Animal source type I collagen liposome as well as preparation method and application thereof

Abstract

The invention discloses an animal source type I collagen liposome as well as a preparation method and application thereof. The method comprises the following steps: respectively dispersing two phospholipids and cholesterol in an organic solvent, carrying out rotary evaporation on the organic solvent, carrying out ultrasonic elution by using a buffer solution, homogenizing to form liposome balloons with uniform particle sizes, dialyzing by using the buffer solution, centrifuging to obtain liposome suspended matters, adding the suspended matters into an animal-derived I-type collagen solution with fixed concentration and pH, and carrying out freeze drying to obtain the collagen liposome. Stirring for a certain time at constant temperature, and centrifuging to obtain a final substance. The collagen coated with the lipidosome has a complete triple helix structure, and the D90 of the lipidosome is 680.51 nm. The liposome can be used for high-end cosmetics, such as essence, emulsion, cream, cream and other products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an animal-derived type I collagen liposome and a preparation method and application thereof. Background Art

[0002] Type I collagen is an important component of mammalian skin, tendons, bones, cartilage tissue, etc., accounting for more than 30% of the total protein in the human body. Type I collagen can effectively accelerate the growth of dermal cells, activate epidermal cells, maintain skin elasticity, and prevent wrinkles. It can also promote skin metabolism, improve skin sagging, and have an anti-aging effect. It can promote skin absorption, nourishment and form a protective film, making the skin particularly moisturizing and smooth. At the same time, it can also fade acne marks and increase blood circulation in the skin. For the human body, the genetic coding similarity between animal-derived type I collagen and human-derived collagen is as high as 90.3%, and has a complete triple helix structure.

[0003] Collagen itself has a large molecular weight, a triple helix structure, a large spatial conformation, and a tendency to gradually increase its molecular size within the pH range acceptable to the skin. When the pH is close to 5.7, the D90 data is close to 10μm. This greatly restricts its application in cosmetics research and development, manufacturing, and market applications. How to promote the transdermal absorption of collagen while ensuring the integrity of the structure has become a difficulty that limits the application of collagen.

[0004] The invention application with publication number CN114053166A discloses a method for preparing acylated type I collagen water light needle, which uses anhydride as a modifier to change the isoelectric point of collagen and its solubility curve, that is, the collagen is soluble under normal physiological pH conditions. In this prior art, a large amount of organic weak polar reagents such as acetone are required as a medium in the reaction process for phase transfer preparation, which is relatively environmentally polluting, and requires recovery treatment such as gas capture and liquefaction, and the process cost is relatively high.

[0005] Liposomes are a nano drug delivery system composed of natural or synthetic liposomes. Currently, common liposome nanoparticles are mostly used as carriers of anti-tumor drugs, most of which are used clinically to treat gastric cancer, esophageal cancer, breast cancer, and various solid tumors. The application of liposomes is rarely seen in the field of collagen.

[0006] And because liposomes can enter the skin through hair follicles, sebaceous glands, sweat glands, etc., collagen uses liposomes as a medium and can more easily pass through the epidermis into the deep layers of the skin and further enter the cells to nourish the cells.

[0007] For example, the invention application with publication number CN117003857A discloses a collagen with rapid absorption effect and its preparation method and application. In order to improve the transdermal absorption performance of collagen, collagen is coated by liposomes. However, the collagen involved in the application is recombinant collagen with a molecular weight of 66.2kDa, and the recombinant collagen itself does not have a triple helix structure. Moreover, the molecular weight is small and it is easier to form liposomes. Animal-derived type I collagen with a larger molecular weight and a complete triple helix structure has better biocompatibility and is more difficult to form liposomes.

[0008] In addition, there is data showing that the similarity between human collagen and bovine collagen is over 90%. Summary of the invention

[0009] In view of the above-mentioned deficiencies in the prior art, the present invention provides an animal-derived type I collagen liposome and a preparation method and application thereof.

[0010] The present invention first provides a method for preparing animal-derived type I collagen liposomes, comprising the following steps:

[0011] (1) pre-dispersing two phospholipids in an organic solvent to obtain a pre-dispersion solution 1; pre-dispersing cholesterol in an organic solvent to obtain a pre-dispersion solution 2; mixing the pre-dispersion solution 1 and the pre-dispersion solution 2, and removing the organic solvent to obtain liposomes;

[0012] (2) dispersing the mixed liposomes in step (1) in a buffer solution with a pH of 6.86 to 8.30 to obtain a nanocapsule dispersion;

[0013] (3) separating and collecting the nanocapsule dispersion obtained in step (2) to obtain nanocapsules;

[0014] (4) Obtaining an animal-derived type I collagen solution having a triple helix structure, and adding the nanocapsule obtained in step (3), mixing, and obtaining the animal-derived type I collagen liposome.

[0015] Preferably, in step (1), the phospholipid is selected from the group consisting of lecithin, soybean lecithin, egg yolk lecithin, cardiolipin, cephalin, and distearoylphosphatidylcholine.

[0016] Preferably, in step (1), the mass ratio of phospholipid to cholesterol is 2.5 to 8:1.

[0017] Preferably, in step (1), the concentration of phospholipids in pre-dispersion liquid 1 is 0.25-0.4 g / mL; and the concentration of cholesterol in pre-dispersion liquid 2 is 0.05-0.2 g / mL.

[0018] Preferably, in step (1), the organic solvent is ethanol. In step (1), the method for removing the organic solvent is at least one of rotary evaporation or vacuum heating.

[0019] Preferably, in step (2), the pH of the buffer solution is 7.40.

[0020] Preferably, in step (2), the buffer is a phosphate buffer or an acetate buffer.

[0021] Preferably, in step (2), the concentration of the nanocapsule dispersion is 0.01 mg / mL-7.5 mg / mL;

[0022] In step (3), the concentration of animal-derived type I collagen in the animal-derived type I collagen solution is 0.8 mg / mL to 9.5 mg / mL;

[0023] In step (4), the mass ratio of the nanocapsule to animal-derived type I collagen is 1:0.075-0.475.

[0024] Preferably, in step (2), a homogenizing device is used for dispersion, and the homogenizing device is at least one of the following: a high-pressure homogenizer, a microfluidizer, an ultrasonic homogenizer, a centrifugal homogenizer, and a rotor cutting homogenizer.

[0025] Preferably, in step (2), after the mixed liposomes in step (1) are dispersed in a buffer solution, they are dialyzed to remove impurities to obtain a nanocapsule dispersion. The pore size used in the dialysis is 10000 kDa to 14000 kDa.

[0026] Preferably, in step (4), the animal-derived type I collagen with a triple helical structure is type I collagen extracted from the Achilles tendon and / or skin of at least one of cattle, pigs, sheep and deer. In the present application, the animal-derived type I collagen with a triple helical structure can be derived from common tissues of commonly used animal sources in the art.

[0027] The present invention further provides animal-derived type I collagen liposomes prepared by the preparation method.

[0028] The present invention also provides application of the animal-derived type I collagen liposome in preparing cosmetics.

[0029] Explanation of the principle of liposome encapsulation of collagen:

[0030] 1. Concept of collagen isoelectric point: Collagen exists in a precipitated state between pH 7.0-7.4, while collagen can exist stably in the form of a solution between pH 2-4.

[0031] 2. Principle of preparation of liposome capsules: When amphiphilic molecules such as phospholipids and sphingolipids are dispersed in the aqueous phase, the hydrophobic tails of these molecules (such as fatty acid chains) tend to gather together and avoid the aqueous phase to reduce contact with water. At the same time, the hydrophilic heads (such as phosphate groups) are exposed to the aqueous phase and form hydrogen bonds and other interactions with water molecules. This self-assembly behavior causes the molecules to form closed vesicles with a bilayer structure, namely liposomes.

[0032] 3. Principle of liposome-loaded collagen: The present invention uses a buffer solution with a pH of 6.86-8.30 and a certain osmotic pressure in the elution process of the liposomes. The interior of the liposomes must contain a certain volume of buffer solution. These liposomes are placed in a collagen solution. Driven by the osmotic pressure, the collagen molecules gradually break through the liposome structure and are attracted to the interior of the liposomes. Under the conditions of neutralization and salting out, the interior of the liposome presents a high-salt and neutral microenvironment. Under this environment, the collagen molecules form collagen fiber nanoparticles and remain stably inside the liposomes.

[0033] Beneficial effects of the present invention:

[0034] The present invention disperses two kinds of phospholipids and cholesterol in an organic solvent respectively, after the organic solvent is rotary evaporated, a buffer is used for ultrasonic elution, homogenized into a liposome capsule with uniform particle size, and a buffer is used for dialysis, and a liposome suspension is centrifuged out, and the suspended matter is added to an animal-derived type I collagen solution with a fixed concentration and pH, and the suspension is stirred at a constant temperature for a certain period of time, and the final substance can be obtained by centrifugation. The liposome-coated collagen of the present invention has a complete triple helical structure, and the D90 of the liposome itself is 680.51nm. The liposome can be used for high-end cosmetics, such as essence water, emulsion, ointment, cream and other products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a graph of SDS-PAGE detection results, wherein the lane "marker" is the standard molecular weight sample, the lane "original sample" is the original sample before collagen coating, and the lane "cleaved sample" is the cleaved sample after collagen coating.

[0036] Figure 2 It is the XRD test result diagram, wherein curve a is the original sample before collagen coating; curve b is the cleavage sample after collagen coating.

[0037] Figure 3 This is the particle size distribution test result of liposomes.

[0038] Figure 4 A diagram showing the markings of the locations on the arm where the samples were applied.

[0039] Figure 5This is a graph showing the SDS-PAGE test results of the recombinant collagen with a molecular weight of 7 kDa used in Example 5, wherein lane "m" is a standard molecular weight sample and lane "1" is a recombinant collagen sample. DETAILED DESCRIPTION

[0040] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0041] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0042] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. In the present invention, the chemical reagents used, products from different manufacturers have no significant effect on the effect. The preferred implementation methods and materials described in the present invention are only for demonstration purposes.

[0043] In the embodiment of the present invention, the collagen solution used is a type I collagen solution, which is derived from a type I collagen solution extracted from bovine Achilles tendon or a type I collagen solution extracted from cowhide. The type I collagen solution extracted from bovine Achilles tendon and the type I collagen solution extracted from cowhide are prepared by referring to the method of Chinese invention patent CN116999619A, which will not be repeated here.

[0044] Example 1

[0045] (1) Place 2 g of egg yolk lecithin and 2 g of distearoylphosphatidylcholine in a beaker at a mass ratio of 1:1 and add 10 mL of ethanol to pre-disperse. The sample is labeled s1. Also, take 0.5 g of cholesterol and add 10 mL of ethanol to pre-disperse in a beaker. The sample is labeled s2. After s1 and s2 are completely dissolved, mix them in an eggplant-shaped bottle and use a rotary evaporator to evaporate the ethanol at 50°C in a vacuum rotary evaporator. The sample is labeled s3.

[0046] (2) Add a PBS buffer solution of pH 7.40 to the eggplant-shaped bottle. Ultrasound is used to remove the upper s3 in the eggplant-shaped bottle, and a microfluidizer is used to disperse it into 0.01 mg / mL nanocapsules with uniform particle size. The sample is labeled as s4.

[0047] (3) The obtained substance s4 was dialyzed with PBS solution in a constant temperature environment for 4 hours to remove impurities. The dialysis bag used was 14000 KDa, and sample s5 was obtained. S5 was centrifuged using a centrifugal device to collect the suspended matter, which was marked as s6.

[0048] (4) 0.5 g of s6 was added to 50 mL of a 0.8 mg / mL type I collagen hydrochloric acid solution of pH 2.20 from an animal source, and stirred at 25° C. for 60 min to obtain a final sample of the substance, which was labeled as s7. After testing, the product had a coverage rate of 76.3%.

[0049] The coverage rate is calculated as follows:

[0050] Coverage = (W 总 -W) / W 总

[0051] Among them, W 总 represents the amount of collagen added; W represents the amount of collagen in the solution after coating.

[0052] The final product was tested and the electrophoresis results of the samples were as follows: Figure 1 As shown, the SDS-PAGE electrophoresis bands are complete, and the sample consists of two α1 and one α2.

[0053] The XRD test results are as follows Figure 2 As shown, XRD shows an obvious peak at θ=31.96, indicating that the animal-derived collagen oil has a complete triple helical structure, and the triple helical structure of collagen is not destroyed before and after coating.

[0054] The particle size analysis results are as follows Figure 3 As shown, D10 = 108.22nm, D50 = 271.27nm, D90 = 680.51nm, and the particle size analysis accurately shows the distribution of particle sizes, that is, about 90% of the liposome particle sizes are less than 680nm.

[0055] Example 2

[0056] (1) Place 1g of egg yolk lecithin and 2g of cephalin in a beaker at a ratio of 1:2 and add 5mL of ethanol to pre-disperse. The sample is labeled s1. Also, take 1g of cholesterol and add 10mL of ethanol to pre-disperse in a beaker. The sample is labeled s2. After s1 and s2 are completely dissolved, mix them in an eggplant-shaped bottle and use a rotary evaporator to evaporate the ethanol in a vacuum rotary at 40°C. The sample is labeled s3.

[0057] (2) Adding a certain pH 8.30 acetate buffer to the eggplant-shaped bottle, oscillating at high frequency to remove the upper s3 in the eggplant-shaped bottle, and using a high-pressure homogenizer to disperse it into 2 mg / mL nanocapsules with uniform particle size. The sample is labeled as s4.

[0058] (3) The obtained substance s4 is dialyzed for 4 h in a constant temperature environment using the acetate solution in step (2) using a 10000 kDa dialysis bag to obtain sample s5. S5 is centrifuged using a centrifuge to collect the suspended matter, which is labeled as s6.

[0059] (4) 0.6 g of s6 was added to 30 mL of animal-derived type I collagen solution with a pH of 2.20 and a content of 9.5 mg / mL, and stirred at 20° C. for 30 min to obtain a final sample of the substance, which was labeled as s7.

[0060] Example 3

[0061] (1) Place 1 g of soybean lecithin and 4 g of egg yolk lecithin in a beaker at a mass ratio of 1:4 and add 20 mL of ethanol to pre-disperse. The sample is labeled s1. Also, take 2 g of cholesterol and add 10 mL of ethanol to pre-disperse in a beaker. The sample is labeled s2. After s1 and s2 are completely dissolved, mix them in an eggplant-shaped bottle, use vacuum heating, and evaporate the ethanol at 78°C. The sample is labeled s3.

[0062] (2) Add a phosphate buffer solution with a pH of 6.86 to the eggplant-shaped bottle. Vortex to remove the upper s3 in the eggplant-shaped bottle, and use a high-pressure homogenizer to disperse it into 7.5 mg / mL nanocapsules with uniform particle size. The sample is labeled as s4.

[0063] (3) The obtained substance s4 is dialyzed using the PBS solution in step (2) for 6 h in a constant temperature environment, and a dialysis bag with a capacity of 14000 kDa is used to obtain sample s5. S5 is centrifuged using a centrifugal device, and the suspended matter is collected and labeled as s6.

[0064] (4) 1.0 g of s6 was added to 15 mL of a pH 3.30, 5.0 mg / mL animal-derived type I collagen solution, and stirred at 20° C. for 60 min to obtain a final sample of the substance, which was labeled as s7.

[0065] Example 4

[0066] The collagen components in the recombinant collagen essence water (Zhejiang G makeup website license number 2024012196) were replaced by s4 and s7 in Example 1, and then the three samples were applied to the arms 1, 2, and 3, respectively. Figure 4 , use the intelligent digital display multi-functional skin detector to test the moisture, oiliness, and elasticity of the skin in three locations at regular intervals.

[0067] Table 1

[0068]

[0069] The results are shown in Table 1. The data in Table 1 indicate that the product with S7 added has better skin moisture content, skin oil content and skin elasticity data at various stages than the original product and the product with only liposome added.

[0070] Example 5

[0071] 0.8 g of s6 in Example 1 was added to 50 mL of collagen solution with a pH of 2.0 and a protein content of 2.0 mg / mL in a hydrochloric acid system, and a recombinant collagen with a molecular weight of 70 kD (invention application with publication number CN117510618A), and stirred at 25°C for 60 min to obtain sample s8. The SDS-PAGE test results of the recombinant collagen are as follows: Figure 5 shown.

[0072] Then add s8 in the same proportion to the recombinant collagen essence water (Zhejiang G makeup website license number 2024012196) as a compatibility.

[0073] This experiment still uses Figure 4 At the positions marked 1 and 2, test the essence water with s8 added and the essence water with s7 added.

[0074] Table 2

[0075]

[0076] The results are shown in Table 2. It can be seen from Table 2 that the elasticity of the skin with the essence water added with s7 is significantly higher than that of the sample with the addition of s8 within 120 minutes. This is obviously due to the special triple helix structure of animal-derived collagen.

Claims

1. A method for preparing animal-derived type I collagen liposomes, characterized in that: The following steps are involved: (1) pre-dispersing two phospholipids in an organic solvent to obtain a pre-dispersion solution 1; pre-dispersing cholesterol in an organic solvent to obtain a pre-dispersion solution 2; mixing the pre-dispersion solution 1 and the pre-dispersion solution 2, and removing the organic solvent to obtain liposomes; (2) dispersing the mixed liposomes in step (1) in a buffer solution with a pH of 6.86 to 8.30 to obtain a nanocapsule dispersion; (3) separating and collecting the nanocapsule dispersion obtained in step (2) to obtain nanocapsules; (4) Obtaining an animal-derived type I collagen solution having a triple helix structure, and adding the nanocapsule obtained in step (3), mixing, and obtaining the animal-derived type I collagen liposome.

2. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (1), the phospholipid is selected from the group consisting of lecithin, soybean lecithin, egg yolk lecithin, cardiolipin, cephalin, and distearoylphosphatidylcholine.

3. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (1), the mass ratio of phospholipid to cholesterol is 2.5 to 8:

1.

4. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (1), the concentration of phospholipids in the pre-dispersion liquid 1 is 0.25-0.6 g / mL; the concentration of cholesterol in the pre-dispersion liquid 2 is 0.05-0.2 g / mL.

5. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (1), the organic solvent is ethanol.

6. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (1), the method for removing the organic solvent is at least one of rotary evaporation or vacuum heating.

7. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (2), the buffer is a phosphate buffer or an acetate buffer.

8. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (2), the concentration of the nanocapsule dispersion is 0.01 mg / mL to 7.5 mg / mL; In step (4), the concentration of animal-derived type I collagen in the animal-derived type I collagen solution is 0.8 mg / mL to 9.5 mg / mL; In step (4), the mass ratio of the nanocapsule to animal-derived type I collagen is 1:0.075-0.

475.

9. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (2), a homogenizing device is used for dispersion, and the homogenizing device is at least one of the following: a high-pressure homogenizer, a microfluidizer, an ultrasonic homogenizer, a centrifugal homogenizer, and a rotor cutting homogenizer.

10. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (2), the mixed liposomes in step (1) are dispersed in a buffer solution, and then dialyzed to remove impurities to obtain a nanocapsule dispersion.

11. The method for preparing animal-derived type I collagen liposomes according to claim 1, characterized in that: In step (4), the animal-derived type I collagen with a triple helix structure is type I collagen extracted from the Achilles tendon and / or skin of at least one of cattle, pigs, sheep and deer.

12. Animal-derived type I collagen liposomes prepared by the preparation method according to any one of claims 1 to 11.

13. Use of the animal-derived type I collagen liposome according to claim 12 in the preparation of cosmetics.

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