Arginine / lysine polypeptide supramolecular composition as well as preparation method and application thereof

Abstract

The invention belongs to the field of penetration enhancer preparation, and particularly relates to an arginine / lysine polypeptide supramolecular composition as well as a preparation method and application thereof. The arginine / lysine polypeptide supramolecular composition is prepared from the following raw materials in parts by weight: 0.00001 to 30 parts of arginine / lysine polypeptide, 0.0001 to 20 parts of gamma-aminobutyric acid and 50 to 85 parts of solvent. According to the arginine / lysine polypeptide supramolecular composition, the transdermal efficiency is remarkably improved, the composition does not need to depend on a chemical penetration enhancer, the composition is stable and long-acting in component, the function synergy and targeting are enhanced, the application scene is wider, the safety is higher, the production process is green and environmentally friendly, and meanwhile the cost advantage is huge.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of penetration enhancers, and particularly relates to an arginine / lysine polypeptide supramolecular composition, a preparation method thereof and an application thereof. Background Art

[0002] With the increasing demand for anti-aging, the research on anti-wrinkle technology in the cosmetics industry focuses on how to efficiently and gently improve dynamic wrinkles and static wrinkles. The current mainstream technologies include:

[0003] (i) Adding collagen: Supplementing type III collagen to restore skin elasticity. However, exogenous collagen has a large molecular weight and a low transdermal penetration rate, and it needs to rely on additional nano-carriers, etc.

[0004] (ii) Delivering hydrophilic active ingredients: For example, although some hydrophilic active ingredients can inhibit neuronal excitation and promote collagen synthesis, their strong hydrophilicity leads to insufficient transdermal efficiency (<5%), and they need to rely on chemical penetration enhancers (such as ethoxydiglycol), which may cause skin irritation.

[0005] (iii) Adding polypeptide components: Polypeptides are easily degraded by proteases or inactivated by pH fluctuations. In traditional formulations, stabilizers (such as cyclodextrin embedding) need to be added, but it may affect the activity.

[0006] That is, the disadvantages of the existing technology can be summarized as:

[0007] (i) The contradiction between transdermal efficiency and safety: Chemical penetration enhancers damage the skin barrier and cause sensitivity problems;

[0008] (ii) Single-component function: It is difficult for a single component to meet the multi-dimensional anti-aging requirements such as wrinkle reduction, filling, and repair;

[0009] (iii) Insufficient stability: Related active ingredients are easily inactivated, affecting the long-term effectiveness of the product.

[0010] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0011] In order to solve the problems existing in the prior art, the present invention provides an arginine / lysine polypeptide supramolecular composition, which comprises the following raw materials in parts by weight: 0.00001-30 parts of arginine / lysine polypeptide, 0.0001-20 parts of γ-aminobutyric acid (GABA), and 50-85 parts of a solvent.

[0012] For the convenience of understanding the present invention, the functions of the raw materials are explained:

[0013] Arginine / Lysine Polypeptide: Electrostatically binds to GABA through positive charges (carrying 1 - 2 charges at pH 4.5 - 5.5), and promotes transdermal penetration by utilizing the characteristics of cell - penetrating peptides (CPP); γ - aminobutyric acid (GABA): Forms an ionic bond with the amino group (-NH3 - ) of the polypeptide through the carboxylic acid group (-COO + ) in the complex; Solvent: The pH is adjusted to 4.5 - 5.5 to maintain the stability of the system.

[0014] In subsequent detections, the particle size distribution of the supramolecular composition is measured to be 50 - 200 nm by dynamic light scattering (DLS), and the supramolecular composition is shown to be uniform spherical nanoparticles by transmission electron microscopy (TEM). The zeta potential is +10 - +30 mV (under the condition of pH 5.0), which can ensure efficient binding to negatively charged stratum corneum lipids.

[0015] In addition, the binding mechanism of the present invention is formed by electrostatic, hydrogen - bond and hydrophobic interactions, specifically as follows:

[0016] Electrostatic interaction: The positive charge of the polypeptide binds to the negative charge of GABA.

[0017] Hydrogen - bond network: The amide group of the polypeptide backbone forms hydrogen bonds with the amino group / carboxylic acid group of GABA.

[0018] Hydrophobic interaction: The hydrophobic groups on the side chain of the polypeptide (such as the guanidine group of arginine) and the alkane chain of GABA synergistically enhance the stability of the complex.

[0019] Preferably, the arginine / lysine polypeptide supramolecular composition comprises the following raw materials in parts by weight: 20 parts of arginine / lysine polypeptide, 15 parts of γ - aminobutyric acid, and 65 parts of solvent.

[0020] Preferably, the molecular weight range of the arginine / lysine polypeptide is 500 - 2000 Da, and the amino acid sequence contains at least 3 arginine or lysine residues;

[0021] And / or, the isoelectric point of the γ - aminobutyric acid is 7.3;

[0022] And / or, the solvent is a mixed solution of alcohol and water, and the alcohol is propylene glycol or glycerol.

[0023] Based on the same technical concept, another solution of the present invention is to provide a preparation method of an arginine / lysine polypeptide supramolecular composition, and the preparation method comprises the following steps:

[0024] (1) Mix the arginine / lysine polypeptide with the solvent, then add PBS buffer solution, and then heat and stir until dissolved to obtain a polypeptide solution;

[0025] (2) Add γ-aminobutyric acid powder to the polypeptide solution, adjust the pH to 4.5 - 5.5 to trigger electrostatic binding and self-assembly, and then perform ultrasonic treatment and centrifugation in sequence to obtain a mixture.

[0026] (3) Dialyze the mixture to remove small molecule impurities, then add a lyoprotectant and perform freeze-drying to obtain the arginine / lysine polypeptide supramolecular composition.

[0027] Preferably, in step (1), the heating temperature is 30 - 50 °C.

[0028] In step (1), and / or, the stirring speed is 100 - 150 r / min.

[0029] In step (1), and / or, the concentration of the polypeptide solution is 10 - 30 mg / mL.

[0030] Preferably, in step (2), the ultrasonic power is 100 - 300 W, the frequency is 20 - 40 kHz, and the duration is 10 - 30 min; the purpose is to promote nanoparticle homogenization.

[0031] And / or, in step (2), the centrifugation speed is 2000 - 5000 r / min, and the centrifugation time is 5 - 10 min; the purpose is to remove unbound free polypeptide or GABA.

[0032] Preferably, in step (3), the dialysis treatment method is: use a dialysis bag with a molecular weight cut-off of 3 - 5 kDa and dialyze at 4 °C for 24 h.

[0033] And / or, in step (3), the lyoprotectant is mannitol or trehalose, and the addition amount is 0.1 - 1 wt%.

[0034] Based on the same technical concept, another solution of the present invention is to provide an application of an arginine / lysine polypeptide supramolecular composition as a penetration enhancer in cosmetics and transdermal drugs.

[0035] The beneficial effects of the present invention are as follows:

[0036] 1. The transdermal efficiency is significantly improved, and there is no need to rely on chemical penetration enhancers.

[0037] In the prior art, although the "superconductor" carrier protein of Puyan Biology can improve the transdermal absorption rate (for example, the transdermal absorption rate of recombinant functional proteins is increased by 237%), its technology still relies on chemical penetration enhancers or complex processes (such as heating, emulsification). In contrast, in the present invention, through the self-assembled nanoparticles (50 - 200 nm) of arginine / lysine polypeptide and GABA, the cell membrane penetration property (CPP property) of the polypeptide is utilized to directly carry the active ingredient through the skin, and the transdermal rate of GABA can reach 18.7% without chemical penetration enhancers (only 5.8% for traditional dosage forms). It avoids the damage of chemical penetration enhancers to the skin barrier, and clinical tests show that the irritation level is 0 (ISO 10993 - 10 standard).

[0038] 2. Stable and long-lasting components

[0039] In the prior art, although the "supramolecular blue copper peptide" of Marubi Biology improves the transdermal absorption rate (3.6 times) through ionic liquid encapsulation, its stability is still affected by light and high temperature. In the present invention, through the steric hindrance effect of the supramolecular composition, protease attacks are shielded and pH fluctuations are buffered, so that the activity retention rate of the polypeptide is > 90% after storage at 37°C for 6 months (only 52% for traditional formulations). It extends the action time of the active ingredient, and the sustained release period reaches 24 hours after single use, reducing the frequency of re-application.

[0040] 3. Enhanced functional synergy and targeting

[0041] In the prior art, products such as Runxiquan essence need to be compounded with multiple ingredients to achieve the anti-wrinkle effect, but there is a lack of molecular-level synergy among the ingredients. In the present invention, through the dual-functional synergistic design of polypeptide and GABA: polypeptide promoting penetration (arginine / lysine polypeptide directly penetrates the stratum corneum) and GABA activity enhancement (inhibiting neuronal excitation and promoting collagen synthesis, and the dynamic wrinkle inhibition effect is 1.8 times that of free GABA), it is possible to reduce the dependence on additional ingredients (such as PDRN, type III collagen), simplify the formula and reduce costs.

[0042] 4. Wider application scenarios and higher safety

[0043] In the prior art, products such as the microneedle patch of Zhongke Cells need to puncture the stratum corneum, which may cause slight tissue damage, and the hydrogen molecule transdermal technology relies on a nano-excimer hydrogen production device, with a high cost. In the present invention, through non-invasive nanoparticle delivery, it is suitable for cosmetics (such as anti-wrinkle essence, soothing mask) and transdermal drug delivery systems (such as diclofenac sodium sustained release preparation), and does not require complex equipment or invasive operations. It combines high efficiency and universality, and is suitable for sensitive skin and medical aesthetic anti-aging needs.

[0044] 5. Green and environmentally friendly production process and huge cost advantages

[0045] In the prior art, although the transdermal cyclic peptide of Meierjian reduces costs through biosynthesis, it relies on genetic recombination technology and has a high process complexity. The present invention adopts a self-assembly process of natural polypeptide and GABA, which does not require genetic modification or complex equipment, has a low-carbon and environmentally friendly production process, and the production efficiency is increased by 80%. The production cost is reduced by 50% compared with traditional chemical synthesis, and consumers can enjoy high-efficiency products at a lower cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0047] Figure 1 It is a real-shot picture before the consumer uses it.

[0048] Figure 2 It is a real-shot picture after the consumer uses it.

[0049] Note: To protect privacy, some areas of the picture are blurred. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0050] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only some embodiments of the present invention, not all of them. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope protected by the present invention.

[0051] Example 1

[0052] This example provides a preparation method of an arginine / lysine polypeptide supramolecular composition, and the preparation method includes the following steps:

[0053] (1) Mix 0.01 g of arginine / lysine polypeptide with 5000 g of a solvent (composed of propylene glycol and water with a mass ratio of 1:1), then add PBS buffer solution, and then heat to 30 °C and stir at a rate of 150 r / min until dissolved to obtain a polypeptide solution;

[0054] (2) Add 0.1 g of γ-aminobutyric acid powder to the polypeptide solution, adjust the pH to 4.5 (close to the isoelectric point of the polypeptide), trigger electrostatic binding and self-assembly, and then perform ultrasonic treatment and centrifugation in sequence to remove unbound free polypeptide or γ-aminobutyric acid to obtain a mixed solution;

[0055] Among them: the power of ultrasound is 100 W, the frequency is 20 kHz, and the duration is 30 min; the rotation speed of centrifugation is 2000 r / min, and the centrifugation time is 10 min;

[0056] (3) Use a dialysis bag with a molecular weight cut-off of 3 kDa and perform dialysis at 4 °C for 24 h to remove small molecule impurities. Then add 0.1 wt% of mannitol freeze-drying protectant, and perform freeze-drying after pre-freezing to obtain a white delivery powder, that is, obtain the arginine / lysine polypeptide supramolecular composition. The activity retention rate after reconstitution is >95%.

[0057] Example 2

[0058] This example provides a preparation method of an arginine / lysine polypeptide supramolecular composition, and the preparation method includes the following steps:

[0059] (1) Mix 30 g of arginine / lysine polypeptide with 85 g of solvent (composed of glycerol and water with a mass ratio of 1:3), add PBS buffer solution, then heat to 50 °C, and stir at a rate of 100 r / min until dissolved to obtain a polypeptide solution;

[0060] (2) Add 20 g of γ-aminobutyric acid powder to the polypeptide solution, adjust the pH to 5.5 (close to the isoelectric point of the polypeptide) to trigger electrostatic binding and self-assembly, and then perform ultrasound and centrifugation in sequence to remove unbound free polypeptide or γ-aminobutyric acid to obtain a mixed solution;

[0061] Among them: the power of ultrasound is 300 W, the frequency is 40 kHz, and the duration is 10 min; the rotation speed of centrifugation is 5000 r / min, and the centrifugation time is 5 min;

[0062] (3) Use a dialysis bag with a molecular weight cut-off of 5 kDa and perform dialysis at 4 °C for 24 h to remove small molecule impurities. Then add 0.1 wt% of mannitol freeze-drying protectant, and perform freeze-drying after pre-freezing to obtain a white delivery powder, that is, obtain the arginine / lysine polypeptide supramolecular composition. The activity retention rate after reconstitution is >95%.

[0063] Example 3

[0064] This example provides a preparation method of an arginine / lysine polypeptide supramolecular composition, and the preparation method includes the following steps:

[0065] (1) Mix 20 g of arginine / lysine polypeptide with 65 g of solvent (composed of glycerol and water with a mass ratio of 1:3), add PBS buffer solution, then heat to 40 °C, and stir at a rate of 120 r / min until dissolved to obtain a polypeptide solution;

[0066] (2) Add 15 g of γ-aminobutyric acid powder to the polypeptide solution, adjust the pH to 5 (close to the isoelectric point of the polypeptide) to trigger electrostatic binding and self-assembly, and then perform ultrasonic treatment and centrifugation in sequence to remove unbound free polypeptides or γ-aminobutyric acid, obtaining a mixed solution;

[0067] Among them: the power of ultrasonic treatment is 200 W, the frequency is 30 kHz, and the duration is 20 min; the rotation speed of centrifugation is 3500 r / min, and the centrifugation time is 7.5 min;

[0068] (3) Use a dialysis bag with a molecular weight cut-off of 4 kDa to perform dialysis at 4 °C for 24 h to remove small molecule impurities, then add 0.1 wt% of mannitol cryoprotectant, and perform freeze-drying after pre-freezing to obtain a white delivery powder, that is, obtain the arginine / lysine polypeptide supramolecular composition, and the activity retention rate after reconstitution is >95%.

[0069] Application examples

[0070] (I) Cosmetics field:

[0071] (1) Apply the supramolecular composition of the present invention to the preparation of anti-wrinkle cosmetics (such as anti-wrinkle essence), and its formula is: arginine / lysine polypeptide supramolecular composition (1% w / w, prepared in Example 1) + sodium hyaluronate (0.5%) + nicotinamide (2%) + polyol solvent (balance).

[0072] The final effect is: the Franz diffusion experiment shows that the transdermal rate of GABA is increased to 18.7% (5.8% in the control group), and the depth of crow's feet wrinkles is reduced by 32% after 28 days of human testing.

[0073] (2) Apply the supramolecular composition of the present invention to the preparation of mask-type cosmetics (such as soothing mask), and its formula is: arginine / lysine polypeptide supramolecular composition (0.5%) + centella asiatica extract (3%) + xanthan gum matrix (balance).

[0074] The final effect is: the clinical test shows that the erythema index is reduced by 42%.

[0075] (II) Transdermal drug delivery field

[0076] Apply the supramolecular composition of the present invention to the preparation of drug-loaded nanoparticles: the arginine / lysine polypeptide supramolecular composition loads 5% diclofenac sodium (model drug), the particle size is 150 nm, the encapsulation efficiency is 88%, and the in vitro sustained release experiment shows that the cumulative release rate in 24 hours is 82%.

[0077] Verification examples

[0078] (I) Verification of transdermal efficiency

[0079] Experimental method: Franz diffusion cell (ex vivo porcine skin), comparing free GABA solution (5% concentration) with the polypeptide supramolecular composition (1% GABA loading).

[0080] Results: The cumulative permeation amounts at 48 hours were 5.8% vs 18.7% respectively, a 3.2-fold increase.

[0081] Principle analysis: Electrostatic self-assembly of arginine / lysine polypeptide and GABA forms nanoparticles (50 - 200 nm), and GABA is directly carried through the skin by the cell membrane penetration property (CPP property) of the polypeptide. In addition, the polypeptide and GABA form a functional synergistic entity through electrostatic binding, with dual activities of promoting permeation (polypeptide) and anti-wrinkle (GABA inhibits nerve signals + promotes collagen synthesis). The inhibitory effect of the composition on dynamic wrinkles is 1.8 times that of free GABA, and it can also reduce the dependence on additional ingredients (such as PDRN). Finally, the nanoparticles of the polypeptide supramolecular composition are slowly released through the lipid channels of the stratum corneum, and combined with the targeted delivery property of the polypeptide, the action time is extended. The release time of the active ingredient is extended to 24 hours after a single use, and the user frequency is reduced by 50%.

[0082] (II) Verification of stability

[0083] Accelerated test: The polypeptide supramolecular composition was stored at 37°C for 6 months, and the retention rate of polypeptide activity determined by HPLC > 90%, while that of the free polypeptide control group was only 52%.

[0084] Enzymatic stability: Incubated in 0.1 mg / mL trypsin solution for 2 hours, the degradation rate of the polypeptide in the complex < 10%, and that of the free polypeptide > 70%.

[0085] Principle analysis: The protease attack is shielded by the steric hindrance effect of the supramolecular composition, and a stable structure is formed at pH 4.5 - 5.5 (the slightly acidic environment of the skin).

[0086] (III) Verification of safety

[0087] Cytotoxicity: The survival rate of HaCaT cells determined by the MTT method > 95% (polypeptide supramolecular composition concentration 0.1 - 1 mg / mL), significantly better than the control group containing ethoxydiglycol (survival rate 82%).

[0088] Human patch test: 30 subjects (including sensitive skin) used the cream containing the complex for 28 days, and 0 cases showed erythema or stinging (ISO 10993 - 10 standard).

[0089] (IV) Pre- and post-use comparison verification

[0090] The pre- and post-use comparison pictures are respectively as Figure 1 、 Figure 2As shown, the usage time is 1 week. From the comparison, it can be seen that there are obvious improvements in wrinkles and other conditions.

[0091] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art in the technical field disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the said claims.

Claims

1. An arginine / lysine polypeptide supramolecular composition, characterized in that, It comprises the following raw materials in parts by weight: arginine / lysine polypeptide 0.00001 - 30 parts, γ-aminobutyric acid 0.0001 - 20 parts, and solvent 50 - 85 parts.

2. The arginine / lysine polypeptide supramolecular composition according to claim 1, wherein It comprises the following raw materials in parts by weight: arginine / lysine polypeptide 20 parts, γ-aminobutyric acid 15 parts, and solvent 65 parts.

3. The arginine / lysine polypeptide supramolecular composition according to claim 1 or 2, characterized in that, The molecular weight range of the arginine / lysine polypeptide is 500 - 2000 Da, and the amino acid sequence contains at least 3 arginine or lysine residues; and / or, the isoelectric point of the γ-aminobutyric acid is 7.3; and / or, the solvent is a mixed solution of alcohol and water, and the alcohol is propylene glycol or glycerol.

4. The preparation method of the arginine / lysine polypeptide supramolecular composition according to any one of claims 1 to 3, characterized in that, The preparation method comprises the following steps: (1) Mix the arginine / lysine polypeptide with the solvent, then add PBS buffer solution, and then heat and stir until dissolved to obtain a polypeptide solution; (2) Add γ-aminobutyric acid powder to the polypeptide solution, adjust the pH to 4.5 - 5.5 to trigger electrostatic binding and self-assembly, and then perform ultrasonic treatment and centrifugation in sequence to obtain a mixed solution; (3) Perform dialysis treatment on the mixed solution to remove small molecule impurities, then add a lyoprotectant and perform freeze-drying to obtain the arginine / lysine polypeptide supramolecular composition.

5. The preparation method of the arginine / lysine polypeptide supramolecular composition according to claim 4, characterized in that, In step (1), the heating temperature is 30 - 50 °C; and / or, in step (1), the stirring speed is 100 - 150 r / min; and / or, in step (1), the concentration of the polypeptide solution is 10 - 30 mg / mL.

6. The preparation method of the arginine / lysine polypeptide supramolecular composition according to claim 4, wherein, In step (2), the power of the ultrasonic treatment is 100 - 300 W, the frequency is 20 - 40 kHz, and the duration is 10 - 30 min; and / or, in step (2), the centrifugation speed is 2000 - 5000 r / min, and the centrifugation time is 5 - 10 min.

7. The preparation method of the arginine / lysine polypeptide supramolecular composition according to claim 4, characterized in that, In step (3), the dialysis treatment method is: use a dialysis bag with a molecular weight cut-off of 3 - 5 kDa and dialyze at 4 °C for 24 h; and / or, in step (3), the lyoprotectant is mannitol or trehalose, and the addition amount is 0.1 - 1 wt%.

8. Use of the arginine / lysine polypeptide supramolecular composition according to claims 1 - 3 as a penetration enhancer in cosmetics and transdermal drugs.

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