A polymer nanomicelle, its preparation method and application

Abstract

This invention belongs to the field of cosmetic technology and provides a polymer nanomicelle, its preparation method, and its application. The polymer nanomicelle of this invention is prepared from an amphiphilic block copolymer and an active ingredient; the structure of the block copolymer is shown in the following formula; the number-average molecular weight of the PEG is 2000-4000; and the number-average molecular weight of the PCL is 1800-13000. This polymer nanomicelle encapsulates a high content of active ingredient, and its active physicochemical properties and sustained-release properties are significantly improved after encapsulation; when combined with the active ingredient, it exhibits better in vitro antioxidant activity and whitening activity, and is widely used in cosmetic active ingredient encapsulation technology.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, and relates to a polymer nanomicelle, its preparation method and application. Background Technology

[0002] As people's awareness continues to improve, they pay more attention to actual efficacy when choosing cosmetics. Today's cosmetics industry is no longer simply about piling on ingredients; instead, it invests more effort in improving efficacy and user experience. In cosmetic research and development, the transdermal absorption of active ingredients has always been a challenge that researchers have been striving to overcome. At the same time, active ingredients often suffer from instability in their physicochemical properties, poor solubility, and dark color, which increases the difficulty of formulation during the research and development process. The introduction of nanotechnology offers a solution. Besides enhancing local skin delivery, nanocarriers have unique advantages in encapsulating active ingredients, improving stability, solubility, and color masking.

[0003] Chinese invention patent CN108524364B discloses a method for encapsulating cosmetic active ingredients in nanocarriers and preparing them. It uses soybean lipids in liquid form with a high concentration of lecithin as a dispersion medium and hydrogenated lecithin with good biocompatibility as an encapsulation material. The prepared cosmetic active ingredient nanocarriers are free of any organic solvents, safe and non-irritating, and can be used in cosmetics. They can also remain stable in aqueous cosmetic systems.

[0004] Chinese invention patent application CN105078776A discloses a carrier for encapsulating active ingredients in cosmetics and its preparation method. The carrier encapsulates soybean lecithin, xanthan gum, glycerin, propylene glycol, and 1,3-butanediol. Each component is sheared under high temperature homogenization to completely break down the cell walls and form a water mist. After cooling, the active ingredient extract is slowly added. This process is simple and effectively utilizes carrier encapsulation technology to encapsulate active ingredients, organically combining multiple effective components and improving the antioxidant capacity of the active ingredients. This effectively enhances the whitening, spot-fading, anti-aging, moisturizing, and skin-firming effects, and ensures that the effective active ingredients are fully absorbed by the skin.

[0005] Among the above technologies, lecithin is used to prepare liposomes. However, due to the presence of multiple unsaturated double bonds in its structure, it is easily oxidized and prone to discoloration under heating conditions, resulting in the instability of liposomes. Using hydrogenated lecithin instead of lecithin as a membrane material improves the stability of liposomes to some extent, but the high cost of hydrogenated lecithin leads to higher production costs for liposomes. In addition, liposomes have problems such as poor dilution stability and difficulty in formulation, which limit the widespread use of liposomes in cosmetics.

[0006] Chinese invention patent CN111821221B discloses a self-assembled micelle containing fragrance from a hydrophilic-hydrophobic polymer and its preparation method. The micelles are core-shell structures formed by the self-assembly of amphiphilic copolymers. The inner core layer is composed of hydrophobic polylactic acid segments, and the outer core layer is composed of hydrophilic polyethylene glycol segments. The fragrance is encapsulated within the inner core layer. This micelle exhibits a sustained-release effect on the fragrance, good stability, and effectively prolongs the fragrance's lasting effect, but the release amount is small.

[0007] To enhance the efficacy of liposomes in cosmetics, it is necessary to improve them to achieve full release of polymer nano-encapsulated active ingredients, improve activity, and shorten action time; at the same time, it should also improve stability and solubility. Summary of the Invention

[0008] To address the problems of dark color, poor stability, and insignificant sustained-release effect of active ingredients in existing cosmetic products, this invention provides a polymer nanomicelle, its preparation method, and its application. The polymer nanomicelle is obtained by reacting a hydrophobic active ingredient with a polyethylene glycol-polycaprolactone block copolymer as a carrier. This polymer nanomicelle has advantages such as high stability and high content of encapsulated active ingredients, and has wide applications in the field of cosmetic technology.

[0009] One of the technical solutions of the present invention is:

[0010] A polymeric nanomicelle is prepared from an amphiphilic block copolymer and an active ingredient; the structure of the block copolymer is shown in the following formula:

[0011] The polymer nanomicelles were prepared from an amphiphilic block copolymer and an active ingredient; the structure of the block copolymer is shown in the following formula:

[0012]

[0013] The preferred structure of the block copolymer is:

[0014]

[0015] Further preferred options are:

[0016]

[0017] The number-average molecular weight of the PEG is 2000-4000.

[0018] Preferably, the molecular weight of the PEG data is 2000.

[0019] The number-average molecular weight of the PCL is 1800-13000.

[0020] Preferably, the number-average molecular weight of the PCL is 8000-12000.

[0021] More preferably, the number-average molecular weight of the PCL fragments at both ends of the PCL-PEG-PCL is 4880, and the number-average molecular weight of the PEG fragment is 2000.

[0022] Preferably, the amphiphilic block copolymer has a core-shell structure.

[0023] Furthermore, the active ingredient is a hydrophobic active ingredient.

[0024] Furthermore, the active ingredient is preferably at least one of the following: peptidotope, phenylethyl resorcinol, 4-butylresorcinol, dimethoxytolyl-4-propylresorcinol, hexylresorcinol, idebenone, coenzyme Q10, resveratrol, tetrahydrocurcumin, hydroxypinazone retinate, and ethyl diiminomethyl guaiacol manganese chloride; more preferably peptidotope, 4-butylresorcinol, or idebenone.

[0025] The second technical solution of the present invention is:

[0026] A method for preparing the above-mentioned polymer nanomicelles is provided, comprising the following steps:

[0027] (1) Dissolve the block copolymer and the active ingredient in an organic solvent to obtain a mixed solution;

[0028] (2) Add the mixed solution to water, heat and stir, and sonicate;

[0029] (3) After removing the organic solvent, filter with a microporous membrane to obtain polymer nanomicelles.

[0030] Furthermore, the mass ratio of the block copolymer to the active ingredient is 5-10:1-2.

[0031] Preferably, the mass ratio of the block copolymer to the active ingredient is 5:2.

[0032] Furthermore, the mass-to-volume ratio of the block copolymer to the organic solvent is 1:200-300 g / mL.

[0033] Preferably, the mass-to-volume ratio of the block copolymer to the organic solvent is 1:200 g / mL.

[0034] Furthermore, the volume ratio of the organic solvent to water is 1:4-5.

[0035] Preferably, the volume ratio of the organic solvent to water is 1:4.

[0036] Furthermore, the organic solvent is acetone.

[0037] Furthermore, the stirring temperature is 40-50℃, the stirring speed is 1000-1200rpm, and the stirring time is 1-2h.

[0038] Preferably, the stirring temperature is 40°C, the stirring speed is 1100 rpm, and the stirring time is 2 hours.

[0039] Furthermore, the ultrasound time is 20-30 minutes; preferably 30 minutes.

[0040] Furthermore, the polymer nanomicelles have a spherical structure with an average particle size of 100-300 nm.

[0041] The third technical solution of the present invention is:

[0042] The application of the above-mentioned polymer nanomicelles or polymer nanomicelles prepared by the above-mentioned preparation method in the field of cosmetics is provided.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The polymer nanomicelles loaded with active substances prepared by the block copolymer and hydrophobic active substances of the present invention have the advantages of high active substance loading content, strong sustained release effect, high freeze-drying stability and high active substance retention rate after freeze-drying and reconstitution.

[0045] 2. The present invention uses a polyethylene glycol-polycaprolactone block copolymer, preferably a PCL-PEG-PCL structure, and by controlling the number-average molecular weight of PEG and PCL fragments in the block copolymer, the content of active substances loaded in polymer nanomicelles and the freeze-drying stability are further improved.

[0046] 3. Compared with the existing technology that first prepares polymer nanomicelles and then encapsulates them, the present invention obtains encapsulated polymer micelles by directly reacting block copolymers with active substances, which is simple to prepare and at the same time results in a higher content of active substances.

[0047] 4. Compared with free active ingredients, its in vitro antioxidant and whitening activities are greatly improved, indicating that there is a strong interaction between the polymer and the active ingredient in the polymer-encapsulated nanomicelles; these polymer nanomicelles with encapsulated active ingredients have wide applications in the preparation of cosmetics with whitening and antioxidant effects. Attached Figure Description

[0048] Figure 1 The diagram shown is a synthetic route for PCL-PEG-PCL in Example 1;

[0049] Figure 2 The image shown is the Fourier transform infrared spectrum of PCL-PEG-PCL in Example 1;

[0050] Figure 3 The image shows the 1H NMR spectrum of PCL-PEG-PCL in Example 1;

[0051] Figure 4 The image shows a gel permeation chromatogram of PCL-PEG-PCL in Example 1;

[0052] Figure 5 The morphology and particle size results of polymer nanomicelles loaded with active ingredients are shown below: A. Example 1; B. Example 2; C. Example 3; D. Comparative Example 1; E. Comparative Example 2; F. Comparative Example 3; G. Comparative Example 4.

[0053] Figure 6 For active ingredients A, peptide Amidato; B, 4-butylresorcinol; C, idebenone content; D, EUK-134 content determination standard curve;

[0054] Figure 7 The in vitro release standard curves are for active ingredients A, peptide Amidato; B, 4-butylresorcinol; and C, idebenone.

[0055] Figure 8 Release curves of polymer nanomicelles loaded with active ingredients; A, Example 1; B, Example 2; C, Example 3; D, Comparative Example 1; E, Comparative Example 2; F, Comparative Example 3;

[0056] Figure 9 Results of HaCaT cell proliferation assessment; A, Idebenone; B, Comparative Example 4 (blank carrier polymer nanomicelles); C, Example 3 (polymer nanomicelles loaded with idebenone);

[0057] Figure 10 The effect of H2O2 on the viability of HaCaT cells;

[0058] Figure 11 The results show the protective effect of the test substance (idebenone group) against H2O2-induced oxidative stress in HaCaT cells; ns indicates no statistical difference, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001;

[0059] Figure 12 The results show the effect of the test substances on the melanin content in B16 cells; A, Peptidotinamide group; B, 4-Butylresorcinol group; ns indicates no statistical difference, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001;

[0060] Figure 13The results show the effect of the test substances on the activity of tyrosinase in B16 cells; A, Peptidotoxin group; B, 4-Butylresorcinol group; ns indicates no statistical difference, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001. Detailed Implementation

[0061] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention. Those skilled in the art can make various changes and modifications to the invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention. It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0062] The following sources of raw materials are provided as examples:

[0063] raw material Purchase manufacturer DMEM high glucose medium Thermo Fisher Scientific RPMI Medium 1640 culture medium GIBCO VivaCell Heat-Inactivated Premium Fetal Bovine Serum GE hyclone Trypsin-EDTA (0.25%) containing phenol red GIBCO Penicillin-Streptomycin Double Antibody Shanghai Beyotime Biotechnology Co., Ltd. Levodopa Shanghai Bohr Chemical Reagent Co., Ltd. Triton X-100 Nanjing Senbeka Biotechnology Co., Ltd. HaCaT cells School of Pharmaceutical and Chemical Engineering, Guangdong Pharmaceutical University B16 cells School of Pharmaceutical and Chemical Engineering, Guangdong Pharmaceutical University

[0064] Example 1

[0065] (1) Preparation of PCL-PEG-PCL amphiphilic block copolymer (synthetic route as follows) Figure 1 As shown):

[0066] Accurately weigh 2.5051 g of PEG 2000 and 12.498 g of ε-caprolactone into a container, fill with nitrogen, add 21 μL of stannous octoate, stir for 30 min, then heat to 130 °C, reflux, and react for 24 h. After the reaction is complete, cool to room temperature, dissolve completely in dichloromethane, slowly add to ice-cold petroleum ether, filter repeatedly three times to obtain a viscous substance, dry under vacuum at 40 °C to constant weight, and store in a sealed container.

[0067] Fourier transform infrared results of PCL-PEG-PCL amphiphilic block copolymer are as follows: Figure 2 As shown, FT-IR (KBr): 1450, 2850, 2950 cm⁻¹ -1 (CH2), 1740cm -1 (C=O);

[0068] The results of the proton nuclear magnetic resonance spectrum are as follows: Figure 3 As shown, 1H NMR (400MHz, CDCl3): δ (ppm) = 1.35, 1.62, 2.28, 3.61, 4.03 (CH2);

[0069] Gel permeation chromatography (GPC) results are as follows Figure 4As shown; the number-average molecular weight of PCL co-linked at both ends of PCL-PEG-PCL is 9760 Daltons; the molecular weight of PCL-PEG-PCL is 11019 Daltons.

[0070] The CMC value was 1.92 × 10⁻³ mg / mL;

[0071] (2) Preparation of polymer nanomicelles loaded with active ingredients:

[0072] Weigh 5 mg of PCL-PEG-PCL amphiphilic block copolymer and 2 mg of peptide amphiphilic polymer, add 1 mL of acetone, and sonicate for 30 min to completely dissolve the PCL-PEG-PCL amphiphilic block copolymer and peptide amphiphilic polymer. Under vigorous stirring at 1100 rpm, add dropwise to 4 mL of ultrapure water with a syringe (waiting for each drop to be completely emulsified before adding more). Stir at 40 °C for 2 h. After stirring, sonicate for 30 min, and completely remove acetone under reduced pressure vacuum at 50 °C. Filter through a 0.45 μm microporous membrane and remove unencapsulated active material by dialysis to obtain the final product.

[0073] Example 2

[0074] The difference from Example 1 is that the active ingredient is 4-butylresorcinol.

[0075] Example 3

[0076] The difference from Example 1 is that the active ingredient is idebenone.

[0077] Comparative Example 1

[0078] (1) The difference from Example 1 is that the preparation of PCL-PEG-PCL amphiphilic block copolymer is as follows: the amount of PEG, ε-caprolactone and stannous octoate added are 7.5010g, 7.7044g and 64μL, respectively.

[0079] The number-average molecular weight of PCL-PEG-PCL with co-linked PCL at both ends is 1970 Daltons; the molecular weight of PCL-PEG-PCL is 4859 Daltons; the CMC value of PCL-PEG-PCL determined by the pyrene fluorescent probe method is 22.51 × 10⁻⁶. -3 mg / mL;

[0080] (2) The preparation of polymer nanomicelles loaded with active substances is the same as in Example 1.

[0081] Comparative Example 2

[0082] The difference from Example 2 is that the polymer nanomicelles were prepared using the amphiphilic block copolymer obtained in step (1) of Comparative Example 1.

[0083] Comparative Example 3

[0084] The difference from Example 3 is that the polymer nanomicelles were prepared using the amphiphilic block copolymer prepared in step (1) of Comparative Example 1.

[0085] Comparative Example 4

[0086] The difference from Example 1 is that no active ingredient is added.

[0087] Comparative Example 5

[0088] The difference from Example 1 is that the active ingredient is the hydrophilic EUK-134.

[0089] Test Example 1: Performance Indicators of Polymer Nanomicelles and Polymer Nanomicelles Encapsulated with Active Ingredients

[0090] (1) The morphology and particle size distribution of polymer nanomicelles loaded with active materials in Examples 1-3 are shown in the figure. Figure 5 A-5C; The morphology and particle size distribution of polymer nanomicelles loaded with active materials in Comparative Examples 1-3 are shown in the figure. Figure 5 D-5F; Comparative Example 4: The morphology and particle size distribution of polymer nanomicelles are shown in the figure. Figure 5 G;

[0091] (2) Determination of active ingredient content in polymer nanomicelles:

[0092] (2-1) Accurately weigh 2.00 mg each of the active ingredients, including peptide Amido, 4-butylresorcinol, idebenone, and EUK-134. Dissolve them in acetonitrile and transfer the solutions to a 10 mL volumetric flask. Make up to the final volume to obtain a 0.2 mg / mL stock solution. Dilute the stock solution to obtain solutions of different concentrations of active ingredients. Measure their UV absorption spectra. After determining the maximum absorption wavelength, measure the absorbance of each sample at that wavelength and plot the standard curve. (See figure) Figure 6 ;

[0093] (2-2) Prepare 0.2 mg / mL polymer nanomicelles loaded with cosmetic active ingredients (polymer nanomicelles loaded with active ingredients in Examples 1-3). Mix the sample with the same volume of acetonitrile, shake well, and sonicate for 30 min. Use a UV-Vis spectrophotometer to measure the absorbance value at the maximum absorption wavelength of the drug. Substitute the absorbance value into the standard curve to calculate the drug concentration, convert the mass of the loaded active ingredient, and substitute the mass into the following formula to calculate the active ingredient content.

[0094] Active ingredient content = (mass of encapsulated active ingredient / total mass of polymer nanomicelles and active ingredient) × 100%.

[0095] The performance indicators of polymer nanomicelles are shown in Table 1;

[0096] Table 1 Performance Indicators of Polymer Nanomicelles

[0097] Group Appearance active ingredients Active ingredient content % Example 1 Round Peptide Anmito 19.08 Example 2 Round 4-Butylresorcinol 10.68 Example 3 Round Idebenone 18.83 Comparative Example 1 Unround Peptide Anmito 5.32 Comparative Example 2 Unround 4-Butylresorcinol 1.59 Comparative Example 3 Unround Idebenone 5.11 Comparative Example 4 Round No additions / Comparative Example 5 Unround EUK-134 0.55

[0098] Test Example 2: In vitro release of polymer nanomicelles loaded with active ingredients

[0099] (1) Accurately weigh 2.00 mg each of the active peptides amido, 4-butylresorcinol, and idebenone, dissolve them in the release medium, transfer them to a 10 mL volumetric flask, and dilute to a final volume to obtain a 0.2 mg / mL stock solution. Dilute the stock solution to obtain solutions of different concentrations of active ingredients, measure their UV absorption spectra, determine the maximum absorption wavelength, and then measure the absorbance of each sample at that wavelength. Plot a standard curve, see [reference needed]. Figure 7 ;

[0100] (2) Place 3 mL of polymer nanomicelles or active substance solution containing the active ingredient into a dialysis bag, then place it in 10 mL of release medium. Release the active ingredient at 100 rpm and 37 °C. Take 2 mL of sample at regular intervals, and then add 2 mL of isothermal blank release medium to the system. Filter each sample through a microporous membrane and measure the UV absorbance. Substitute the absorbance into the standard curve to calculate the concentration of the active ingredient in the medium solution at that time point. Substitute the absorbance into the following formula to calculate the cumulative release amount at each sampling point, calculate the cumulative release percentage, and plot the in vitro release curve. The results are shown in [Figure number missing]. Figure 8 ;

[0101]

[0102] r: Cumulative release of active ingredient; V e V0: Replacement volume of the release medium; C: Total volume of the release medium; i : Concentration of the released solution during the i-th displacement sampling; m drug : Total mass of active material encapsulated in polymer nanomicelles; n: Number of times the release medium is replaced;

[0103] pass Figure 8 It can be seen that Examples 1-3 ( Figure 8 A-8C) polymer nanomicelles loaded with peptide-enriched polymers, 4-butylresorcinol, and idebenone exhibit a slower release rate compared to free active ingredient solutions, demonstrating slow-release properties; while comparative examples 1-3 ( Figure 8 D-8F polymer nanomicelles loaded with active ingredients exhibit burst release, with subsequent release rates similar to those of free active ingredient solutions.

[0104] Test Example 3: Lyophilization Stability Study of Polymer Nanomicelles Encapsulated with Active Ingredients

[0105] (1) Lyophilized and reconstituted polymer nanomicelles loaded with active ingredients

[0106] Weigh 150 mg of hydroxypropyl-β-cyclodextrin and add it to 3 mL of a 0.05 mg / mL polymer nanomicelle solution containing the active ingredient. Shake to mix well, freeze, and then lyophilize to obtain a lyophilized powder. Add 3 mL of ultrapure water to the sample and shake for 5 min to reconstitute.

[0107] (2) Investigation on the retention rate of active substances after lyophilization and reconstitution

[0108] The lyophilized and reconstituted sample was placed in a dialysis bag and dialyzed multiple times to remove unencapsulated active substances. After concentration, the active substance content was measured, and the retention rate of active substances after lyophilization and reconstitution was calculated using the following formula; the results are shown in Table 2.

[0109] Retention rate of active ingredient after lyophilization and reconstitution (%) = (Mass of active ingredient encapsulated in polymer nanomicelles after lyophilization / Mass of active ingredient encapsulated in polymer nanomicelles before lyophilization) × 100%.

[0110] Table 2. Retention rate of active materials after freeze-drying of polymer nanomicelles loaded with active materials.

[0111]

[0112] As shown in Table 2, the polymer nanomicelle samples loaded with active ingredients in Examples 1-3 can form loose cake-like products after freeze-drying. After reconstitution, the morphology of the nanoparticles can be restored and they have a good particle size distribution. The retention rate of active ingredients is over 80%, and the stability is good.

[0113] In contrast, the polymer nanomicelles loaded with active ingredients in Comparative Examples 1-3 exhibited the highest retention rate of active ingredients after lyophilization and reconstitution, at just over 20%; their reconstitution stability was also poor.

[0114] In contrast, the active ingredient retention rate of the hydrophilic EUK-134 polymer nanomicelles loaded in Comparative Example 4 was only 6.8% after lyophilization and reconstitution. Test Example 4: Investigation of the improvement in active physicochemical properties after loading with polymer nanomicelles.

[0115] (1) Comparison of the state of Peptidone powder and polymer nanomicelles loaded with an equal amount of Peptidone (Example 1) in ultrapure water; Peptidone is a white powder; Peptidone has low solubility in water and is still difficult to dissolve after shaking; The same mass of Peptidone can be uniformly dispersed in water after being loaded with polymer nanomicelles; This proves that the solubility of Peptidone in water increases after being loaded with polymer nanomicelles.

[0116] (2) Comparison of the state of 4-butylresorcinol powder and polymer nanomicelles loaded with an equal amount of 4-butylresorcinol (Example 2) after being placed indoors (25±3℃) for 4 days; 4-butylresorcinol is a white transparent needle-like crystal, which turns red after being placed indoors for 4 days and changes from solid to liquid; the polymer nanomicelles loaded with 4-butylresorcinol are uniformly distributed before and after placement, and only some water evaporates after 4 days. Fourier transform infrared spectroscopy confirmed that the structure of 4-butylresorcinol changed after being placed indoors for 4 days, while the structure of 4-butylresorcinol loaded with polymer nanomicelles did not change.

[0117] (3) Comparison of idebenone powder, polymer nanomicelles loaded with the same amount of idebenone (Example 3), and the state of idebenone in water and acetonitrile; idebenone is an orange-yellow powder; idebenone is sparingly soluble in water; the same mass of idebenone can be uniformly dispersed in water after being loaded with polymer nanomicelles, and it appears pale yellow; idebenone dissolves in acetonitrile, and it appears golden yellow in the dissolved state; this proves that the solubility of idebenone in water increases after being loaded with polymer nanomicelles, while masking the original color.

[0118] Test Example 5: Evaluation of the in vitro antioxidant activity of polymer nanomicelles loaded with active ingredients

[0119] (1) Determination of HaCaT cell proliferation rate

[0120] HaCaT cells were digested and their density was adjusted to 10 × 10⁶. 4 Cells were seeded at a density of 100 μL / well in a 96-well plate. After cell attachment, 100 μL of different concentrations of the test substance solution (idebenone, unloaded polymer nanomicelles, and idebenone-loaded polymer nanomicelles) were added to each well in the experimental group, with three replicates for each concentration. The average value was taken. The control group was treated with 100 μL of DMEM. Cells were cultured for 24 h. After culture, the culture medium was discarded, and 100 μL of 0.5 mg / mL MTT solution was added to each well. Cells were cultured for 4 h, and the MTT solution was discarded. 100 μL of dimethyl sulfoxide was added to each well, and the cells were shaken for 20 min. The absorbance at 492 nm was measured, and the cell proliferation rate was calculated according to the following formula. The results are shown in the figure. Figure 9 ;

[0121] Cell proliferation rate (%) = (OD) 492 (Experimental well)-OD 492 (blank hole) / (OD) 492 (Reference Hole)-OD 492 (Blank hole) × 100;

[0122] Depend on Figure 9It can be seen that the polymer nanomicelles loaded with idebenone have a stronger HaCaT cell proliferation rate than idebenone and polymer nanomicelles without active ingredients, and the HaCaT cell proliferation rate decreases significantly with the increase of the concentration of polymer nanomicelles loaded with idebenone.

[0123] (2) Establishment of a model of H2O2-induced oxidative stress damage in HaCaT cells

[0124] HaCaT cells were digested and their density was adjusted to 10 × 10⁶. 4 Cells were cultured at concentrations of 100 μmol / mL, divided into a control group (no treatment) and groups subjected to H2O2 damage at different final concentrations (100, 200, 300, 400, and 500 μmol / L). Cell proliferation rate was measured after 24 h of culture. Results are shown in […]. Figure 10 When the final concentration of H2O2 was 300 μmol / L, the survival rate of HaCaT cells was 51.08%, and 300 μmol / L H2O2 was selected as the damage concentration of HaCaT cells.

[0125] (3) Protective effect of the test substance against H2O2-induced oxidative stress in HaCaT cells

[0126] HaCaT cells were digested and their density was adjusted to 10 × 10⁶. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and cultured overnight. After cell attachment, the culture medium was aspirated. 100 μL of different concentrations of the test substance solution were added to the experimental group (Example 3), with 3 replicates for each concentration. 100 μL of DMEM was added to the control and damage groups, and the cells were incubated for 2 h. H2O2 with a final concentration of 300 μmol / L was added to the experimental and damage groups, while the same volume of DMEM was added to the control group. Cell proliferation rate was measured after 24 h of culture.

[0127] See results Figure 11 Polymer nanomicelles loaded with idebenone at concentrations of 5.2 μg / mL, 10.4 μg / mL, and 20.8 μg / mL showed significant protective effects against H2O2-induced oxidative stress damage in HaCaT cells. At all three concentrations, polymer nanomicelles loaded with idebenone exhibited better antioxidant activity than the free active idebenone. The corresponding concentrations of the free active idebenone (equivalent to 0.92 μg / mL, 1.84 μg / mL, and 3.67 μg / mL) showed protective effects only at high concentrations; the other two groups showed no significant difference compared to the H2O2-damaged group. The blank carrier polymer nanomicelles (concentrations of 4.28 μg / mL, 8.56 μg / mL, and 17.12 μg / mL, respectively) showed no significant difference compared to the H2O2-damaged group.

[0128] In summary, under the condition of consistent idebenone active ingredient content, idebenone-loaded polymer nanomicelles have a protective effect against H2O2-induced oxidative stress damage in HaCaT cells, while the free active ingredient idebenone has weaker antioxidant activity at this concentration. Furthermore, the cell survival rate of the blank carrier polymer nanomicelles at all three concentrations was basically consistent with that of the H2O2-damaged group, indicating that the blank carrier polymer nanomicelles themselves do not possess antioxidant activity. This suggests that the superior antioxidant activity of idebenone-loaded polymer nanomicelles is a result of the synergistic effect between the polymer nanomicelles and the active ingredient.

[0129] Test Example 6: In vitro whitening activity evaluation of polymer nanomicelles loaded with active ingredients

[0130] (1) Effect of the test substance on melanin content in B16 cells

[0131] The cells were digested and their density was adjusted to 10 × 10⁻⁶. 4 Cells were seeded at a density of 1.5 mL / well in 6-well plates, and culture medium was added to a final volume of 2 mL. Cells were cultured overnight. After cell attachment, the supernatant was discarded. 2 mL of different concentrations of the test substance solution (Examples 1 and 2) were added to the experimental groups. The control group received no sample, and the positive group received arbutin. After 48 hours of culture, the culture medium was discarded, the cells were washed once with PBS, digested with trypsin, and then 2–3 mL of PBS was added to homogenize the cells and adjust the density to 10 × 10⁶ cells / well. 4 Approximately 10 cells / mL; 1 mL of cell suspension was transferred to three parallel centrifuge tubes, and the supernatant was discarded after centrifugation; 200 μL of PBS was added to resuspend the cells, and 1 mL of ethanol / ether solution (v / v = 1 / 1) was added to dissolve the opaque non-melanin particles. After incubation at room temperature for 15 min, the cells were centrifuged and the supernatant was discarded; then 1 mL of 10% DMSO-1mol / L NaOH solution was added, and the cells were dissolved in an 80℃ water bath for 30 min; the cells were transferred into 96-well plates, with three replicates per concentration. The absorbance at 405 nm was measured using a microplate reader, and the relative melanin content was calculated according to the following formula. The results are shown in [Figure number missing]. Figure 12 ;

[0132] Relative melanin content (%) = (OD) 405 (Experimental group) / OD 405 (Control group) × 100

[0133] Depend on Figure 12As shown in Figure A, compared with the control group, the peptide-encapsulated polymer nanomicelles of Example 1 at concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL significantly reduced melanin content. Compared with the corresponding concentrations of free active peptide-encapsulated peptide-encapsulated polymer nanomicelles (equivalent to 0.18 μg / mL, 0.36 μg / mL, and 0.72 μg / mL), the effect of peptide-encapsulated polymer nanomicelles in reducing melanin content was more significant. There was no significant difference between the control group and the blank carrier polymer nanomicelles (corresponding concentrations of 0.82 μg / mL, 1.64 μg / mL, and 3.28 μg / mL), indicating that the blank carrier polymer nanomicelles had no effect on inhibiting melanin production.

[0134] Depend on Figure 12 As shown in Figure B, compared with the control group, the 4-butylresorcinol polymer nanomicelles of Example 2 at concentrations of 15.6 μg / mL, 31.3 μg / mL, and 62.5 μg / mL significantly reduced melanin content. Compared with the corresponding concentrations of free active 4-butylresorcinol (equivalent to 1.67 μg / mL, 3.34 μg / mL, and 6.68 μg / mL), the 4-butylresorcinol polymer nanomicelles had a more significant effect on reducing melanin content. There was no significant difference between the control group and the blank carrier polymer nanomicelles (corresponding concentrations of 13.9 μg / mL, 27.9 μg / mL, and 55.8 μg / mL), indicating that the blank carrier polymer nanomicelles had no effect on inhibiting melanin production.

[0135] In summary, when the contents of the active peptide Anmido and 4-butylresorcinol are the same, the polymer nanomicelles loaded with the active ingredient can inhibit melanin content more significantly than the free active ingredient. In addition, the melanin content of the blank carrier polymer nanomicelles is basically the same as that of the control group, indicating that the blank carrier polymer nanomicelles themselves cannot inhibit melanin production.

[0136] (2) Effect of test substance on tyrosinase activity in B16 cells

[0137] B16 cells were digested and their cell density was adjusted to 10 × 10⁶. 4Cells were seeded at 100 μL / mL in 96-well plates and cultured overnight. After cell adhesion, 100 μL of different concentrations of test substance solution (Examples 1 and 2) were added to each well, with three replicates for each concentration. Arbutin was used as the positive group, and culture medium was added to the control group. After 48 h, the culture medium was discarded, and the cells were washed once with PBS. 50 μL of 1% Triton-X100 aqueous solution was added to each well, and the cells were quickly placed in a -80°C freezer for 30 min. After freezing, the cells were thawed at 37°C to completely rupture them. Then, 100 μL of 0.1% levodopa solution was added to each well, and the reaction was carried out at 37°C for 2 h. The absorbance at 475 nm was measured using a microplate reader, and the tyrosinase activity was calculated according to the following formula. The results of Examples 1 and 2 are shown in the figures below. Figure 13 ;

[0138] Tyrosinase activity (%) = (OD 475 (Experimental group) / OD 475 (Control group) × 100

[0139] Depend on Figure 13 As shown in Figure A, compared with the control group, the peptide-encapsulated polymer nanomicelles of Example 1 at concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL significantly inhibited tyrosinase activity; and the tyrosinase activity gradually decreased with increasing concentration. Compared with the corresponding concentrations of free active peptide-encapsulated peptide-encapsulated peptide-encapsulated peptide-encapsulated polymer nanomicelles (equivalent to 0.18 μg / mL, 0.36 μg / mL, and 0.72 μg / mL), the inhibitory effect of peptide-encapsulated polymer nanomicelles on tyrosinase was more significant. There was no significant difference between the control group and the blank carrier polymer nanomicelles (corresponding concentrations of 0.82 μg / mL, 1.64 μg / mL, and 3.28 μg / mL), indicating that the blank carrier polymer nanomicelles had no inhibitory effect on tyrosinase activity.

[0140] Depend on Figure 13 As shown in Figure B, compared with the control group, the 4-butylresorcinol polymer nanomicelles of Example 2 at concentrations of 15.6 μg / mL, 31.3 μg / mL, and 62.5 μg / mL significantly inhibited tyrosinase activity, with the activity gradually decreasing as the concentration increased. Compared with the corresponding concentrations of free 4-butylresorcinol (equivalent to 1.67 μg / mL, 3.34 μg / mL, and 6.68 μg / mL), the 4-butylresorcinol polymer nanomicelles showed a more significant inhibitory effect on tyrosinase activity. There was no significant difference between the control group and the blank carrier polymer nanomicelles (corresponding concentrations of 13.9 μg / mL, 27.9 μg / mL, and 55.8 μg / mL), indicating that the blank carrier polymer nanomicelles had no inhibitory effect on tyrosinase activity.

[0141] In summary, when the contents of the active ingredient peptide Amidato and 4-butylresorcinol are the same, the polymer nanomicelles loaded with the active ingredient have a more significant inhibitory effect on tyrosinase activity compared with the free active ingredient. Furthermore, the tyrosinase activity of the blank carrier polymer nanomicelles is basically the same as that of the control group, indicating that the blank carrier polymer nanomicelles themselves cannot inhibit tyrosinase activity. This suggests that the superior inhibitory effect of the peptide Amidato-loaded polymer nanomicelles and the 4-butylresorcinol-loaded polymer nanomicelles on tyrosinase activity is due to the synergistic effect between the polymer nanomicelles and the active ingredient.

[0142] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A polymer nanomicelle, characterized in that, The polymer nanomicelles were prepared from an amphiphilic block copolymer and an active ingredient; the structure of the block copolymer is shown in the following formula: The PEG has a number-average molecular weight of 2000; the PCL has a number-average molecular weight of 8000-12000. The active ingredient is a hydrophobic active ingredient, specifically a peptide, idebenone, or 4-butylresorcinol.

2. The polymer nanomicelles according to claim 1, characterized in that, The number-average molecular weight of the PCL fragment in the PCL-PEG-PCL is 4880, and the number-average molecular weight of the PEG fragment is 2000.

3. The polymer nanomicelles according to claim 1, characterized in that, The amphiphilic block copolymer has a core-shell structure.

4. The method for preparing polymer nanomicelles according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Dissolve the block copolymer and the active ingredient in an organic solvent to obtain a mixed solution; (2) Add the mixed solution to water, heat and stir, and sonicate; (3) After removing the organic solvent, filter with a microporous membrane to obtain polymer nanomicelles.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the block copolymer to the active ingredient is 5-10:1-2; the mass-to-volume ratio of the block copolymer to the organic solvent is 1:200-300 g / mL; and the volume ratio of the organic solvent to water is 1:4-5.

6. The preparation method according to claim 4, characterized in that, In step (2), the stirring temperature is 40-50℃, the stirring speed is 1000-1200rpm, and the stirring time is 1-2h; the ultrasonic time is 20-30min.

7. The preparation method according to claim 4, characterized in that, The polymer nanomicelles have a spherical structure and an average particle size of 100-300 nm.

8. The application of polymer nanomicelles according to any one of claims 1-3 or prepared by any one of claims 4-7 in the field of cosmetics.

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