Multi-target penetration and slow-release anti-aging collagen nanocarrier and preparation method thereof

Abstract

This invention belongs to the A61K technical field, and more specifically relates to a multi-target, penetration-enhancing, sustained-release anti-aging collagen nanocarrier and its preparation method. The first aspect of this invention provides a multi-target, penetration-enhancing, sustained-release anti-aging collagen nanocarrier, the components of which include at least: surfactant, hydroxyl-containing compound, polypeptide, collagen, and water. Nanoencapsulation refers to bilayer vesicles composed of polymeric materials, which can encapsulate active ingredients within nanoparticles with a diameter of nanometers. This invention employs nanoencapsulation technology to encapsulate recombinant composite collagen (Ⅰ+Ⅲ+XⅦ), beauty composite peptides, hydroxyethyl deacetylated chitosan, and other active ingredients; thereby achieving comprehensive and precise replenishment and collagen promotion.

Description

Technical Field

[0001] This invention belongs to the field of A61K technology, and more specifically relates to a multi-target collagen nanocarrier for promoting penetration and slow-release anti-aging and its preparation method. Background Technology

[0002] Women begin losing collagen from the age of 20, with the peak loss occurring around age 25. Statistics show that by age 40, collagen levels typically drop to half of what they were at age 18. Collagen significantly impacts skin hydration, smoothness, firmness, and skin age. How to increase collagen levels in the skin of women over 25 is a challenge currently facing both consumers and researchers.

[0003] Currently, some researchers and related whitening and anti-aging products have emerged, aiming to enhance the whitening or anti-aging effects of the human body through product use. However, due to the skin barrier, the active ingredients in the formula are unable to exert their actual efficacy. For example, whitening ingredients targeting melanin or anti-aging ingredients targeting fibroblasts have specific targets that are difficult to penetrate the skin and be absorbed due to the skin barrier, thus failing to exert their effects.

[0004] On the other hand, there are still some traditional methods to promote penetration at this stage, such as

[0005] 1) Through physical permeation enhancement, such as thermal permeation enhancement by laser and radiofrequency, or electropermeation enhancement by electroporation and mechanical permeation enhancement by microneedles;

[0006] 2) Through formulation technologies: encapsulation technology, microemulsion technology;

[0007] 3) Permeation enhancers: By affecting intercellular lipids and their fluidity, they promote the transdermal absorption of active ingredients.

[0008] The above-mentioned methods for enhancing penetration often involve a certain degree of irritation, which limits their application in formulations.

[0009] To address the current shortcomings, researchers have developed new methods to promote collagen absorption. Chinese invention patent CN113599281A discloses a collagen polypeptide carboxymethyl chitosan nanoparticle sustained-release particle and its manufacturing method. This patent involves extracting collagen to prepare small-molecule collagen polypeptides, which are then encapsulated to form collagen polypeptide carboxymethyl chitosan nanoparticles. However, the patent does not specify how to promote collagen absorption, nor can it prove this effect. Chinese invention patent CN115778844A provides a polypeptide composition, its preparation method, and its uses. This patent proposes using a combination of multiple polypeptides to prepare a new composition that effectively promotes the transdermal absorption of polypeptide substances. While the patent mentions the polypeptide composition forming nanoparticles, it does not specify the particle size of the nanoparticles, making it impossible to determine their transdermal absorption effect. Summary of the Invention

[0010] To address the aforementioned technical problems, a first aspect of the present invention provides a multi-target, permeation-enhancing, sustained-release, and anti-aging collagen nanocarrier, comprising at least: a surfactant, a hydroxyl-containing compound, a polypeptide, collagen, and water.

[0011] In some preferred embodiments, the surfactant includes at least one of PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, polyol surfactant, cetearyl alcohol polyether-20, and stearyl alcohol polyether-6.

[0012] More preferably, the surfactant includes PEG-40 hydrogenated castor oil.

[0013] In some preferred embodiments, the polyol surfactants include, but are not limited to, 1,2-hexanediol, 1,2-pentanediol, 1,2-octanediol, 1,8-octanediol, 1,2-heptanediol, 1,7-heptanediol, and 1,10-decanediol.

[0014] More preferably, the polyol surfactant can be selected from 1,2-hexanediol.

[0015] In some preferred embodiments, the weight ratio of the PEG-40 hydrogenated castor oil to the polyol surfactant is (10~30):(5~20).

[0016] More preferably, the weight ratio of the PEG-40 hydrogenated castor oil to 1,2-hexanediol is (10~30):(5~20).

[0017] More preferably, the weight ratio of the PEG-40 hydrogenated castor oil to 1,2-hexanediol is 15:7.554.

[0018] During the experiment, the inventors discovered that the entire system became turbid during the process of mixing the raw materials to encapsulate the active ingredients. This directly affected the success of the collagen nanocarrier experiment described in this application. To address this issue, the inventors investigated the raw materials and ultimately found that changing the ratio of PEG-40 hydrogenated castor oil and 1,2-hexanediol directly affected the turbidity. Theoretically, the inventors believed that the ratio of PEG-40 hydrogenated castor oil and 1,2-hexanediol, as surfactants, could be any. However, during their investigation, they discovered that when… The inventors speculate that a weight ratio of PEG-40 hydrogenated castor oil to 1,2-hexanediol of 15:7.554 is necessary to avoid turbidity in the system. They suggest this is because, at this ratio, the hydrophilic and lipophilic groups on the surfaces of PEG-40 hydrogenated castor oil and 1,2-hexanediol can achieve a greater balance, forming strong hydrogen bonds with other hydroxyl-containing compounds in the system. This results in a tighter binding of collagen and peptides within the system, reducing the frequency of collisions between active ingredients and thus preventing turbidity caused by their aggregation.

[0019] In some preferred embodiments, the collagen is recombinant collagen.

[0020] More preferably, the recombinant collagen is a recombinant of I+III+XVII collagen.

[0021] More preferably, the recombinant collagen is a combination of type I, type III, and type XVII collagen, consisting of 100 kDa type I collagen, 5 kDa type III collagen, and 23.8 kDa type XVII collagen, respectively.

[0022] The recombinant collagen was purchased from Chuangjian Medical. According to the biomimetic skin technology, the ratio of recombinant collagen I+III+XVII is (1-5):(2-10):1; more preferably, the ratio of recombinant collagen I+III+XVII is 2.5:6.5:1.

[0023] In some preferred embodiments, the polypeptide includes at least one of tripeptide-1, acetyl hexapeptide-8, palmitoyl tripeptide-8, and acetyl tetrapeptide-5.

[0024] More preferably, the polypeptide may be selected from tripeptide-1 and palmitoyl tripeptide-8.

[0025] In some preferred embodiments, the hydroxyl-containing compounds include, but are not limited to, 1,2-pentanediol, ethanol, isopropanol, glycerol, propylene glycol, 1,2-butanediol, 1,4-butanediol, and butanediol.

[0026] More preferably, the hydroxyl-containing compound is a combination of 1,2-pentanediol, glycerol, and butanediol.

[0027] More preferably, the weight ratio of 1,2-pentanediol, glycerol, and butanediol is 1.45:0.45:0.45.

[0028] In some preferred embodiments, the amount of the hydroxyl-containing compound is 8-15 wt% of the surfactant.

[0029] During the experiment, the applicant discovered that, given a fixed type and amount of surfactant, changing the amount of hydroxyl-containing compounds affected the stability of collagen nanocarriers in aqueous solution and their cycling stability at -10℃ / 40℃. The reason for this phenomenon is speculated to be that 1,2-pentanediol, glycerol, and butanediol form hydrogen bonds with hydroxyl groups in the system through their hydroxyl groups. At the same time, the alkyl groups in the structures of 1,2-pentanediol, glycerol, and butanediol, in a certain quantity, promote the formation of collagen I+III+XVII by the surfactant, avoiding sedimentation caused by random molecular collisions during the -10℃ / 40℃ temperature cycling process, thus ensuring stability in high and low temperature environments of -10℃ / 40℃.

[0030] In some preferred embodiments, the components also include isopropyl palmitate and p-hydroxyacetophenone.

[0031] In some preferred embodiments, the components also include high molecular weight polysaccharides.

[0032] More preferably, the high molecular weight polysaccharide is hydroxyethyl deacetylated chitosan.

[0033] More preferably, the hydroxyethyl deacetylated chitosan has a molecular weight of 2000 Da and a particle size of about 30 nm, and the hydroxyethyl deacetylated chitosan was purchased from the Chinese Academy of Sciences.

[0034] During the experiment, the applicant discovered that the use of PEG-40 hydrogenated castor oil and 1,2-hexanediol in this application can improve the stability of collagen nanocarriers. In particular, when the weight ratio of PEG-40 hydrogenated castor oil to 1,2-hexanediol is 15:7.55, it ensures that the prepared nanocarriers achieve a particle size of 100-400 nm, with a PDI < 0.4, and no precipitation occurs. The applicant speculates that this phenomenon is due to the poor water solubility of the peptides and recombinant collagen present in this application, which improves with the addition of PEG-40 hydrogenated castor oil. The addition of oil improves the solubility, especially when 1,2-hexanediol is added in batches. When the weight ratio of PEG-40 hydrogenated castor oil to 1,2-hexanediol is 15:7.55, the interaction between hydroxyl groups in the system ensures a higher viscosity at the interface between the surfactant and the peptides and recombinant collagen, promoting collagen encapsulation. At the same time, it limits the movement rate of the encapsulated recombinant collagen and other active substances such as peptides, ensuring their stability in the aqueous system and avoiding precipitation. This results in a carrier particle size of 100-400 nm and a PDI < 0.4.

[0035] A second aspect of the present invention provides a method for preparing a multi-target, penetration-enhancing, sustained-release, anti-aging collagen nanocarrier, comprising the following steps:

[0036] (1) In a separate container, add a portion of the surfactant and stir at medium speed for 5-10 minutes until the mixture is homogeneous to obtain mixture A for later use;

[0037] (2) Weigh out isopropyl palmitate and set aside;

[0038] (3) Weigh 10wt% surfactant, p-hydroxyacetophenone, hydroxyl-containing compound, and water, and dissolve them by heating below 50°C to obtain mixture B, which is then set aside.

[0039] (4) Weigh out the remaining components except for the surfactant, add water and dissolve completely to obtain mixture C;

[0040] (5) Mix mixture A, isopropyl palmitate, and the remaining surfactant and stir for 5-10 minutes to obtain mixture D;

[0041] (6) Mix mixture C, mixture B and mixture D together and stir for 5-10 minutes, then let stand.

[0042] (7) Take samples for testing to check color, taste, viscosity, pH and state. Discharge the material after passing the test.

[0043] Beneficial effects:

[0044] The present invention has the following positive advancements:

[0045] 1. Nanoencapsulation refers to bilayer vesicles composed of polymer materials, which can encapsulate active ingredients within nanoparticles with a diameter of nanometers. This invention utilizes nanoencapsulation technology to encapsulate active ingredients such as recombinant complex collagen (Ⅰ+Ⅲ+XⅦ), beauty complex peptides, and hydroxyethyl deacetylated chitosan; thereby achieving comprehensive and precise replenishment and promotion of collagen production.

[0046] 2. The recombinant composite collagen (Ⅰ+Ⅲ+XⅦ) humanized collagen used in this invention has a scientific and reasonable ratio, which allows it to penetrate the skin from different molecular weights, thereby achieving precise anti-aging care for the skin from the outside.

[0047] 3. The added polypeptides in this invention activate fibroblasts to generate collagen and elastin; inhibit the release of the neurotransmitter acetylcholine, weaken muscle contraction, and increase elastin activity.

[0048] 4. The hydroxyethyl deacetylated chitosan in this invention, in conjunction with recombinant complex collagen (Ⅰ+Ⅲ+XⅦ) and peptides, can efficiently penetrate the stratum corneum barrier and enter the dermis, thereby enhancing the permeability of the nano-encapsulation and achieving the reduction of wrinkles on the skin surface;

[0049] 5. The multi-target, penetration-enhancing, sustained-release anti-aging collagen nanocarrier of this invention can effectively regulate the release rate of active ingredients and increase their ability to permeate biological membranes. It reduces skin irritation of active ingredients, increases their stability, improves transdermal absorption and conversion, and increases skin retention.

[0050] 6. In the research process, this invention creatively explored a method to stabilize hydroxyethyl deacetylated chitosan, recombinant composite collagen, and polypeptides in the system, ensuring that precipitation and aggregation do not occur during use, and achieving stability during temperature cycling at -10℃ / 40℃, thus guaranteeing the effectiveness of its use.

[0051] 7. Through the research of this invention, it is ensured that the prepared carrier is a nanocarrier with a particle size range of 100-400nm and PDI < 0.4, truly realizing the penetration and efficacy of the product at the nano level. Attached Figure Description

[0052] Figure 1 The protective effects of free collagen, collagen nanocarriers, and the multi-target collagen nanocarrier of Example 1 of this application on the HSF cell oxidative damage model are shown in the figure.

[0053] Figure 2The diagram shows the improvement of facial wrinkles in volunteers after 0 and 28 days using the product in the experimental group and placebo group in Example 1; a and b represent the test results of the placebo group using the cream without the multi-target collagen nanocarrier prepared in the application on 0 and 28 days, respectively; c and d represent the test results of the experimental group using the cream with the multi-target collagen nanocarrier on 0 and 28 days, respectively.

[0054] Figure 3 This is a picture of the actual product in Example 1;

[0055] Figure 4 The images show the sedimentation and turbidity of the comparative products during preparation. A1 corresponds to comparative 1, A2 corresponds to comparative 2, and A3 corresponds to comparative 3. Detailed Implementation

[0056] Example

[0057] Example 1

[0058] A multi-target collagen nanocarrier for promoting penetration and sustained release anti-aging, the components and contents of which are shown in Table 1 below:

[0059] Table 1:

[0060]

[0061] Comparative Example

[0062] Comparative Example 1

[0063] A multi-target collagen nanocarrier for promoting penetration and sustained release anti-aging, the components and contents of which are shown in Table 2 below:

[0064] Table 2:

[0065]

[0066] Comparative Example 2

[0067] A multi-target collagen nanocarrier for promoting penetration and sustained release anti-aging, the components and contents of which are shown in Table 3 below:

[0068] Table 3:

[0069]

[0070] Comparative Example 3

[0071] A multi-target collagen nanocarrier for promoting penetration and sustained release anti-aging, the components and contents of which are shown in Table 4 below:

[0072] Table 4:

[0073]

[0074] Example 1 and Comparative Examples 1-3 provide a method for preparing a multi-target, penetration-enhancing, sustained-release, and anti-aging collagen nanocarrier, comprising the following steps:

[0075] (1) In a separate container, first add component PEG-40 hydrogenated castor oil and 30wt% 1,2-hexanediol, stir at medium speed for 6 minutes, and mix evenly to obtain mixture A, which is ready for use.

[0076] (2) Weigh out isopropyl palmitate and set aside;

[0077] (3) Weigh 10wt% of 1,2-hexanediol, p-hydroxyacetophenone and butanediol, add 10wt% of water, heat at 45℃ to dissolve, and set aside;

[0078] (4) Weigh out the residue excluding 1,2-hexanediol, add water to dissolve it completely, and obtain mixture C;

[0079] (5) Mix mixture A, isopropyl palmitate, and the remaining 1,2-hexanediol and stir for 7 min to obtain mixture D;

[0080] (6) Mix mixture C, mixture B and mixture D together, stir for 8 minutes, and let stand;

[0081] (7) Take samples for testing to check color, taste, viscosity, pH and state. Discharge the material after passing the test.

[0082] Performance testing:

[0083] 1. Particle size test:

[0084] The Z-average potential and particle size distribution were measured using a 90Plus PALS high-sensitivity Zeta potential and particle size analyzer. 1 mL of the sample from Example 1 was added to a plastic cuvette, the lid was closed, and the cuvette was inserted into the sample well. The measurement wavelength was set to 658 nm, the scattering angle to 173°, the equilibration time to 60 s, and the interval time to 10 s. The software automatically recorded the particle size grouping data. Each sample was scanned three times, and the test was repeated twice to obtain the Z-average value and PDI value.

[0085]

[0086] The particle size test results show that the particle size of the carrier provided by this invention is 100-400 nm, which is within the standard range of 10-1000 nm for nanocarriers, and PDI < 0.4.

[0087] 2. Protective effect against oxidative damage to human skin fibroblasts (HSF) cells.

[0088] A cell oxidative damage model was established by treating human skin fibroblasts (HSF) with 800 μM H2O2 for 24 hours. In the experiment, collagen concentration was used as a reference standard to investigate the protective effects of free collagen, collagen nanocarriers, and multi-target collagen nanocarriers on the HSF cell oxidative damage model. The results are shown in [Figure number missing]. Figure 1 ;

[0089] Through performance testing and Figure 1 Data from the study showed that free collagen only has a protective effect against oxidatively damaged cells at high concentrations. Collagen nanocarriers and multi-target collagen nanocarriers exhibited significantly better cell protection than free collagen; co-delivery nanocarriers, under the combined action of multiple active ingredients, showed even better protection against oxidatively damaged cells.

[0090] 3. Human Skin Aging Test

[0091] A skin trial of collagen nanocarriers was conducted on 10 volunteers (aged 40 to 50 years). Subjects used a cream containing 5 wt% of the multi-target collagen nanocarriers prepared in Example 1 of this application, twice daily for 28 consecutive days. After 28 days, siloxane was applied to the skin surface of the volunteer's test area to create a reverse-replicated silicone film that conforms to the texture of human skin. The texture on the silicone film was analyzed using a laser scanning microscope. The results are shown in [Figure 1]. Figure 2 : Figure 2 a and b represent test graphs for the placebo group after 0 days and 28 days of using the cream without the multi-target collagen nanocarriers prepared in the application; Figure 2 c and d represent the test results of the cream using multi-target collagen nanocarriers in the experimental group at 0 days and 28 days, respectively.

[0092] Placebo group: Ten volunteers (aged 40 to 50) used a cream without the proposed multi-target collagen nanocarrier twice daily for 28 days.

[0093] Experimental results: After 28 days, the skin of volunteers who used the multi-target collagen nanocarrier cream showed a significant reduction in both wrinkle depth and wrinkle length compared to the placebo group.

[0094] 4. Cold and heat resistance stability test

[0095] The multi-target collagen nanocarriers prepared in the examples and comparative examples of this application were subjected to cold and heat resistance tests under five conditions: room temperature (25℃), high temperature (40℃), refrigeration (4℃), freezing (-10℃), and cycling (-10℃ / 40℃). Comparative observations were conducted under the five conditions in the first, second, third, and fourth weeks to determine their stability. The test categories and judgment criteria are shown in Table 5, and the test results of Example 1 and Comparative Examples 1-3 are recorded in Tables 6, 7, 8, and 9, respectively.

[0096] Table 5:

[0097]

[0098]

[0099]

[0100]

[0101]

Claims

1. A multi-target collagen nanocarrier for promoting penetration and sustained release anti-aging, characterized in that, The components include at least: surfactants, hydroxyl-containing compounds, peptides, collagen, and water; The surfactants include PEG-40 hydrogenated castor oil and polyol surfactants; The polyol surfactant is 1,2-hexanediol; The weight ratio of the PEG-40 hydrogenated castor oil to 1,2-hexanediol is 15:7.

554. The collagen mentioned is recombinant collagen; The polypeptide includes at least one of tripeptide-1, acetyl hexapeptide-8, palmitoyl tripeptide-8, and acetyl tetrapeptide-5; The hydroxyl-containing compound is a combination of 1,2-pentanediol, glycerol, and butanediol; the weight ratio of 1,2-pentanediol, glycerol, and butanediol is 1.45:0.45:0.

45. The amount of the hydroxyl-containing compound used is 8-15 wt% of the surfactant.

2. The multi-target, penetration-enhancing, sustained-release, and anti-aging collagen nanocarrier according to claim 1, characterized in that, The components also include isopropyl palmitate and p-hydroxyacetophenone.

3. A method for preparing a multi-target, penetration-enhancing, sustained-release, anti-aging collagen nanocarrier according to claim 2, characterized in that, Includes the following steps: (1) In a separate container, first add component PEG-40 hydrogenated castor oil and 30wt% 1,2-hexanediol, stir at medium speed for 6 minutes, and mix evenly to obtain mixture A, which is ready for use; (2) Weigh out isopropyl palmitate and set aside; (3) Weigh 10wt% of 1,2-hexanediol, p-hydroxyacetophenone and butanediol, add 10wt% of water, heat at 45℃ to dissolve, and obtain mixture B for later use; (4) Weigh the remaining substance excluding 1,2-hexanediol, add water to dissolve it completely, and obtain mixture C; (5) Mix mixture A, isopropyl palmitate, and the remaining 1,2-hexanediol for 7 min to obtain mixture D; (6) Mix mixture C, mixture B and mixture D together, stir for 8 minutes, and let stand; (7) Take samples for testing to check color, taste, viscosity, pH and state. Discharge the material after passing the test.

Images