A collagen carrier, collagen nanoparticles and a preparation method and application thereof

Abstract

The present application relates to the technical field of supramolecular technology, in particular to a collagen carrier, a collagen nanoparticle and a preparation method and application thereof, the collagen carrier is prepared from collagen, a stabilizer, an oil phase solvent, glycerol and water, raw materials are widely available and easy to obtain, the cost is low, no toxic solvent is contained, and the environment and human body are friendly, the collagen nanoparticle is loaded with active ingredients based on the above collagen carrier, meanwhile, through the specific preparation method provided in the present application, the collagen carrier and the collagen nanoparticle are easy to prepare and scale up production, and have great development and application value.

Description

Technical Field

[0001] This invention relates to the field of supramolecular technology, and in particular to a collagen carrier, collagen nanoparticles, their preparation methods and applications. Background Technology

[0002] Collagen is a major component of the extracellular matrix and is the most abundant and widely distributed protein in animals, primarily found in the connective tissues of mammals. In human tissues, collagen accounts for 70-80% of dry weight, mainly of type I and type III. In infants' skin, collagen is primarily type III, while in the skin of young adults and the elderly, it is primarily type I. Type I collagen determines the skin's contour support, while type III collagen determines skin elasticity and smoothness. The levels of type I and type III collagen are closely related to skin youthfulness, wound repair, cell support, and regeneration. Because epithelial damage and skin aging are accompanied by significant collagen loss, the main way to repair the skin is to replenish collagen significantly. For example, recombinant human type III collagen is a type of collagen produced through fermentation using genetic engineering technology. Studies have shown that exogenous supplementation with recombinant human type III collagen can effectively increase the level of type III collagen in the wound area, thereby inhibiting the excessive secretion of type I collagen that causes abnormal tissue proliferation and scar formation, and reducing pigmentation.

[0003] Collagen, as a biomaterial, possesses excellent biocompatibility, low immunogenicity, and the ability to promote cell growth and wound repair. Therefore, it is frequently added as an active ingredient in topical skincare products. However, it is well known that collagen has a large molecular weight, making it difficult to directly reach the dermis to improve skin condition. Simultaneously, due to its unique structure and function, collagen also holds significant potential as a carrier for active ingredients. Currently, collagen carriers are mostly prepared in the form of collagen membranes, collagen sponges, and microspheres. These carriers, such as collagen microspheres, often use chemical cross-linking agents to solidify the collagen during preparation. For example, Chinese invention patent CN 108785128 A discloses a polypropylene glycol-modified hydrolyzed collagen liposome and its preparation method. This method uses polyethylene glycol to modify hydrolyzed collagen liposome films, but the preparation process involves mixed solvents including chloroform. Therefore, the irritation and potential harm of the final product require further verification. In addition, Chinese invention patent application CN 101868298 A discloses a method for preparing microspheres based on natural extracellular matrix. This method effectively avoids the implantation of toxic chemical cross-linking agents that may cause cytotoxicity and calcification. It prepares collagen microspheres loaded with active ingredients by combining water-oil emulsification and photochemical cross-linking. It adopts relatively mild production conditions. However, the collagen microspheres prepared by this method have a large particle size and the preparation process is complicated and not easy to implement. Therefore, it still has certain drawbacks. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a collagen carrier, collagen nanoparticles, their preparation method, and applications, thereby overcoming the deficiencies in the prior art.

[0005] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a collagen carrier, which is prepared from the following raw materials in parts by weight: 0.5 to 10 parts of collagen, 0.1 to 10 parts of stabilizer, 5 to 25 parts of oil phase solvent, 2 to 10 parts of glycerol and 80 to 97.5 parts of water.

[0007] Preferably, the collagen is any one of collagen, collagen extract, hydrolyzed collagen, soluble collagen, deer bone collagen, protoplasmic collagen, and recombinant type III human collagen.

[0008] Preferably, the oil phase solvent is an alcohol solvent, and more preferably any one or a combination of ethanol, propylene glycol, and butanediol.

[0009] More preferably, the alcohol solvent is selected from any one or more combinations of ethanol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,2-pentanediol, 1,5-pentanediol, or 1,6-hexanediol.

[0010] Preferably, the stabilizer is selected from any one or more combinations of cationic stabilizers, and is preferably selected from: myristyltrimethylammonium bromide, stearyltrimethylammonium bromide, lauryltrimethylammonium bromide, dicocarbamate dimethylammonium chloride, PEG-15 cococarbamate methylammonium chloride, PEG-2 cococarbamate methylammonium chloride, PEG-5 stearylammonium chloride, PG-hydroxyethylcellulose cococarbamate dimethylammonium chloride, PG-hydroxyethylcellulose stearyldimethylammonium chloride, PPG-25 diethylmethylammonium chloride, ricinoleic acid amamidopropyltrimethylammonium chloride, locust bean gum hydroxypropyltrimethylammonium chloride, locust bean hydroxypropyltrimethylammonium chloride, soybean oil-based trimethylammonium chloride, soybean oil amamidopropyl benzyldimethylammonium chloride. Ammonium, starch hydroxypropyltrimethylammonium chloride, diC12-15 alkyldimethylammonium chloride, didecyldimethylammonium chloride, dicetyldimethylammonium chloride, ditallowyldimethylammonium chloride, dihydroxypropylPEG-5 linoleylammonium chloride, octadecyltrimethylammonium chloride, distearate dimethylammonium chloride, distearate ethyldimethylammonium chloride, dipalmitoyl ethyldimethylammonium chloride, panthenol hydroxypropyl stearyldimethylammonium chloride, guar gum hydroxypropyltrimethylammonium chloride, cetearyldimethylammonium chloride, cetearyldimethylammonium chloride, polymethacrylamide propyltrimethylammonium chloride, cassia gum hydroxypropyltrimethylammonium chloride, tallowyltrimethylammonium chloride, dextran hydroxypropyltrimethylammonium chloride, hydroxypropyl guar gum Hydroxypropyltrimethylammonium chloride, hydroxypropyl bis-hydroxyethyldimethylammonium chloride, hydroxypropyl distearate dimethylammonium chloride, hydroxypropyl oxidized starch PG-trimethylammonium chloride, hydroxycetylhydroxyethyldimethylammonium chloride, hydroxyethyl betainepropyl dimethylammonium chloride, hydroxyethyl oleyl dimethylammonium chloride, ginseng hydroxypropyltrimethylammonium chloride, cinnamamidopropyltrimethylammonium chloride, lactamidopropyltrimethylammonium chloride, tricerylmethylammonium chloride, behenylbenzyldimethylammonium chloride, behenyltrimethylammonium chloride, behenamidopropyl PG-dimethylammonium chloride, behenyloxy PG-trimethylammonium chloride, dodecanebenzyltrimethylammonium chloride, dodecanehexadecyltrimethylammonium chloride, bishydroxyethylbis-hydroxypropyl stearyl chloride Ammonium, octyldodecyltrimethylammonium chloride, cocoyltrimethylammonium chloride, cocamidopropyl PG-dimethylammonium chloride, stearyltrimethylammonium chloride, stearamide-propyl dimethylbenzylammonium chloride, stearoxypropyltrimethylammonium chloride, oleylbenzyldimethylammonium chloride, oleamide-propyl PG-dimethylammonium chloride, lauryl methyl glucetol polyether-10-hydroxypropyl dimethylammonium chloride, lauryltrimethylammonium chloride, lauroyl PG-trimethylammonium chloride, palmitamide-propyltrimethylammonium chloride, or any one or more combinations thereof, or trimethyl-2,3-diolenooxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), dimethyl-2-trifluoroacetic acid.3-Dioleoyloxypropyl-2-(2-Sperminecarbamoylamino)ethylammonium (DOSPA), Trimethyldodecylammonium bromide (DTAB), Trimethyltetradecylammonium bromide (TTAB), Trimethylhexadecylammonium bromide (CTAB), Dimethylbisoctadecylammonium bromide (DDAB), Dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide (DORI), Dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide (DORIE), Dimethyl-3-hydroxypropyl-2,3-dioleoyloxypropylammonium bromide (DORIE-HP), Dimethyl-4-hydroxybutyl-2,3-dioleoyloxypropylammonium bromide The following are possible combinations of one or more of the following: dioleoyl-5-hydroxypentyl-2,3-dioleenooxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl-2,3-dihexadecopropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide (DMRIE), N-(2-argininoyl)-N',N'-dioctadecylglycineamide (DOGS), and 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC).

[0011] More preferably, the stabilizer is selected from any one or more of myristyltrimethylammonium bromide, stearyltrimethylammonium bromide, lauryltrimethylammonium bromide, discoyldimethylammonium chloride, and distearyldimethylammonium chloride, or from any one or more combinations of trimethylhexadecylammonium bromide (CTAB), dimethyl dioctadecylammonium bromide (DDAB), trimethyl-2,3-diolenopropylammonium chloride (DOTMA), and trimethyl-2,3-dioleoylpropylammonium bromide (DOTAP).

[0012] It should be noted that, when implementing this invention, the type of stabilizer used can be selected according to the intended use of the collagen supramolecular body to ensure compliance with relevant regulatory requirements.

[0013] Secondly, the present invention also provides a method for preparing the collagen carrier described in the first aspect above, the method specifically comprising the following steps:

[0014] S1: Prepare an aqueous phase and an organic phase separately, wherein the organic phase includes an oil phase solvent and a stabilizer, and the aqueous phase includes collagen, glycerol and water;

[0015] S2: Mix the above aqueous phase and organic phase, and prepare the primary emulsion by high-speed shearing;

[0016] S3: The above colostrum is homogenized under high pressure to prepare a solution system containing collagen carrier.

[0017] Preferably, the preparation method of the collagen carrier further includes step S4, wherein S4 is: separating the collagen carrier from the solution system containing the collagen carrier.

[0018] Furthermore, the high-speed shearing time is 1 to 5 minutes, preferably 1 to 4 minutes; the high-speed shearing speed is 5000 to 12000 rpm.

[0019] Preferably, the high-pressure homogenization conditions are: homogenization pressure of 300-800 bar, and homogenization cycle number of 3-8 times.

[0020] Thirdly, the present invention also provides a collagen nanoparticle, which includes the collagen carrier provided in the first aspect above, and further includes an active ingredient loaded on the collagen carrier.

[0021] Preferably, the active ingredient is a small molecule drug or a cosmetic ingredient.

[0022] More preferably, the small molecule drug is selected from retinoic acid, isotretinoin, tazarotene, adapalene, bezarotine, mupirocin, fusidic acid, compound polymyxin B, erythromycin, ofloxacin, clindamycin metronidazole, and benzoyl peroxide.

[0023] More preferably, the cosmetic raw material is selected from any one or more of the following: ascorbyl glucoside, glyceryl glucoside, ascorbic acid, tetrahydrocurcumin, salicylic acid, tocopherol, ferulic acid, resveratrol, panthenol, glutathione, ceramide, arbutin, astaxanthin, nicotinamide, 4-butylresorcinol, phloretin, hydroxypropyltetrahydropyranotriol, ergothioneine, ectoine, polyquaternium-51, hydroxypinazone retinate, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl hexapeptide-12, palmitoyl tripeptide-8, nonapeptide-1, and palmitoyl pentapeptide-4.

[0024] Fourthly, the present invention also provides a method for preparing collagen nanoparticles as described in the third aspect above, specifically including the following steps:

[0025] A1: Prepare an aqueous phase and an organic phase separately, wherein the organic phase includes an oil phase solvent and a stabilizer, and the aqueous phase includes collagen, glycerol, and water; the active ingredient may be added to any one or both of the aqueous and organic phases;

[0026] A2: Mix the above aqueous phase and organic phase, and prepare the primary emulsion by high-speed shearing;

[0027] A3: The above colostrum was homogenized under high pressure to prepare a solution system containing collagen nanoparticles.

[0028] Furthermore, the above-mentioned method for preparing collagen nanoparticles further includes step A4, wherein A4 specifically involves separating collagen nanoparticles from the solution system containing collagen nanoparticles.

[0029] Fifthly, the present invention further provides the use of the collagen carrier provided in the first aspect above, and the collagen nanoparticles provided in the third aspect above, in any aspect of the preparation of food, health products, cosmetics, skin care products, feed or medicines for human or animal use.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] To address the shortcomings of existing technologies, this invention provides a collagen carrier prepared from collagen, a stabilizer, an oil-phase solvent, glycerol, and water. The raw materials are widely available, inexpensive, and do not contain toxic solvents, making it environmentally and human-friendly. Furthermore, the specific preparation method provided by this invention facilitates easy preparation and scale-up production. In addition, the resulting collagen carrier exhibits good stability and uniform particle size, making it suitable as a carrier for loading various active ingredients and possessing significant development and application value.

[0032] Furthermore, this invention provides a collagen nanoparticle comprising the aforementioned collagen carrier and an active ingredient loaded onto the collagen carrier. This active ingredient can be diverse, thus enabling the collagen carrier and collagen nanoparticles to be applied in multiple fields. For example, when the collagen nanoparticles provided by this invention are applied in the preparation of skincare products or topical formulations, the collagen itself, acting as a carrier, can exert certain effects. Simultaneously, it can effectively improve the transdermal absorption of various active ingredients, thereby reducing the dosage of active ingredients, decreasing allergic reactions, and improving safety. Attached Figure Description

[0033] Figure 1 This is a photograph of the appearance of the solution system containing collagen nanoparticles prepared in Example 3 of the present invention.

[0034] Figure 2 This is a transmission electron microscope image of the collagen nanoparticles prepared in Example 3 of the present invention;

[0035] Figure 3 The particle size test results of the collagen nanoparticles prepared in Example 2 of this invention;

[0036] Figure 4 The particle size test results are for the collagen nanoparticles prepared in Example 3 of this invention.

[0037] Figure 5The particle size test results are for the collagen nanoparticles prepared in Example 4 of this invention.

[0038] Figure 6 The particle size test results are for the collagen nanoparticles prepared in Example 5 of this invention.

[0039] Figure 7 The particle size test results are for the collagen nanoparticles prepared in Example 6 of this invention.

[0040] Figure 8 The particle size test results are for the collagen nanoparticles prepared in Example 7 of this invention.

[0041] Figure 9 The particle size test results of collagen provided in Comparative Example 2 of this invention;

[0042] Figure 10 The particle size test results are for the collagen mixture provided in Comparative Example 3 of this invention.

[0043] Figure 11 The particle size test results of the collagen nanoparticles provided in Comparative Example 4 of this invention;

[0044] Figure 12 The results of particle size stability test of collagen nanoparticles prepared in Example 2 of this invention;

[0045] Figure 13 The encapsulation efficiency and stability test results of the collagen nanoparticles prepared in Example 2 of this invention;

[0046] Figure 14 The results of the comparative experiment on the skin retention amount of each group provided in Example 8 of the present invention. Detailed Implementation

[0047] The embodiments of the technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. Unless otherwise specified, the test methods used in the following embodiments are conventional methods; materials, reagents, or instruments whose manufacturers are not specified are commercially available reagents and materials, and conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer.

[0048] Example 1

[0049] This embodiment provides a collagen carrier and its preparation method, wherein the collagen carrier is prepared according to the following steps:

[0050] S1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0051] The organic phase includes 10 parts of 1,3-butanediol and 4 parts of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP). The above-mentioned raw materials are heated to a set temperature of 60°C and stirred to dissolve and mix thoroughly to obtain the organic phase.

[0052] The aqueous phase consists of 10 parts of recombinant type III human collagen, 10 parts of glycerin, and water (to bring the total weight of the aqueous phase to 100 parts). Weigh the above-mentioned parts of raw materials, mix them, heat to the set temperature of 60°C, and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0053] S2: The organic phase obtained above is added to the aqueous phase and sheared at high speed for 2 minutes at a speed of 12000 rpm to obtain the primary emulsion;

[0054] S3: The colostrum obtained above is homogenized under high pressure at 800 bar and cycled 5 times to obtain a solution system containing collagen carrier.

[0055] S4: Evaporate the solvent under reduced pressure at temperatures below 45°C to obtain the collagen carrier.

[0056] Example 2

[0057] This embodiment is the second preparation embodiment of the present invention. This embodiment provides a solution system containing collagen nanoparticles and its preparation method. The collagen nanoparticles include a collagen carrier and a small molecule drug, retinoic acid, loaded on the collagen carrier. The solution system containing collagen nanoparticles is prepared according to the following steps:

[0058] A1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0059] The organic phase includes 5 parts of ethanol, 0.1 parts of trimethylhexadecylammonium bromide (CTAB), and 0.025 parts of retinoic acid. The above-mentioned raw materials are heated to a set temperature of 40°C and stirred until fully dissolved to obtain the organic phase.

[0060] The aqueous phase consists of 0.5 parts of recombinant type III human collagen, 2 parts of glycerol, and water (to bring the total weight of the aqueous phase to 100 parts). Weigh the above-mentioned parts of raw materials, mix them, heat to the set temperature of 40°C, and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0061] A2: The organic phase obtained above is added to the aqueous phase and sheared at high speed for 2 minutes at a speed of 5000 rpm to obtain the primary emulsion;

[0062] A3: The above colostrum was homogenized under high pressure at 300 bar and cycled 3 times to obtain a solution system containing collagen nanoparticles.

[0063] Example 3

[0064] This embodiment is the third preparation embodiment of the present invention. This embodiment provides a solution system containing collagen nanoparticles. The collagen nanoparticles include a collagen carrier and isotretinoin, a small molecule drug, loaded on the collagen carrier. The solution system containing collagen nanoparticles is prepared according to the following steps:

[0065] A1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0066] The organic phase includes 25 parts of 1,2-propanediol, 0.05 parts of isotretinoin, and 2.0 parts of dimethyl dioctadecyl ammonium bromide (DDAB). The above-mentioned raw materials are heated to a set temperature of 60°C and stirred to dissolve and mix thoroughly to obtain the organic phase.

[0067] The aqueous phase consists of 10 parts of recombinant type III human collagen, 8 parts of glycerin, and water (to bring the total weight of the aqueous phase to 100 parts). Weigh the above-mentioned parts of raw materials, mix them, heat to the set temperature of 60°C, and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0068] A2: The organic phase obtained above was added to the aqueous phase and sheared at high speed for 4 minutes at a speed of 7000 rpm to obtain the primary emulsion;

[0069] A3: The colostrum obtained above was subjected to high-pressure homogenization at 600 bar and cycled 8 times to obtain a solution system containing collagen nanoparticles.

[0070] Example 4

[0071] This embodiment is the fourth preparation embodiment of the present invention. This embodiment provides a solution system containing collagen nanoparticles. The collagen nanoparticles include a collagen carrier and a small molecule drug, tazarotene, loaded on the collagen carrier. The solution system containing collagen nanoparticles is prepared according to the following steps:

[0072] A1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0073] The organic phase includes 15 parts of 1,3-butanediol, 0.1 parts of tazarotene, and 5 parts of trimethyl-2,3-dioleoxypropylammonium chloride (DOTMA). The above-mentioned raw materials are heated to a set temperature of 60°C and stirred to dissolve and mix thoroughly to obtain the organic phase.

[0074] The aqueous phase consists of 6 parts of recombinant type III human collagen, 6 parts of glycerol, and water (to bring the total weight of the aqueous phase to 100 parts). Weigh the above-mentioned parts of raw materials, mix them, heat to the set temperature of 60°C, and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0075] A2: The organic phase obtained above is added to the aqueous phase and sheared at high speed for 2 minutes at a speed of 8000 rpm to obtain the primary emulsion;

[0076] A3: The colostrum obtained above was subjected to high-pressure homogenization at 500 bar and cyclicated 6 times to obtain a solution system containing collagen nanoparticles.

[0077] Example 5

[0078] This embodiment is the fifth preparation embodiment of the present invention. This embodiment provides a solution system containing collagen nanoparticles. The collagen nanoparticles include a collagen carrier and a small molecule drug adapalene loaded on the collagen carrier. The solution system containing collagen nanoparticles is prepared according to the following steps:

[0079] A1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0080] The organic phase includes 10 parts of 1,3-butanediol, 0.1 parts of adapalene, and 4 parts of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP). The above-mentioned raw materials are heated to a set temperature of 60°C and stirred until fully dissolved to obtain the organic phase.

[0081] The aqueous phase consists of 10 parts of recombinant type III human collagen, 10 parts of glycerin, and water (to bring the total weight of the aqueous phase to 100 parts). Weigh the above-mentioned parts of raw materials, mix them, heat to the set temperature of 60°C, and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0082] A2: The organic phase obtained above is added to the aqueous phase and sheared at high speed for 2 minutes at a speed of 12000 rpm to obtain the primary emulsion;

[0083] A3: The colostrum obtained above was subjected to high-pressure homogenization at 800 bar and cycled 5 times to obtain a solution system containing collagen nanoparticles.

[0084] Example 6

[0085] This embodiment is the sixth preparation embodiment of the present invention. This embodiment provides a solution system containing collagen nanoparticles. The collagen nanoparticles include a collagen carrier and a small molecule drug, bezarotine, loaded on the collagen carrier. The solution system containing collagen nanoparticles is prepared according to the following steps:

[0086] The above-mentioned collagen product solution loaded with palmitoyl tripeptide-1 is prepared according to the following steps:

[0087] A1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0088] The organic phase includes 15 parts of 1,3-butanediol, 1 part of bezarotine, and 10 parts of trimethylhexadecylammonium bromide (CTAB). The above-mentioned raw materials are heated to a set temperature of 55°C and stirred to dissolve and mix thoroughly to obtain the organic phase.

[0089] The aqueous phase consists of 10 parts of recombinant type III human collagen, 10 parts of glycerin, and water (to bring the weight of the aqueous phase to 100 parts). Weigh the above-mentioned parts of raw materials, mix them, heat to the set temperature of 55°C, and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0090] A2: The organic phase obtained above is added to the aqueous phase and sheared at high speed for 2 minutes at a speed of 12000 rpm to obtain the primary emulsion;

[0091] A3: The colostrum obtained above was subjected to high-pressure homogenization at 800 bar and cycled 5 times to obtain a solution system containing collagen nanoparticles.

[0092] Example 7

[0093] This embodiment is the seventh preparation embodiment of the present invention. This embodiment provides a solution system containing collagen nanoparticles. The collagen nanoparticles include a collagen carrier and small molecule drugs isotretinoin and fusidic acid loaded on the collagen carrier. The solution system containing collagen nanoparticles is prepared according to the following steps:

[0094] A1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0095] The organic phase includes 25 parts of propylene glycol, 0.05 parts of isotretinoin, 2 parts of fusidic acid, and 2 parts of dimethyl dioctadecyl ammonium bromide (DDAB). The above-mentioned raw materials are heated to a set temperature of 60°C and stirred to dissolve and mix thoroughly to obtain the organic phase.

[0096] The aqueous phase consists of 10 parts of recombinant type III human collagen, 8 parts of glycerin, and water (to bring the total weight of the aqueous phase to 100 parts). Weigh the above-mentioned parts of raw materials, mix them, heat to the set temperature of 60°C, and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0097] A2: The organic phase obtained above is added to the aqueous phase and sheared at high speed for 1 min at a speed of 12000 rpm to obtain the primary emulsion;

[0098] A3: The colostrum obtained above was subjected to high-pressure homogenization at 600 bar and cycled 5 times to obtain a solution system containing collagen nanoparticles.

[0099] It should be noted that in all of the above embodiments 2 to 7, step A4 can also be included. Step A4 involves separating the collagen carrier from the solution system containing collagen nanoparticles. This step A4 can be added as needed, and the methods used for A4 include, but are not limited to, solvent evaporation, centrifugation, lyophilization, and spray drying.

[0100] Comparative Example 1

[0101] This embodiment is the first comparative example of the present invention. This comparative example provides an isotretinoin solution and its preparation method. The isotretinoin solution is prepared according to the following method:

[0102] Weigh 0.05 parts of isotretinoin and uniformly disperse it in ethanol at 60℃ to prepare a solution with a concentration of 0.05 parts.

[0103] Comparative Example 2

[0104] This embodiment is the second comparative example of the present invention. This comparative example provides a collagen solution and its preparation method. The collagen solution is prepared according to the following method:

[0105] The recombinant type III human collagen is dissolved in water at 60°C, and the aqueous solution is dissolved evenly at 60°C to obtain the final product.

[0106] Comparative Example 3

[0107] This embodiment is the third comparative example of the present invention. This comparative example provides collagen nanoparticles prepared by conventional methods. The specific preparation steps of the collagen nanoparticles are as follows:

[0108] B1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0109] Weigh out 25 parts of propylene glycol, 0.05 parts of isotretinoin, and 2.0 parts of dimethyl dioctadecyl ammonium bromide (DDAB), heat to a set temperature of 60°C, stir to dissolve and mix thoroughly to obtain an organic phase.

[0110] Weigh 10 parts of recombinant type III human collagen, 8 parts of glycerin, and water (to make up to 100 parts of the aqueous phase). Heat to the set temperature of 60°C and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0111] B2: Preparation of the mixture solution:

[0112] The organic phase obtained above was added to the aqueous phase and stirred to prepare a collagen mixture solution loaded with isotretinoin.

[0113] Comparative Example 4

[0114] This embodiment is the fourth comparative example of the present invention. This comparative example provides collagen nanoparticles prepared by conventional methods. The specific preparation steps of the collagen nanoparticles are as follows:

[0115] C1: Prepare the aqueous phase and organic phase separately, specifically as follows:

[0116] Weigh out 25 parts of chloroform, 0.05 parts of isotretinoin, and 2.0 parts of dimethyl dioctadecyl ammonium bromide (DDAB), heat to a set temperature of 60°C, stir to dissolve and mix thoroughly to obtain an organic phase.

[0117] Weigh 10 parts of recombinant type III human collagen, 8 parts of glycerin, and water (to make up to 100 parts of the aqueous phase). Heat to the set temperature of 60°C and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0118] C2: Preparation of colostrum:

[0119] The organic phase obtained above was added to the aqueous phase and sheared at high speed for 4 minutes at a speed of 7000 rpm to obtain the colostrum.

[0120] C3: High-pressure homogenization:

[0121] The colostrum obtained above was subjected to high-pressure homogenization at 600 bar for 8 cycles to obtain collagen nanoparticles loaded with isotretinoin.

[0122] C4: The above solution was evaporated at 40°C for 20 minutes until the solvent was completely evaporated, thus obtaining isotretinoin collagen nanoparticles.

[0123] Example 8

[0124] This embodiment is a test embodiment of the present invention, including:

[0125] 8.1 Appearance Test

[0126] The solution system containing collagen nanoparticles prepared in Example 2 was photographed, as shown below. Figure 1 As shown.

[0127] The solution system containing collagen nanoparticles prepared in Example 2 was diluted with distilled water, and then the sample was aspirated by a capillary tube and blown onto a support grid. After staining, washing, and drying, it was observed using a transmission electron microscope, and the results were photographed and recorded. The results are as follows: Figure 2 As shown.

[0128] 8.2 Particle size determination

[0129] The solutions containing collagen nanoparticles prepared in Examples 2-7 were diluted, and the particle size was determined using a collagen nanoparticle zeta potential analyzer (Anton Paar Co., Ltd., Litesizer 500).

[0130] The particle size test results of the collagen nanoparticles prepared in Examples 2-7 and Comparative Examples 2-3 are as follows: Figures 3-8 , Figures 9-10 As shown in Table 1, the experimental data for each group are as follows:

[0131] Table 1. Particle size determination results of Examples 2-7 and Comparative Examples 2-4

[0132]

[0133] 8.3 Appearance Stability Test

[0134] The solution systems containing collagen nanoparticles prepared in Examples 2-7 and Comparative Example 4 were each divided into four portions and stored at 4°C, room temperature light irradiation (RT), 45°C, and -25°C, respectively. The appearance changes were recorded on the day of preparation (0) and 30 days later, as shown in Table 2.

[0135] Table 2 Appearance Stability Observation Table

[0136]

[0137]

[0138] The experimental results above show that Comparative Example 3 was unstable after 30 days, indicating that the solution system containing collagen nanoparticles prepared by the method of the present invention has better stability than the solution system containing collagen nanoparticles prepared by using chloroform as solvent.

[0139] 8.4 Particle size stability test

[0140] The solution system containing collagen nanoparticles obtained in Example 2 was placed under conditions of 4°C, room temperature light exposure (RT), 45°C, and -25°C, respectively. The particle size change was measured on the day of preparation (0 represents 0) and 30 days later. The test results are as follows: Figure 11 As shown.

[0141] The experimental results above show that the collagen-containing nanoparticles prepared by the method of the present invention have good thermodynamic stability within one month.

[0142] 8.5 Encapsulation efficiency and stability testing

[0143] 8.5.1 HPLC Test Method

[0144] Chromatographic conditions:

[0145] Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol: 0.05% glacial acetic acid = 90:10; flow rate: 1 mL / min; detection wavelength: 353 nm; column temperature: 30 °C; injection volume: 20 μl.

[0146] Establishing the standard curve:

[0147] Weigh an appropriate amount of isotretinoin into a brown volumetric flask, dissolve it in methanol, dilute to volume, and prepare a concentration gradient solution. Analyze the solution using HPLC. Plot a standard curve with the mass concentration of isotretinoin on the x-axis and the peak area on the y-axis. The linear regression equation is y = 154.79x - 45.561(R²). 2=0.9991), and showed good linearity in the range of 0.5-20 μg / ml.

[0148] 8.5.2 Encapsulation efficiency determination method and results

[0149] The Sephadex G50 gel column (1.5cm*30cm) was packed using standard methods. 0.2ml of the test sample was loaded onto the column, eluted with water, and collected in fractions. The free drug in the eluent was determined using the HPLC method described in 6.5.1 above, according to (W... 总 -W 游离 ) / W 总 *Encapsulation efficiency was calculated at 100%. Test samples included the solution system containing collagen nanoparticles obtained on the day of preparation in Example 2, and the above-mentioned solution system containing collagen nanoparticles stored for 30 days at 4°C, room temperature light exposure (RT), 45°C, and -25°C, respectively. The changes in encapsulation efficiency were observed.

[0150] Test results are as follows Figure 12 As shown in the results, the encapsulation efficiency of collagen nanoparticles decreases slightly under high temperature conditions, especially at 45°C, where the encapsulation efficiency drops to 85.1%. However, overall, the encapsulation efficiency of the collagen nanoparticles of the present invention remains relatively stable over one month.

[0151] 8.6 Comparison of skin retention

[0152] 8.6.1 Experimental Methods

[0153] Experimental group:

[0154] Before the experiment, a pigskin model with a thickness of 300±50 μm was prepared using a skin grafting scalpel. This model was then cut into small circular pieces the size of the receiving pool and placed in physiological saline for later use. Phosphate-buffered saline (pH=7.4) was used as the receiving medium. During the experiment, the skin model was fixed between the release pool and the receiving pool, with the stratum corneum side facing the release pool and the dermis side facing the receiving pool, ensuring close contact between the skin and the receiving solution, and preventing air bubbles from forming. Subsequently, a certain amount of the solution system containing collagen nanoparticles obtained in Example 2 was added to the skin surface. The temperature of the receiving pool was maintained at 37±0.5℃, and a magnetic magnet was placed inside the receiving pool, rotating at 300 rpm throughout the experiment.

[0155] Eight hours after the in vitro transdermal treatment, the skin was removed, and the residual solution on the surface was rinsed off with pure water. The skin from the transdermal site was taken, minced, and homogenized with 1.5 ml of ethanol. The mixture was sonicated for 1 hour to extract ascorbate glucoside from the skin. The homogenate was transferred to a centrifuge tube, vortexed, and centrifuged at 12,000 rpm for 10 minutes. The residue was extracted again with 1 ml of ethanol. The two supernatants were combined, mixed, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed according to the HPLC test method provided in 6.5.1 above. This is the transdermal retention of isotretinoin solution.

[0156] In this embodiment, a control group was also set up. The control group used the solution systems obtained from Comparative Examples 1, 3, and 4 as test samples. The transdermal retention of isotretinoin was determined in accordance with the experimental group. Both the control group and the experimental group had three replicates.

[0157] The results show that there was no significant difference in skin retention between Comparative Example 1 and Comparative Example 3, indicating that the simple mixing method cannot improve the skin penetration of collagen. Compared with Comparative Example 1, the skin retention of isotretinoin in Example 2 was 4.4 times that of Comparative Example 1, indicating that the collagen nanoparticles formed in this invention have a significant penetration-enhancing effect. Compared with Comparative Example 4, the skin retention of isotretinoin in Example 2 was 2.1 times that of Comparative Example 4, indicating that the collagen nanoparticles formed by the preparation process of this invention have a better penetration-enhancing effect than Comparative Example 4, proving that the collagen nanoparticles prepared by the preparation process of this invention have a better transdermal effect.

[0158] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A collagen carrier, characterized in that, It is prepared from the following raw materials in parts by weight: 0.5-10 parts collagen, 0.1-10 parts stabilizer, 5-25 parts oil phase solvent, 2-10 parts glycerol, and 80-97.5 parts water. The oil phase solvent is selected from one or more of 1,2-propanediol, 1,3-butanediol, and ethanol; The stabilizer is selected from one or more of dimethyl dioctadecylammonium bromide, trimethyl-2,3-diolenopropylammonium chloride, trimethyl-2,3-dioleoylpropylammonium bromide, and trimethyl hexadecylammonium bromide. The preparation methods of collagen carriers include: S1: Prepare an aqueous phase and an organic phase separately, wherein the organic phase includes an oil phase solvent and a stabilizer, and the aqueous phase includes collagen, glycerol and water; S2: The aqueous phase and organic phase are mixed, and a primary emulsion is prepared by high-speed shearing; S3: The colostrum is homogenized under high pressure to prepare a solution system containing collagen carrier, and the process is repeated 3 to 8 times.

2. The collagen carrier as described in claim 1, characterized in that: It also includes step S4, which is: separating the collagen carrier from the solution system containing the collagen carrier.

3. The method for preparing a collagen carrier as described in claim 1, characterized in that: The high-speed shearing time is 1 to 5 minutes; the high-speed shearing speed is 5000 to 12000 rpm.

4. The method for preparing a collagen carrier as described in claim 1, characterized in that, The high-pressure homogenization conditions are: homogenization pressure of 300-800 bar.

5. A collagen nanoparticle, characterized in that: The collagen carrier includes the collagen carrier according to any one of claims 1 to 4, and further includes the active ingredient loaded on the collagen carrier.

6. The method for preparing collagen nanoparticles as described in claim 5, characterized in that, Including the following steps: A1: Prepare an aqueous phase and an organic phase separately, wherein the organic phase comprises 5-25 parts of oil phase solvent and 0.1-10 parts of stabilizer, and the aqueous phase comprises 0.5-10 parts of collagen, 2-10 parts of glycerol and 80-97.5 parts of water; the active ingredient may be added to any one or both of the aqueous and organic phases; A2: Mix the above aqueous phase and organic phase, and prepare the primary emulsion by high-speed shearing; A3: The above colostrum was subjected to high-pressure homogenization to prepare a solution system containing collagen nanoparticles, and the process was repeated 3-8 times. The oil phase solvent is selected from one or more of 1,2-propanediol, 1,3-butanediol, and ethanol; The stabilizer is selected from one or more of dimethyl dioctadecylammonium bromide, trimethyl-2,3-diolenopropylammonium chloride, trimethyl-2,3-dioleoylpropylammonium bromide, and trimethyl hexadecylammonium bromide.

7. The use of the collagen carrier according to any one of claims 1 to 4, or the collagen nanoparticles according to claim 5, in any aspect of the preparation of food, health products, cosmetics, feed, or pharmaceuticals for human or animal use.

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