Nano collagen nucleic acid peptide as well as preparation method and application thereof

Abstract

The invention discloses a nano collagen nucleic acid peptide as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method comprises the following steps: S1, dissolving a polylactic acid-glycolic acid copolymer in ethyl acetate, and carrying out ultrasonic treatment to obtain a solution A; s2, sequentially dissolving polysorbate-20, III type collagen and DNA (deoxyribonucleic acid) sodium into water to obtain a solution B; s3, adding the solution B into the solution A, performing ultrasonic emulsification to obtain a mixed solution C, and performing centrifugation after stirring to obtain supernate; and S4, respectively adding tocopherol, ursolic acid and polyethylene glycol 2000 into the supernate, dissolving in absolute ethyl alcohol, carrying out vacuum drying, centrifuging, and extruding to obtain the nano collagen nucleic acid peptide. According to the invention, the III type collagen and the DNA sodium are prepared into the nanoparticles, so that the transdermal absorption effect of the nanoparticles in the application of a product for repairing skin is improved, and the bioavailability of the nanoparticles in vivo is improved, thereby achieving a better repairing effect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a nano-collagen nucleic acid peptide, its preparation method, and its application. Background Technology

[0002] In recent years, the market size of functional skincare products has been increasing year by year. According to questionnaire survey data analysis, efficacy and ingredients are the primary considerations for consumers when purchasing skincare products. 82% of consumers focus on efficacy, 71% focus on ingredients, and 58% focus on both. Skin anti-aging is a perennial hot topic in functional skincare and medical aesthetics, and its key lies in repairing the damaged skin barrier. Skin is increasingly susceptible to various problems such as dryness, aging, inflammation, and barrier damage due to external environmental stimuli and internal factors. Strengthening the skin barrier can effectively defend against external stimuli and reduce moisture loss. Therefore, repairing the skin barrier is the first step in anti-aging. By choosing skincare products suitable for your skin type, you can provide the skin with the necessary nutrients and moisture, repair the damaged epidermis, activate cell regeneration, and restore the skin's health and radiance. Only by repairing the skin barrier can a stable environment be provided for the skin, maximizing the effectiveness of anti-aging products, which will open up new possibilities for achieving youthful and healthy skin.

[0003] The efficacy of repair products depends on their repairing ingredients. Type III collagen is a crucial component of skin structure, possessing excellent biocompatibility and biodegradability. By supplementing and repairing collagen, it can promote skin elasticity, hydration, and firmness. Sodium DNA, also known as polydeoxynucleotide, is an active ingredient that promotes cell regeneration and repair. By acting at the cellular level, it can stimulate skin cell metabolism and promote collagen synthesis, thereby achieving skin repair and regeneration. However, both type III collagen and sodium DNA are large molecules, far exceeding 500 Daltons, making transdermal absorption difficult. Therefore, developing a raw material that can both repair damaged skin and promote transdermal absorption is an urgent need. Summary of the Invention

[0004] The purpose of this invention is to provide a nano-collagen nucleic acid peptide, its preparation method, and its application, to solve the problems existing in the prior art. By preparing type III collagen and sodium DNA into nanoparticles, the transdermal absorption effect in skin care products is improved, and its bioavailability in vivo is enhanced, thereby achieving a better repair effect.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing nano-collagen nucleic acid peptides, comprising the following steps:

[0007] S1. Dissolve the polylactic acid-glycolic acid copolymer in ethyl acetate and sonicate to obtain solution A;

[0008] S2. Polysorbate-20, type III collagen and sodium DNA are dissolved in water sequentially to obtain solution B;

[0009] S3. Add solution B to solution A, emulsify by sonication to obtain mixed solution C, stir and centrifuge to obtain supernatant;

[0010] S4. Tocopherol, ursolic acid and polyethylene glycol 2000 were added to the supernatant and dissolved in anhydrous ethanol. After vacuum drying, the mixture was centrifuged and extruded to obtain the nano-collagen nucleic acid peptide.

[0011] In the polylactic acid-glycolic acid copolymer, the molar ratio of lactic acid to glycolic acid is 75:25.

[0012] Further, in step S1, the mass-to-volume ratio of the polylactic acid-glycolic acid copolymer to the ethyl acetate is 10 mg: 1 mL.

[0013] Further, in step S2, the mass ratio of the polysorbate-20, the type III collagen, and the sodium DNA is 44:(5-15):(5-15).

[0014] Furthermore, in step S3, the ultrasonic emulsification time is 2 minutes.

[0015] Furthermore, in step S3, the stirring conditions are 1250 rpm for 3 hours; the centrifugation conditions are 10000 rpm for 5 minutes.

[0016] Further, in step S4, the mass ratio of the tocopherol, the ursolic acid, and the polyethylene glycol 2000 is 2:5:1.

[0017] Furthermore, in step S4, the extrusion conditions are 0.75 kPa, passing through a 50 nm film, and repeated twice.

[0018] The present invention also provides a nano-collagen nucleic acid peptide prepared by the above preparation method.

[0019] This invention also provides the application of the aforementioned nano-collagen nucleic acid peptide in the preparation of skin care and repair products.

[0020] The present invention discloses the following technical effects:

[0021] This invention uses polylactic acid-glycolic acid copolymer (PLA-glycolic acid copolymer) as the active ingredient of the carrier material. Compared with the phospholipid molecules of traditional nanocarriers, it has better stability and uniformity, effectively protecting the drug within the carrier from the influence of the external environment. This promotes uniform drug distribution and release, prolongs the drug's effective period, and improves its bioavailability. Simultaneously, by adjusting the material structure and ratio, sustained drug release is achieved, extending the drug's duration of action and improving its persistence.

[0022] This invention prepares type III collagen and sodium DNA into nanoparticles, which can promote drug penetration through the skin barrier, improve drug permeability, increase drug absorption efficiency, and precisely deliver drugs to target tissues or cells, thereby improving drug targeting and bioavailability and enhancing efficacy.

[0023] This invention combines type III collagen and sodium DNA, which synergistically enhance each other's efficacy, resulting in better therapeutic effects. The combined use of tocopherol and ursolic acid allows them to regenerate each other, enhancing their antioxidant capacity, combating free radical damage, and protecting cells from oxidative damage. Furthermore, ursolic acid promotes collagen synthesis, while tocopherol protects collagen from oxidative damage; their combined use promotes skin repair and regeneration. Tocopherol and ursolic acid synergistically promote skin moisturizing and anti-aging effects, improving skin health.

[0024] The nano-collagen nucleic acid peptides provided by this invention possess physical properties such as stability and uniformity, controllable sustained release, and transdermal absorption. Simultaneously, they exhibit multiple biological benefits including antioxidant, repair, and anti-aging effects. They can be applied to the preparation of skincare product raw materials related to anti-aging, antioxidant, transdermal absorption, and controllable sustained release. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Transmission electron microscope images of images 1-3 under no load, magnified 10,000 times;

[0027] Figure 2 Transmission electron microscope images of samples 1-4 magnified 30,000 times;

[0028] Figure 3 The expression levels of the COL1A1 gene in each group were detected by qRT-PCR.

[0029] Figure 4The expression levels of COL3A1 gene in each group were detected by qRT-PCR;

[0030] Figure 5 The expression levels of the MMP2 gene in each group were detected by qRT-PCR.

[0031] Figure 6 The expression levels of the MMP9 gene in each group were detected by qRT-PCR.

[0032] Figure 7 The results of ROS content detection in each group were obtained using a real-time microplate reader.

[0033] Figure 8 Results of ELISA detection of ColⅠ protein expression levels in each group;

[0034] Figure 9 The results are for the transdermal absorption test of the active ingredients (the sum of type III collagen and sodium DNA) in each sample group. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Unless otherwise specified, "room temperature" and "room temperature" in the embodiments of this invention refer to 15-30℃.

[0041] Unless otherwise specified, the "%" mentioned in this invention refers to a percentage by mass.

[0042] In this embodiment of the invention, PLGA refers to poly(lactic-co-glycolic acid) copolymer, which is composed of lactic acid and glycolic acid in a molar ratio of 75:25; PEG-2000 refers to polyethylene glycol 2000; all raw materials, unless otherwise specified, can be obtained from commercial sources.

[0043] Example

[0044] Preparation process of PLGA nanoparticles:

[0045] PLGA nanoparticle empty carriers 1-3 and PLGA nanoparticle collagen nucleopeptide samples 1-4 were prepared according to the raw material formulation ratios in Table 1.

[0046] Table 1 Raw Material Ratio

[0047]

[0048] The preparation methods for empty loads 1 to 3 are as follows:

[0049] 1) Weigh PLGA and dissolve it in 1 mL of ethyl acetate, then sonicate until dissolved;

[0050] 2) Dissolve polysorbate-20 in 2 mL of water;

[0051] 3) Add the solution from 2) to 1) and sonicate for 2 minutes;

[0052] 4) After ultrasonic emulsification, the mixed solution was stirred at 1250 rpm for 3 hours in a fume hood, then centrifuged at 10000 rpm for 5 minutes, and the supernatant was collected.

[0053] 5) Add PEG-2000 and dissolve it in 0.45 mL of anhydrous ethanol;

[0054] 6) Dry in a vacuum drying oven at 40℃ for 10 minutes;

[0055] 7) Centrifuge at 4℃, 12000 rpm for 30 min;

[0056] 8) Extrusion (0.75 kPa, 50 nm film, repeated twice) to obtain empty nanoparticles.

[0057] The preparation methods for collagen nucleic acid peptide samples 1-4 are as follows:

[0058] 1) Weigh PLGA and dissolve it in 1 mL of ethyl acetate, then sonicate until dissolved;

[0059] 2) Dissolve polysorbate-20, type III collagen, and sodium DNA in 2 mL of water;

[0060] 3) Add the solution from 2) to 1) and sonicate for 2 minutes;

[0061] 4) After ultrasonic emulsification, the mixed solution was stirred at 1250 rpm for 3 hours in a fume hood, then centrifuged at 10000 rpm for 5 minutes, and the supernatant was collected.

[0062] 5) Add tocopherol (vitamin E), ursolic acid (which has the effect of softening the stratum corneum and promoting absorption) and PEG-2000 to the supernatant in sequence, and dissolve in 0.45 mL of anhydrous ethanol;

[0063] 6) Dry in a vacuum drying oven at 40℃ for 10 minutes;

[0064] 7) Centrifuge at 4℃, 12000 rpm for 30 min;

[0065] 8) Extrusion (0.75 kPa, 50 nm membrane, repeated twice) to obtain collagen nucleic acid peptide sample nanoparticles.

[0066] Effect verification example

[0067] I. Characterization and Detection

[0068] 1. Transmission electron microscopy (TEM) image

[0069] The detection steps are as follows: Use a pipette to drop 10 μL of sample onto a copper grid and allow it to precipitate for 1 minute. Absorb the excess liquid with filter paper. Then, add 10 μL of uranium acetate to the sample area, absorb the excess liquid with filter paper, and allow it to dry at room temperature for several minutes. Finally, perform electron microscopy imaging at 80-120 kV to obtain the electron microscopy imaging results, such as... Figure 1-2 As shown ( Figure 1 The values ​​in 'ac' represent the TEM results magnified 10,000 times for unloaded mode 1, unloaded mode 2, and unloaded mode 3, respectively. Figure 2 The images in the image above are TEM results of samples 1, 2, 3, and 4 magnified 30,000 times, respectively.

[0070] 2. Particle size

[0071] The particle size determination procedure is as follows: The particle sizes of unloaded samples 1–3 and samples 1–4 were determined using dynamic light scattering (DLS) with a Nicomp Z3000 nanoparticle size analyzer (USA). 1000 μL of sample was diluted to 3000 μL with PBS buffer of the same concentration, and the particle size was measured at 300 kHz and room temperature. The results are shown in Table 2.

[0072] Table 2. Particle size detection results

[0073] serial number Average particle size (d.nm) Unloaded 1 280.36±1.44 Unloaded 2 130.52±1.00 Unloaded 3 103.08±0.52 Sample 1 108.23±0.37 Sample 2 107.31±0.19 Sample 3 101.63±0.45 Sample 4 98.02±0.28

[0074] Depend on Figure 1-2 As shown in Table 3, the optimal ratio of PLGA to polysorbate-20 without loading is 10:44 (empty loading 3). The particle size of the collagen nucleic acid peptide samples prepared under this ratio is all <150nm. Among them, the average particle size of sample 4 is 98.02±0.28nm, which is the optimal particle size.

[0075] 3. Encapsulation rate

[0076] Sample preparation: take the free supernatant of different collagen nucleic acid peptide nanoparticle samples (samples 1-4) prepared in the examples: centrifuge at 4℃, 15000rpm for 20min, and take the supernatant for testing;

[0077] Detection method: High performance liquid chromatography (HPLC).

[0078] Chromatographic condition 1 (Type III collagen):

[0079] Column: Reversed-phase Hypersil Gold C18 (250mm × 4.6mm, 5μm);

[0080] Mobile phase: A is an aqueous solution containing 5% acetonitrile and 0.05% trifluoroacetic acid (TFA), and B is acetonitrile;

[0081] The detection wavelength was 220 nm, the flow rate was 0.1 mL / min, and the column temperature was 35 °C. Good linearity was observed in the range of 1 mg / mL to 20 mg / L: Y = 3290.28X + 58.25, RL 2 =0.9996, detection limit is 0.01 mg / mL.

[0082] Chromatographic condition 2 (sodium DNA):

[0083] Column: Reversed-phase Hypersil Gold C18 (250mm × 4.6mm, 5μm);

[0084] Mobile phase: A is a solution containing 0.025mM Tris-HCl, with pH adjusted to 8.0 using 1M NaOH; B is a solution of 0.025mM Tris-HCl + 1M NaCl, with pH adjusted to 8.0 using 1M NaOH.

[0085] The detection wavelength was 260 nm, the flow rate was 0.7 mL / min, and the column temperature was 60 °C. Good linearity was observed in the range of 1 mg / mL to 20 mg / L: Y = 2314.15X + 82.12, RL 2 =0.9965, detection limit is 0.01 mg / mL.

[0086] Calculation formula:

[0087] Encapsulation efficiency = (Total drug amount - Free drug amount) / Total drug amount × 100%.

[0088] The encapsulation efficiency test results are shown in Table 3.

[0089] Table 3 Encapsulation efficiency test results

[0090] serial number Type III collagen / mg Sodium DNA / mg Encapsulation rate Sample 1 0 0 0 Sample 2 11.2 4.5 78.5％ Sample 3 3.3 13.7 85.0％ Sample 4 8.9 9.2 90.5％

[0091] II. Efficacy Testing

[0092] 1. SIPS (Short-Incidence Premature Aging Model)

[0093] We established a stress-induced premature aging model of human diploid fibroblasts (SIPS), measured the expression of collagen and matrix metalloproteinases in fibroblasts at the gene and protein levels, investigated the role of matrix metalloproteinases in regulating collagen degradation, and studied the anti-aging effects of bioactive substances.

[0094] 1.1 Experimental Methods:

[0095] ① Cell selection: Primary isolation and culture of human skin fibroblasts (HSF), selecting 4th generation cells for experiments, with cell confluence of more than 60%, cultured in 96-well plates or 35mm cell culture dishes;

[0096] ② Sample processing:

[0097] The cells used in the experiment in ① were randomly divided into 7 groups (negative control group, blank control group, positive control group, test group 1 (1% sample 1), test group 2 (1% sample 2), test group 3 (1% sample 3) and test group 4 (1% sample 4)).

[0098] Positive control group cells were cultured in 100 μM VE cell culture medium;

[0099] Cells from the negative control group, blank control group, and test groups 1-4 were cultured in standard cell culture medium (DMEM basal medium with low glucose (GlutaMAX)). TM The cells were cultured in DMEM (10% fetal bovine serum (Gibco), antibiotic-free). In this case, 1% of sample 1, 1% of sample 2, 1% of sample 3 and 1% of sample 4 were added to the cell culture medium of test groups 1-4 respectively.

[0100] ③ Add H2O2 to the cell culture medium of each group (excluding the blank control group) to make the concentration 10μM, and treat for a total of 7 days. Replace the cell culture medium with a new one on the 3rd day.

[0101] ④ Collect cells, detect gene expression of COL1A1, COL3A1, MMP2 and MMP9 by qRT-PCR, and detect ROS content by fluorescence quantitative ELISA reader;

[0102] ⑤ Collect the supernatant and use ELISA to detect the protein expression of ColⅠ.

[0103] Step ④ of the qRT-PCR reaction includes the following operations:

[0104] (1) Extraction of total RNA

[0105] Prepare 1.5 mL AXYGEN RNase-free EP tubes in advance and label them accordingly;

[0106] After changing the medium and culturing for 3 hours, aspirate the original cell culture medium and wash each well twice with 1 mL of PBS to remove residual cell culture medium.

[0107] Remove the PBS, add 1 mL of NucleoZOL lysis reagent to each well, and after fully lysing the cells, transfer all the cell lysis buffer to a labeled 1.5 mL EP tube (one tube per well, with corresponding labels, and try to transfer as much as possible to reduce residue);

[0108] Add 400 μL of RNase-free water (or DEPC water) to each tube, shake the sample vigorously for 15 seconds, incubate at room temperature for 15 minutes, centrifuge at 12000×g for 15 minutes, and aspirate the supernatant containing RNA (approximately 75%, 800 μL) into a new 1.5 mL EP tube with the corresponding label.

[0109] Add an equal volume (700 μL) of isopropanol to a new EP tube, incubate at room temperature for 10 min, centrifuge at 12000×g for 10 min, and usually there will be white granular RNA precipitate at the bottom of the tube;

[0110] Remove the supernatant, add 600 μL of 75% ethanol (analytical grade anhydrous ethanol and DEPC water, freshly prepared) to the EP tube, centrifuge at 8000×g for 3 min, remove the supernatant, repeat this washing step, remove the 75% ethanol, and air dry the RNA appropriately.

[0111] Add 20 μL of RNase-free dd H2O (or DEPC water) to each EP tube to redissolve the RNA. Vortex for 2–5 minutes to ensure complete dissolution. For long-term storage, store at -80°C.

[0112] (2) RNA purity and concentration detection

[0113] The purity and concentration of RNA samples were detected using an ultra-micro spectrophotometer. The detection volume for each sample was 3–4 μL, and the relevant data on the display screen were recorded.

[0114] OD 260 / OD 280 A value in the range of 1.9 to 2.1 indicates that the RNA purity is acceptable, and the RNA sample can be used for subsequent experiments;

[0115] RNA concentration (μg / mL) = OD 260 ×Dilution factor×40, dilute the RNA with TE buffer at pH=8.0 to achieve an RNA concentration in the range of 200–1000 μg / mL.

[0116] (3) Reverse transcription

[0117] The kit used was the StarScript II First-strand cDNA Synthesis Kit With gDNARemover (reverse transcription stage), and the entire process was performed on ice.

[0118] To detect RNA concentration, a total RNA concentration of 50 ng to 2 μg is usually used; 1 μg is used here.

[0119] Thaw the template RNA and cDNA synthesis kit on ice and vortex to mix.

[0120] Prepare the reaction system for removing genomic DNA (i.e. gDNA) from RNA (Table 4);

[0121] Table 4 gDNA Remover System

[0122] reagents Added amount RNA templates (13) 1μg 5×gDNA Remover Reaction buffer 1.6μL gDNA Remover 0.5μL <![CDATA[RNase-free dd H2O or DEPC water]]> To8μL

[0123] After mixing the above reaction system using an eight-tube bundle (including cap), briefly centrifuge at 37°C for 5 minutes.

[0124] After the gDNA Remover system reaction is complete, prepare the cDNA reverse transcription system (Table 5). According to the number of tubes, first prepare the corresponding volumes of other total systems except for the 8 μL gDNA Remover system, and then add the other total systems to each well of the eight-tube array, so that the final volume of each tube is 8 μL + 12 μL = 20 μL.

[0125] Table 5 cDNA reverse transcription system

[0126] reagents Added amount 2×RT Reaction buffer 10μL Primer mix(random & OligodT) 1μL StarScriptII RT mix 1μL RNA template after gDNA Remover treatment 8μL

[0127] The above reverse transcription system was mixed, briefly centrifuged, incubated at 42°C for 15–30 min, and then heated at 85°C for 5 min to inactivate StarScript II RTmix.

[0128] After the reaction is complete, the resulting cDNA can be placed on ice for subsequent experiments or stored at -20°C.

[0129] (4) qRT-PCR reaction

[0130] The primers used in the qRT-PCR reaction are shown in Table 6.

[0131] Table 6 qRT-PCR reaction primers

[0132]

[0133]

[0134] After synthesizing primers according to the sequence information in Table 6, dilute them to a concentration of 10 μM with RNase-free dd H2O or DEPC water and store them at -20℃. Thaw them on ice together with other reagents before use.

[0135] The qRT-PCR reaction used a 2×RealStar Green Power Mixture (PCR amplification stage) kit, and the entire process was performed on ice. The cDNA template, forward and reverse primers for PCR, and the qRT-PCR kit were thawed on ice and vortexed before use. The reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.

[0136] Table 7 qRT-PCR reaction system

[0137] reagents Added amount Forward primer (10 μM) 0.5μL Reverse primer (10 μM) 0.5μL 2×RealStar Green Power Mixture 10μL RNase-free dd H2O or DEPC water 8μL cDNA template 1μL

[0138] Table 8 qRT-PCR reaction procedure

[0139]

[0140] The experiment was performed on the corresponding real-time PCR instrument, and the data was recorded; data processing was performed based on the internal reference gene and the gene to be tested (2). -△△CT Then, the data from the blank control group were normalized and plotted.

[0141] Step ④, detecting ROS content, includes the following operations:

[0142] DHE red fluorescent dye (5mM), diluted the stock solution to 5μM with serum-free medium, and injected into 96-well plates at a rate of 1×10⁶ cells per well. 5 Add 200 μL of staining solution (working solution concentration 5 μM) and incubate at 37°C in the dark for 30-40 min. Then, remove the staining solution, wash twice with PBS, and detect with a fluorescence microplate reader (excitation wavelength 518 nm, emission wavelength 610 nm).

[0143] Formula for calculating relative intracellular ROS:

[0144] Relative intracellular ROS (%) = [A(detection) - A(solvent)] / [A(induction) - A(solvent)] × 100

[0145] A (Detection): Fluorescence intensity of wells containing cells, CCK-8 solution, and the sample solution to be tested.

[0146] A (solvent): Fluorescence intensity in pores containing culture medium and CCK-8 solution but without cells.

[0147] A (Induction): Fluorescence intensity of wells containing cells, CCK-8 solution, and no sample solution.

[0148] Step ⑤, the process of detecting ColⅠ protein expression, includes the following operations:

[0149] 1) Remove the reagent kit 30 minutes before the experiment and allow it to return to room temperature;

[0150] 2) Add 100 μL of standard and sample to each well, and cover with adhesive tape. Incubate at 37°C for 2 hours.

[0151] 3) Discard the liquid with a pipette, add 100 μL of biotinylate antibody to each well. Cover with a thin strip of gel and incubate at 37°C for 1 hour;

[0152] 4) Discard the liquid with a pipette, add 200 μL of washing solution to each well, let stand for 2 min, discard the washing solution, and repeat the washing operation 2 to 3 times;

[0153] 5) Add 100 μL of enzyme-labeled avidin to each well. Cover with a new gel strip and incubate at 37°C for 1 hour.

[0154] 6) Discard the liquid and repeat step 4) 5 times;

[0155] 7) Add 90 μL of TMB substrate solution to each well. Incubate at 37°C in the dark for 15-30 min;

[0156] 8) Add 50 μL of stop solution to each well, and gently tap the plate to mix thoroughly;

[0157] 9) Immediately measure the OD value using an ELISA reader at a wavelength of 450nm (within 5 minutes).

[0158] 1.2 Experimental Results

[0159] (1) qRT-PCR detection results:

[0160] The results are as follows Figures 3-6 As shown.

[0161] Depend on Figure 3 and Figure 4 It was found that, compared with the negative control group, the expression levels of collagen production-related genes (COL1A1 and COL3A1) in all test groups and the positive control group (100 μM VE) were significantly increased, with the most significant increase in the expression level of collagen-promoting genes in sample 4; Figure 5 and Figure 6 It can be seen that, compared with the negative control group, the expression levels of collagen loss-related genes (MMP21 and MMP9) in each test group and the positive control group (100μMVE) were significantly downregulated. Among them, the expression level of collagen loss-related genes in sample 4 decreased most significantly, proving that sample 4 has excellent skin anti-aging effects.

[0162] (2) ROS detection results

[0163] The results are as follows Figure 7 As shown, compared with the negative control group, the ROS expression levels in each test group and the positive control group (100 μM VE) were significantly downregulated. Among them, the ROS expression level of sample 4 was reduced most significantly, which proves that it has excellent antioxidant effect.

[0164] (3) ColⅠ test results

[0165] The results are as follows Figure 8 As shown, compared with the negative control group, the collagen expression levels in each test group and the positive control group (100 μM VE) were significantly increased, with the most significant increase in type I collagen expression in sample 4.

[0166] 2. Transdermal Absorption Living Skin Model (TDDS)

[0167] 2.1 Experimental Methods

[0168] Pigskin from the abdomen of Bama miniature pigs (thickness <1mm, 0.8-1mm was selected in this study) was selected. Fresh skin was generally used within 24 hours of extraction and could be stored for a short period up to -4℃. For long-term storage, it should be stored at -80℃, and theoretically, the storage period should not exceed 3 months.

[0169] Before conducting transdermal experiments, a skin integrity assessment should be performed. The specific steps are as follows:

[0170] 1) Secure the skin, receiving pool, and supply pool, wherein the receiving pool is not filled with any liquid.

[0171] 2) Add an appropriate amount (3 mL) of physiological saline or PBS solution to the supply tank.

[0172] 3) If no significant subcutaneous fluid titration or outflow occurs after 30 minutes, it indicates that the skin barrier function is intact; otherwise, it indicates that the skin barrier function is impaired.

[0173] Skin with intact skin barrier function was selected for transdermal absorption experiments. The steps are as follows:

[0174] 1) Lay the pigskin flat and use surgical scissors to cut a circular piece of skin with a radius of about 1.5cm;

[0175] 2) Fill the receiving pool with 6 mL of physiological saline, place the rotor inside, make the surface of the physiological saline adhere to the skin, and fix the receiving pool with a rack;

[0176] 3) Start the water bath to control the temperature at 32±1℃, and the rotor speed is 220rpm;

[0177] 4) Add 3 mL of the test sample (samples 1-4) to the supply cell and record the time;

[0178] 5) During sample preparation, the liquid in the supply tank should be checked periodically, and physiological saline should be added in a timely manner to fill the original volume.

[0179] 6) After incubation for 0.25, 0.5, 1, 2, 4, 6, 12 and 24 hours, collect samples from the receiving pool and the supply pool;

[0180] 7) Prepare samples and store them at 4℃ for a short period of time;

[0181] 8) The sustained-release amount of type III collagen and sodium DNA was tested using high performance liquid chromatography (HPLC).

[0182] 2.2 Experimental Results

[0183] Specific results are as follows Figure 9The transdermal absorption of the active ingredients (the sum of type III collagen and sodium DNA) in each of the tested sample groups increased significantly. Among them, sample 4 had the best transdermal absorption effect, which was higher than that of samples 2 and 3, and much higher than that of sample 1.

[0184] In summary, based on the particle size of samples 1-3 and TEM analysis, the optimal ratio of PLGA to polysorbate-20 in the empty payload is 10:44 (m:m). Based on the encapsulation efficiency of samples 1-4, and verification using the SIPS and TDDS models, the optimal efficacy ratio of type III collagen to sodium DNA nanopeptides is 10:10 (m:m). Collagen nanoparticles prepared under these conditions exhibit significant anti-aging (promoting collagen production and inhibiting collagen degradation), antioxidant (downregulating ROS), transdermal absorption, and controllable sustained release (TDDS curve) effects, making them suitable for preparing related skincare product ingredients.

[0185] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing nano-collagen nucleic acid peptides, characterized in that, Includes the following steps: S1. Dissolve the polylactic acid-glycolic acid copolymer in ethyl acetate and sonicate to obtain solution A; S2. Polysorbate-20, type III collagen and sodium DNA are dissolved in water sequentially to obtain solution B; S3. Add solution B to solution A, emulsify by sonication to obtain mixed solution C, stir and centrifuge to obtain supernatant; S4. Tocopherol, ursolic acid and polyethylene glycol 2000 were added to the supernatant and dissolved in anhydrous ethanol. After vacuum drying, the mixture was centrifuged and extruded to obtain the nano-collagen nucleic acid peptide. In the polylactic acid-glycolic acid copolymer, the molar ratio of lactic acid to glycolic acid is 75:

25.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of the polylactic acid-glycolic acid copolymer to the ethyl acetate is 10 mg: 1 mL.

3. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of polysorbate-20, type III collagen and sodium DNA is 44:(5-15):(5-15).

4. The preparation method according to claim 1, characterized in that, In step S3, the ultrasonic emulsification time is 2 minutes.

5. The preparation method according to claim 1, characterized in that, In step S3, the stirring conditions are 1250 rpm for 3 hours; the centrifugation conditions are 10000 rpm for 5 minutes.

6. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of the tocopherol, the ursolic acid, and the polyethylene glycol 2000 is 2:5:

1.

7. The preparation method according to claim 1, characterized in that, In step S4, the extrusion conditions are 0.75 kPa, passing through a 50 nm membrane, and repeated twice.

8. A nano-collagen nucleic acid peptide prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the nano-collagen nucleic acid peptide as described in claim 8 in the preparation of skin repair products.

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