm, recess width 495μm, recess depth 250μm, and base height 120μm.

[0026] This parameter combination can achieve the minimum piercing force while obtaining a higher piercing depth, thus significantly improving the overall performance of the microneedle.

[0027] Step 2: Prepare a mold according to the parameters determined in Step 1. Use PDMS with good flexibility as the material of the microneedle template. Mix the prepolymer and the curing agent in a ratio of 10:1 (m / m) at room temperature and add them to a beaker. Stir the mixture evenly by turning it up and down with a glass rod. Then remove the bubbles under vacuum conditions, and then pour it into the mold, place it under vacuum conditions for 20 minutes, and then put it into an oven to cure at 60°C for 1 hour. Gently peel it off from the metal microneedle to obtain the PDMS microneedle template.

[0028] Mix the recombinant humanized collagen solution and the hyaluronic acid solution in proportion. The final concentration of the recombinant humanized collagen reaches 0.1mg / mL, and the hyaluronic acid concentration is 15%. Pour the mixed solution into the tip part of the PDMS mold, scrape off the excess solution on the surface, and then place the mold in a vacuum environment for 30 minutes. After it dries naturally at room temperature, inject a 15% hyaluronic acid solution into the micro-mold to prepare the backing layer. After completing the above steps and drying, the double-layer microneedle is formed.

[0029] Microneedle Characterization

[0030] To observe the drug distribution, the fluorescent dye Rhodamine B was added during the preparation of microneedles, and the preparation process was exactly the same as the above operation. Ordinary microneedles and octopus microneedles were prepared through the same procedure, and the hyaluronic acid concentration was 15%.

[0031] The bionic microneedles were arranged in a 10×10 array. The microneedle morphology was complete and the structure was uniform. Their height was 560±50μm, and the tip spacing was 1.1mm. Conical, octopus-shaped, and bionic microneedles all exhibited excellent mechanical strength and did not bend or break during the process of piercing the skin. The bionic microneedles showed the highest adhesion strength, significantly superior to the other two microneedle structures. By calculating the remaining length ratio of the three types of microneedles after acting on the skin, it was found that the bionic microneedles had the highest dissolution ratio and showed the best dissolution performance.

[0032] To further verify the bilayer structure design of the microneedles and the distribution of collagen in the microneedles, the fluorescent dye Rhodamine B was incorporated into the microneedle tip solution. Observation with a fluorescence microscope showed that the tip part of the microneedles uniformly presented red fluorescence, while the basal part had almost no fluorescence signal.

[0033] The skin treated with microneedles was subjected to HE staining. The microneedles formed sharp conical needle hole morphologies in the skin, proving that the microneedles pierced the skin. Further, small animal in vivo imaging was used to evaluate the transdermal drug delivery ability of the microneedles. The fluorescent dye indocyanine green was loaded into the microneedles as a tracer. After the microneedles were pressed on the mouse skin and then removed, the mice were placed in an in vivo imaging chamber for fluorescence image acquisition. The skin surface treated with microneedles carried fluorescence. Quantitative analysis of the fluorescence signal showed that the fluorescence intensity gradually decreased over time, and the fluorescence could be maintained subcutaneously for at least 48h, indicating that the prepared microneedles had excellent transdermal drug delivery efficiency and could achieve a continuous drug release effect for up to 48h.

[0034] After being stored at 4℃ for three weeks, the circular dichroism spectra of collagen in the microneedles were basically coincident with the initial spectra, both showing a positive peak near 220nm and a negative peak near 190nm, which was consistent with the characteristic peak positions of the collagen triple helix structure reported in the literature. However, starting from the fourth week, the peak intensity in the circular dichroism spectra gradually decreased, accompanied by a slight blue shift phenomenon, indicating that there were subtle changes in the triple helix structure. Under room temperature conditions, the secondary structure of collagen in the microneedles remained relatively stable within the first week, but by the second week, its secondary structure had undergone a significant transformation. In a high-temperature environment of 55℃, the activity of collagen was rapidly lost and the triple helix structure was damaged. In summary, the recombinant humanized collagen microneedles can be hermetically stored at 4℃ for at least 1 month, and can maintain stability for one week under room temperature environment.

[0035] In vitro experiments have demonstrated the anti-aging effect of microneedles at the cellular level.

[0036] HSF cells were seeded into 6-well plates, and collagen raw materials or collagen microneedles were added to the culture medium to make the collagen concentration in the culture medium 0, 0.01, 0.05, 0.1, 0.5 mg / mL, and incubated for 24 h. The cells were irradiated with ultraviolet light for 10 min, and then incubated for another 6 h. The group without ultraviolet irradiation was set as the control group. The content of ROS in the cells was detected using a reactive oxygen species detection kit; the activities of antioxidant enzymes in the cells were detected using a glutathione peroxidase detection kit, a catalase detection kit, and a total SOD activity detection kit (Beyotime); the mRNA expressions of P21, P16, P53, TNF-α, and MMP were detected by real-time quantitative fluorescence PCR; the protein expressions of P21, P16, and H3-K9 were detected by Western Blot.

[0037] Recombinant humanized collagen can effectively reduce the accumulation of reactive oxygen species (ROS) induced by ultraviolet irradiation and enhance the activities of antioxidant enzymes (such as SOD, glutathione peroxidase, and catalase). Meanwhile, microneedle treatment significantly inhibited the expressions of senescence-related genes (P21, P16, P53) and inflammatory factors (TNF-α, MMP-9), and also inhibited the expressions of senescence proteins (P21, P16, H3-K9).

[0038] In vivo experiments have demonstrated the anti-aging effect of biomimetic microneedles.

[0039] In vivo experiments have demonstrated the anti-aging effect of microneedles at the mouse level.

[0040] A photoaging mouse model was constructed and administered simultaneously. At the end of the eighth week, the mice were sacrificed by cervical dislocation, and then the full-thickness skin of the experimental area on their backs was quickly removed. After carefully peeling off the connective tissue and subcutaneous fat in the skin, the skin was quickly cut into three parts, one for measuring skin moisture content, one for measuring the content of antioxidant enzymes in the skin, and the other was fixed in 4% paraformaldehyde solution for paraffin embedding and sectioning. Then, HE staining, Masson staining, immunofluorescence staining (collagen, elastin), and immunohistochemical (IL-6, TNFα, MMP) staining were performed in sequence.

[0041] In animal experiments, microneedles significantly increased the water content, dermal layer thickness, and collagen fiber content of the skin of photoaging mice. Through multiple mechanisms such as moisturizing, antioxidant, promoting the expression of collagen and elastin, and inhibiting inflammatory reactions, it significantly delayed skin aging. Compared with the traditional application method and conical microneedles, the biomimetic microneedles of the present invention have a better anti-aging effect.

[0042] The above content of the present invention is only a preferred embodiment of the present invention and is not used to limit the implementation of the present invention. Those of ordinary skill in the art can easily make corresponding adaptations or modifications according to the main concept and spirit of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope required by the claims.

Claims

1. A method for preparing recombinant humanized collagen bionic microneedles for skin anti-aging, characterized in that: The following steps are involved: Step 1: Use Abaqus software to design the shape of the microneedle, use finite element analysis to optimize the geometric parameters, and make a polymethyl methacrylate male mold and a polydimethylsiloxane female mold; Step 2: Mix recombinant humanized collagen and hyaluronic acid and inject them into a polydimethylsiloxane mold, and prepare recombinant humanized collagen bionic microneedles through a step-by-step micro-molding method.

2. The method for preparing the recombinant humanized collagen bionic microneedle according to claim 1, characterized in that: The step-by-step micro-molding method is specifically: The prepolymer and the curing agent are mixed in a ratio of 10:1 (m / m), bubbles are removed under vacuum conditions, poured into the mold, cured under vacuum conditions, and finally peeled off from the PMMA mold to obtain a PDMS microneedle template; The recombinant humanized collagen solution and the hyaluronic acid solution were mixed in proportion, the recombinant humanized collagen concentration was 0.1 mg / mL, and the hyaluronic acid concentration was 15%, and the mixed solution was poured into the tip part of the PDMS mold, and the mold was placed in a vacuum environment; After it dries, 15% of the solution is injected into the micro mold to prepare a backing layer, and after drying, the double-layer microneedles are formed.

3. A recombinant humanized collagen bionic microneedle for skin anti-aging prepared by the method according to any one of claims 1 to 2, characterized in that: The microneedle comprises a needle tip, a base and a recessed chamber, wherein the base and the recessed chamber are made of hyaluronic acid, and recombinant humanized collagen is evenly distributed on the needle tip.

4. The recombinant humanized collagen bionic microneedle according to claim 3, characterized in that: The microneedle has a height of 500-1000 μm, a diameter of 250-500 μm, a needle tip spacing of 700-1200 μm, a recess chamber width of 385-693 μm, a recess chamber depth of 100-250 μm, and a base height of 100-250 μm.

5. The recombinant humanized collagen bionic microneedle according to claim 4, characterized in that: The microneedle has a height of 800 μm, a diameter of 250 μm, a needle tip spacing of 1200 μm, a recess chamber width of 495 μm, a recess chamber depth of 250 μm, and a base height of 120 μm.

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