Anti-aging compound polypeptide solid-in-oil nanosuspension for external use and preparation method of anti-aging compound polypeptide solid-in-oil nanosuspension

Abstract

The invention discloses an anti-aging compound polypeptide solid-in-oil nanosuspension for external use and a preparation method thereof, and belongs to the technical field of medicines. The compound polypeptide solid-in-oil nanosuspension comprises a water-soluble polypeptide drug, a surfactant and an oil phase matrix, the water-soluble polypeptide drug is wrapped by the surfactant, and is uniformly dispersed in the oil-phase matrix after being freeze-dried to form the compound polypeptide solid-in-oil nanosuspension; the water-soluble polypeptide drugs comprise snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4, and the average particle size of the drugs in the compound polypeptide solid-in-oil nanosuspension is smaller than 300 nm. The compound polypeptide solid-in-oil nanosuspension has the advantages of being stable in anhydrous phase, good in permeability, high in encapsulation efficiency, good in activity and the like, and is suitable for percutaneous delivery of hydrophilic drugs; the prescription of the solid-in-oil nanosuspension does not contain water, so that the preparation of a stable polypeptide preparation is facilitated, and the solid-in-oil nanosuspension is convenient to store; the composition can be used for anti-aging cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an anti-aging topical compound polypeptide oil-in-solid nano-suspension and a preparation method thereof. Background Art

[0002] With the increase of age, the skin gradually loses its elasticity, showing wrinkles and signs of aging. Among many anti-aging ingredients, peptides have attracted much attention due to their unique functions and effects. Currently, the polypeptides commonly used in anti-aging products are mainly divided into three categories according to different mechanisms: signal peptides, neurotransmitter inhibitory peptides, and carrier peptides; among them, dipeptide diaminobutyroyl benzylamide diacetate, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 have attracted much attention due to their good effects. Dipeptide diaminobutyroyl benzylamide diacetate and acetyl hexapeptide-8, as neurotransmitter inhibitors, reduce muscle contraction by inhibiting the release of the neurotransmitter acetylcholine, thereby smoothing wrinkles; Bgok et al. confirmed the interaction between dipeptide diaminobutyroyl benzylamide diacetate and matrix metalloproteinase (MMP) and Sirtuin (SIRT1) through computer simulation methods, and showed in vitro that it can effectively remove free radicals that age the skin. Acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4, as signal peptides, can promote the synthesis of collagen, elastin, and hyaluronic acid, enhance skin elasticity and barrier function, improve skin firmness and elasticity, and reduce wrinkles; among them, acetyl tetrapeptide-11 also has certain antioxidant and anti-inflammatory effects, and can neutralize free radicals to a certain extent. Polypeptides have the characteristics of instability, are not easy to store, and are water-soluble drugs with large molecular weights, making it difficult to penetrate the skin for absorption; although the combined use of five polypeptide drugs can achieve better anti-skin aging effects, due to the large molecular weights of the five drugs and the repulsion between their hydrophilicity and the lipid barrier of the skin, they will be hindered by poor transdermal performance and the stratum corneum and it is difficult to exert their efficacy; in addition, the direct use of a physical mixture of the five polypeptide drugs is difficult to achieve a drug sustained-release effect.

[0003] The oil-in-solid suspension encapsulates hydrophilic drugs through the action of hydrophobic surfactants, and after freeze-drying, a drug-surfactant complex is obtained. A preparation obtained by uniformly dispersing the complex in an oil phase has the advantages of improving the stability of drugs, controlling drug release, better exerting efficacy, simultaneously loading multiple drugs in the oil phase, and realizing the development of compound preparations. However, the particle size of the oil-in-solid suspension affects the transdermal delivery effect of hydrophilic drugs. Therefore, the oil-in-solid nano-suspension can provide a simple and feasible new strategy for improving the transdermal delivery of peptide drugs. Summary of the Invention

[0004] Aiming at the problems of stability and skin permeability of compound polypeptide drugs in preparations, the purpose of the present invention is to provide an anti-aging topical compound polypeptide oil-in-solid nano-suspension and its preparation method. The compound polypeptide drug is prepared into an oil-in-solid nano-suspension, which is suitable for the transdermal delivery of hydrophilic drugs, can better maintain the activity of the drug, improve stability, overcome the problem that the large molecular weight of the drug makes it difficult to cross the skin barrier, and can control the sustained and slow release of the drug, better exert its efficacy, obtain good transdermal absorption effect, and thus achieve good anti-skin aging effect.

[0005] The present invention provides an anti-aging topical compound polypeptide oil-in-solid nano-suspension (hereinafter referred to as compound polypeptide oil-in-solid nano-suspension), which includes water-soluble polypeptide drugs, surfactants and oil phase matrices; the water-soluble polypeptide drugs are wrapped by surfactants and evenly dispersed in the oil phase matrices after freeze-drying, forming a compound polypeptide oil-in-solid nano-suspension.

[0006] Furthermore, the water-soluble polypeptide drugs include syn-ake, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4. By weight, the proportion of the water-soluble polypeptide drugs in the compound polypeptide oil-in-solid nano-suspension is 0.1%; the average particle size of the drugs in the compound polypeptide oil-in-solid nano-suspension is < 300 nm.

[0007] Furthermore, the water-soluble polypeptide drugs are wrapped by surfactants in the form of a drug mixture and suspended in the oil phase matrix after freeze-drying.

[0008] Furthermore, each drug in the water-soluble polypeptide drugs is separately wrapped by a surfactant and simultaneously suspended in the oil phase matrix after freeze-drying.

[0009] Furthermore, the surfactant is a hydrophobic surfactant. By weight, the proportion of the hydrophobic surfactant in the compound polypeptide oil-in-solid nano-suspension is 1% - 4%; the hydrophobic surfactant is sucrose erucate, sucrose laurate or soybean phospholipid or a mixture thereof;

[0010] The oil phase matrix is jojoba oil, isopropyl myristate or medium-chain triglyceride or a mixture thereof. By weight, the proportion of the oil phase matrix is 95.8% - 98.9%.

[0011] Even further, the surfactant also includes a hydrophilic surfactant. By weight, the proportion of the hydrophilic surfactant in the compound polypeptide oil-in-solid nano-suspension is ≤ 0.2%;

[0012] The hydrophilic surfactant is sucrose monolaurate, sucrose stearate or sucrose palmitate or a mixture thereof.

[0013] The present invention provides a method for preparing an anti-aging topical compound polypeptide oil-in-solid nano-suspension. Five water-soluble drugs, namely, snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4, are dissolved in water. Optionally, a hydrophilic surfactant is added and allowed to dissolve before use. Separately, a hydrophobic surfactant is weighed and added to cyclohexane. Then, the aqueous phase of the mixed drug-hydrophilic surfactant or the mixed drug and the organic phase of cyclohexane-hydrophobic surfactant are mixed in a vial, and homogenized using a high-speed shear machine to encapsulate the drugs in the hydrophobic surfactant, obtaining a W / O emulsion. The W / O emulsion is pre-frozen using liquid nitrogen and then placed in a freeze dryer to sublime and remove the aqueous phase and organic solvent phase under vacuum, obtaining a freeze-dried complex of drug-surfactant. The freeze-dried complex is dispersed in an oil-phase matrix to obtain the compound polypeptide oil-in-solid nano-suspension. During the homogenization process, the drug and the surfactant pass through a very narrow gap under high pressure, resulting in a high flow rate, causing the material to be subjected to a strong shear force, thereby enabling the two to be fully mixed and preparing an emulsion at the micron level.

[0014] The present invention also provides a method for preparing an anti-aging topical compound polypeptide oil-in-solid nano-suspension. Snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide are separately dissolved in water, and a hydrophilic surfactant is separately added and allowed to dissolve before use. Separately, a hydrophobic surfactant is weighed and added to cyclohexane. The aqueous phases of snake venom-like tripeptide-hydrophilic surfactant, acetyl hexapeptide-8-hydrophilic surfactant, acetyl tetrapeptide-9-hydrophilic surfactant, acetyl tetrapeptide-11-hydrophilic surfactant, and palmitoyl pentapeptide-hydrophilic surfactant are respectively mixed with the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, and homogenized using a high-speed shear machine to separately encapsulate each drug in the hydrophobic surfactant, obtaining a W / O emulsion. Each W / O emulsion is pre-frozen using liquid nitrogen and then placed in a freeze dryer for freeze-drying to remove the aqueous phase and organic phase, respectively obtaining freeze-dried complexes of snake venom-like tripeptide-surfactant, acetyl hexapeptide-8-surfactant, acetyl tetrapeptide-9-surfactant, acetyl tetrapeptide-11-surfactant, and palmitoyl pentapeptide-surfactant. The five freeze-dried complexes are simultaneously dispersed in an oil-phase matrix to obtain the compound polypeptide oil-in-solid nano-suspension.

[0015] Further, the mass ratio of the drug to water in the aqueous phase containing snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide is 1:200, and the mass ratio of the hydrophobic surfactant to cyclohexane in the organic phase of cyclohexane-hydrophobic surfactant is 1:(10 - 40);

[0016] During the homogenization process, the rotation speed of the high-speed shearer is 20,000 rpm, and the time is 2 min - 3 min.

[0017] The present invention also provides an application of the anti-aging topical compound polypeptide oil-in-solid nano-suspension for preparing anti-aging skin care products.

[0018] The present invention has the following advantages and effects:

[0019] 1. The compound polypeptide oil-in-solid nano-suspension of the present invention has the advantages of anhydrous phase stability, good permeability, high encapsulation rate, etc., and is suitable for the transdermal delivery of hydrophilic drugs;

[0020] 2. By preparing the drug-surfactant complex, the dispersibility of water-soluble drugs in the oily matrix is improved, and the dispersibility of drugs in oils is not affected;

[0021] 3. The oil-in-solid nano-suspension enables the polypeptide to exhibit slow release, and this drug release behavior can cleverly prolong the action time of the drug on the body surface, better meet the usage requirements of transdermal preparations, and improve the transdermal absorption efficacy of the polypeptide;

[0022] 4. The compound polypeptide oil-in-solid nano-suspension has stability. The formulation of the oil-in-solid nano-suspension does not contain water, which is more conducive to preparing a stable polypeptide preparation and convenient for storage; and the drug particle size is controlled at the nanoscale. This nanoscale lipophilic carrier can better maintain the activity of the drug, improve stability, overcome the problem that the large molecular weight of the drug makes it difficult to cross the skin barrier, and is more conducive to permeating through the skin and being absorbed by the human body. Description of the Drawings

[0023] Figure 1 It is the in vitro release test diagram of the compound polypeptide oil-in-solid nano-suspension in Example 1 of the present invention;

[0024] Figure 2 It is the in vitro transdermal test diagram of the compound polypeptide oil-in-solid nano-suspension in pig skin in Example 1 of the present invention;

[0025] Figure 3 It is the in vitro retention test diagram of the compound polypeptide oil-in-solid nano-suspension in pig skin in Example 1 of the present invention;

[0026] Figure 4 It is the HE staining diagram for efficacy evaluation in Example 1 of the present invention, where: (a) is the normal group, (b) is the model group, and (c) is the Example 1 group;

[0027] Figure 5 It is the Masson staining diagram for efficacy evaluation in Example 1 of the present invention, where: (a) is the normal group, (b) is the model group, and (c) is the Example 1 group;

[0028] Figure 6HE staining image for efficacy evaluation in Comparative Example 2 of the present invention;

[0029] Figure 7 Masson staining image for efficacy evaluation in Comparative Example 2 of the present invention. Detailed implementation manners

[0030] The present invention will be described in detail below in conjunction with embodiments.

[0031] Embodiment 1

[0032] An anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention comprises water-soluble polypeptide drugs, surfactants and an oil-phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal), each polypeptide drug is respectively wrapped by a surfactant, and after freeze-drying, the five freeze-dried complexes are simultaneously and uniformly dispersed in the oil-phase matrix jojoba oil to form a compound polypeptide oil-in-solid nano-suspension; the surfactants include the hydrophilic surfactant sucrose monolaurate and the hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drugs in the compound polypeptide oil-in-solid nano-suspension is 145.9±18.5 nm; by weight ratio, in the compound polypeptide oil-in-solid nano-suspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil-phase matrix is 98.8%.

[0033] A preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention: Weigh 2 mg of each of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4, dissolve them separately in 0.4 g of ultrapure water, and add 2 mg of the hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Co., Ltd., Japan) respectively and wait for dissolution for later use. Separately weigh five portions of 20 mg of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Co., Ltd., Japan) and add them to 0.8 g of cyclohexane respectively. Each aqueous phase of the drug-hydrophilic surfactant is mixed with the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize with a high-speed shearer at 20000 rpm for 2 min to wrap each drug in the hydrophobic surfactant to obtain a W / O emulsion. Then use liquid nitrogen for freezing, and then place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase to obtain five drug-surfactant complexes. The five complexes are simultaneously dispersed in 9.88 g of the oil-phase matrix jojoba oil to obtain a compound polypeptide oil-in-solid nano-suspension.

[0034] Performance evaluation:

[0035] (1) In vitro release evaluation of the drug:

[0036] The in vitro release experiment of the compound polypeptide oil-in-solid nanosuspension was carried out by the vertical diffusion cell method. 0.5 g of the compound polypeptide oil-in-solid nanosuspension (equivalent to a total of 0.5 mg of five polypeptides) was accurately weighed and evenly coated on a nylon membrane with a pore size of 0.45 μm. 12 mL of ultrapure water was used as the release medium, the rotation speed was 350 r / min, and the temperature was 32 ± 0.5 °C. Samples of 2 mL were taken at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h (while replenishing an equal volume of blank medium), filtered through a 0.22 μm microporous membrane, 0.3 mL of the initial filtrate was discarded, and the subsequent filtrate was taken as the test solution; the concentrations of AKE, Arg, AcTP1, AcTP2, and Pal were calculated by the external standard method, and the cumulative release amount calculation formula was as follows:

[0037]

[0038] Where: Q: cumulative release amount (μg), V0: diffusion cell volume (12 mL), V: volume of each sample replenishment (2 mL), C n : drug concentration in the receiving solution at the nth sampling, C i : drug concentration in the receiving solution before the nth time, S: effective permeation area (1.77 cm 2 ).

[0039] The prescription of this example is shown in Table 1. As Figure 1 shown, the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the degassed ultrapure water in this prescription, and the cumulative release amount data are shown in detail in Table 2.

[0040] Table 1 Prescription of the compound polypeptide oil-in-solid nanosuspension in Example 1

[0041] Prescription Components Content (g) Additive Function Snake Venom-like Tripeptide 0.002 Main Drug Acetyl Hexapeptide-8 0.002 Main Drug Acetyl Tetrapeptide-9 0.002 Main Drug Acetyl Tetrapeptide-11 0.002 Main Drug Palmitoyl Pentapeptide 0.002 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant Jojoba Oil 9.88 Oil Phase Matrix Total Prescription 10

[0042] Table 2 Cumulative release amounts of the compound polypeptide oil-in-solid nanosuspension prescription in Example 1 in degassed ultrapure water

[0043]

[0044] (2) In vitro percutaneous permeation amount evaluation of the drug:

[0045] The percutaneous penetration experiment of the compound polypeptide oil-in-solid nanosuspension was carried out using a vertical diffusion device. The percutaneous penetration amount of the drug through porcine skin was investigated. 0.5 g of each group of preparations was evenly spread on the skin, and the supply cell mouth was covered with a glass slide. The receiving cell was filled with 12 mL of receiving medium to ensure that there were no bubbles between the skin and the receiving medium. The temperature was set at 32 ± 0.5 °C and the rotation speed was adjusted to 350 rpm. 5 mL of samples were taken at 1, 2, 4, 8, 12, and 24 h respectively, and an equal volume and temperature of blank receiving medium was supplemented at the same time. The supernatant in the taken samples was filtered through a 0.22 μm filter membrane, the initial filtrate was discarded, and the subsequent filtrate was injected into LC-MS / MS for analysis. The concentrations of AKE, Arg, AcTP1, AcTP2, and Pal were calculated by the external standard method. The formula for calculating the cumulative percutaneous penetration amount is as follows:

[0046]

[0047] Where: Q: Cumulative penetration amount (μg), V0: Diffusion cell volume (12 mL), V: Volume of each sample replenishment (2 mL), C n : Drug concentration in the receiving liquid at the nth sampling, C i : Drug concentration in the receiving liquid before the nth sampling, S: Effective permeation area (1.77 cm 2 ).

[0048] As Figure 2 shown, the skin cumulative penetration amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the prescription of this example in degassed ultrapure water, and the cumulative penetration amount data are shown in Table 3 in detail.

[0049] Table 3 Skin cumulative penetration amounts of the compound polypeptide oil-in-solid nanosuspension prescription in Example 1 in degassed ultrapure water

[0050]

[0051] (3) Evaluation of the in vitro skin retention amount of the drug:

[0052] An in vitro skin retention experiment of compound polypeptide oil-in-solid nanosuspension was carried out using a vertical diffusion device. The retention amount of the drug in the epidermis and dermis of porcine skin for 24 hours was investigated. After the in vitro transdermal experiment was completed, the skin surface was gently wiped with lint-free paper to remove the residual preparation on the surface, and then rinsed with absolute ethanol, and blotted dry with filter paper. Cut the skin within the effective diffusion area, and perform 20 paste-peeling operations on the skin surface layer with Scotch test tape to remove the stratum corneum of the skin. Finally, gently separate the epidermal layer and the dermal layer of the skin with forceps, put the epidermal layer and the shredded dermal layer into EP tubes respectively, add 4 mL of methanol, vortex for 5 minutes, and then ultrasonicate for 30 minutes under ice bath conditions to extract the drug components in the skin. Take the supernatant and centrifuge at 10000 r / min for 5 minutes, filter through a 0.22 μm microporous filter membrane, discard the initial filtrate, and inject the subsequent filtrate into LC-MS / MS for analysis. The concentrations of AKE, Arg, AcTP1, AcTP2, and Pal were calculated by the external standard method. The calculation formula for the drug retention amount per unit area of skin is as follows:

[0053] X = CV / S

[0054] Where: C: drug concentration in the extraction solution (μg / mL), V: volume of the extraction solution (mL), S: effective permeation area (cm 2 ).

[0055] As Figure 3 shown, the skin retention amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the prescription of this example in degassed ultrapure water. The retention amount data are shown in Table 4 for details.

[0056] Table 4 Skin retention amounts of the compound polypeptide oil-in-solid nanosuspension prescription of Example 1 in degassed ultrapure water

[0057]

[0058] (4) Efficacy evaluation:

[0059] SPF-grade male Kunming mice with a body weight of (30 g - 40 g) were selected, and pharmacodynamic observations were carried out according to the normal group (blank control), model group (modeling without drug administration), and Example 1 group (modeling and drug administration).

[0060] 1) HE staining:

[0061] The pathological changes of skin tissues were observed by HE staining. Through the combination of hematoxylin and eosin, the structures of cell nuclei, cytoplasm, and extracellular matrix could be clearly shown. After the administration was completed, about 2 cm × 2 cm of mouse skin tissues were taken and fixed in 4% paraformaldehyde, followed by dehydration with a series of ethanol and transparentization with xylene. Finally, they were embedded in conventional paraffin and made into sections for HE staining. The thicknesses of the epidermis and dermis were measured using Image-Pro Plus 6.0 software, and the data were analyzed.

[0062] As Figure 4 shown, for each group of HE-stained sections, they were observed under a 200-fold microscope respectively. The measurement results of the epidermis and dermis thicknesses are shown in Table 5. The results showed that the epidermis of mice in the normal group was relatively flat, the boundary between the epidermis and dermis was obvious and complete. The thickness of the epidermis was 42.84 ± 4.4 μm, and the thickness of the dermis was 628.06 ± 22.98 μm. In contrast, the epidermis of the model group was rough and uneven. The thickness of the epidermis decreased by about 51%, and the thickness of the dermis decreased by about 16%, indicating that D-galactose could significantly reduce the thicknesses of the epidermis and dermis of mice and successfully establish a model. Compared with the model group, the epidermis thickness in Example 1 increased by about 1 time, and the dermis was thickened more, increasing by about 19%. This indicated that the compound polypeptide oil-in-water solid nano-suspension in this example could effectively increase the thicknesses of both the epidermis and dermis of mice and improve the thinning of skin thickness caused by D-galactose modeling.

[0063] Table 5 Thicknesses of dermis and epidermis in the HE staining of the efficacy evaluation test in Example 1

[0064] Group <![CDATA[Dosing dose (mg·kg -1 ·d -1 )]]> Epidermal Thickness (μm) Dermal Thickness (μm) Normal Group - 42.84±4.41 628.06±22.98 Model Group - <![CDATA[20.94±2.54 △ΔΔ△ > <![CDATA[430.01±75.28 △Δ△ > Example 1 200 <![CDATA[40.62±7.28 **** > <![CDATA[628.97±87.69 *** >

[0065] Note: △ Compared with the normal group, △ P < 0.05, △△ P < 0.01, △Δ△ P < 0.001, △ΔΔ△ P < 0.0001;

[0066] * Compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0067] 2) Masson staining:

[0068] Masson staining is a commonly used histological staining method. After staining, collagen fibers appear blue or green, muscle fibers appear red, and cell nuclei appear dark blue or black. Components are distinguished according to the color of collagen fibers under a polarized light microscope. After the administration ended, approximately 2 cm × 2 cm of mouse skin tissue was taken and fixed in 4% paraformaldehyde, followed by dehydration with a series of ethanol and xylene transparency treatment. Finally, it was embedded in conventional paraffin and made into sections for Masson staining. The area ratio of collagen fibers was statistically analyzed using Image-Pro Plus 6.0 software.

[0069] As Figure 5 shown, for each group of Masson-stained sections, observations were made under a 200-fold microscope. The measurement results of the collagen fiber ratio are shown in Table 6. The results showed that in the dermis of the skin of normal group mice, the collagen fibers presented a uniform thickness, were arranged tightly and regularly, and the collagen fiber content was 61.68 ± 3.95%. However, the collagen fibers in the skin of the model group mice were disordered, loose and missing, and decreased by about 30% compared with the normal group, indicating that D-galactose could damage the collagen fibers in the skin and the model was successfully established. Compared with the model group, the number of collagen fibers in Example 1 showed a significant increase, increasing by about 59%, and the arrangement was relatively tight. It shows that the compound polypeptide oil-in-water nano-suspension of this example can effectively reduce the damage of D-galactose to skin collagen fibers, increase the proportion of collagen fibers in the skin, and has a certain anti-aging effect.

[0070] In this example, the method of separate encapsulation was adopted, that is, the freeze-dried complexes of each drug-surfactant were prepared separately first, and then these complexes were dispersed in the same oil phase, which could avoid the interaction between each polypeptide and further improve the stability of the drug.

[0071] Table 6 Collagen fiber ratio of Masson staining in the efficacy evaluation test of Example 1

[0072] Group <![CDATA[Dose administered (mg·kg -1 ·d -1 )]]> Collagen Fiber Ratio (%) Normal Group - <![CDATA[61.68±3.95 △ΔΔ△ > Model Group - 42.93±3.81 Example 1 200 <![CDATA[68.10±3.86 **** >

[0073] Note: △ Compared with the normal group, △ P < 0.05, △△ P < 0.01, △Δ△ P < 0.001, △ΔΔ△ P < 0.0001;

[0074] * Compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0075] In summary, the present invention prepares a solid-in-oil nano-suspension by encapsulating polypeptide drugs in surfactants, which can avoid the contact of drugs with external moisture, air, etc., and improve the drug stability; by preparing polypeptide drugs into a solid-in-oil nano-suspension, the nano-scale drugs are more conducive to drug release and transdermal delivery, thereby improving the therapeutic effect; the addition of hydrophilic surfactants makes the particle size of the preparation smaller and more uniformly dispersed in the oil phase; the pharmacodynamic experiments prove that the preparation in Example 1 can effectively treat skin aging.

[0076] Example 2

[0077] An anti-aging topical compound polypeptide solid-in-oil nano-suspension of the present invention comprises a water-soluble polypeptide drug, a surfactant and an oil phase matrix; the water-soluble polypeptide drug comprises snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal). The five polypeptide drug mixtures are encapsulated by surfactants and uniformly dispersed in the oil phase matrix jojoba oil after freeze-drying to form a compound polypeptide solid-in-oil nano-suspension; the surfactant comprises a hydrophilic surfactant sucrose monolaurate and a hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drug in the compound polypeptide solid-in-oil nano-suspension is 173.6±23.3 nm; by weight ratio, in the compound polypeptide solid-in-oil nano-suspension, the proportion of the water-soluble polypeptide drug is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil phase matrix is 98.8%.

[0078] A preparation method of an anti-aging topical compound polypeptide solid-in-oil nano-suspension of the present invention: weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Add 0.01 g of the hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Co., Ltd., Japan), and wait for it to dissolve before use. Another 0.1 g of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Co., Ltd., Japan) is added to 4 g of cyclohexane. Then, the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant are mixed in a vial, and homogenized with a high-speed shearing machine at 20,000 rpm for 2 min to encapsulate the drug in the hydrophobic surfactant to obtain a W / O emulsion. Then, it is frozen with liquid nitrogen and then freeze-dried in a freeze dryer for 24 h to remove the aqueous phase and the organic solvent phase to obtain a drug-surfactant complex. The complex is dispersed in 9.88 g of the oil phase matrix jojoba oil to obtain a compound polypeptide solid-in-oil nano-suspension.

[0079] In vitro release evaluation of the drug:

[0080] The formulation of this example is shown in Table 7, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water for this formulation are shown in Table 8.

[0081] Table 7 Formulation of the compound polypeptide oil-in-solid nanosuspension of Example 2

[0082] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant Jojoba Oil 9.88 Oil Phase Matrix Total Prescription 10

[0083] Table 8 Cumulative release amounts of the formulation of the compound polypeptide oil-in-solid nanosuspension of Example 2 in degassed ultrapure water

[0084]

[0085] Example 3

[0086] An anti-aging topical compound polypeptide oil-in-solid nanosuspension of the present invention comprises water-soluble polypeptide drugs, surfactants, and an oil phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2), and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by surfactants and uniformly dispersed in the oil phase matrix jojoba oil after freeze-drying to form a compound polypeptide oil-in-solid nanosuspension; the surfactants include the hydrophilic surfactant sucrose monolaurate and the hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drugs in the oil-in-solid nanosuspension is 232.9 ± 15.8 nm; by weight ratio, in the compound polypeptide oil-in-solid nanosuspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 2%, and the proportion of the oil phase matrix is 97.8%.

[0087] Preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Add 0.01 g of hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan), and set it aside after dissolution. Separately, weigh 0.2 g of hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan) and add it to 4 g of cyclohexane. Then, mix the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize it with a high-speed shear machine at 20,000 rpm for 2 minutes to encapsulate the drug in the hydrophobic surfactant, obtaining a W / O type emulsion. Then, freeze it with liquid nitrogen and place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase, obtaining a drug-surfactant complex. Disperse the complex in 9.78 g of oil phase matrix jojoba oil to obtain the compound polypeptide oil-in-solid nano-suspension.

[0088] In vitro release evaluation of the drug:

[0089] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 9, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in its prescription are shown in Table 10.

[0090] Table 9 Prescription of the compound polypeptide oil-in-solid nano-suspension in Example 3

[0091] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.2 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant Jojoba Oil 9.78 Oil Phase Matrix Total Prescription 10

[0092] Table 10 Cumulative release amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 3 in degassed ultrapure water

[0093]

[0094] Example 4

[0095] An anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention comprises water-soluble polypeptide drugs, surfactants and an oil-phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by surfactants and evenly dispersed in the oil-phase matrix jojoba oil after freeze-drying to form a compound polypeptide oil-in-solid nano-suspension; the surfactants include the hydrophilic surfactant sucrose monolaurate and the hydrophobic surfactant sucrose laurate. The hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drugs in the oil-in-solid nano-suspension is 268.3 ± 31.5 nm; by weight ratio, in the compound polypeptide oil-in-solid nano-suspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 4%, and the proportion of the oil-phase matrix is 95.8%.

[0096] A preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Add 0.01 g of the hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan), and wait for it to dissolve before setting aside. Weigh another 0.4 g of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan) and add it to 4 g of cyclohexane. Then mix the aqueous phase of the mixed drugs-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize them with a high-speed shearer at 20,000 rpm for 2 min to wrap the drugs in the hydrophobic surfactant to obtain a W / O emulsion. Then use liquid nitrogen for freezing, and then place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase to obtain a drug-surfactant complex. Disperse the complex in 9.58 g of the oil-phase matrix jojoba oil to obtain a compound polypeptide oil-in-solid nano-suspension.

[0097] In vitro release evaluation of drugs:

[0098] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 11, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2 and Pal in degassed ultrapure water in its prescription are shown in Table 12.

[0099] Table 11 Prescription of the compound polypeptide oil-in-solid nano-suspension in Example 4

[0100] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.4 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant Jojoba Oil 9.58 Oil Phase Matrix Total Prescription 10

[0101] Cumulative Release of the Prescription of Compound Polypeptide Oil-in-Solid Nanosuspension in Example 4 in Degassed Ultrapure Water

[0102]

[0103] Example 5

[0104] An anti-aging topical compound polypeptide oil-in-solid nanosuspension of the present invention comprises a water-soluble polypeptide drug, a surfactant, and an oil-phase matrix; the water-soluble polypeptide drug comprises snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2), and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by the surfactant and uniformly dispersed in the oil-phase matrix jojoba oil after freeze-drying to form a compound polypeptide oil-in-solid nanosuspension; the surfactant comprises a hydrophilic surfactant sucrose monolaurate and a hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drug in the oil-in-solid nanosuspension is 167.7±15.5 nm; by weight ratio, in the compound polypeptide oil-in-solid nanosuspension, the proportion of the water-soluble polypeptide drug is 0.1%, the proportion of the hydrophilic surfactant is 0.2%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil-phase matrix is 98.7%.

[0105] A preparation method of an anti-aging topical compound polypeptide oil-in-solid nanosuspension of the present invention: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Add 0.02 g of the hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan), and wait for it to dissolve before setting aside. Another 0.1 g of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan) is added to 4 g of cyclohexane. Then, the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant are mixed in a vial, and homogenized using a high-speed shearer at 20000 rpm for 2 minutes to wrap the drug in the hydrophobic surfactant, obtaining a W / O emulsion. Then, it is frozen with liquid nitrogen and then placed in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase, obtaining a drug-surfactant complex. The complex is dispersed in 9.87 g of the oil-phase matrix jojoba oil to obtain a compound polypeptide oil-in-solid nanosuspension.

[0106] In vitro release evaluation of the drug:

[0107] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 13, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in the prescription are shown in Table 14.

[0108] Table 13 Prescription of the compound polypeptide oil-in-solid nanosuspension in Example 5

[0109] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.02 Hydrophilic Surfactant Jojoba Oil 9.87 Oil Phase Matrix Total Prescription 10

[0110] Table 14 Cumulative release amounts of the prescription of the compound polypeptide oil-in-solid nanosuspension in Example 5 in degassed ultrapure water

[0111]

[0112] Example 6

[0113] An anti-aging topical compound polypeptide oil-in-solid nanosuspension of the present invention comprises water-soluble polypeptide drugs, surfactants, and an oil phase matrix; the water-soluble polypeptide drugs include syn-ake (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2), and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by surfactants and uniformly dispersed in the oil phase matrix jojoba oil after freeze-drying to form a compound polypeptide oil-in-solid nanosuspension; the surfactants include the hydrophilic surfactant sucrose monolaurate and the hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drugs in the oil-in-solid nanosuspension is 183.6 ± 22.8 nm; by weight ratio, in the compound polypeptide oil-in-solid nanosuspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.05%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil phase matrix is 98.85%.

[0114] Preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide), dissolve them in 2 g of ultrapure water, add 0.005 g of hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan), and set aside after dissolution. Separately, weigh 0.1 g of hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan), add it to 4 g of cyclohexane, then mix the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, homogenize it with a high-speed shearer at 20000 rpm for 2 min to encapsulate the drug in the hydrophobic surfactant, obtaining a W / O emulsion. Then, freeze it with liquid nitrogen and place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase, obtaining a drug-surfactant complex. Disperse the complex in 9.885 g of oil phase matrix jojoba oil to obtain the compound polypeptide oil-in-solid nano-suspension.

[0115] In vitro release evaluation of the drug:

[0116] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 15, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in its prescription are shown in Table 16.

[0117] Table 15 Prescription of the compound polypeptide oil-in-solid nano-suspension in Example 6

[0118] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.005 Hydrophilic Surfactant Jojoba Oil 9.885 Oil Phase Matrix Total Prescription 10

[0119] Table 16 Cumulative release amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 6 in degassed ultrapure water

[0120]

[0121] Example 7

[0122] An anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention comprises water-soluble polypeptide drugs, surfactants and an oil-phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by surfactants and evenly dispersed in the oil-phase matrix jojoba oil after freeze-drying to form a compound polypeptide oil-in-solid nano-suspension; the surfactants include the hydrophilic surfactant sucrose monolaurate and the hydrophobic surfactant sucrose erucate. The average particle size of the compound polypeptide drugs in the oil-in-solid nano-suspension is 248.2±22.3 nm; by weight ratio, in the compound polypeptide oil-in-solid nano-suspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil-phase matrix is 98.8%.

[0123] A preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Add 0.01 g of the hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan), and wait for it to dissolve before use. Weigh another 0.1 g of the hydrophobic surfactant sucrose erucate (trade name: ER290; Mitsubishi Food Corporation, Japan) and add it to 4 g of cyclohexane. Then mix the aqueous phase of the mixed drugs with the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize it with a high-speed shearer at 20000 rpm for 2 min to wrap the drugs in the hydrophobic surfactant to obtain a W / O emulsion. Then use liquid nitrogen for freezing, and then place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase to obtain a drug-surfactant complex. Disperse the complex in 9.88 g of the oil-phase matrix jojoba oil to obtain a compound polypeptide oil-in-solid nano-suspension.

[0124] Performance evaluation:

[0125] (1) In vitro release evaluation of drugs:

[0126] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 17, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2 and Pal in degassed ultrapure water in its prescription are shown in Table 18.

[0127] Table 17 Prescription of the compound polypeptide oil-in-solid nano-suspension in Example 7

[0128] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Erucate 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant Jojoba Oil 9.88 Oil Phase Matrix Total Prescription 10

[0129] Cumulative release amount of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 7 in degassed ultrapure water

[0130]

[0131]

[0132] (2) Evaluation of in vitro percutaneous permeation amount of the drug:

[0133] According to the in vitro percutaneous permeation experiment method and the cumulative permeation amount calculation formula in Example 1, the skin cumulative permeation amounts of AKE, Arg, AcTP1, AcTP2 and Pal in the prescription of this example in degassed ultrapure water are shown in Table 19.

[0134] Table 19 Skin cumulative permeation amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 7 in degassed ultrapure water

[0135]

[0136] (3) Evaluation of in vitro skin retention amount of the drug:

[0137] According to the in vitro skin retention experiment method and the retention amount calculation formula in Example 1, the skin retention amounts of AKE, Arg, AcTP1, AcTP2 and Pal in the prescription of this example in degassed ultrapure water are shown in Table 20.

[0138] Table 20 Skin retention amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 7 in degassed ultrapure water

[0139]

[0140] Example 8

[0141] An anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention comprises water-soluble polypeptide drugs, a surfactant and an oil phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by the surfactant and uniformly dispersed in the oil phase matrix jojoba oil after freeze-drying to form a compound polypeptide oil-in-solid nano-suspension; the surfactant includes the hydrophilic surfactant sucrose monolaurate and the hydrophobic surfactant soy lecithin. The average particle size of the compound polypeptide drugs in the oil-in-solid nano-suspension is 256.3 ± 11.2 nm; by weight ratio, in the compound polypeptide oil-in-solid nano-suspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil phase matrix is 98.8%.

[0142] Preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide), dissolve them in 2 g of ultrapure water, add 0.01 g of hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan), and set aside after dissolution. Separately, weigh 0.1 g of hydrophobic surfactant soy lecithin, add it to 4 g of cyclohexane, then mix the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, homogenize at 20000 rpm for 2 min using a high-speed shear machine, wrap the drug in the hydrophobic surfactant to obtain a W / O emulsion, then freeze it with liquid nitrogen, and then place it in a freeze dryer for freeze drying for 24 h to remove the aqueous phase and the organic solvent phase, obtaining a drug-surfactant complex. Disperse the complex in 9.88 g of the oil phase matrix jojoba oil to obtain the compound polypeptide oil-in-solid nano-suspension.

[0143] Performance evaluation:

[0144] (1) In vitro release evaluation of the drug:

[0145] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 21, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in its prescription are shown in Table 22.

[0146] Table 21 Prescription of the compound polypeptide oil-in-solid nano-suspension in Example 8

[0147] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Soybean Phospholipid 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant Jojoba Oil 9.88 Oil Phase Matrix Total Prescription 10

[0148] Table 22 Cumulative release amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 8 in degassed ultrapure water

[0149]

[0150] (2) In vitro percutaneous permeation amount evaluation of the drug:

[0151] According to the in vitro percutaneous permeation experiment method and the cumulative permeation amount calculation formula in Example 1, the skin cumulative permeation amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in the prescription of this example are shown in Table 23.

[0152] Table 23 Skin cumulative permeation amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 8 in degassed ultrapure water

[0153]

[0154]

[0155] (3) Evaluation of in vitro skin retention amount of the drug:

[0156] According to the in vitro skin retention experiment method and the retention amount calculation formula in Example 1, the skin retention amounts of AKE, Arg, AcTP1, AcTP2 and Pal in the prescription of this example in degassed ultrapure water are shown in Table 24.

[0157] Table 24 Skin retention amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 8 in degassed ultrapure water

[0158]

[0159] Example 9

[0160] An anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention comprises water-soluble polypeptide drugs, surfactants and an oil phase matrix; the water-soluble polypeptide drugs include syn-ake (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by surfactants and uniformly dispersed in the oil phase matrix jojoba oil after freeze-drying to form a compound polypeptide oil-in-solid nano-suspension; the surfactants include the hydrophilic surfactant sucrose stearate and the hydrophobic surfactant sucrose laurate. The average particle size of the compound polypeptide drugs in the oil-in-solid nano-suspension is 173.1 ± 14.7 nm; by weight ratio, in the compound polypeptide oil-in-solid nano-suspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil phase matrix is 98.8%.

[0161] Preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide), dissolve them in 2 g of ultrapure water, add 0.01 g of hydrophilic surfactant sucrose stearate (trade name: S1570; Mitsubishi Food Co., Ltd., Japan), and set aside after dissolution. Weigh another 0.1 g of hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Co., Ltd., Japan), add it to 4 g of cyclohexane, then mix the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, homogenize it with a high-speed shear machine at 20,000 rpm for 2 min to encapsulate the drug in the hydrophobic surfactant, obtaining a W / O emulsion. Then, freeze it with liquid nitrogen and place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase, obtaining a drug-surfactant complex. Disperse the complex in 9.88 g of oil phase matrix jojoba oil to obtain the compound polypeptide oil-in-solid nano-suspension.

[0162] In vitro release evaluation of the drug:

[0163] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 25, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in its prescription are shown in Table 26.

[0164] Table 25 Prescription of the compound polypeptide oil-in-solid nano-suspension in Example 9

[0165] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Stearate 0.01 Hydrophilic Surfactant Jojoba Oil 9.88 Oil Phase Matrix Total Prescription 10

[0166] Table 26 Cumulative release amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 9 in degassed ultrapure water

[0167]

[0168] Example 10

[0169] An anti-aging topical compound polypeptide solid-in-oil nano-suspension of the present invention comprises water-soluble polypeptide drugs, surfactants and an oil-phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by surfactants and evenly dispersed in the oil-phase matrix jojoba oil after freeze-drying to form a compound polypeptide solid-in-oil nano-suspension; the surfactants include the hydrophilic surfactant sucrose palmitate and the hydrophobic surfactant sucrose laurate. The average particle size of the compound polypeptide drugs in the solid-in-oil nano-suspension is 184.8±14.8nm; by weight ratio, in the compound polypeptide solid-in-oil nano-suspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil-phase matrix is 98.8%.

[0170] A preparation method of an anti-aging topical compound polypeptide solid-in-oil nano-suspension of the present invention: Weigh 0.01g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4 (2mg of each polypeptide), dissolve them in 2g of ultrapure water, add 0.01g of the hydrophilic surfactant sucrose palmitate (trade name: P1570; Mitsubishi Food Co., Ltd., Japan), and set aside after dissolution. Another 0.1g of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Co., Ltd., Japan) is weighed and added to 4g of cyclohexane. Then, the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant are mixed in a vial, homogenized by a high-speed shearer at 20000rpm for 2min to wrap the drug in the hydrophobic surfactant to obtain a W / O emulsion. Then, it is frozen with liquid nitrogen and then freeze-dried in a freeze dryer for 24h to remove the aqueous phase and the organic solvent phase to obtain a drug-surfactant complex. The complex is dispersed in 9.88g of the oil-phase matrix jojoba oil to obtain a compound polypeptide solid-in-oil nano-suspension.

[0171] In vitro release evaluation of drugs:

[0172] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 27, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2 and Pal in degassed ultrapure water in its prescription are shown in Table 28.

[0173] Table 27 Prescription of the compound polypeptide solid-in-oil nano-suspension in Example 10

[0174] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Palmitate 0.01 Hydrophilic Surfactant Jojoba Oil 9.88 Oil Phase Matrix Total Prescription 10

[0175] Cumulative Release of the Prescription of the Compound Polypeptide Oil-in-Solid Nanosuspension in Example 10 in Degassed Ultrapure Water

[0176]

[0177] Example 11

[0178] An anti-aging topical compound polypeptide oil-in-solid nanosuspension of the present invention comprises water-soluble polypeptide drugs, a surfactant and an oil phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2) and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by the surfactant and uniformly dispersed in the oil phase matrix isopropyl myristate (IPM) after freeze-drying to form a compound polypeptide oil-in-solid nanosuspension; the surfactant includes a hydrophilic surfactant sucrose monolaurate and a hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drugs in the oil-in-solid nanosuspension is 167.9±20.6 nm; by weight ratio, in the compound polypeptide oil-in-solid nanosuspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil phase matrix is 98.8%.

[0179] A preparation method of an anti-aging topical compound polypeptide oil-in-solid nanosuspension of the present invention is as follows: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Add 0.01 g of the hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan) and wait for it to dissolve before use. Separately weigh 0.1 g of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan) and add it to 4 g of cyclohexane. Then mix the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize them with a high-speed shearer at 20000 rpm for 2 min to wrap the drug in the hydrophobic surfactant to obtain a W / O emulsion. Then use liquid nitrogen for freezing, and then place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase to obtain a drug-surfactant complex. Disperse the complex in 9.88 g of the oil phase matrix IPM to obtain a compound polypeptide oil-in-solid nanosuspension.

[0180] In vitro release evaluation of the drug:

[0181] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 29, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in the prescription are shown in Table 30.

[0182] Table 29 Prescription of the compound polypeptide oil-in-solid nanosuspension in Example 11

[0183] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant IPM 9.88 Oil Phase Matrix Total Prescription 10

[0184] Table 30 Cumulative release amounts of the prescription of the compound polypeptide oil-in-solid nanosuspension in Example 11 in degassed ultrapure water

[0185]

[0186] Example 12

[0187] An anti-aging topical compound polypeptide oil-in-solid nanosuspension of the present invention comprises water-soluble polypeptide drugs, surfactants, and an oil phase matrix; the water-soluble polypeptide drugs include syn-ake (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2), and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by surfactants and uniformly dispersed in medium-chain triglyceride (MCT) in the oil phase matrix after freeze-drying to form a compound polypeptide oil-in-solid nanosuspension; the surfactants include the hydrophilic surfactant sucrose monolaurate and the hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drugs in the oil-in-solid nanosuspension is 213.5 ± 16.4 nm; by weight ratio, in the compound polypeptide oil-in-solid nanosuspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophilic surfactant is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil phase matrix is 98.8%.

[0188] Preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension of the present invention: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Add 0.01 g of hydrophilic surfactant sucrose monolaurate (trade name: L1695; Mitsubishi Food Corporation, Japan), and set aside after dissolution. Weigh another 0.1 g of hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan) and add it to 4 g of cyclohexane. Then mix the aqueous phase of the mixed drug-hydrophilic surfactant and the organic solvent phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize it with a high-speed shearer at 20,000 rpm for 2 min to encapsulate the drug in the hydrophobic surfactant, obtaining a W / O emulsion. Then use liquid nitrogen for freezing, and then place it in a freeze dryer for freeze-drying for 24 h to remove the aqueous phase and the organic solvent phase, obtaining a drug-surfactant complex. Disperse the complex in 9.88 g of oil phase matrix MCT to obtain the compound polypeptide oil-in-solid nano-suspension.

[0189] In vitro release evaluation of the drug:

[0190] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 31, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in its prescription are shown in Table 32.

[0191] Table 31 Prescription of the compound polypeptide oil-in-solid nano-suspension in Example 12

[0192] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Sucrose Monolaurate 0.01 Hydrophilic Surfactant MCT 9.88 Oil Phase Matrix Total Prescription 10

[0193] Table 32 Cumulative release amounts of the prescription of the compound polypeptide oil-in-solid nano-suspension in Example 12 in degassed ultrapure water

[0194]

[0195] Example 13

[0196] An anti-aging topical compound polypeptide solid-in-oil nano-suspension of the present invention comprises water-soluble polypeptide drugs, a surfactant, and an oil-phase matrix; the water-soluble polypeptide drugs include snake venom-like tripeptide (AKE), acetyl hexapeptide-8 (Arg), acetyl tetrapeptide-9 (AcTP1), acetyl tetrapeptide-11 (AcTP2), and palmitoyl pentapeptide-4 (Pal). The mixture of the five polypeptide drugs is wrapped by the surfactant and uniformly dispersed in the oil-phase matrix jojoba oil after freeze-drying to form a compound polypeptide solid-in-oil nano-suspension; the surfactant is a hydrophobic surfactant sucrose laurate, and the hydrophobic sucrose laurate can also act as a stabilizer. The average particle size of the compound polypeptide drugs in the solid-in-oil nano-suspension is 213.5 ± 16.4 nm; by weight ratio, in the compound polypeptide solid-in-oil nano-suspension, the proportion of the water-soluble polypeptide drugs is 0.1%, the proportion of the hydrophobic surfactant is 1%, and the proportion of the oil-phase matrix is 98.9%.

[0197] A preparation method of an anti-aging topical compound polypeptide solid-in-oil nano-suspension of the present invention: Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 2 g of ultrapure water. Separately weigh 0.1 g of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan) and add it to 4 g of cyclohexane. Then mix the water phase containing the drugs with the organic phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize it at 20000 rpm for 2 min using a high-speed shearing machine to wrap the drugs in the hydrophobic surfactant to obtain a W / O emulsion. Then use liquid nitrogen for freezing and then place it in a freeze dryer for freeze-drying for 24 h to remove the water phase and the organic solvent phase to obtain a drug-surfactant complex. Disperse the complex in 9.89 g of the oil-phase matrix jojoba oil to obtain a compound polypeptide solid-in-oil nano-suspension.

[0198] In vitro release evaluation of the drug:

[0199] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the prescription of this example is shown in Table 33, and the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in degassed ultrapure water in its prescription are shown in Table 34.

[0200] Table 33 Prescription of the compound polypeptide solid-in-oil nano-suspension in Example 13

[0201] Prescription Components Content (g) Additive Function Five Kinds of Polypeptides 0.01 Main Drug Sucrose Laurate 0.1 Hydrophobic Surfactant Jojoba Oil 9.89 Oil Phase Matrix Total Prescription 10

[0202] Table 34 Cumulative release amounts of the prescription of the compound polypeptide solid-in-oil nano-suspension in Example 13 in degassed ultrapure water

[0203]

[0204] Comparative Example 1

[0205] A preparation method of a physical mixture of compound polypeptides, with the same prescription as in Example 2, except that it is only prepared into a physical mixture of compound polypeptides without adopting the preparation process of oil-in-solid nano-suspension;

[0206] Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide), dissolve them in 2 g of ultrapure water, add 0.1 g of the hydrophobic surfactant sucrose laurate (trade name: L195; Mitsubishi Food Corporation, Japan), and disperse the complex in 9.89 g of the oil phase matrix jojoba oil.

[0207] Performance evaluation:

[0208] (1) In vitro release evaluation of the drug:

[0209] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the degassed ultrapure water in the prescription of this Comparative Example 1 are shown in Table 35.

[0210] Table 35 Cumulative release amounts of the physical mixture prescription of the compound polypeptides in Comparative Example 1 in degassed ultrapure water

[0211]

[0212]

[0213] After the drug of the present invention is prepared into an oil-in-solid nano-suspension, the release becomes significantly slower, without the burst release phenomenon of the physical mixture, achieving a sustained release effect.

[0214] (2) In vitro percutaneous permeation amount evaluation of the drug:

[0215] According to the in vitro percutaneous permeation experiment method and the cumulative permeation amount calculation formula in Example 1, the skin cumulative permeation amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the degassed ultrapure water in the prescription of this Comparative Example 1 are shown in Table 36.

[0216] Table 36 Skin cumulative permeation amounts of the physical mixture prescription of the compound polypeptides in Comparative Example 1 in degassed ultrapure water

[0217]

[0218] After the drug of the present invention is prepared into an oil-in-solid nano-suspension, it can effectively increase the cumulative percutaneous permeation amount of the drug, which is significantly better than the physical mixture.

[0219] (3) In vitro skin retention amount evaluation of the drug:

[0220] According to the in vitro skin retention experiment method and the retention amount calculation formula in Example 1, the skin retention amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the prescription of Comparative Example 1 in degassed ultrapure water are shown in Table 37.

[0221] Table 37 Skin retention amounts of the physical mixture prescription of the compound polypeptide in Comparative Example 1 in degassed ultrapure water

[0222]

[0223] In the examples and Comparative Example 1 of the present invention, the drugs were retained in both the dermis and the epidermis, indicating that the compound polypeptide drugs can diffuse in the skin; however, the retention amount of the drug in the epidermis in Comparative Example 1 was significantly higher than that in the dermis, while in the examples, the retention amount of the drug in the dermis was significantly higher than that in the epidermis, indicating that the solid-in-oil nano-suspension can effectively deliver the drug to the dermis layer. And compared with Comparative Example 1, the overall retention amount of the drug in Example 1 was significantly increased, indicating that the solid-in-oil nano-suspension can make the drug retain more in the skin.

[0224] Comparative Example 2

[0225] A preparation method of an aqueous solution preparation of a compound polypeptide. Weigh 0.01 g of snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11, and palmitoyl pentapeptide-4 (2 mg of each polypeptide) and dissolve them in 9.99 g of ultrapure water, and add an appropriate amount of hyaluronic acid and sodium benzoate to obtain an aqueous solution preparation of the compound polypeptide.

[0226] (1) In vitro release evaluation of the drug:

[0227] According to the in vitro release experiment method and the cumulative release amount calculation formula in Example 1, the cumulative release amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the prescription of Comparative Example 2 in degassed ultrapure water are shown in Table 38.

[0228] Table 38 Cumulative release amounts of the aqueous solution prescription of the compound polypeptide in Comparative Example 2 in degassed ultrapure water

[0229]

[0230] After the drug of the present invention is prepared into a solid-in-oil nano-suspension, the release is significantly slowed down, there is no burst release phenomenon of the aqueous solution, and a sustained release effect is achieved.

[0231] (2) In vitro percutaneous permeation amount evaluation of the drug:

[0232] According to the in vitro percutaneous penetration experiment method and the cumulative penetration amount calculation formula in Example 1, the skin cumulative penetration amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the prescription of Comparative Example 2 in degassed ultrapure water are shown in Table 39.

[0233] Table 39 Skin cumulative penetration amounts of the aqueous solution prescription of the compound polypeptide in Comparative Example 2 in degassed ultrapure water

[0234]

[0235] After the drug of the present invention is prepared into an oil-in-solid nanosuspension, the percutaneous cumulative penetration amount of the drug can be effectively increased, which is significantly better than that of the aqueous solution.

[0236] (3) Evaluation of in vitro skin retention amount of the drug:

[0237] According to the in vitro skin retention experiment method and the retention amount calculation formula in Example 1, the skin retention amounts of AKE, Arg, AcTP1, AcTP2, and Pal in the prescription of Comparative Example 2 in degassed ultrapure water are shown in Table 40.

[0238] Table 40 Skin retention amounts of the aqueous solution prescription of the compound polypeptide in Comparative Example 2 in degassed ultrapure water

[0239]

[0240] In the examples of the present invention and Comparative Example 2, the drug is retained in both the dermis and the epidermis, indicating that the compound polypeptide drug can diffuse in the skin; however, the retention amount of the drug in the epidermis in Comparative Example 2 is significantly higher than that in the dermis, while in the examples, the retention amount of the drug in the dermis is significantly higher than that in the epidermis, indicating that the oil-in-solid nanosuspension can effectively deliver the drug to the dermis layer; and compared with Comparative Example 2, the overall retention amount of the drug in Example 1 is significantly increased, indicating that the oil-in-solid nanosuspension can make the drug retain more in the skin.

[0241] Pharmacodynamic evaluation:

[0242] SPF-grade male Kunming mice with a body weight of (30 g - 40 g) were selected, and pharmacodynamic observations were carried out according to the normal group (blank control), the model group (modeling without drug administration), and the Comparative Example 2 group (modeling and drug administration). HE staining and Masson staining were carried out according to the experimental method in Example 1. As Figure 6 shown is the HE staining diagram, and as Figure 7 shown is the Masson staining; the data statistics of the fiber area ratio were carried out by using the data analysis method in Example 1, as shown in Table 41 and Table 42 in detail.

[0243] Table 41 Dermis and epidermis thicknesses of HE staining in the pharmacodynamic evaluation test of Comparative Example 2

[0244] Group <![CDATA[Dosing dose (mg·kg -1 ·d -1 )]]> Epidermal Thickness (μm) Dermal Thickness (μm) Normal Group - 42.84±4.41 628.06±22.98 Model Group - <![CDATA[20.94±2.54 △ΔΔ△ > <![CDATA[430.01±75.28 △Δ△ > Control Example 2 200 <![CDATA[29.01±4.55 * > <![CDATA[605.36±66.35 ** >

[0245] Note: Compared with the normal group, △ P < 0.05, △△ P < 0.01, △Δ△ P < 0.001, △ΔΔ△ P < 0.0001;

[0246] * Compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0247] Table 4 Proportion of collagen fibers by Masson staining in the pharmacodynamic evaluation test of Comparative Example 2

[0248] Group <![CDATA[Dosing dose (mg·kg -1 ·d -1 )]]> Collagen Fiber Ratio (%) Normal Group - <![CDATA[61.68±3.95 △ΔΔ△ > Model Group - 42.93±3.81 Control Example 2 200 <![CDATA[53.50±7.38 ** >

[0249] Note: Compared with the normal group, △ P < 0.05, △△ P < 0.01, △Δ△ P < 0.001, △ΔΔ△ P < 0.0001;

[0250] * Compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0251] Compared with the model group, the epidermal thickness of Comparative Example 2 increased by about 41%, the dermis thickened by about 14%, and the collagen fiber content increased by about 25%, all of which were lower than those of Example 1 of the present invention.

[0252] Compared with the aqueous solution preparation of the compound polypeptide of Comparative Example 2, after using the oil-in-solid nano-suspension of the compound polypeptide of the present invention, the epidermal and dermal thicknesses of mice increased more significantly, and the number of collagen fibers in the dermis was significantly increased, indicating that the present invention can improve the drug efficacy.

Claims

1. An anti-aging topical compound polypeptide oil-in-solid nano-suspension, characterized in that, It includes water-soluble polypeptide drugs, surfactants and an oil-phase matrix; the water-soluble polypeptide drugs are wrapped by surfactants and evenly dispersed in the oil-phase matrix after freeze-drying to form a compound polypeptide solid-in-oil nano-suspension.

2. The anti-aging topical compound polypeptide oil-in-solid nano-suspension according to claim 1, characterized in that, The water-soluble polypeptide drugs include syn-tetrapeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4. By weight, the proportion of the water-soluble polypeptide drugs in the compound polypeptide solid-in-oil nano-suspension is 0.1%; the average particle size of the drugs in the compound polypeptide solid-in-oil nano-suspension is <300 nm.

3. The anti-aging topical compound polypeptide oil-in-solid nano-suspension according to claim 2, wherein, The water-soluble polypeptide drugs are wrapped by surfactants in the form of a drug mixture and suspended in the oil-phase matrix after freeze-drying.

4. The anti-aging topical compound polypeptide oil-in-solid nano-suspension according to claim 2, wherein Each drug in the water-soluble polypeptide drugs is respectively wrapped by surfactants and simultaneously suspended in the oil-phase matrix after freeze-drying.

5. The anti-aging topical compound polypeptide oil-in-solid nano-suspension according to claim 1, characterized in that, The surfactant is a hydrophobic surfactant. By weight, the proportion of the hydrophobic surfactant in the compound polypeptide solid-in-oil nano-suspension is 1%-4%; the hydrophobic surfactant is sucrose erucate, sucrose laurate, soybean phospholipid or a mixture thereof; The oil-phase matrix is jojoba oil, isopropyl myristate or medium-chain triglyceride or a mixture thereof. By weight, the proportion of the oil-phase matrix in the solid-in-oil nano-suspension is 95.8%-98.9%.

6. The anti-aging topical compound polypeptide oil-in-solid nano-suspension according to claim 5, characterized in that The surfactant also includes a hydrophilic surfactant. By weight, the proportion of the hydrophilic surfactant in the compound polypeptide solid-in-oil nano-suspension is ≤0.2%; the hydrophilic surfactant is sucrose monolaurate, sucrose stearate, sucrose palmitate or a mixture thereof.

7. A preparation method of the anti-aging topical compound polypeptide oil-in-solid nano-suspension according to any one of claims 1-6, characterized in that, It includes the following steps: Dissolve syn-tetrapeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide-4, five water-soluble drugs, in water. Optionally, add a hydrophilic surfactant and wait for it to dissolve for later use. Weigh another hydrophobic surfactant and add it to cyclohexane. Then mix the water phase of the mixed drugs-hydrophilic surfactant or the mixed drugs with the organic phase of cyclohexane-hydrophobic surfactant in a vial, and use a high-speed shearer for homogenization to wrap the drugs in the hydrophobic surfactant to obtain a W / O emulsion. Use liquid nitrogen to pre-freeze the W / O emulsion, and then place it in a freeze dryer to sublime and remove the water phase and the organic solvent phase under vacuum to obtain a freeze-dried complex of the drug-surfactant. Disperse the freeze-dried complex in the oil-phase matrix to obtain a compound polypeptide solid-in-oil nano-suspension.

8. A preparation method of the anti-aging topical compound polypeptide oil-in-solid nano-suspension according to claim 1, characterized in that, It includes the following steps: Dissolve the snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide in water respectively, add hydrophilic surfactants respectively and wait for dissolution before use. Separately weigh hydrophobic surfactants and add them to cyclohexane. The aqueous phases of snake venom-like tripeptide-hydrophilic surfactant, acetyl hexapeptide-8-hydrophilic surfactant, acetyl tetrapeptide-9-hydrophilic surfactant, acetyl tetrapeptide-11-hydrophilic surfactant, and palmitoyl pentapeptide-hydrophilic surfactant are mixed with the organic phase of cyclohexane-hydrophobic surfactant in a vial, and homogenize using a high-speed shear machine. Each drug is respectively encapsulated in the hydrophobic surfactant to obtain a W / O emulsion. Use liquid nitrogen to pre-freeze each W / O emulsion, and then place it in a freeze dryer for freeze-drying to remove the aqueous phase and organic solvent phase, respectively obtaining freeze-dried complexes of snake venom-like tripeptide-surfactant, acetyl hexapeptide-8-surfactant, acetyl tetrapeptide-9-surfactant, acetyl tetrapeptide-11-surfactant, and palmitoyl pentapeptide-surfactant. Disperse the five freeze-dried complexes in an oil-phase matrix simultaneously to obtain a compound polypeptide oil-in-solid nanosuspension.

9. The preparation method of an anti-aging topical compound polypeptide oil-in-solid nano-suspension according to claim 7 or 8, characterized in that, The mass ratio of the drug to water in the aqueous phase containing snake venom-like tripeptide, acetyl hexapeptide-8, acetyl tetrapeptide-9, acetyl tetrapeptide-11 and palmitoyl pentapeptide is 1:200, and the mass ratio of the hydrophobic surfactant to cyclohexane in the organic phase of cyclohexane-hydrophobic surfactant is 1:(10 - 40); During the homogenization process, the rotation speed of the high-speed shear machine is 20000 rpm, and the time is 2 min - 3 min.

10. Use of the anti-aging topical compound polypeptide oil-in-solid nano-suspension according to any one of claims 1-6, characterized in that, Used for preparing anti-aging skin care products.

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