CN108714111B

CN108714111B - Cell-penetrating peptide-acetyl hexapeptide nanoemulsion and preparation method thereof

Info

Publication number: CN108714111B
Application number: CN201810461431.6A
Authority: CN
Inventors: 滕肇慧; 谭铖洸; 杨永鹏; 董萍; 丁克祥; 朱晓亮; 左夏林; 丁宇
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-05-05
Filing date: 2018-05-05
Publication date: 2022-05-24
Anticipated expiration: 2038-05-05
Also published as: CN108714111A

Abstract

The invention relates to the fields of skin aging resistance, beauty, skin care, health care, daily cosmetics and the like, and discloses a permeable-skin-absorbable cell penetrating peptide-acetyl hexapeptide nanoemulsion and a preparation method thereof. Firstly, designing and artificially synthesizing the cell-penetrating peptide-acetyl hexapeptide and then preparing the nano-emulsion. The invention adopts the synergistic effect of the nano-emulsion and the cell-penetrating peptide modification technology to improve the transdermal permeability and the transdermal absorption effect of the acetyl hexapeptide, thereby reducing the degradation of the peptide, maintaining the stability and the bioactivity of the molecular structure of the peptide, and improving the solubility and the effectiveness of the peptide as a beauty purpose medicament or an effective component. The acetyl hexapeptide nanoemulsion modified by the cell-penetrating peptide can reduce S/C which is easy to cause skin irritation and allergy, can improve application safety and comfortableness, is prepared at normal temperature, does not need heating and energy supply, can be produced in large quantities without special equipment, is beneficial to industrialization, environmental protection and energy saving.

Description

Cell-penetrating peptide-acetyl hexapeptide nanoemulsion and preparation method thereof

Technical Field

The invention relates to the fields of human face beautifying, skin care and health care, skin anti-aging cosmetic and other related subjects, in particular to a cell-penetrating peptide carrying acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) nanoemulsion which can be used for facial skin beautifying and can be permeated and absorbed transdermally and a preparation method thereof.

Background

1. Background of the related art for skin barrier function and transdermal absorption thereof

The human Skin (Skin) is the largest tissue organ covering the body surface, not only has the physiological functions of protecting the whole body, and secreting, excreting, sensing, metabolizing and the like, but also is the most important natural barrier outside the human body, and can prevent various tissues and organs in the body from being soaked into the body and invading the body by external physical, chemical, mechanical and pathogenic microorganisms and other substances, and simultaneously prevent the water, electrolyte and other substances in the body from being lost outside the body. In fact, these physiological functions (including skin barrier function) possessed by human skin are closely related to their specific tissue structure. Because the skin tissue of the human body is divided into three layers from outside to inside, namely an epidermal layer, a dermal layer and a subcutaneous tissue. The outermost epidermal layer microstructure can be divided into basal cell layer, spinous cell layer, granular layer, stratum lucidum and stratum corneum from inside to outside (but there is no stratum lucidum structure in the epidermal layer of facial skin tissue). The human epidermis is about 0.070-0.120mm thick and mainly comprises keratin cells and dendritic cells. At the epidermal layer, the transdermal penetration and absorption of various functional ingredients are mainly related to the keratinization process of keratin cells and the degree thereof. The dermis layer of the middle layer of the skin tissue is a structural tissue between the epidermis and the subcutaneous tissue, and is interwoven with collagen fibers and elastic fibers which belong to connective tissues to form reticular fibers. Under an electron microscope, the dermis is in a fiber mesh structure, collagen fibers and elastic fibers are mutually interwoven and criss-cross, amorphous matrixes are filled in the dermis, and skin appendages, abundant nerves, blood vessels, lymphatic vessels and the like are arranged in the dermis. The subcutaneous tissue of the lowest layer of skin tissue is located below the dermis, a loose tissue containing a large number of fat cells. In the subcutaneous tissue, there are sweat glands, blood vessels, lymphatic vessels, etc. In general, there are abundant capillaries in the dermis layer in the skin tissue, and the functional components such as penetration reach the dermis and are rapidly absorbed, and the absorption of the transdermal functional components can rapidly enter the blood circulation. The stratum corneum, the outermost layer of the epidermis, is the primary barrier to the entry of various functional ingredients into the skin tissue due to its high degree of keratinization, low water content, and encapsulation by the extracellular matrix of lipids. Therefore, one of the key factors of transdermal penetration and transdermal absorption of various functional ingredients is mainly how they penetrate the stratum corneum, the outermost layer of skin tissue.

Theoretically, there are two possible ways for the in vitro transdermal penetration and absorption of various functional components: (1) it is absorbed into the systemic circulation by capillary vessels through the epidermal route, penetrating through the stratum corneum and the layers of the epidermis into the dermis, which is the main route of transdermal penetration and absorption of the active ingredients. In this pathway, the drug can pass through the stratum corneum cells to reach the active epidermis, and can also pass through the stratum corneum intercellular spaces to reach the active epidermis. Because of the too great a resistance to cell diffusion in the stratum corneum, the active ingredients diffuse mainly from the cells through the stratum corneum. Stratum corneum cells are multilayered lipid bilayers formed by lipid molecules, the hydrophilic part of the lipid molecule combines with water molecules to form an aqueous region, the hydrocarbon chain part of the lipid molecule forms a hydrophobic region, polar drug molecules permeate through the aqueous region between stratum corneum cells, and nonpolar drug molecules permeate through the hydrophobic region. (2) By the hair follicles, sebaceous glands and sweat glands: namely, the functional components are possible to permeate and absorb transdermally through the accessory organs of the skin tissues of the human body, such as hair follicles, sebaceous glands and sweat glands, and thus play corresponding biological and physiological roles.

From the functional role of cosmetics with cosmetic function, the exertion and the obtaining of the cosmetic function are closely related to the percutaneous penetration and the percutaneous absorption of functional components, and the action mechanism related to the percutaneous penetration and the percutaneous absorption mainly comprises the following hypotheses at present: firstly, the integrity of the stratum corneum of the skin is directly destroyed through a physical method, so that the effective components directly enter the stratum corneum below the damaged part on the skin, such as iontophoresis penetration promotion, electroporation penetration promotion, ultrasonic penetration promotion, laser penetration promotion, microneedle penetration promotion, thermal burn, dermabrasion and the like; secondly, the ordered arrangement of lipid bilayers in the skin cuticle is changed through the chemical method, the liquidity of the lipid bilayers is increased to a certain degree, and drug molecules can smoothly pass through from loose intervals of the cuticle, such as dimethyl sulfoxide, azone compounds, liposomes, amino acids and derivatives thereof, surfactants, dendritic macromolecules, volatile oil mint oil, eucalyptus oil, turpentine oil and the like, and some natural products and the like, in addition, terpenes, alkaloids, lactones, alcohols and the like can also enable functional components to permeate into the skin; thirdly, the binding capacity of keratin on the surface of the skin and water is improved through physical or chemical action, the hydration of the stratum corneum of the skin is changed, and the functional ingredients permeate into the skin along with the hydration; fourthly, the skin accessory organs are affected by physical or chemical actions, such as penetration of the transdermal enhancer into the sebaceous gland duct to dissolve sebum or adipogenic differentiated cells on the glandular cavity wall, reduce the hydrophobicity in the sebaceous gland duct and promote ionic drugs to permeate the skin. The penetration enhancer enlarges openings of sweat glands and hair follicles by softening the stratum corneum, thereby facilitating the passage and further absorption of the functional ingredients through the skin. The related methods of percutaneous penetration and percutaneous absorption adopted at present are related to the method.

Transdermal delivery or topical routes of administration, while encountering the technical bottleneck of the natural barrier of human skin, also represent a very valuable transport pathway and permeation route for the drug and functional ingredient of interest. Transdermal delivery or external skin routes of administration are simpler and more convenient, safer and more convenient than other modes of administration. Meanwhile, the mode is less painful, the first pass effect of the liver can be avoided, and the risk of disease transmission caused by the reuse of the injection needle tube is reduced. Especially, in the fields of medical cosmetology and cosmetics, the acceleration of the percutaneous permeation and the transdermal absorption of the effective components has important significance and application value.

2. Nanoemulsion and related technical background of its application

The concept of Nanoemulsions (NE) originated in the forties of the last century for the first time and was first proposed by Schulman and Hoar, etc.; schulman also proposed the concept of formally naming Nanoemulsion (NE) as "Microemulsion (ME)" in 1959, and thus the naming of Nanoemulsion (NE) or Microemulsion (ME) has been used so far. However, it is really applied as a drug delivery carrier for transdermal administration in the medical field, which was first discovered, researched, applied and publicly reported in 1974 by doctor Aitwood et al. In the last 90 s, NE or ME has become a hot spot of medical and pharmaceutical research as a transdermal drug delivery system, in 2000, NE or ME has become a research field with huge application potential in the international medical field, and in 2006, NE or ME has formally appeared in the fields of skin care, health care and efficacy beauty cosmetics.

Although the scientific formation mechanism and the transdermal action mechanism of NE or ME are complicated, the basic system composition and the constituent components of NE or ME mainly comprise water phase, oil phase, emulsifier/co-emulsifier or surfactant/co-surfactant and other pseudo-ternary phase liquids, and the pseudo-ternary phase liquids can be prepared by adopting related instruments and methods under the condition of normal temperature according to the proper proportion relationship determined by scientific design and experiments to form a thermodynamically stable colloid dispersion system. Under normal conditions, the NE or ME basic system has clear and transparent appearance, slight opalescence, good dispersibility, good fluidity, low liquid viscosity, isotropy, uniform particle size distribution and diameter range of 10-110 nm. NE or ME is a scientific theory that NE or ME formation mechanism can be completely and exactly stated and explained, such as NE or ME system negative interfacial tension formation mechanism, mixed film formation theory, geometric arrangement theory and R ratio theory are difficult to be completely and scientifically explained and illustrated, and the negative interfacial tension formation mechanism generally considered and agreed in the same field of science at present can be said to be a theory and explanation which are currently considered to be more scientific and reasonable. The core of the negative interfacial tension forming theory and the key point thereof are that the interfacial tension mechanism of NE or ME plays a crucial role in the spontaneous and autonomous formation dynamic process, namely, the emulsifier or surfactant can cause the reduction of the oil-in-water (O/W) interfacial tension, and the addition of a proper amount of co-emulsifier or co-surfactant can cause the continuous reduction of the O/W interfacial tension until the interfacial tension reaches a negative value, namely, the negative interfacial tension. The negative interfacial tension promotes the system to spontaneously expand the interface, so that the emulsifier or surfactant and the co-emulsifier or co-surfactant are continuously added to be adsorbed on the interface until the interfacial tension is gradually balanced and the interfacial tension value returns to zero. It is due to the above-mentioned instantaneously occurring negative interfacial tension formation, which induces the spontaneous expansion of the system interface and the autonomous formation of NE or ME. When NE or ME gradually forms coalescence, the interfacial area of the NE or ME is gradually reduced, and a new negative interfacial tension is formed, and the new negative interfacial tension causes the interfacial tension value of the system to return to zero, so that the aggregation of the NE or ME is resisted, and the stability of the NE or ME is maintained and supported.

With the continuous elucidation of the mechanism of NE or ME formation and its academic theory, NE or ME has been considered as one of the most excellent transdermal drug or functional ingredient delivery vehicles at present. The novel ternary phase liquid has a plurality of application characteristics and action advantages, can improve the solubility, the fusion degree and the bearing capacity of functional medicinal components with different attributes, and can effectively act on epidermal cornified layers, granular layers, acanthocyte layers, skin appendages and the like through an NE or ME system formed by the pseudo ternary phase liquid, so that the transdermal penetration and transdermal absorption of the functional medicinal components with different attributes are promoted, increased and accelerated, and the maximum biological effect of the functional medicinal components is exerted. The high-efficiency biological effect of the effective components is closely related to the bioavailability, the transdermal penetration and the transdermal absorption amount thereof. Therefore, NE or ME is now widely used not only in many novel pharmaceutical preparations but also in some high-grade cosmetics with skin caring effect as a highly effective transdermal drug delivery vehicle. The skin-care nano-emulsion has the advantages of skin whitening, freckle removing, repairing, wrinkle resisting, nourishing, moisturizing, sun-screening, desensitizing, anti-allergy, anti-aging, anti-oxidation, pain relieving, anesthesia, hemostasis and the like, and has a new peak and popular trend when being applied to skin care, beauty and health care, medical beauty and minimally invasive beauty and plastic.

3. Cell-penetrating peptides and related technical background of their use

Cell-penetrating peptide (CPP) is a transdermal delivery carrier which is not applied to skin cosmetics at present, and in the field of medicine, the CPP is a short peptide which can determine functional components such as target protein or polypeptide drugs and can promote the functional components to penetrate through the skin, has a carrying function and a transdermal delivery function, and can be one of the most ideal transdermal delivery carriers for carrying the functional components such as the polypeptide drugs to permeate and absorb through the skin at present. The skin care product has no damage or stimulation or anaphylactic reaction to skin, has no independent pharmacological activity and action, has relatively stable molecular structure and physical and chemical properties, and has good connection, matching and compatibility with functional components of target protein or polypeptide drugs carried by the skin care product; the transdermal patch has the characteristics and advantages of quick response and long action on a target site, is one of simple, convenient and effective transdermal permeation and transdermal absorption ways of target proteins or polypeptide drugs and the like entering the systemic circulation through a skin surface way, is also another beneficial supplement to the traditional administration way and transdermal way, is expected to play a role in the research and development and application of certain high-tech, high-grade and high-efficiency functional skin anti-aging, skin care and health care, beauty cosmetics and other preparations in the future, and has wide development and application prospects and promising social and economic benefits.

The biological function, native conformation, molecular structure and amino acid composition of the cell-penetrating or transdermal peptides are substantially similar. It has been reported in the literature that cell-penetrating peptides can penetrate the cell membrane directly into different types of cells by carrying the target drug (protein, polypeptide, nucleic acid and larger particle polymer, etc.) of biological macromolecules, and even can carry some biological macromolecular substances directly across the blood-brain barrier. The research on the action mechanism of the cell penetrating peptide shows that the cell penetrating peptide can be actively or passively taken up by living cells at 37 ℃ and can enter the living cells at 4 ℃, and the cell penetrating peptide is considered to be a kinetic mechanism which is independent of receptors, energy and endocytosis. In recent years, researchers have proposed a new membrane penetrating mechanism, and it is believed that its cell penetrating effect is based on clathrin-mediated endocytosis, clathrin wraps the endocavity membrane to make it break away from the cell membrane and directly enter the cytoplasm, this process may attract and interact with the positive and negative charges of cell surface negatively charged glycosaminoglycan and positively charged membrane penetrating peptide, and the direct electrostatic interaction of the positively charged amino acid residue on the surface of the membrane penetrating peptide and the negatively charged proteoglycan or glycosaminoglycan (such as heparan sulfate, heparin, etc.) on the surface of the cell membrane is one of the key factors necessary for its membrane penetrating effect and its uptake by cells. The transmembrane phenomenon of the membrane-penetrating peptide combined with a target drug with the molecular weight of more than 3 ten thousand daltons is considered to be a lipid raft endocytosis effect formed by nonspecific large vesicles, and the endocytosis is initiated and mediated by actin filaments. The poly-arginine in the cell-penetrating peptide rich in arginine residues can be directly combined with a small molecular target drug, and is promoted to penetrate a cell membrane to enter a cell through electrostatic action or hydrogen bonding effect, when the poly-arginine is combined with negative charges on the surface of the cell, an ion pair can be formed, and is translocated under the influence of a membrane potential and crosses the cell membrane to enter cytoplasm, and then the target drug combined with the cell-penetrating peptide is released into the cytoplasm to play a biological role.

The cell-penetrating peptide can carry active ingredients such as macromolecular protein or polypeptide drugs to penetrate a cell membrane with a double-lipid structure to enter cells, and the transdermal peptide can carry the same or similar target drugs or active ingredients to penetrate an epidermal layer to enter the dermis. The biological membrane and the cell membrane of the organism are both double-layer lipid structures, although the lipid membrane at the outermost layer of the skin consists of fatty acid, cholesterol and ceramide, the lipid membrane is also lipid and both have negative charges, and the structure is also suitable for the action mechanism of the cell penetrating peptide. The transdermal peptide is likely to create a transient open transdermal delivery channel for macromolecular functional components or macromolecular drugs such as target proteins or polypeptides and the like to achieve systemic blood circulation. The scholars have covalently linked cyclosporine to the cell-penetrating peptide poly-arginine heptamer, and found that the capability of percutaneous permeation and percutaneous absorption of the drug and the effect of controlling the acute inflammatory reaction are both remarkably improved, while the capability of percutaneous permeation and transdermal absorption and acute inflammatory reaction inhibition of the cyclosporine without modification of the cell-penetrating peptide are far from each other. It has also been discovered that non-covalent conjugation of specific mimetic protein or polypeptide components to arginine-rich intracellular carrier peptides can also enhance the transdermal penetration and absorption of these macromolecular mimetic protein or polypeptide drugs in non-covalently conjugated form. Such polypeptides having cell membrane penetrating and skin penetration enhancing effects, also known as protein transduction domains, can not only penetrate cell membranes, but also carry certain desired drugs into skin tissues. Transdermal peptides and cell-penetrating peptides (transduction domains) share a common structural feature, namely being rich in positively charged arginine and lysine residues, and being capable of transporting binding peptides, oligonucleotides, liposomes, and the like across mammalian cell membranes. The special arginine-rich structure can also generate the effect of directly penetrating through a skin membrane through the charge effect of arginine side groups and charged components in a cell membrane, and the arginine-rich structure can play the biological effect and the physiological function of the functional component by instantaneously opening a phospholipid membrane on the surface of the skin to form a polar instantaneous channel so as to ensure that the functional component is carried to be permeated and absorbed into the skin through the skin. While basic protein transduction domains such as cell-penetrating peptides are thought to enter mammalian cells by virtue of cellular pinocytosis, which may be accomplished by virtue of cell membrane recesses, membrane receptors, clathrin, etc., along with covalently linked polypeptide or protein drugs. Moreover, at present, the researchers apply the in vivo phage display technology to the research of the transdermal enhancer, and find an active short peptide which is composed of 11 amino acids and can effectively transport the protein drugs for transdermal penetration. The short peptide and insulin are simply mixed in physiological saline and are coated on the skin of a rat with a diabetes model, so that a good blood sugar reducing effect can be achieved. In addition, the short peptides can help various other proteins or hormones penetrate the skin barrier into the skin tissue. Preliminary mechanistic studies show that the short peptide may carry macromolecular protein or polypeptide drugs to penetrate the skin and enter the circulation after acting on skin tissues and temporarily opening the lipid membrane of the skin barrier and corresponding channels (including intercellular spaces, hair follicle pathways and the like) after combining positive and negative charges of the short peptide. Another student has discovered a natural pore-forming peptide that increases skin permeability, which when used in conjunction with a skin surfactant, enhances skin permeability and penetration. In addition, the researchers design to artificially synthesize the peptide analogue by a polypeptide solid phase synthesis method, and modify the molecular structure of the existing transdermal peptide, namely change the composition of amino acid residues in the transdermal peptide, increase the cation number and change the position by scanning lysine or arginine, and also can achieve the effect of improving the transdermal penetration and transdermal absorption effects. Therefore, researchers think that macromolecular proteins, polypeptides and nucleic acid drugs which cannot be or are difficult to be transdermally administered are likely to penetrate the function and effect of a skin plasma membrane when being covalently linked to a cell-penetrating peptide poly-arginine heptamer, and that functional components and target drugs of the proteins or polypeptides which seem to have low transdermal penetration and transdermal absorption rates or are theoretically thought to be difficult to be transdermally penetrated and absorbed can enter skin tissues to exert good biological effect and clinical treatment effect. The theoretical basis and experimental results provide an innovative approach for transdermal drug delivery, and also provide a brand new thought, research method and novel product for the research, development and application of functional cosmetic, in particular to functional cosmetic containing macromolecular bioactive functional components (protein, polypeptide, enzyme, nucleic acid) and the like.

4. Related technical background of beauty peptide and application thereof

Peptides (peptides), also known to scholars as peptides, are largely divided into polypeptides and oligopeptides, which are biologically active substances that are condensed from two or more amino acids and linked by peptide or amide bonds, which are intermediates of proteins and fragments of molecular structures between amino acids and proteins. Peptide substances are also called bioactive polypeptides, because they are characterized by high biological activity and biodiversity compared to other substances in the body. Peptides or peptides are composed of more than 20 natural amino acids in different arrangement modes and forms through peptide chains, namely, the peptides or peptides are formed by complex linear and annular structures from two peptides to polypeptides, the peptides or peptides are all multifunctional compounds derived from protein intermediates and natural conformations and are also one of the most basic and important substances forming life, the peptides or peptides play important biological effects in organisms in a natural biological activity mode, and various life activities, metabolic processes, physiological functions and the like in the bodies are almost all guided and regulated by active polypeptides or proteins consisting of specific amino acid sequences. Moreover, the species, content and biological activity of the polypeptide or peptide are closely related to the structure and function of human skin tissue, and play very important biological and physiological roles under certain conditions, such as skin tissue and cell division, proliferation, differentiation, chemotaxis, migration, aggregation, angiogenesis, microcirculation, pigmentation, synthesis and regulation of skin tissue protein, and the like.

In the fields of medical cosmetology, cosmetics and fine chemistry, oligopeptides consisting of two amino acids are generally called di-peptides, while those consisting of 3 amino acids are called tri-peptides, and so on, and can be divided into tetra-, penta-, hexa-, etc. Particularly, with the continuous development of polypeptide synthesis technology and the continuous disclosure of the effects and functions of peptides composed of different amounts of amino acids on skin, peptides with various structures are gradually used for facial beauty, skin care, health care, skin aging delaying and skin aging resistance, especially peptides with unique biological effects, small amount of amino acid components, simple molecular structure, small molecular weight and less than ten peptides, such as pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide and derivatives thereof, palmitoyl pentapeptide-3, acetyl hexapeptide-3, acetyl tetrapeptide-5, palmitoyl tripeptide-1, palmitoyl tetrapeptide-3 and carnosine, and the like, are continuously disclosed in applied beauty cosmetics and are popular and popular.

However, in the eighties of the 20 th century, researchers have introduced the concept of proteolytic fragments or protein fragments, i.e., macromolecular protein materials that are hydrolyzed into polypeptides or oligopeptides by proteolytic or fermentation engineering methods. However, these macromolecular proteins are degraded by hydrolysis to give complex products, and the derived polypeptides are varied in molecular weight. Because polypeptide fragments and sequences are difficult to select, the classification and the type of the polypeptide are difficult to determine, active selection and definition of the polypeptide name are difficult, and purification of a single polypeptide is more difficult. Therefore, the hydrolyzed or fermented protein decomposition products or derivatives, which are actually the mixture of various proteins, polypeptides and amino acids, cannot be said to be true cosmetic peptides. The cosmetic peptides really related to skin beauty can be traced back to copper peptide (also called as copper peptide) discovered by Dr.Loren Pickart et al in 1973, the copper peptide not only can reduce scar tissue generation, but also can stimulate active healing of damaged skin tissue, and the copper peptide is also discovered by America to improve skin aging condition, and the copper peptide is applied to human skin repair, wrinkle resistance, hair hyperplasia and the like. In addition, there are several cosmetic peptides, mainly including: (1) signal peptide: palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-3, myristoyl pentapeptide-11, palmitoyl hexapeptide, hexapeptide-9, and the like; (2) neurotransmitter inhibitory peptides: dipeptide snake toxin, pentapeptide-3, acetyl hexapeptide-3, acetyl octapeptide-1, etc.; (3) carrying out peptide analogous: copper peptide, etc.; (4) other anti-wrinkle peptides: palmitoyl dipeptide-5, hexapeptide-8, hexapeptide-10, and the like. Among them, the most widely used and most effective hexapeptide, also known as acetyl hexapeptide-3, which is known as Argileline and is also known as botulinum toxin, is a peptide consisting of 6 amino acids, which can relax muscles by slowing down the force of muscle contraction, reduce or inhibit the occurrence of dynamic wrinkles and eliminate fine lines.

5. Botulinum toxin (hexapeptide) and related technical background for its use

Hexa peptide is also called botulinum toxin or botulinum toxin in medical cosmetology at present, and the synthetic derivative of the hexa peptide is Acetyl hexa peptide-3, the English name of the hexa peptide is Acetyl hexa peptide-3, the name of the hexa peptide is Argireline, and the hexa peptide is one of skin anti-wrinkle effective components which are most widely applied in cosmetology at present. The hexapeptide is a chemically synthesized skin external anti-wrinkle functional component, the skin anti-wrinkle mechanism of the hexapeptide is basically the same as that of botulinum toxin A used for anti-wrinkle injection, namely the hexapeptide is similar to a relaxant of facial skin muscles and can selectively act on peripheral cholinergic nerve (motor nerve) peripheral tips to inhibit release of nerve medium acetylcholine from a presynaptic membrane at a nerve muscle junction contact point (the strongest point), so that the transmission of nerve medium is effectively blocked, muscle relaxation paralysis is caused, muscle fibers cannot contract, muscle tension is reduced, muscle spasm is relieved, facial expression muscles cannot play a role, the facial muscles are relaxed, wrinkles formed by movement of the facial expression muscles are reduced or eliminated, and the purpose of smoothing dynamic lines, static lines and fine lines is achieved. Meanwhile, the botulinum toxin (hexapeptide) can also effectively prevent wrinkles generated by excessive release of catecholamine, reduce wrinkles formed by repeated traction of facial muscles, thereby effectively relieving and inhibiting the contraction and activity of forehead raising lines, crow's feet lines, glabellar lines, transverse nasal lines, back nasal lines and wrinkles peripheral muscles, helping muscles relax, restoring the elastic tissues of the skin to smooth lines, and further reducing or eliminating the fine lines of the face. With aging, collagen fibers of aged skin become straight, fibrin bundles become more parallel to the skin surface, the skin stretching margin is greatly reduced, the elasticity is reduced, and the skin becomes sagging without tension, thereby generating wrinkles. Argileline, the earliest developed by Lipotec corporation, was patented internationally as early as 2002 and demonstrated that it was able to locally block neurotransmission, muscle contraction information, affect cortical neurotransmission, and relax facial muscles to smooth dynamic, static, and fine lines. Lipotec even thought Argireline could be expected to have a comparable effect to botulinum toxin type A, but avoided the pain and cost of botulinum toxin type A injection. Almost at the same time, Sederma also produces a cosmetic anti-wrinkle peptide, Palmitoyl Pentapeptide-3 (palmityl Pentapeptide-3), which is also the most widely used at present, but the cosmetic peptide has a different anti-wrinkle action mechanism from hexapeptide, which is achieved by locally stimulating and promoting the production of type I and type III collagen and fibronectin, thereby increasing skin thickness and reducing fine wrinkles. Acetyl hexapeptide (Argireline) has the function of resisting dynamic wrinkles of the face, mainly because the acetyl hexapeptide can block the nerve transmission muscle contraction information on the face, influence the skin sac nerve conduction, relax the facial muscles and smooth dynamic lines, static lines and fine lines, and even the scholars think that the acetyl hexapeptide can be used for external application through skin to avoid the defects and defects that the conventional botulinum toxin needs to be injected and the use cost is high. Therefore, acetyl hexapeptide (Argireline) is one of the most widely used products in beauty peptide, and is one of the skin anti-aging and facial anti-wrinkle cosmetics which are considered to be the most promising and vital by the beauty cosmetic industry to date. Most of the currently used hexapeptides are synthesized by 6 amino acids, fluorenylmethyloxycarbonyl (Fmoc) can be used as a protecting group of a amino group during artificial synthesis, and basic compounds can be used as deprotection agents by virtue of the characteristics of stability to acid and easiness in action with alkali. Fmoc is used as a protecting group of amino acid, carboxyl of the first amino group of the hexapeptide is connected with solid carrier amino Resin (Rjnk Amide MBHA Resin) in a covalent bond mode to form a product Fmoc-Arg (pbf) -Rink Amide MBHA Resin as a starting point of polypeptide synthesis, the polypeptide to be synthesized is subjected to extension condensation peptide-grafting reaction one by one on the carrier Resin to finally synthesize an ER-6-NH2 peptide chain, the Resin is removed by acid hydrolysis of the peptide chain to obtain a crude product of a target product, and then the crude product is identified and purified to obtain a final product.

6. The related art has the defects or shortcomings at present:

the optimal treatment effect of the skin external medicine and the optimal cosmetic effect of the functional cosmetics are closely related to the added target medicine and the added functional components, and the key nodes of the added target medicine and the added functional components can possibly play the optimal biological effect and application effect only through the maximum transdermal penetration and transdermal absorption. At present, gel used as a skin external medicament and a cosmetic in domestic and foreign markets is not only rare but also various in variety and cannot be mastered. Although these gels have certain characteristics and advantages compared with the traditional emulsions and creams, the gel completely meets the quality standards of external application to human skin and facial application, completely meets the good appearance and physicochemical characteristics of common gels, and has good spreadability and comfort for external application, the common gels not only have larger particle size, but also have the characteristics of promoting the transdermal penetration and transdermal absorption of active ingredients and medicines therein, so that the expected ideal therapeutic effect and beauty effect are difficult to achieve in practical application.

The nano-emulsion is regarded as one of the good transdermal drug delivery carriers at present, the results in the aspects of theoretical research and application research are continuously published, and some nano-emulsions even have obtained stage results and breakthrough progress, particularly, the nano-emulsion has ultra-micro particle size, reaches the nano-scale standard, has uniform particle distribution, good dispersion and good solubilization and containment effects, particularly has the characteristics and advantages of accelerating the promotion of the transdermal permeation and transdermal effect of target drugs and functional components, and the like, and is concerned, regarded and favored by people. However, most of the most classical nanoemulsion basic systems at present consist of pseudo-ternary phase liquids such as an oil phase, a water phase, a surfactant/cosurfactant and the like, and the formed nanoemulsion system has the defects of obvious greasy feeling, slight irritation and tingling feeling, unpleasant peculiar smell, less comfort in smearing, poor storage stability of the preparation for a long time and the like or prominent defects after external application to the skin and facial smearing, in particular, in order to achieve and maintain the negative interface tension, namely negative interface tension, the adsorption of the emulsifier or surfactant and the co-emulsifier or co-surfactant on the interface is continuously increased, so that a classic nanoemulsion basic system contains a higher concentration of the surfactant/co-surfactant, and the relative sensitivity of the nanoemulsion to skin irritation, especially to the destructive effect of functional components such as proteins or polypeptides, and the like, becomes a technical bottleneck and a main obstacle of the application of the nanoemulsion in the fields of skin external preparations and beauty cosmetics. Moreover, the transdermal penetration and transdermal absorption effects of the nanoemulsion are influenced and interfered by a plurality of factors, including the composition, the composition ratio, the types and the drug-loading rate of the functional components, the penetration concentration gradient and the like, which are problems and problems to be solved and overcome by the existing nanoemulsion preparation.

Most of the functional components of the traditional skin anti-wrinkle cosmetics are nutrients, humectants and the like biochemically extracted from animals, plants and marine organisms, and the addition of skin anti-wrinkle peptides (hexapeptides) with certain biological activity provides a brand-new research and development direction and idea for the skin anti-wrinkle biological cosmetics. The present hexapeptide has been used as a botulinum toxin substitute in high-grade cosmetic products and skin beautifying and anti-wrinkle products, is one of the beauty peptides with more definite effect, most extensive application and smaller molecular weight as the botulinum toxin-like, for example, transdermal administration can reach corresponding action sites, and the botulinum toxin can inhibit synthesis of SNARE receptors and excessive release of catecholamine and acetylcholine of skin to exert local blocking of neurotransmission muscle contraction and influence skin sac nerve conduction after being applied to facial skin for a certain time, so that facial muscle is relaxed, and the effects of inhibiting or relieving dynamic wrinkles, static wrinkles, facial fine lines and the like are achieved. However, the application of the polypeptide still has some defects and problems, for example, the polypeptide as water-soluble polypeptide has small molecular weight, but is still difficult to exert the due biological effect through skin, even if the application is assisted by some penetration enhancer capable of promoting the transdermal absorption, the theoretically expected effect can not be achieved, the clinical manifestation is that the anti-wrinkle reaction is slow, and the curative effect is not obvious. In addition, the peptide structure of the hexapeptide leads the hexapeptide to have poor stability in common water-soluble media or aqueous solution liquid, and functional groups and active centers of the hexapeptide are easy to degrade and destroy in the aqueous solution. When the hexapeptide is added into the conventional preparation (such as aqua, essence, emulsion, cream, ointment, gel and the like) to be applied to the surface of the skin or applied to the face, the problems of difficult percutaneous penetration and percutaneous absorption and the like exist, particularly, the target position of the action required by the hexapeptide in the skin application is deeper, and the effect of the percutaneous penetration and the percutaneous absorption must be better to play the due biological action and physiological function.

Transdermal peptides or cell-penetrating peptides belong to peptide structures, have the same poor stability in water-soluble media or liquid preparations, and are easy to degrade or destroy in aqueous solutions; and the pure transdermal peptide or cell penetrating peptide has no special pharmacological activity and curative effect per se; in addition, the transdermal or membrane-penetrating peptide must be linked or carried with and matched with certain highly effective target drugs and functional components to exert corresponding biological effects and physiological functions. Particularly, the application of the functional component in the skin beautifying cosmetic as a carrier for connecting the functional component is not reported at present, and the experimental research on the actual effects of the skin permeation and the transdermal absorption in vitro after the functional component is connected or carried with the skin beautifying cosmetic is not reported at present.

Therefore, the invention designs a preparation which can reduce the defects of the application technology, is suitable for modern skin beautifying application and can be better permeated and absorbed through the skin in a percutaneous way, namely the nano emulsion of the cell-penetrating peptide carrying acetyl hexapeptide (the cell-penetrating peptide-hexapeptide) and the preparation method thereof.

Disclosure of Invention

The invention mainly aims to make up, improve, enhance and innovate for various defects in the background art. The invention specifically provides a cell-penetrating peptide-carried acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) nanoemulsion which can be permeated and absorbed through skin and has more stable molecular structure and bioactivity, and a preparation method thereof. In order to achieve the above purpose, the invention adopts the following specific design and technical scheme:

1. the design of the invention

The technology of the invention is divided into two parts: firstly, completing the design and synthesis of a cell-penetrating peptide carrying acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) molecular structure and an artificial synthesis method; secondly, the design and preparation of the raw material composition, the proportion and the preparation method of the nano-emulsion of the cell-penetrating peptide carrying acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) are completed.

2. The present invention

According to the design thought of the invention, the membrane-penetrating peptide-acetyl hexapeptide nanoemulsion and the preparation method thereof adopt a technical scheme and a specific implementation mode which are divided into two parts.

The first part is that the cell-penetrating peptide of the invention carries acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) molecular structure and artificial synthesis method and analysis and identification:

the amino acid molecular structure sequence of the membrane-penetrating peptide-acetyl hexapeptide artificially synthesized by adopting the chemical solid-phase synthesis method is Ac-EEMQRRYGRKKRRQRRR. The artificial chemical synthesis route and the process flow are as follows: selecting a resin carrier as dichloro resin, wherein active sites on the resin are halogen chloride, firstly swelling the resin in polypeptide solid phase synthesis, then reacting C-terminal carboxyl of a first amino acid with the active site chloride on the resin, carrying out dehydration condensation to join a second amino acid after the first amino acid is connected on the resin, and then removing Fmoc protection after the condensation is finished. Repeating the operation according to the designed amino acid sequence, sequentially connecting the rest amino acids, completing N-terminal acetylation, and finally cutting the polypeptide from the resin by using a cutting reagent to form naked carboxyl. The preparation method comprises the following steps: (1) swelling of the resin: weighing 1.5g of dichloro resin (0.08 mmol of synthesized target peptide, 0.4mmol/g of substitution degree of the dichloro resin, and 7.5 times of excess amount, so that the required mass of the dichloro resin is 0.08/0.4 × 7.5 ═ 1.5g), placing into a reaction column, adding 20 ml of DCM into the reaction column, oscillating for 30min, and activating for later use. (2) Linking the first amino acid: DCM solvent was removed by suction filtration through a sand core, 1.05 times the molar amount of resin Fmoc-L-Arg (Pbf) -OH was added, 10 times the molar amount of resin DIEA was added, and finally a small amount of DMF was added for dissolution and shaking for 1 h. After the reaction was complete, the reaction was washed 6 times with DMF and DCM alternately. (3) Deprotection: 20 ml of 20% piperidine/DMF solution was added and the mixture was removed after 5 min. 20 ml of 20% piperidine/DMF solution was added, and the mixture was shaken for 15 min. (4) Analyzing and detecting: and (3) pumping out the piperidine solution, taking dozens of resins, washing with ethanol for three times, adding ninhydrin, pyridine and phenol, heating at 105-110 ℃ for 5min, changing the color into dark blue to be a positive reaction, and continuing to prepare the next amino acid, wherein if the color is not changed, the next amino acid is negative, and deprotection is required again. (5) Cleaning for the first time: the mixture was washed twice with 15ml of DMF, 15ml of methanol and 15ml of DMF in this order. (6) Condensation reaction: Fmoc-L-Arg (Pbf) -OH with 3 times of resin molar weight and HBTU with 3 times of resin molar weight were added, and dissolved in a small amount of DMF, and DIEA with 10 times of resin molar weight was immediately added and reacted for 30 min. (7) And (3) cleaning for the second time: the mixture was washed twice with 15ml of DMF, 15ml of methanol and 15ml of DMF in this order. (8) Extension of peptide chain: repeating the above steps to sequentially connect the rest amino acids.

TABLE 1 conditions of amino acids used in the Artificial Synthesis procedure

(9) Acetylation (AC) reaction and condensation of peptides: acetylation is the last part of the cell-penetrating peptide-acetyl hexapeptide, acetic anhydride with 3 times of resin molar weight and pyridine with 3 times of resin molar weight are added into a reaction column, and the reaction is carried out for 30 min. After AC ligation, the entire peptide synthesis was completed. Entering the final shrink phase, deprotection is not required here since AC is not Fmoc protected. Peptide contraction: the reaction was washed with DMF 3 times, DCM 3 times, MeOH 3 times, and finally the peptide resin was drained. (10) Deprotection of amino acid side chains and resin cleavage: the amino acid sequence of the cell-penetrating peptide-acetyl hexapeptide is Ac-EEMQRRYGRKKRRQRRR, wherein the side chain protecting groups contained in the amino acid sequence are: pbf, Trt, Boc and Tbu, which are unstable under acidic conditions, and TFA is used for resin cleavage, so that deprotection and resin cleavage can be performed simultaneously. Preparing 15ml of cutting fluid, wherein the volume ratio of each component is as follows: TFA (94.5%), water (2%), EDT (2.5%), TIS (1%). The resin was charged into a flask and shaken at a constant temperature (30 ℃ C.) for 2 hours. The lysate is blown dry as much as possible with nitrogen, then poured into a centrifuge tube, and slowly poured into ether. Sealing and placing in a centrifuge for 5min, pouring out the supernatant, and collecting the white solid below. Washing with ether for 6 times, and volatilizing at normal temperature to obtain crude peptide. (11) Purification by High Performance Liquid Chromatography (HPLC): dissolving: the crude peptide was placed in a vessel and dissolved completely with 30-50ml of 50% acetonitrile in water, possibly with gentle sonication for 2 min. Filtering: the lysate was filtered through a 0.45 μm filter. Analysis: 3 μ l of the solution was taken and analyzed by analytical grade HPLC for the crude product for subsequent preparation. The mobile phase is water and acetonitrile, time is 30min, gradient elution is carried out, HPLC is balanced for 5min by using an initial gradient, then sample injection is carried out, and the initial gradient is as follows: water 95%, acetonitrile 5%, end gradient: 5% of water and 95% of acetonitrile. Fourthly, preparation: and (5) carrying out sample injection preparation and purification on the dissolved sample. Preparative HPLC equilibrated for 10min with an initial gradient: water 95%, acetonitrile 5%, end gradient: 25% of water, 75% of acetonitrile and 40min of gradient time. The sample from the detector is collected. Identifying: the collected samples were sampled and subjected to purity and mass spectrometric identification. (12) Analyzing and identifying: identification and analysis of mass spectrum: ESI-MS is utilized to carry out mass spectrum identification, namely, the actual molecular weight of the synthesized peptide is measured, the actual molecular weight value is compared with the theoretical molecular weight of 2431.8, the molecular weight errors actually measured by mass spectrum analysis are within plus or minus 2 of the allowable error or the molecular weights of the both are completely consistent, and the successful synthesis of the cell-penetrating peptide-acetyl hexapeptide can be proved. HPLC analysis: adopting an LC3000 high performance liquid chromatograph, and the chromatographic analysis conditions are as follows: c18, reverse phase, 4.6mm x 150mm, gradient elution. The initial gradient was 5% A + 95% B, the final gradient was 30% A + 70% B, the time was 30min, the flow rate was 1.0ml/min, the UV detection wavelength was 214nm, and the sample size was 10. mu.l. Mobile phase a was 0.1% trifluoroacetic acid in 100% acetonitrile and mobile phase B was 0.1% trifluoroacetic acid in 100% water. And (4) observing the pure product, analyzing the highest peak and the peak with the largest area by HPLC analysis, and detecting the purity of the cell-penetrating peptide-acetyl hexapeptide.

The second part is a raw material composition proportion and a preparation method of the cell-penetrating peptide-carrying acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) nanoemulsion and a transdermal test:

1) the membrane-penetrating peptide-acetyl hexapeptide nanoemulsion and the preparation method thereof are characterized in that the membrane-penetrating peptide-acetyl hexapeptide nanoemulsion consists of the following raw materials in percentage by weight:

7.32 to 10.75 percent of isopropyl myristate, 17.53 to 20.12 percent of caprylic capric polyethylene glycol glyceride, 5.84 to 6.71 percent of polyglycerol fatty acid ester, 64.54 to 68.97 percent of double distilled water and 0.01 to 0.2 percent of cell-penetrating peptide-acetyl hexapeptide.

2) The optimal weight percentage of the raw materials for preparing the cell-penetrating peptide-acetyl hexapeptide nanoemulsion is as follows according to claim 1:

7.66 percent of isopropyl myristate, 17.53 percent of caprylic capric acid polyethylene glycol glyceride, 5.84 percent of polyglycerol fatty acid ester, 68.87 percent of double distilled water and 0.1 percent of cell penetrating peptide-acetyl hexapeptide.

A method for preparing a cell-penetrating peptide-carried acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) nanoemulsion comprises the following steps:

1) according to the proportion of the raw materials of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion, isopropyl myristate is used as an oil phase (A) for later use;

2) mixing acetyl hexapeptide and a proper amount of distilled water according to the proportion of the raw materials of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion to completely dissolve the acetyl hexapeptide, and adding the distilled water to obtain a water phase (C) for later use;

3) according to the proportion of the raw materials of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion, the caprylic capric acid polyethylene glycol glyceride and the polyglycerol fatty acid ester are placed in another container in a ratio of 3: 1, and are fully and uniformly stirred to form an S/C mixture (B) for standby;

4) then according to the proportion that the oil phase (A), the S/C mixture (B) and the water phase (C) are equal to 7.66: 23.37: 68.97, the S/C mixture (B) and the water phase (C) are respectively taken and sequentially added into a container of the oil phase (A);

5) selecting a timing constant-temperature magnetic stirrer at room temperature and rotating at 200 rpm/min^-1Magnetically stirring for 30min at the rotating speed to obtain the cell-penetrating peptide-acetyl hexapeptide nanoemulsion.

6) The detection result of the Malvern particle size analyzer shows that: the Malvern particle size determination result shows that the average particle size of the acetyl hexapeptide nanoemulsion is between 24.62 and 163.2 nm.

7) The FRANZ method skin penetration experiment research shows that: the skin penetration rate of the cell-penetrating peptide-acetyl hexapeptide in the nano-milk is 0.066% -0.18%.

3. Characteristic of the invention

(1) According to the fact that medical or medicinal cell-penetrating peptide or transdermal peptide has basically similar physicochemical properties and biological characteristics, namely the biological characteristics of stable physicochemical properties, obvious cell-penetrating or transdermal effects and the like, the cell-penetrating peptide with excellent cell-penetrating and cell-penetrating effects is successfully connected with acetyl hexapeptide, and the cell-penetrating peptide with the functions of transdermal penetration and transdermal absorption is artificially synthesized to carry the acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide).

(2) The present domestic and foreign research proves that the cell penetrating peptide which penetrates the barrier plasma membrane of the skin surface and penetrates the double lipid layer structure of the cell membrane carries acetyl hexapeptide (cell penetrating peptide-acetyl hexapeptide) as a biological active component and a functional target polypeptide which can be added into skin cosmetic, and then the cell penetrating peptide is matched with a nano-emulsion system which is proved by the present research and is generally considered to have one of high-efficiency transdermal drug delivery systems, and simultaneously the double effects of the cell penetrating peptide and the nano-emulsion are exerted, so that the synergistic interaction and the combined synergistic effect are scientifically and reasonably exerted, and the cosmetic hexapeptide with the good skin cosmetic anti-wrinkle effect can form better situations and functions of transdermal penetration and transdermal absorption.

(3) In order to overcome the relative instability and the characteristic that most polypeptide substances (including beauty peptides, hexapeptides and the like) are easy to degrade in aqueous solution or other liquid phases and lose the biological activity and the efficacy, acetylated acetyl hexapeptides are adopted to replace naked hexapeptides and are connected or modified by cell-penetrating peptides, and the cell-penetrating peptides-acetyl hexapeptides are prepared into a nano-emulsion type, namely the cell-penetrating peptides-acetyl hexapeptides nano-emulsion, so that the beauty efficacy component acetyl hexapeptides can maintain good biological activity and physiological functions and keep good transdermal penetration and transdermal absorption functions. Moreover, compared with a pure aqueous solution, the cell-penetrating peptide-acetyl hexapeptide is less prone to be degraded in the nano-emulsion preparation, and the bioactivity, the storage time and the effective period of the cell-penetrating peptide are longer and longer than those of the cell-penetrating peptide in the pure aqueous solution.

(4) Because the cell-penetrating peptide-acetyl hexapeptide nanoemulsion is prepared based on a nanoemulsion basic system and a nanoemulsion modern technology, compared with a liquid preparation prepared by a conventional or traditional method, the cell-penetrating peptide-acetyl hexapeptide nanoemulsion is smaller in particle size, more uniform in distribution and dispersion, better in appearance and sense and more comfortable to apply externally.

(5) The penetrating peptide-acetyl hexapeptide nanoemulsion adopts a high-efficiency transdermal transfer carrier nanoemulsion basic system, and the effective component acetyl hexapeptide is combined or connected with a penetrating peptide which can penetrate skin and penetrate membrane, so that the penetrating peptide-acetyl hexapeptide nanoemulsion has better transdermal penetration and transdermal absorption effects.

(6) Because the cell-penetrating peptide-acetyl hexapeptide nanoemulsion has better transdermal penetration and transdermal absorption effects, the cosmetic biological effect of the active ingredient acetyl hexapeptide can be better exerted and embodied.

4. The innovation point of the invention

(1) The cell-penetrating peptide which is designed and synthesized and can be added into beauty cosmetics is connected with and carries acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) and is applied as a human skin beauty effect component and a target polypeptide, and at present, no report is found at home and abroad, and no similar product exists, so that the invention has innovation.

(2) The beauty-care effect nanoemulsion containing the transmembrane peptide connection and carrying the acetyl hexapeptide (the transmembrane peptide-acetyl hexapeptide) added in the pseudo-ternary phase nanoemulsion basic system designed and prepared by the invention enables the activity of the acetyl hexapeptide to be more stable, and the transdermal permeation and transdermal absorption effects to be better.

5. The invention has the advantages of

(1) The cosmetic functional component of the invention, namely the hexapeptide, is a target polypeptide, the molecular structure of the hexapeptide is modified into acetyl hexapeptide through acetylation, the application physicochemical property of the hexapeptide in the cosmetic is more stable, and the biological effect is better.

(2) The invention connects the cell-penetrating peptide with the acetyl hexapeptide, so that the transdermal penetration and transdermal absorption of the acetyl hexapeptide of the target functional component can be improved, and the cosmetic effect in actual application can be improved.

(3) The acetyl hexapeptide is added into the traditional preparation (such as aqua, essence, emulsion, cream, ointment, gel and the like) for external application on the face, and the expected transdermal penetration and transdermal absorption effects are difficult to achieve. The invention connects the cell-penetrating peptide with the acetyl hexapeptide, and matches and combines the cell-penetrating peptide with the high-efficiency transdermal delivery carrier nanoemulsion system, so that the cell-penetrating peptide can exert a skin-penetrating effect, the nanoemulsion system can also exert a permeation promoting effect of transdermal delivery of the acetyl hexapeptide, the advantages of the cell-penetrating peptide and the nanoemulsion system are complementary, the functions of the cell-penetrating peptide and the acetyl hexapeptide are complementary, the transdermal permeation and the transdermal absorption of the target drug and the hexapeptide of the functional component are better, and the cosmetic effect in practical application is better.

(4) The basic nanoemulsion system in the nanoemulsion is optimized by a pseudo-ternary phase diagram, the proportion of the adopted water phase is improved, the proportion of the adopted oil phase is reduced, and the new blank nanoemulsion basic system is prepared, so that the basic nanoemulsion system has the advantages of finer texture, more uniform dispersion, more tiny particles, nano-level standard particle size and good distribution of nano particles, and the comfort of facial coating is improved.

(5) The nano-emulsion is of an oil-in-water type, so that the cell-penetrating peptide carrying target drugs and functional components has better solubility, solubilization and inclusion effects.

(6) In the nano-emulsion system prepared by the invention, the addition amount of the surfactant/cosurfactant or the emulsifier/co-emulsifier is obviously reduced compared with that of the surfactant/cosurfactant or the emulsifier/co-emulsifier used in the conventional nano-emulsion documents (including patent documents), so that the tingling feeling and the skin irritation of the facial application are obviously reduced, and the use safety is obviously improved.

(7) The nano-emulsion is not only of an oil-in-water type, but also has obviously reduced addition ratio of the oil phase in the pseudo-ternary phase to the surfactant/cosurfactant, so that the greasy feeling and the peculiar smell feeling of the conventional nano-emulsion are basically eliminated, and the satisfaction degree of facial painting is obviously improved.

(8) Both acetyl hexapeptide and cell-penetrating peptide belong to bioactive components with polypeptide structures, the stability of the acetyl hexapeptide and the cell-penetrating peptide in a water-soluble medium or an aqueous solution is poor, and functional groups and active centers of the acetyl hexapeptide and the cell-penetrating peptide are easily degraded or destroyed in the aqueous solution. The nano-emulsion preparation is similar to microspheres, so that the cell-penetrating peptide such as protein or polypeptide functional components can be wrapped with the acetyl hexapeptide of target drugs and functional components, the stability of the nano-emulsion preparation is improved, and the destructiveness of physicochemical properties is obviously reduced, so that the biological effect and the effect of facial application of the nano-emulsion preparation are reasonably improved.

(9) The components, the composition proportion, the types of the functional components, the drug loading rate, the osmotic concentration gradient and the like of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion are more beneficial to percutaneous permeation and transdermal absorption, and the problems and difficulties which need to be solved and overcome by the conventional liquid preparation are partially solved.

(10) No matter the cell-penetrating peptide or the transdermal peptide has no special beauty effect or drug effect, the invention skillfully connects or carries the target drug with beauty function and the functional component-acetyl hexapeptide, so that the cell-penetrating peptide has the functions of skin penetration and beauty.

(11) In addition, compared with the existing cosmetic preparation formulation, the invention also has the following beneficial effects: compared with other cosmetic formulations, the nanoemulsion prepared by the technology has higher stability and prolonged validity period; secondly, the main functional components in the nano milk prepared by the invention have better transdermal absorption effect and higher transdermal absorption rate; the nano-emulsion prepared by the technology has better application safety; the nano-emulsion prepared by the technology is prepared at room temperature, so that the nano-emulsion has better biological activity and biological effect on thermolabile cell-penetrating peptide-acetyl hexapeptide; the nanoemulsion prepared by the technology of the invention has better appearance and better actual use comfort. The nano-emulsion prepared by the technology has the advantages of simple preparation components, simple preparation process, easy actual operation, high efficiency and rapidness in preparation, no need of special instruments and equipment, no need of external energy supply, no need of heating and no environmental pollution. The resource consumption and waste are reduced, and the product cost is greatly reduced.

It will thus be seen that the present invention not only overcomes and solves the above-mentioned deficiencies or drawbacks associated with the prior art in the application of modern skin cosmetics and skin care, but also achieves further beneficial results or benefits through the present technology.

Description of the drawings:

FIG. 1 is a process flow and a technical route chart of artificial synthesis of cell-penetrating peptide-acetyl hexapeptide.

FIG. 2 is a schematic diagram of the molecular structure of cell-penetrating peptide-acetyl hexapeptide.

FIG. 3 is a mass spectrometric identification, analysis, detection and identification pattern of cell-penetrating peptide-acetyl hexapeptide.

FIG. 4 is the HPLC analysis detection pattern of the cell-penetrating peptide-acetyl hexapeptide.

FIG. 5 is a process flow and a technical route chart for preparing the cell-penetrating peptide-acetyl hexapeptide nanoemulsion.

FIG. 6 is the analysis chart of the particle size distribution determination of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion.

Fig. 7 shows the transdermal absorption rate of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion.

Examples of the embodiments

One example of implementation:

the preparation method of the cell-penetrating peptide carrying acetyl hexapeptide (cell-penetrating peptide-acetyl hexapeptide) comprises the following steps:

1 experimental part

1.1 Main raw materials and reagents

Fmoc-Arg (Pbf) -OH (arginine), Fmoc-Gln (Trt) -OH (glutamine), Fmoc-Lys (Boc) -OH (lysine), Fmoc-Gly-OH (glycine), Fmoc-Tyr (Tbu) -OH (tyrosine), Fmoc-Met-OH (methionine), Fmoc-Glu (Tbu) -OH (glutamic acid), Ac (acetic anhydride: pyridine ═ 1: 1), 2-Chlorotrityl Chloride Resin (2 Chloride Resin), DMF (N, N-dimethylformamide), DCM (dichloromethane), acetonitrile, HBTU (benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate), DIEA (N, N-diisopropylethylamine), TFA (trifluoroacetic acid), TIS (triisopropylsilane), EDT (1, 2-ethanedithiol), Ether, piperidine, ethanol, ninhydrin, phenol, pyridine.

1.2 Main instruments

TDL-50 (desk type low-speed large-capacity centrifuge) (Changzhou Meixiang apparatus Co., Ltd.), HY-2 (speed-regulating multipurpose oscillator) (Changzhou Lange apparatus manufacturing Co., Ltd.), FD-1A-50 (vacuum freeze dryer) (Nanjing Pusen apparatus Co., Ltd.), SHZ-D (III) type (circulating water vacuum pump) (Ponci apparatus science and technology Co., Ltd.), an electronic balance (Changzhou Shapin precision apparatus Co., Ltd.), a polypeptide synthesizer, industrial nitrogen, LC3000 type high performance liquid chromatograph (Beijing Keruihai scientific apparatus Co., Ltd.), and Agilent 6120 LC/MS.

1.3 solid phase Synthesis method and procedure

1.3.1 synthetic route process flow: the method comprises the steps of selecting 2-chloro resin as a resin carrier, selecting halogen chlorine as an active site on the resin, firstly swelling the resin during polypeptide solid phase synthesis, then reacting C-terminal carboxyl of a first amino acid with the active site chlorine on the resin, carrying out dehydration condensation to join a second amino acid after the first amino acid is connected on the resin, and then removing Fmoc protection after the condensation is finished. Repeating the operation according to the designed amino acid sequence, sequentially connecting the rest amino acids, completing N-terminal acetylation, and finally cutting the polypeptide from the resin by using a cutting reagent to form naked carboxyl.

1.3.2 swelling of the resin: weighing 1.5g of dichloro resin (0.08 mmol of synthesized target peptide, 0.4mmol/g of substitution degree of the dichloro resin, and 7.5 times of excess, so that the required mass of the dichloro resin is 0.08/0.4 × 7.5 ═ 1.5g), placing into a reaction column, adding 20 ml of DCM into the reaction column, oscillating for 30min, and activating for later use.

1.3.3 linking the first amino acid DCM solvent was removed by sand core suction filtration, 1.05 times resin molar amount of Fmoc-L-Arg (Pbf) -OH was added, 10 times resin molar amount of DIEA was added, finally a small amount of DMF was added for dissolution, and shaking was carried out for 1 h. After the reaction was complete, the reaction was washed 6 times with DMF and DCM alternately.

1.3.4 deprotection 20 ml of 20% piperidine/DMF solution was added and after 5min was aspirated. 20 ml of 20% piperidine/DMF solution was added, and the mixture was shaken for 15 min.

1.3.5 detecting, pumping out the piperidine solution, taking dozens of resins, washing with ethanol for three times, adding ninhydrin, pyridine and phenol one drop by drop, heating at 105-110 ℃ for 5min, taking the color of the resin turning dark blue as a positive reaction, and continuing to take next amino acid, if the color is not changed, taking the resin as a negative, and needing to remove the protection again.

1.3.6 initial washes were performed twice with 15ml of DMF, 15ml of methanol, and 15ml of DMF, respectively.

1.3.7 condensation, 3 times of resin molar weight of Fmoc-L-Arg (Pbf) -OH and 3 times of resin molar weight of HBTU were added, and all were dissolved in a small amount of DMF, and 10 times of resin molar weight of DIEA was immediately added and reacted for 30 min.

1.3.8 second washing: the mixture was washed twice with 15ml of DMF, 15ml of methanol and 15ml of DMF in this order.

1.3.9 extension of the peptide chain the procedure was repeated as above, with the remaining amino acids being successively ligated.

Table 2 amino acids used in the synthesis of cell-penetrating peptide-acetyl hexapeptide in the present invention

1.3.10 AC reaction and peptide shrink acetylation are the last part of the dermatan-hexapeptide, adding 3 times of resin molar amount of acetic anhydride and 3 times of resin molar amount of pyridine into the reaction column, and reacting for 30 min. After AC ligation, the entire peptide synthesis was completed. Entering the final shrink phase, deprotection is not required here since AC is not Fmoc protected. Peptide contraction: the reaction was washed with DMF 3 times, DCM 3 times, MeOH 3 times, and finally the peptide resin was drained.

1.3.11 the amino acid side chain deprotection and resin cutting transdermal peptide-hexapeptide has the amino acid sequence of Ac-EEMQRRYGRKKRRQRRR, wherein the side chain protecting groups comprise: pbf, Trt, Boc and Tbu, which are unstable under acidic conditions, and TFA is used for resin cleavage, so that deprotection and resin cleavage can be performed simultaneously. Preparing 15ml of cutting fluid, wherein the volume ratio of each component is as follows: TFA (94.5%), water (2%), EDT (2.5%), TIS (1%). The resin was charged into a flask and shaken at a constant temperature (30 ℃ C.) for 2 hours. The lysate is blown dry as much as possible with nitrogen, then poured into a centrifuge tube, and slowly poured into ether. Sealing and placing in a centrifuge for 5min, pouring out the supernatant, and collecting the white solid below. Washing with ether for 6 times, and volatilizing at normal temperature to obtain crude peptide.

1.3.12 HPLC purification (1) dissolution the crude peptide was placed in a vessel and dissolved completely with 30-50ml of 50% strength aqueous acetonitrile, possibly with gentle sonication for 2 min. (2) The resulting solution was filtered through a 0.45-. mu.m filter. (3) Mu.l of the solution was analyzed by analytical grade HPLC for crude product for subsequent preparation. The mobile phase is water and acetonitrile, time is 30min, gradient elution is carried out, HPLC is balanced for 5min by using an initial gradient, then sample injection is carried out, and the initial gradient is as follows: water 95%, acetonitrile 5%, end gradient: 5% of water and 95% of acetonitrile. (4) Preparation the dissolved sample was ready for injection. Preparative HPLC equilibrated for 10min with an initial gradient: water 95%, acetonitrile 5%, end gradient: 25% of water, 75% of acetonitrile and 40min of gradient time. The sample from the detector is collected.

1.3.13 characterization samples were taken from the collected samples and tested for purity and mass spectra.

2 analytical identification

2.1 mass spectrometric identification analysis: the ESI-MS is utilized to carry out mass spectrum identification on the actual molecular weight of the cell-penetrating peptide-acetyl hexapeptide, and 5 target peaks with different charges are obtained through mass spectrum detection and analysis, wherein the target peaks are respectively as follows:

[M+3H]3H⁺a mass to nucleus ratio of 811.4, a measured molecular weight of 811.4 x 3-3 x 2431.2,

[M+4H]4H⁺a mass to nucleus ratio of 608.9, a measured molecular weight of 608.9 x 4-2431.6,

[M+5H]5H⁺has a mass-to-nucleus ratio of 487.4, a measured molecular weight of 487.4 × 5-5 ═ 2432.0,

[M+6H]6H⁺the nucleus ratio is 406.3, the molecular weight is 406.3 x 6-6 ═ 2431.8,

[M+7H]7H⁺the mass to nucleus ratio was 348.4, with a measured molecular weight of 348.4 × 7-7 ═ 2431.8. The theoretical molecular weight of the cell-penetrating peptide, acetyl hexapeptide, is also just 2431.8. Usually, the mass spectrum actually measures the allowable error of the molecular weight and the theoretical molecular weightThe difference is + -2. The theoretical molecular weight and the actual molecular weight of the cell-penetrating peptide-acetyl hexapeptide are consistent, so that the cell-penetrating peptide-acetyl hexapeptide synthesized by the invention can be proved to be the cell-penetrating peptide. The invention relates to a mass spectrometric identification analysis detection identification map of cell-penetrating peptide-acetyl hexapeptide (see figure 3 for details in the description of the attached drawings).

2.1 high performance liquid phase (HPLC) analytical detection: the invention adopts an LC3000 high performance liquid chromatograph, and the chromatographic analysis conditions are as follows: c18, reverse phase, 4.6mm x 150mm, gradient elution. The initial gradient was 5% A + 95% B, the final gradient was 30% A + 70% B, the time was 30min, the flow rate was 1.0ml/min, the UV detection wavelength was 214nm, and the sample size was 10. mu.l. HPLC analysis detection is carried out on the cell-penetrating peptide-acetyl hexapeptide with the mobile phase A of 0.1 percent trifluoroacetic acid in 100 percent acetonitrile and the mobile phase B of 0.1 percent trifluoroacetic acid in 100 percent water, and the detection results show that: HPLC analysis of the pure product of the cell-penetrating peptide-acetyl hexapeptide determines that the highest peak of the retention time 8.219 is the peak with the largest area, and the purity of the synthesized product is calculated to reach 98.76% through the integration of the peak of the synthesized product. HPLC analysis detection pattern of the cell-penetrating peptide-acetyl hexapeptide (see figure 3 for details in the description of the attached drawings).

The second embodiment is as follows:

a cell-penetrating peptide-acetyl hexapeptide nanoemulsion and a preparation method thereof (taking 100g of preparation as an example):

1. the main raw materials are as follows:

preparing raw materials: isopropyl myristate (IPM), caprylic capric polyethylene glycol glyceride (Labrasol), polyglycerin fatty acid ester (plural Oleique CC497), double distilled water; ② main functional components: cell-penetrating peptide-acetyl hexapeptide;

2. main preparation equipment

One in ten thousand electronic balance, one in one thousand medicine balance, liquid fast mixer, timing constant temperature magnetic stirrer, quartz tube and glass double water distiller, large capacity low temperature centrifuge, sterile filter, container of various specifications, flask and beaker.

3. The main preparation process

Accurately weighing 7.66g of isopropyl myristate by a one-thousandth drug balance, and immediately placing the isopropyl myristate in a washed and disinfected 100mL clean triangular flask. And marked clearly as oil phase (A) with a marker pen

Secondly, 100mg of the cell-penetrating peptide-acetyl hexapeptide is weighed by a one-ten-thousandth precision analytical balance and is placed in a 100mL clean triangular flask which is sterilized by washing. Directly adding 40mL of double distilled water into the 100mL Erlenmeyer flask containing various functional components, covering, and quickly placing the Erlenmeyer flask on a liquid quick mixer; opening the liquid quick mixer, adjusting to II grade, mixing well to dissolve completely, 3-5min later, closing the liquid quick mixer, taking off the Erlenmeyer flask, and clearly marking with color marker as water phase (C).

③ according to the designed caprylic capric polyethylene glycol glyceride: respectively and accurately weighing 17.53 g of caprylic capric acid polyethylene glycol glyceride and 5.84 g of polyglycerol fatty acid ester, and placing the two into a washed 100mL clean triangular flask; capping and quickly placing the Erlenmeyer flask on a liquid flash mixer, opening the liquid flash mixer and adjusting to II, mixing well to form an emulsifier/co-emulsifier (S/C) mixture, after 3-5 minutes, closing the liquid flash mixer, removing the Erlenmeyer flask and clearly marking the Erlenmeyer flask as S/C mixture (B) by a marker.

Fourthly, directly adding the water phase (C) into a 100mL triangular flask in the oil phase (A) according to the proportion that the oil phase (A), the S/C mixture (B) and the water phase (C) are equal to 7.66: 23.37: 68.97; then, the S/C mixture (B) formed by thoroughly mixing caprylic/capric macrogol glyceride and polyglyceryl fatty acid ester was directly added to the 100mL Erlenmeyer flask in which the oil phase (a) and the water phase (C) were mixed.

Fifthly, immediately selecting a timing constant-temperature magnetic stirrer at room temperature of 25 ℃, and rotating at 200 rpm/min^-1Magnetically stirring for 30 min.

Sixthly, closing the timing constant-temperature magnetic stirrer, taking down a 100mL triangular flask, observing that the appearance of the flask is clear, transparent, good in fluidity, good in dispersity and obvious in visible opalescence, and thus obtaining 100g of the penetrating peptide carrying acetyl hexapeptide nanoemulsion.

The embodiments of the present invention are described only for the preferred embodiments of the present invention, and not for the limitation of the concept and scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the design concept of the present invention shall fall into the protection scope of the present invention, and the technical content of the present invention which is claimed is fully set forth in the claims.

Claims

1. A preparation method of a cell-penetrating peptide-acetyl hexapeptide nanoemulsion is characterized by comprising the following raw materials in percentage by weight:

7.66 percent of isopropyl myristate, 17.53 percent of caprylic capric acid polyethylene glycol glyceride, 5.84 percent of polyglycerol fatty acid ester, 68.87 percent of double distilled water and 0.1 percent of cell-penetrating peptide-acetyl hexapeptide;

the preparation method comprises the following steps:

firstly, designing a molecular structure and a sequence of a cell-penetrating peptide-acetyl hexapeptide, and artificially synthesizing:

(1) weighing 1.5g of dichloro resin, putting the dichloro resin into a reaction column, then adding 20 ml of DCM into the reaction column, and oscillating for 30min to complete the swelling and activation of the dichloro resin for standby;

(2) filtering off DCM solvent by sand core, adding Fmoc-L-Arg (Pbf) -OH with the molar weight being 1.05 times of that of the resin, then adding DIEA with the molar weight being 10 times of that of the resin, finally adding DMF for dissolving, and oscillating for 1 h; after the reaction is finished, alternately cleaning the solution for 6 times by using DMF and DCM to complete the connection of the first amino acid;

(3) adding 20 ml of 20% piperidine/DMF solution, pumping off after 5min, adding 20 ml of 20% piperidine/DMF solution, and oscillating for 15min to finish deprotection reaction;

(4) taking out piperidine solution, taking dozens of resins, washing with ethanol for three times, adding ninhydrin, pyridine and phenol, heating at 105-110 ℃ for 5min, turning dark blue to be a positive reaction, and performing deprotection again if the reaction is not a positive reaction;

(5) washing with 15ml of DMF, 15ml of methanol and 15ml of DMF respectively twice in sequence;

(6) adding Fmoc-L-Arg (Pbf) -OH with the molar weight 3 times that of the resin and HBTU with the molar weight 3 times that of the resin, dissolving the mixture by using a small amount of DMF, immediately adding DIEA with the molar weight 10 times that of the resin, and reacting for 30 min;

(7) then washing the mixture twice with 15ml of DMF, 15ml of methanol and 15ml of DMF in sequence respectively;

(8) repeating the operation according to the method, namely sequentially connecting the rest amino acids according to the amino acid structure sequence Ac-EEMQRRYGRKKRRQRRR of the cell-penetrating peptide-acetyl hexapeptide so as to complete the extension of the peptide chain;

(9) adding acetic anhydride with 3 times of resin molar weight and pyridine with 3 times of resin molar weight into the reaction column, and reacting for 30 min; after the acetyl group was grafted, the synthesis of the entire peptide was completed and the peptide was contracted: washing with DMF for 3 times, washing with DCM for 3 times, washing with methanol for 3 times, and draining off peptide resin;

(10) preparing 15ml of cutting fluid, wherein the volume ratio of each component is as follows: 94.5% TFA, 2% water, 2.5% EDT and 1% TIS; putting the resin into a flask, oscillating at constant temperature of 30 ℃ for 2h, drying the lysate with nitrogen as much as possible, pouring the lysate into a centrifuge tube, slowly pouring diethyl ether, sealing, centrifuging in a centrifuge for 5min, pouring off the supernatant, and taking a white solid below; washing with diethyl ether for 6 times, and volatilizing at normal temperature to obtain crude peptide;

(11) purification was performed by HPLC method, i.e.: dissolving: putting the crude peptide into a vessel, and completely dissolving the crude peptide by using 30-50ml of acetonitrile aqueous solution with the concentration of 50%, and slightly performing ultrasonic treatment for 2 min; and (3) filtering: filtering the dissolved solution with 0.45 μm filter membrane; and (3) analysis: 3 μ l of the solution was taken for analytical grade HPLC analysis of the crude product for subsequent preparation; the mobile phase was water and acetonitrile for 30min, gradient elution was performed by equilibrating the HPLC with an initial gradient for 5min and then injecting the sample, the initial gradient being: water 95%, acetonitrile 5%, end gradient: 5% of water and 95% of acetonitrile; preparation: preparing a sample injection preparation for the dissolved sample; preparative HPLC equilibrated for 10min with an initial gradient: water 95%, acetonitrile 5%, end gradient: 25% of water, 75% of acetonitrile and 40min of gradient time; collecting a sample from the detector; and (3) identification: sampling the collected sample and identifying the purity and the mass spectrum;

secondly, the preparation method of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion comprises the following steps:

1) taking isopropyl myristate as an oil phase A for later use according to the proportion of the raw materials of the cell-penetrating peptide-acetyl hexapeptide nanoemulsion;

2) mixing the cell-penetrating peptide-acetyl hexapeptide with a proper amount of distilled water to be completely dissolved, and adding the distilled water to be used as a water phase C for later use;

3) mixing caprylic capric acid polyethylene glycol glyceride, polyglycerol fatty acid ester equal to 3: 1, placing the mixture in another container, and fully and uniformly stirring to form a mixture B of caprylic/capric polyethylene glycol glyceride and polyglycerol fatty acid ester for later use;

4) then adding the oil phase A, the caprylic/capric polyethylene glycol glyceride, the polyglycerol fatty acid ester mixture B and the water phase C into the oil phase A container in sequence, wherein the water phase C is equal to the proportion of 7.66: 23.37: 68.97;

5) selecting a timing constant-temperature magnetic stirrer at room temperature and 200rpm^-1Stirring for 30min by magnetic force at the rotating speed to obtain the cell-penetrating peptide-acetyl hexapeptide nanoemulsion.