A polypeptide composition for treating skin diseases and preventing aging and a preparation method thereof

Abstract

The present application relates to the field of pharmaceutical preparations, in particular to a kind of polypeptide composition for treating skin diseases and preventing aging and preparation method thereof.The polypeptide composition includes the following components: palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl esters, acetyl heptapeptide-4, oligopeptide-1, hexapeptide-9, hexapeptide-11 and hydroxypropyl chitosan, glycerol, bamboo leaf essential oil, squalane, modified nanocellulose, deionized water and other additives.The present application constructs double emulsion system by three different solvent systems of hydroxypropyl chitosan, squalane and modified nanocellulose, opens the polypeptide channel of skin by squalane, promotes polypeptide to penetrate stratum corneum, and penetrates into deep skin tissue;Hydroxypropyl chitosan and modified nanocellulose are connected by Schiff base reaction after demulsification, and in-situ crosslinking film is rapidly carried out on the skin surface, which can effectively protect the wound surface of acne site.The present application has the effects of soothing skin, treating acne and anti-aging.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, specifically to a polypeptide composition for treating skin diseases and preventing aging, and its preparation method. Background Technology

[0002] Acne is a chronic inflammatory skin disease of the pilosebaceous unit that can damage one's appearance. The earliest morphological change in the development of acne is the abnormal keratinization of the follicular epithelium, leading to the formation of microcomedones, which are invisible to the naked eye. Impaired sebum secretion and the progressive enlargement of microcomedones form clinically visible comedones. Comedones can resolve spontaneously or develop into inflammatory acne lesions. If the follicle opening is blocked, sebum cannot be secreted, resulting in whiteheads. As the comedones expand, the pilosebaceous duct ruptures, leading to inflammatory lesions. If the lesion is superficial, a pustule forms, which ruptures within a few days, drains pus, and the skin heals without scarring. If the lesion is deeper, the skin presents as firm, red papules. If the deep inflammation progresses, nodules, cysts, sinuses, and fistulas can form, leaving scars of varying appearances after the inflammatory lesions heal. Acne damages the skin barrier, further leading to collagen loss and resulting in signs of skin aging.

[0003] Aging is driven by multiple molecular pathways and biochemical events, and can be divided into two types: endogenous aging and exogenous aging. Endogenous aging, also known as natural aging, occurs as we age. Cellular metabolism declines, collagen and elastic fibers in the dermis break down, the skin barrier function decreases, and the function of skin appendages such as sweat glands and sebaceous glands weakens. This leads to reduced skin elasticity and tension regulation, a decrease in subcutaneous fat, and a lack of support in the dermis, resulting in sagging and wrinkles. Endogenous aging is generally related to one's genetic makeup and is therefore irreversible. Exogenous aging, on the other hand, is mainly caused by external factors such as ultraviolet radiation, smoking, and lack of sleep, leading to premature aging phenomena such as uneven skin tone and deep wrinkles. It is closely related to the environment and therefore can be prevented and mitigated.

[0004] In the two major skin care fields of anti-aging and repair, peptides have become a research hotspot in the treatment of skin diseases and anti-aging due to their high bioactivity and good skin compatibility. Chinese patent CN112716826B discloses an anti-wrinkle composition composed of acetyl hexapeptide-8, tripeptide-1, palmitoyl tetrapeptide-7, and palmitoyl pentapeptide-4. This composition, through a specific ratio, achieves a long-lasting wrinkle-reducing effect, but its efficacy is limited to improving static wrinkles and enhancing elasticity, without addressing acne-prone skin problems. Chinese patent CN113018210B discloses a skin repair composition, its preparation method, and its application. Through a complex system of transdermal peptides, oligopeptide-1, and plant extracts, it demonstrates significant effects in barrier repair, particularly in alleviating redness in sensitive skin and healing burn wounds. However, this technology focuses on barrier repair and wound healing, without addressing the properties required for anti-aging such as collagen regeneration and elasticity enhancement, and therefore cannot address the problems of skin aging.

[0005] Therefore, to address the difficulty in synergistically addressing anti-aging and acne-prone skin improvement in the field of skin care, a peptide composition is needed that can improve both skin problems, while also taking into account the activity limitations of the peptide composition. Acne is often accompanied by problems such as damaged skin barrier and local wounds, which places additional demands on the bactericidal ability of the peptide composition components and the physical protective effect on damaged areas. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide composition for treating skin diseases and preventing aging, and its preparation method. This composition utilizes a water-in-oil-in-water dual emulsion system to achieve the coexistence of water-soluble and lipid-soluble polypeptides in the same stable system, alleviating the problem of incompatibility between polypeptide components. The polypeptide components, along with other components, can penetrate deep into the dermis, synergistically alleviating acne-prone skin and exerting anti-aging effects. A protective film formed by in-situ crosslinking of modified nanocellulose and hydroxypropyl chitosan provides a physical barrier for damaged wounds, preventing infection at the damaged site. Based on the differences in the solubility characteristics of polypeptide components, this invention constructs a dual emulsion system to achieve antibacterial and anti-inflammatory effects, barrier repair, and anti-aging effects against wrinkles, skin laxity, and collagen loss in acne-prone skin.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a polypeptide composition for treating skin diseases and preventing aging. The polypeptide composition is a water-in-oil-in-water double emulsion, which includes an inner aqueous phase, an oil phase, and an outer aqueous phase. The inner aqueous phase includes water-soluble polypeptides, hydroxypropyl chitosan, and deionized water. The oil phase includes fat-soluble polypeptides, bamboo leaf essential oil, Span-80, and squalane. The outer aqueous phase includes modified nanocellulose and an aqueous glycerol solution. The modified nanocellulose is obtained by grafting carboxymethyl cellulose with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide), and an aldehyde crosslinking agent.

[0008] Further, the mass ratio of the inner aqueous phase, oil phase, and outer aqueous phase is (2-5):(3-5):(40-45); the mass ratio of water-soluble peptides, hydroxypropyl chitosan, and deionized water in the inner aqueous phase is (0.066-0.07):1:(6.934-18.93); the mass ratio of fat-soluble peptides, bamboo leaf essential oil, Span-80, and squalane in the oil phase is (4-7):1:(3.6-6):(155-592.3); and the mass ratio of modified nanocellulose and glycerol aqueous solution in the outer aqueous phase is 1:(179-449).

[0009] Further, the water-soluble polypeptides include oligopeptide-1, hexapeptide-9, and hexapeptide-11, with a mass ratio of (5-25):(1-4):(1-4); the lipid-soluble polypeptides include palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, and acetyl heptapeptide-4, with a mass ratio of 1:(1-5):(5-10); and the mass ratio of glycerol to deionized water in the glycerol aqueous solution is (2-3):(86.5-87.8).

[0010] To achieve phased intervention throughout the entire process of acne development and post-healing aging, six polypeptide components were selected: oligopeptide-1, hexapeptide-9, hexapeptide-11, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, and acetyl heptapeptide-4. In the early stages of acne, palmitoyl tetrapeptide-7 can inhibit key inflammatory factors such as IL-6 and TNF-α, while the antioxidant effect of oligopeptide-1 can scavenge excess free radicals. The synergistic effect of the two can effectively control the inflammatory response and prevent acne from worsening. In the middle stage of acne, the pustular stage, hexapeptide-11 promotes the proliferation and migration of keratinocytes, and hexapeptide-9 promotes the synthesis of dermal structural proteins, together accelerating wound healing and strengthening the skin barrier. In the post-acne recovery stage, acetyl heptapeptide-4 inhibits the excessive release of neurotransmitters and inhibits the formation of dynamic wrinkles, acetyl dipeptide-1 cetyl ester improves the quality of elastic fibers and enhances skin elasticity, and oligopeptide-1 promotes collagen regeneration and reduces pigmentation through antioxidant effects. This improves the aging problems such as sagging, pigmentation, and wrinkles left after acne recovery, achieving full-stage intervention from anti-inflammatory repair to anti-aging and whitening.

[0011] Furthermore, the aldehyde crosslinking agent includes adipic acid dihydrazide and glutaraldehyde.

[0012] Secondly, the present invention provides a method for preparing a polypeptide composition for treating skin diseases and preventing aging, comprising the following steps:

[0013] S1: Add water-soluble peptides and hydroxypropyl chitosan to deionized water and stir at 200-500 rpm for 5-10 min to obtain the inner aqueous phase;

[0014] S2: Add bamboo leaf essential oil and Span-80 to squalane, stir at 200-500 rpm for 5-10 min, then add fat-soluble peptides, stir at 300-600 rpm for 4-8 min to obtain the oil phase;

[0015] S3: Homogenize the oil phase at a speed of 8000-10000 rpm for 3-5 min. Add the internal aqueous phase to the oil phase and continue homogenizing at 8000-10000 rpm for 2-4 min to obtain the primary emulsion.

[0016] S4: Disperse the modified nanocellulose in an aqueous glycerol solution and stir at 300-600 rpm for 5-10 min to obtain the external aqueous phase;

[0017] S5: Homogenize the external aqueous phase at a speed of 2000-4000 rpm for 2-4 min, add the primary emulsion, and homogenize at 2000-4000 rpm for 5-10 min to obtain a water-in-oil-in-water double emulsion.

[0018] S6: Add the post-addition phase solution to the water-in-oil-in-water double emulsion, stir evenly, and obtain a polypeptide composition for treating skin diseases and preventing aging.

[0019] Further, in step S4, the preparation method of the modified nanocellulose includes the following steps:

[0020] A1: Disperse carboxymethyl cellulose in PBS (phosphate buffer), stir to suspend, add EDC, stir at 400-600 rpm for 45-75 min, adjust pH to 5.5-6.0 with hydrochloric acid solution, centrifuge and wash to obtain EDC-grafted carboxymethyl cellulose;

[0021] A2: Disperse EDC-grafted carboxymethyl cellulose in deionized water, add NHS, adjust the pH to 5.8-6.2 with sodium hydroxide solution, stir the reaction to obtain an NHS-EDC-grafted carboxymethyl cellulose suspension;

[0022] A3: Add adipic acid dihydrazide solution to the NHS-EDC grafted carboxymethyl cellulose suspension, stir, wash and dialyze, add glutaraldehyde solution, adjust the pH to 5.8-6.8 with sodium hydroxide solution, stir to react, and obtain modified cellulose suspension;

[0023] A4: Adjust the pH of the modified cellulose suspension to 7-9 with sodium hydroxide solution, add sodium cyanoborohydride in an ice-water bath, stir at room temperature, adjust the pH to 6.5-7.0 with hydrochloric acid solution, dialyze, homogenize under high pressure, and collect the supernatant by centrifugation to obtain the modified nanocellulose dispersion.

[0024] Further, in A1, the mass ratio of carboxymethyl cellulose, PBS buffer, and EDC is 1:(4.5-5.5):50; the concentration of the PBS buffer is 0.01~0.05mol / L, the pH of the PBS buffer is 7.2-7.4, and the concentration of the hydrochloric acid solution is 0.05-0.1mol / L.

[0025] Furthermore, in A2, the mass ratio of EDC-grafted carboxymethyl cellulose, deionized water, and NHS is 1:(30-50):(2.5-3.5), the stirring speed of the stirring reaction is 800-1000 rpm, and the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L.

[0026] Further, in A3, the concentration of the adipic acid dihydrazide solution is 8-12 wt.%, the concentration of the glutaraldehyde solution is 20-30 wt.%, the volume ratio of the glutaraldehyde solution, the adipic acid dihydrazide solution, and the NHS-EDC grafted carboxymethyl cellulose dispersion is 1:(2.4-3.6):(3-5), and the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L; in A4, the mass ratio of sodium cyanoborohydride and the modified cellulose suspension is (0.5-0.7):(30-50), the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L, and the concentration of the hydrochloric acid solution is 0.05-0.1 mol / L.

[0027] Further, the added phase solution comprises an aqueous solution of potassium sorbate and an aqueous solution of nicotinamide; the concentration of the aqueous solution of potassium sorbate is 5-10 wt.%; the concentration of the aqueous solution of nicotinamide is 40-50 wt.%; and the mass ratio of the aqueous solution of potassium sorbate, the aqueous solution of nicotinamide, and the water-in-oil-in-water double emulsion is (1.5-2):(5-6):100.

[0028] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0029] This invention leverages the differences in the solubility properties of peptides, using a two-phase solvent system of squalane and hydroxypropyl chitosan aqueous solution to load lipid-soluble and water-soluble peptides, respectively. Further, an oil-in-water dual emulsion system is constructed using emulsification technology, isolating the modified nanocellulose with reactive aldehyde groups on its surface from the amino-containing active peptides, preventing Schiff base reactions during storage that could lead to peptide inactivation. During use, the system demulsifies under body temperature and massage, releasing squalane that promotes transdermal peptide penetration. The modified nanocellulose and hydroxypropyl chitosan cross-link to form a film on the skin surface via a Schiff base reaction, blocking external contaminants and reducing the risk of secondary infection. Furthermore, the bamboo leaf essential oil released after demulsification synergistically exerts antibacterial effects with the antibacterial peptides, and the released niacinamide provides antioxidant and skin-brightening effects. The modified nanocellulose of this invention is obtained by chemically modifying carboxymethyl cellulose using an EDC / NHS catalytic system. First, EDC reacts with the carboxyl group of carboxymethyl cellulose to form an O-acyl isourea intermediate, which then reacts with NHS to generate a stable NHS ester. Next, it reacts with the primary amino group at one end of adipic acid dihydrazide to form an amide bond. Finally, glutaraldehyde reacts with the amino group at the other end in a Schiff base reaction, and the aldehyde group is reduced to a stable CN bond by sodium cyanoborohydride. This successfully links the reactive aldehyde group to the cellulose backbone through stable covalent bonds, resulting in modified nanocellulose. Attached Figure Description

[0030] Figure 1 The present invention provides a process flow diagram for preparing a polypeptide composition for treating skin diseases and preventing aging. Detailed Implementation

[0031] The technical solution of the present invention will be described in detail below through specific embodiments. It should be understood that the embodiments of the present invention and the specific features in the embodiments are detailed descriptions of the technical solution of the present invention, rather than limitations on the technical solution of the present invention. In the absence of conflict, the embodiments of the present invention and the technical features in the embodiments can be combined with each other.

[0032] The term "and / or" simply describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. Additionally, the character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0033] The chemical reagents used in the embodiments and comparative examples of this invention are all commercially available products and have not undergone further purification or processing.

[0034] Example 1

[0035] like Figure 1 As shown, a method for preparing a polypeptide composition for treating skin diseases and preventing aging includes the following steps:

[0036] For ease of operation and calculation, oligopeptide-1, hexapeptide-9, and hexapeptide-11 were prepared into solutions with a concentration of 1 wt.% using deionized water as the solvent; palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, and acetyl heptapeptide-4 were prepared into liquids with a concentration of 1 wt.% using squalane as the solvent.

[0037] Step S1: Take 20g of oligopeptide-1 solution and stir at 280rpm. Add 5g of hexapeptide-9 solution and 5g of hexapeptide-11 solution in sequence. Stir until the system is homogeneous. Then add 5g of hydroxypropyl chitosan and stir until the hydroxypropyl chitosan dissolves. Add deionized water to make the total mass 40g and continue stirring for 7min to obtain the inner aqueous phase.

[0038] Step S2: Take 40g of squalane, stir at 400rpm, add 0.3g of bamboo leaf essential oil and 1.2g of Span-80, stir for 7min, after the system is homogeneous, add 2g of palmitoyl tetrapeptide-7 liquid, 2g of acetyl dipeptide-1 cetyl ester liquid and 6g of acetyl heptapeptide-4 liquid, add squalane to make the total mass 60g, and continue stirring at 400rpm for 5min to obtain the oil phase.

[0039] Step S3: Take 30g of oil phase, homogenize at 9000rpm for 3min, slowly add 20g of aqueous phase, and continue homogenizing at 9000rpm for 3min to obtain the primary emulsion.

[0040] Step S4: Add 6g of glycerol to 60g of deionized water and stir at 150rpm until the glycerol is completely dissolved. Add 100g of modified nanocellulose dispersion with a concentration of 1wt.% and add deionized water to make the total mass 180g. Stir at 400rpm for 7min to obtain the external aqueous phase.

[0041] The preparation method of modified nanocellulose is as follows:

[0042] A1: Disperse 1g of carboxymethyl cellulose in PBS buffer at pH 7.2 and stir at 500 rpm to form a carboxymethyl cellulose suspension. Add 5g of EDC in batches and adjust the pH to 5.8 with 0.05mol / L hydrochloric acid solution. Continue stirring for 60min, then centrifuge at 8000 rpm for 5min and wash 6 times to remove unreacted EDC to obtain EDC-grafted carboxymethyl cellulose.

[0043] A2: Resuspend the EDC-grafted carboxymethyl cellulose in deionized water at 900 rpm. The mass ratio of EDC-grafted carboxymethyl cellulose to deionized water is 1:45 to form a uniform suspension. Add 3g of NHS and adjust the pH to 6.0 with 0.05mol / L sodium hydroxide solution. Stir continuously for 60min to obtain the NHS-EDC-grafted carboxymethyl cellulose suspension.

[0044] A3: Add 30 mL of 10 wt.% adipic acid dihydrazide solution to 45 mL of NHS-EDC grafted carboxymethyl cellulose suspension, stir at 650 rpm for 3 h, and dialyze the reaction solution in deionized water for 48 h, changing the water every 6 h; add 10 mL of 25 wt.% glutaraldehyde solution to the dialyzed reaction solution, adjust the pH to 6.5 with 0.05 mol / L sodium hydroxide solution, stir at 900 rpm for 8 h to obtain modified cellulose suspension;

[0045] A4: Place 40g of modified cellulose suspension in an ice-water bath, adjust the pH of the system to 8.0 with 0.05mol / L sodium hydroxide solution, slowly add 0.6g of sodium cyanoborohydride in batches of 0.1g each, and stir until no solids remain. After the sodium cyanoborohydride is added, remove the system from the ice-water bath and stir at 500rpm for 8h. Adjust the pH of the system to 6.8 with 0.05mol / L hydrochloric acid solution, and dialyze in deionized water for 60h, changing the water every 4h. The dialyzed reaction solution is homogenized twice each at 0bar, 100bar, 200bar, 300bar and 400bar. The homogenized solution is centrifuged at 8000rpm for 8min, and the supernatant is collected to obtain the modified nanocellulose dispersion.

[0046] Step S5: Take 90g of external aqueous phase, homogenize at 3000rpm for 3min, slowly add 10g of primary emulsion, and continue homogenizing at 3000rpm for 6min to obtain a water-in-oil-in-water double emulsion.

[0047] Step S6: Add 5g of 50wt.% nicotinamide solution and 2g of 7.5wt.% potassium sorbate solution to 100g of water-in-oil-in-water double emulsion, respectively, and stir until the system is homogeneous to obtain a polypeptide composition for treating skin diseases and preventing aging.

[0048] Example 2

[0049] like Figure 1 As shown, a method for preparing a polypeptide composition for treating skin diseases and preventing aging includes the following steps:

[0050] For ease of operation and calculation, oligopeptide-1, hexapeptide-9, and hexapeptide-11 were prepared into solutions with a concentration of 1 wt.% using deionized water as the solvent; palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, and acetyl heptapeptide-4 were prepared into liquids with a concentration of 1 wt.% using squalane as the solvent.

[0051] Step S1: Take 10g of oligopeptide-1 solution, stir at 200rpm, add 2g of hexapeptide-9 solution and 2g of hexapeptide-11 solution in sequence, stir until the system is homogeneous, add 2g of hydroxypropyl chitosan, stir until the hydroxypropyl chitosan dissolves, add deionized water to the total mass of 40g, and continue stirring for 10min to obtain the inner aqueous phase.

[0052] Step S2: Take 40g of squalane, stir at 300rpm, add 0.1g of bamboo leaf essential oil and 0.6g of Span-80, stir for 10min, after the system is homogeneous, add 1g of palmitoyl tetrapeptide-7 liquid, 1g of acetyl dipeptide-1 cetyl ester and 5g of acetyl heptapeptide-4 liquid, add squalane to make the total mass 60g, and continue stirring at 300rpm for 8min to obtain the oil phase.

[0053] Step S3: Take 30g of oil phase, homogenize at 8000rpm for 5min, slowly add 20g of inner aqueous phase, and continue homogenizing at 8000rpm for 4min to obtain the primary emulsion.

[0054] Step S4: Add 4g of glycerol to 60g of deionized water and stir at 100rpm until the glycerol is completely dissolved. Add 100g of 0.4wt.% modified nanocellulose dispersion and add deionized water to bring the total mass to 180g. Stir at 300rpm for 10min to obtain the external aqueous phase.

[0055] The preparation method of modified nanocellulose is as follows:

[0056] A1: Disperse 1g of carboxymethyl cellulose in PBS buffer at pH 7.2 and stir at 400 rpm to form a carboxymethyl cellulose suspension. Add 4.5g of EDC in batches and adjust the pH to 5.5 with 0.07mol / L hydrochloric acid solution. Continue stirring for 45min, then centrifuge at 6000 rpm for 8min and wash 6 times to remove unreacted EDC, to obtain EDC-grafted carboxymethyl cellulose.

[0057] A2: Resuspend the EDC-grafted carboxymethyl cellulose in deionized water at 800 rpm. The mass ratio of EDC-grafted carboxymethyl cellulose to deionized water is 1:50 to form a uniform suspension. Add 2.5 g of NHS and adjust the pH to 5.8 with 0.07 mol / L sodium hydroxide solution. Stir continuously for 45 min to obtain the NHS-EDC-grafted carboxymethyl cellulose suspension.

[0058] A3: Add 24 mL of 12 wt.% adipic acid dihydrazide solution to 50 mL of NHS-EDC grafted carboxymethyl cellulose suspension, stir at 600 rpm for 4 h, and dialyze the reaction solution in deionized water for 36 h, changing the water every 10 h; add 10 mL of 30 wt.% glutaraldehyde solution to the dialyzed reaction solution, adjust the pH to 5.8 with 0.07 mol / L sodium hydroxide solution, stir at 800 rpm for 12 h to obtain modified cellulose suspension;

[0059] A4: Place 30g of modified cellulose suspension in an ice-water bath, adjust the pH of the system to 7.0 with 0.07mol / L sodium hydroxide solution, slowly add 0.5g of sodium cyanoborohydride in batches of 0.1g each, and stir until no solids remain. After the sodium cyanoborohydride is added, remove the system from the ice-water bath and stir at 400rpm for 12h. Adjust the pH of the system to 6.5 with 0.07mol / L hydrochloric acid solution, and dialyze in deionized water for 72h, changing the water every 6h. The dialyzed reaction solution is homogenized twice each at 0bar, 100bar, 200bar, 300bar and 400bar. The homogenized solution is centrifuged at 7500rpm for 10min, and the supernatant is collected to obtain the modified nanocellulose dispersion.

[0060] Step S5: Take 90g of external aqueous phase, homogenize at 2000rpm for 4min, slowly add 10g of primary emulsion, and continue homogenizing at 2000rpm for 10min to obtain a water-in-oil-in-water double emulsion.

[0061] Step S6: Add 5g of 40wt.% nicotinamide solution and 2g of 5wt.% potassium sorbate solution to 100g of water-in-oil-in-water double emulsion, respectively, and stir until the system is homogeneous to obtain a polypeptide composition for treating skin diseases and preventing aging.

[0062] Example 3

[0063] like Figure 1 As shown, a method for preparing a polypeptide composition for treating skin diseases and preventing aging includes the following steps:

[0064] For ease of operation and calculation, oligopeptide-1, hexapeptide-9, and hexapeptide-11 were prepared into solutions with a concentration of 1 wt.% using deionized water as the solvent; palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, and acetyl heptapeptide-4 were prepared into liquids with a concentration of 1 wt.% using squalane as the solvent.

[0065] Step S1: Take 50g of oligopeptide-1 solution, stir at 300rpm, add 8g of hexapeptide-9 solution and 8g of hexapeptide-11 solution in sequence, stir until the system is homogeneous, add 10g of hydroxypropyl chitosan, stir until the hydroxypropyl chitosan dissolves, add deionized water to the total mass of 80g, and continue stirring for 5min to obtain the inner aqueous phase.

[0066] Step S2: Take 40g of squalane, stir at 500rpm, add 0.5g of bamboo leaf essential oil and 1.8g of Span-80, stir for 5min, and after the system is homogeneous, add 5g of palmitoyl tetrapeptide-7 liquid, 5g of acetyl dipeptide-1 cetyl ester and 10g of acetyl heptapeptide-4 liquid, and add squalane to make the total mass 80g. Stir continuously at 600rpm for 4min to obtain the oil phase.

[0067] Step S3: Take 30g of oil phase, homogenize at 10000rpm, slowly add 30g of inner aqueous phase, and continue homogenizing at 10000rpm for 2min to obtain the primary emulsion.

[0068] Step S4: Add 10g of glycerol to 60g of deionized water and stir at 200rpm until the glycerol is completely dissolved. Add 100g of modified nanocellulose dispersion with a concentration of 1.2wt.% and add deionized water to make the total mass 180g. Stir at 600rpm for 5min to obtain the external aqueous phase.

[0069] The preparation method of modified nanocellulose is as follows:

[0070] A1: Disperse 1g of carboxymethyl cellulose in PBS buffer at pH 7.4 and stir at 600 rpm to form a carboxymethyl cellulose suspension. Add 5.5g of EDC in batches and adjust the pH of the system to 6.0 with 0.1mol / L hydrochloric acid solution. Continue stirring for 75min, then centrifuge at 9000 rpm for 3min and wash 5 times to remove unreacted EDC to obtain EDC-grafted carboxymethyl cellulose.

[0071] A2: Resuspend the EDC-grafted carboxymethyl cellulose in deionized water at 1000 rpm. The mass ratio of EDC-grafted carboxymethyl cellulose to deionized water is 1:30 to form a uniform suspension. Add 3.5 g of NHS and adjust the pH to 6.2 with 0.1 mol / L sodium hydroxide solution. Stir continuously for 75 min to obtain the NHS-EDC-grafted carboxymethyl cellulose suspension.

[0072] A3: Add 36 mL of 8 wt.% adipic acid dihydrazide solution to 30 mL of NHS-EDC grafted carboxymethyl cellulose suspension, stir at 800 rpm for 2 h, and dialyze the reaction solution in deionized water for 60 h, changing the water every 6 h; add 10 mL of 20 wt.% glutaraldehyde solution to the dialyzed reaction solution, adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution, and stir at 1000 rpm for 6 h to obtain modified cellulose suspension;

[0073] A4: Place 50g of modified cellulose suspension in an ice-water bath, adjust the pH of the system to 9.0 with 0.1mol / L sodium hydroxide solution, slowly add 0.7g of sodium cyanoborohydride in batches of 0.1g each, and stir until no solids remain. After the sodium cyanoborohydride is added, remove the system from the ice-water bath and stir at 600rpm for 4h. Adjust the pH of the system to 7.0 with 0.1mol / L hydrochloric acid solution, and dialyze in deionized water for 48h, changing the water every 4h. The dialyzed reaction solution is homogenized twice each at 0bar, 100bar, 200bar, 300bar and 400bar. The homogenized solution is centrifuged at 8500rpm for 5min, and the supernatant is collected to obtain the modified nanocellulose dispersion.

[0074] Step S5: Take 80g of external aqueous phase, homogenize at 4000rpm for 2min, slowly add 20g of primary emulsion, and continue homogenizing at 4000rpm for 5min to obtain a water-in-oil-in-water double emulsion.

[0075] Step S6: Add 6g of 50wt.% nicotinamide solution and 2g of 10wt.% potassium sorbate solution to 100g of water-in-oil-in-water double emulsion, respectively, and stir until the system is homogeneous to obtain a polypeptide composition for treating skin diseases and preventing aging.

[0076] Comparative Example 1

[0077] A method for preparing a polypeptide composition for treating skin diseases and preventing aging, which differs from Example 1 in that the polypeptide composition lacks an external aqueous phase, while the other operating steps and process parameters are exactly the same as in Example 1.

[0078] Comparative Example 2

[0079] A method for preparing a polypeptide composition for treating skin diseases and preventing aging, which differs from Example 1 in that unmodified nanocellulose is used as the external aqueous phase, while other operating steps and process parameters are exactly the same as in Example 1.

[0080] Comparative Example 3

[0081] A method for preparing a polypeptide composition for treating skin diseases and preventing aging, which differs from Example 1 in that the hydroxypropyl chitosan in the aqueous phase is replaced with xanthan gum, while the other operating steps and process parameters are exactly the same as in Example 1.

[0082] Comparative Example 4

[0083] A method for preparing a polypeptide composition for treating skin diseases and preventing aging, which differs from Example 1 in that the polypeptide composition lacks an oil phase, while the other operating steps and process parameters are exactly the same as in Example 1.

[0084] Comparative Example 5

[0085] A method for preparing a polypeptide composition for treating skin diseases and preventing aging, which differs from Example 1 in that the polypeptide composition lacks an internal aqueous phase, while the other operating steps and process parameters are exactly the same as in Example 1.

[0086] Performance testing:

[0087] Emulsion stability test: Take 10g of emulsion and place it in the same capped glass bottle. Tighten the cap and place it at ambient temperature. Record whether the emulsion breaks at time intervals of 0, 1, 3, and 7 to determine the storage stability of the emulsion. Evaluate the emulsion stability of the polypeptide compositions prepared in Examples 1-3 and Comparative Examples 1-3 and Comparative Examples 5 of this invention. Comparative Example 4 is not included in the emulsion stability test because it lacks an oil phase and the other two phases are miscible aqueous phases.

[0088] Film-forming performance test: Take 1 mL of emulsion, simulate the usage scenario, and spread it in a clean petri dish. Observe the film-forming speed of the emulsion under ambient temperature and humidity, record the film-forming time of each group, and evaluate the film-forming performance of the peptide compositions prepared in Examples 1-3 and Comparative Examples 1-5 of this invention.

[0089] Biosafety testing: The effects of the examples and comparative examples prepared in this invention on the viability of human immortalized keratinocytes (HaCaT) were evaluated using the Cell Counting Kit-8 (CCK-8). Cell activity was quantitatively analyzed by measuring absorbance using an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the biosafety of the polypeptide compositions prepared in Examples 1-3 and Comparative Examples 1-5 of this invention.

[0090] Anti-aging test: Using HaCaT cells as a model, the expression levels of proteins corresponding to moisturizing-related genes such as HAS1 were detected by ELISA to evaluate the anti-aging effects of the polypeptide compositions prepared in Examples 1-3 and Comparative Examples 4-5 of this invention; the reagent used in the blank control group was physiological saline.

[0091] Anti-inflammatory test: Using HaCaT as a cell model, cells were stimulated with inflammatory factors to establish an in vitro inflammation model; the contents of key inflammatory factors IL-1β, IL-6 and TNF-α in the cell culture supernatant were detected by ELISA to evaluate the anti-inflammatory effect of the polypeptide compositions prepared in Examples 1-3 and Comparative Examples 4-5 of this invention; physiological saline was used as a blank control group.

[0092] Table 1. Emulsion stability test results for each example and comparative example.

[0093]

[0094] According to the data in Table 1, no demulsification was found in Examples 1-3 and Comparative Examples 3 and 5 during the seven-day storage period. Compared with Comparative Examples 1 and 2, the dual emulsion system prepared by the present invention has good storage stability.

[0095] Comparative Example 1 is a traditional single-emulsion system prepared through homogenization. In this system, the peptides in the aqueous phase come into direct contact with other substances, leading to physical aggregation or chemical degradation during storage. This causes rapid emulsion stratification and a significant decrease in storage stability. The interfacial film formed by small-molecule emulsifiers such as Tween-80 is weaker than that of solid nanoparticles like nanocellulose, making it prone to flocculation and aggregation, ultimately resulting in demulsification. In Comparative Example 2, the unmodified nanocellulose, due to its nanoscale effect, can still aggregate on the surface of the primary emulsion droplets through electrostatic adsorption to form a stable dual-emulsion system with good storage stability. The water-in-oil-in-water emulsion is doubly stabilized by both Tween-80 and modified nanocellulose. Therefore, the xanthan gum in the inner aqueous phase of Comparative Example 3 does not affect the storage stability of the water-in-oil-in-water dual emulsion, which remained stable during a seven-day short-term storage test. Comparative Example 5 lacked an internal aqueous phase. After homogenization, the external aqueous phase and oil phase formed an oil-in-water emulsion. This is because the modified nanocellulose in the external aqueous phase, acting as nanoscale colloidal particles, worked together with Tween-80 in the oil phase to stabilize the emulsion. The nanoscale colloidal particles, after aggregating at the water-oil interface, can form a dense structure, effectively stabilizing the emulsion droplets and preventing aggregation.

[0096] Table 2. Film-forming performance test results of each embodiment and comparative example.

[0097] Example 1 15 Example 2 21 Example 3 23 Comparative Example 1 -- Comparative Example 2 51 Comparative Example 3 67 Comparative Example 4 25 Comparative Example 5 24

[0098] According to the data in Table 2, the film-forming time of Examples 1-3 and Comparative Examples 4-5 is shorter than that of Comparative Examples 1-3, which proves that the dual emulsion prepared by the present invention has good film-forming properties and can quickly form a thin film with a barrier effect on the skin surface during use.

[0099] Comparative Example 1 is a single emulsion system. One minute after coating, the water evaporated, leaving an uneven layer of oily substance. It did not form a network membrane structure; it was merely a physical accumulation and lacked the integrity of a functional network membrane. In Comparative Example 2, the outer aqueous phase was unmodified cellulose. After emulsification, it formed a thin film structure with the hydroxypropyl chitosan in the inner aqueous phase through physical entanglement and hydrogen bonding. However, this structure had low strength and easily disintegrated, failing to meet the requirements for adhering to the skin surface and blocking external dust and bacteria. In Comparative Example 3, the inner aqueous phase was xanthan gum. It could not connect with the modified cellulose in the outer aqueous phase to form a network structure through a Schiff base reaction. Instead, the peptides in the inner aqueous phase reacted with the aldehyde groups of the modified cellulose, becoming largely fixed within the modified cellulose. This significantly delayed peptide release, which was detrimental to the rapid anti-inflammatory and analgesic effects on acne-prone skin. The high viscosity of the xanthan gum in the inner aqueous phase further delayed film formation.

[0100] Table 3 Biosafety test results for each embodiment

[0101] Example 1 112.60±0.32 Example 2 105.57±0.87 Example 3 108.56±1.15 Comparative Example 1 95.36±0.61 Comparative Example 2 92.34±0.77 Comparative Example 3 97.59±0.27 Comparative Example 4 101.29±1.38 Comparative Example 5 100.72±0.55 Blank control 98.17±1.36

[0102] As shown in Table 3, the survival rate of HaCaT cells in Examples 1-3 all exceeded 105%, indicating that the polypeptide compositions prepared according to the present invention will not cause serious biosafety issues in further in vivo applications and can also promote cell growth. The survival rates of HaCaT cells in Comparative Examples 1-3 were all above 92%, but below 100%, not significantly different from the blank control group (98.17% ± 1.36%), proving that Comparative Examples 1-3 had no significant biotoxicity to HaCaT cells. However, due to their poor emulsion stability, the polypeptides in the compositions could not fully exert their effects and could not promote cell growth. The survival rates of HaCaT cells in Comparative Examples 4-5 were all above 100%, but below 105%, proving that they had no significant biotoxicity to HaCaT cells and could promote cell growth to a certain extent, but the promoting effect was not as good as that in Examples 1-3.

[0103] Table 4. Anti-aging cell test results for each example

[0104] Example 1 584.53±0.76 145.93±0.66 Example 2 558.69±0.37 136.61±0.92 Example 3 559.45±0.42 141.33±1.21 Comparative Example 4 534.53±0.29 131.33±0.27 Comparative Example 5 517.95±0.36 129.65±0.78 Blank control 513.48±0.98 127.95±0.52

[0105] As shown in Table 4, the expression levels of type I collagen and fibronectin in Examples 1-3 and Comparative Example 4 were all higher than those in the blank control group, indicating that the polypeptide composition prepared in this invention can enhance the expression of collagen and other proteins, thereby achieving barrier repair and anti-aging effects. The expression levels of type I collagen and fibronectin in Comparative Example 5 were slightly higher than those in the saline-treated blank control group, but significantly lower than those in Examples 1-3 and Comparative Example 4. This demonstrates the important role of the components in the aqueous phase in promoting the expression of type I collagen and fibronectin, proving that the water-soluble polypeptides in the aqueous phase mainly play an anti-aging role in the polypeptide composition.

[0106] Table 5. Anti-inflammatory cell test results for each embodiment.

[0107] Example 1 403±57 705±77 1056±178 Example 2 421±72 728±93 1139±146 Example 3 453±86 739±102 1246±127 Comparative Example 4 1436±197 3218±156 4035±294 Comparative Example 5 517±102 783±88 1275±156 Blank control 1735±236 3578±298 4372±375

[0108] As shown in Table 5, Examples 1-3 all exhibited significant inhibitory effects on the expression of three key inflammatory factors: IL-1β, IL-6, and TNF-α. In Comparative Example 4, the expression of these three key inflammatory factors was significantly higher than in other comparative examples, but slightly lower than in the blank control group, indicating that the components in the oil phase had a significant inhibitory effect on the inflammatory factors IL-1β, IL-6, and TNF-α. Although the polypeptide components in the aqueous phase could inhibit inflammatory factors to some extent, the effect was not as good as that of the polypeptide components in the oil phase. Comparative Example 5 also showed a significant inhibitory effect on the expression of inflammatory factors, but not as good as the complete polypeptide composition, because the oligopeptide-1 in the aqueous phase has a certain antioxidant effect, which can assist the other components in inhibiting the inflammatory factors. Inhibition of TNF-α expression indicates that the peptide composition can alleviate the initial initiation of inflammation and apoptosis, demonstrating its effective anti-inflammatory effect in the early stages of acne. Inhibition of IL-1β expression targets acne-related inflammatory pathways, improving follicular hyperkeratosis and severe local inflammation, and showing good anti-inflammatory effects on acne that rapidly progresses to a purulent state, effectively preventing further spread of inflammation. Inhibition of IL-6 expression regulates the activation of immune cells, helping to control further deterioration of inflammation. The peptide composition prepared in this invention can effectively inhibit the activation of multiple inflammatory signaling pathways in vivo, exhibiting good anti-inflammatory effects and demonstrating good efficacy in alleviating acne problems at different stages.

[0109] In summary, this invention utilizes the differences in the solubility characteristics of peptides to construct their respective solvent systems. Under the action of Tween-80 and modified nanocellulose, a stable dual-emulsion system is formed, resulting in a peptide composition that can achieve a synergistic effect of improving acne-prone skin and anti-aging. Targeted modification of carboxymethyl cellulose ensures that, during storage, the modified nanocellulose and hydroxypropyl chitosan are separated by the oil phase and cannot come into contact, remaining stable individually. During use, external influences cause the dual-emulsion system to demulsify, and the modified nanocellulose in the aqueous phase rapidly spreads to the application site. Subsequently, the oil phase is released, where squalane promotes the expansion of the intercellular spaces in the stratum corneum, which is beneficial for improving the transdermal penetration rate of peptides. Finally, the inner aqueous phase is released, in which peptides are delivered to the deeper layers of the skin through the channels opened by squalane to exert their effects. Hydroxypropyl chitosan rapidly cross-links with the modified nanocellulose spread on the application site, utilizing the excellent film-forming properties of hydroxypropyl chitosan to quickly form a film, achieving protection of damaged areas and preventing infection and inflammatory reactions. The glycerin in the outer aqueous phase provides a moist environment that can promote the skin's self-repair of damaged areas.

[0110] The embodiments of the present invention have been described above. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A polypeptide composition for treating skin diseases and preventing aging, characterized in that, The polypeptide composition is a water-in-oil-in-water double emulsion, which includes an inner aqueous phase, an oil phase, and an outer aqueous phase. The inner aqueous phase includes water-soluble polypeptides, hydroxypropyl chitosan, and deionized water; the oil phase includes fat-soluble polypeptides, bamboo leaf essential oil, Span-80, and squalane; and the outer aqueous phase includes modified nanocellulose and an aqueous glycerol solution. The mass ratio of the inner aqueous phase, oil phase, and outer aqueous phase is (2-5):(3-5):(40-45); the mass ratio of water-soluble peptides, hydroxypropyl chitosan, and deionized water in the inner aqueous phase is (0.066-0.07):1:(6.934-18.93); the mass ratio of fat-soluble peptides, bamboo leaf essential oil, Span-80, and squalane in the oil phase is (4-7):1:(3.6-6):(155-592.3); and the mass ratio of modified nanocellulose and glycerol aqueous solution in the outer aqueous phase is 1:(179-449). The water-soluble polypeptides include oligopeptide-1, hexapeptide-9, and hexapeptide-11, with a mass ratio of (5-25):(1-4):(1-4); the lipid-soluble polypeptides include palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, and acetyl heptacapeptide-4, with a mass ratio of 1:(1-5):(5-10); the mass ratio of glycerol to deionized water in the glycerol aqueous solution is (2-3):(86.5-87.8). The preparation method of the modified nanocellulose includes the following steps: A1: Disperse carboxymethyl cellulose in PBS buffer, stir to suspend, add EDC, stir at 400-600 rpm for 45-75 min, adjust pH to 5.5-6.0 with hydrochloric acid solution, centrifuge and wash to obtain EDC-grafted carboxymethyl cellulose; A2: Disperse EDC-grafted carboxymethyl cellulose in deionized water, add NHS, adjust the pH to 5.8-6.2 with sodium hydroxide solution, stir the reaction to obtain an NHS-EDC-grafted carboxymethyl cellulose suspension; A3: Add adipic acid dihydrazide solution to the NHS-EDC grafted carboxymethyl cellulose suspension, stir, wash and dialyze, add glutaraldehyde solution, adjust the pH to 5.8-6.8 with sodium hydroxide solution, stir to react, and obtain modified cellulose suspension; A4: Adjust the pH of the modified cellulose suspension to 7-9 with sodium hydroxide solution, add sodium cyanoborohydride in an ice-water bath, stir at room temperature, adjust the pH to 6.5-7.0 with hydrochloric acid solution, dialyze, homogenize under high pressure, and collect the supernatant by centrifugation to obtain the modified nanocellulose dispersion.

2. The method for preparing a polypeptide composition for treating skin diseases and preventing aging according to claim 1, characterized in that, Includes the following steps: S1: Add water-soluble peptides and hydroxypropyl chitosan to deionized water and stir at 200-500 rpm for 5-10 min to obtain the inner aqueous phase; S2: Add bamboo leaf essential oil and Span-80 to squalane, stir at 200-500 rpm for 5-10 min, then add fat-soluble peptides, stir at 300-600 rpm for 4-8 min to obtain the oil phase; S3: Homogenize the oil phase at a speed of 8000-10000 rpm for 3-5 min. Add the internal aqueous phase to the oil phase and continue homogenizing at 8000-10000 rpm for 2-4 min to obtain the primary emulsion. S4: Disperse the modified nanocellulose in an aqueous glycerol solution and stir at 300-600 rpm for 5-10 min to obtain the external aqueous phase; S5: Homogenize the external aqueous phase at a speed of 2000-4000 rpm for 2-4 min, add the primary emulsion, and homogenize at 2000-4000 rpm for 5-10 min to obtain a water-in-oil-in-water double emulsion. S6: Add the post-addition phase solution to the water-in-oil-in-water double emulsion, stir evenly, and obtain a polypeptide composition for treating skin diseases and preventing aging.

3. The method for preparing a polypeptide composition for treating skin diseases and preventing aging according to claim 2, characterized in that, In A1, the mass ratio of carboxymethyl cellulose, PBS buffer, and EDC is 1:(4.5-5.5):50; the concentration of the PBS buffer is 0.01~0.05mol / L, the pH of the PBS buffer is 7.2-7.4, and the concentration of the hydrochloric acid solution is 0.05-0.1mol / L.

4. The method for preparing a polypeptide composition for treating skin diseases and preventing aging according to claim 2, characterized in that, In A2, the mass ratio of EDC-grafted carboxymethyl cellulose, deionized water, and NHS is 1:(30-50):(2.5-3.5), the stirring speed of the stirring reaction is 800-1000 rpm, and the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L.

5. The method for preparing a polypeptide composition for treating skin diseases and preventing aging according to claim 2, characterized in that, In A3, the concentration of the adipic acid dihydrazide solution is 8-12 wt.%, the concentration of the glutaraldehyde solution is 20-30 wt.%, and the volume ratio of the glutaraldehyde solution, the adipic acid dihydrazide solution, and the NHS-EDC grafted carboxymethyl cellulose dispersion is 1:(2.4-3.6):(3-5). The concentration of the sodium hydroxide solution is 0.05-0.1 mol / L. In A4, the mass ratio of sodium cyanoborohydride and the modified cellulose suspension is (0.5-0.7):(30-50), the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L, and the concentration of the hydrochloric acid solution is 0.05-0.1 mol / L.

6. The method for preparing a polypeptide composition for treating skin diseases and preventing aging according to claim 2, characterized in that, The added phase solution comprises an aqueous solution of potassium sorbate and an aqueous solution of nicotinamide; the concentration of the aqueous solution of potassium sorbate is 5-10 wt.%; the concentration of the aqueous solution of nicotinamide is 40-50 wt.%; and the mass ratio of the aqueous solution of potassium sorbate, the aqueous solution of nicotinamide, and the water-in-oil-in-water double emulsion is (1.5-2):(5-6):100.

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