CN116327610A - Botulinum-like polypeptide nano micelle and preparation method and application thereof - Google Patents
Botulinum-like polypeptide nano micelle and preparation method and application thereof Download PDFInfo
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- CN116327610A CN116327610A CN202211447850.7A CN202211447850A CN116327610A CN 116327610 A CN116327610 A CN 116327610A CN 202211447850 A CN202211447850 A CN 202211447850A CN 116327610 A CN116327610 A CN 116327610A
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0291—Micelles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/41—Particular ingredients further characterized by their size
- A61K2800/413—Nanosized, i.e. having sizes below 100 nm
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
- A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention belongs to the technical field of cosmetics, and particularly relates to a botulinum-like polypeptide nano micelle and a preparation method and application thereof. Botulinum-likeThe amino acid sequence of the polypeptide is as follows: R-Glu-Asn-Arg-Ser-Phe-Met-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala-NH 2 R is myristoyl or palmitoyl. The botulinum-like polypeptide prepared by the method is added into a solution, and self-assembled into the botulinum-like polypeptide nano micelle. The botulinum-like polypeptide has anti-wrinkle effect, can be self-assembled into nano micelle in solution, can be quickly transdermal and dissociated in physiological salt environment after transdermal, can enter nerve cells, and has the advantages of anti-wrinkle effect, high biocompatibility, high safety and double anti-wrinkle effect compared with the hexapeptide which is a product sold in the market. The nano micelle can be used as a carrier, and the hydrophilic shell can load a plurality of bioactive molecules with hydrophilic activity, so that other effects are added on the effects of resisting wrinkle of the botulinum-like.
Description
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to a botulinum-like polypeptide nano micelle and a preparation method and application thereof.
Background
Wrinkles are the first sign of skin aging, and as the age increases, the facial muscle contractions and muscle forces decrease, so does the skin tone, and the elastic skin begins to wrinkle. The occurrence of this phenomenon has various intrinsic and extrinsic causes, mainly due to the gradual decrease of the regeneration capacity of the human body itself with the increase of age; external inducement includes ultraviolet rays, electromagnetic radiation, long-term stay up and the like. The superposition of these internal and external causes can lead to facial skin to show the characteristics of increased wrinkles, loose skin, great loss of collagen, etc.
The generation of wrinkles is unavoidable due to natural aging and the like, but can be delayed by an anti-wrinkle mode, and the prior art adopts a mode of injecting botulinum toxin type A to remove wrinkles. Botulinum toxin type A is a neurotoxin protein produced by botulinum during reproduction and is 150kD in size and acts in a manner that inhibits the release of peripheral motor nerve ending presynaptic membrane acetylcholine, thereby causing muscle relaxant paralysis and inhibiting muscle contraction. Botulinum toxins also present a number of problems when used as wrinkle-removing products. Firstly, botulinum toxin needs to act by injection and the duration of effect is limited after one injection; second, botulinum toxin is expensive and has a relatively small audience size; most importantly, improper handling and use can cause the user to leave sequelae such as facial stiffness, facial paralysis, etc.
With the progress of research, the appearance of the hexapeptide greatly improves the anti-wrinkle safety, and compared with botulinum toxin, the hexapeptide has no stimulation, safety, no operation risk, no age skin requirement and no injection, and is not easy to leave sequelae such as facial stiffness, facial paralysis and the like. Hexapeptide is the most classical polypeptide against expression lines, which acts as an antiwrinkle agent by competing with SNAP-25 to interfere with SNARE protein ternary complex formation, inhibiting excessive release of neurotransmitters. The 10% emulsion is smeared on the canthus for 30 days, can reduce the depth of the canthus fish tail vein by 30%, and is a very effective anti-wrinkle active substance. There are other anti-wrinkle polypeptides in the market, such as velocin, which can reduce eye wrinkles by 49% and skin roughness by 47% when used over a 28 day period; a snake venom peptide is a small molecule active peptide simulating snake venom toxin, and can effectively reduce the generation of wrinkles. And the facial expression cannot be influenced, and through efficacy verification experiments, 25ppm of snake venom peptide-like solution is used for cultured cells, the muscle contraction rate is reduced to 36% after one minute, and the muscle contraction rate is reduced to 82% after 2 hours, so that an instant wrinkle removing effect can be achieved.
The above-mentioned hexapeptide and the other products such as the Weilos peptide are mostly short peptides, and the longer the peptide chain is, the more difficult the peptide chain is to penetrate the skin barrier. For example, CN113402586a discloses a polypeptide and its application as a muscle paralysis relaxant or a dermatological anti-wrinkle agent, the polypeptide sequence is: R-Glu-Glu-Met-Gln-Arg-Arg-Ala, wherein R is at least one selected from Ser, glu, phe, met, arg, asn, glu and Leu, and is the shortest octapeptide and the longest 15 peptide from the aspect of polypeptide structure. When the amino acid sequence exceeds 10, the skin penetrability is necessarily affected, and when it is 15 peptide, the transdermal property is further weakened; therefore, although the pentadecapeptide of the type A botulinum toxin prepared by the patent has good stability, permeability and affinity and shows good biocompatibility, is used for facial expression myoparalysis relaxation, and has the effects of improving facial contours and eliminating or reducing wrinkles, the skin penetrability is reduced, most of the pentadecapeptide stays in the stratum corneum, and the part penetrating through the skin into epidermis and dermis is very little, so that the bioavailability is greatly reduced, the efficacy is limited, and the transdermal problem after peptide chain extension is not practically solved. Thus, the existing peptide products on the market can increase the penetrability of the peptide products by reducing the chain length of the peptide, and meanwhile, the functional sites on the peptide chain are abandoned, so that the polypeptide efficacy is reduced.
In order to improve the penetrability of the polypeptide, CN113512092A discloses that the polypeptide nano-hybrid has good effect of eliminating or reducing wrinkles. The polypeptide nanometer hybrid has a structure of [ Au-S-S-pentadecapeptide ] n, and the amino acid sequence of the pentadecapeptide is SEFMRNELEEMQRRA. The pentadecapeptide is modified by Cys and then is prepared into a hybrid with nano gold, and the pentadecapeptide is delivered transdermally by means of a nano gold carrier. According to the technical scheme, the method comprises the following steps: the final form of the nanometer hybrid is an acidic solution form, the nanometer hybrid contains hydroxyethyl piperazine ethyl sulfonic acid and chloroauric acid, the pH value is relatively low, the nanometer gold is an exogenous substance, when the nanometer hybrid is applied to injection preparations, polypeptide and gold are easily dissociated, gold ions lack to pass through skin barriers, certain allergic hidden dangers exist, and the allergic hidden dangers can cause skin breaking, micro-damage, serrated cell phagocytosis induction, inflammation and other series of problems.
Polypeptide nano-micelles are widely applied to the field of polypeptide medicaments, and have been reported in the aspect of cancer treatment. CN112426537a discloses a polypeptide nano micelle, a preparation method and application thereof. The polypeptide nano micelle is formed by self-assembly of polyethylene glycol phospholipid (PEG-PE) and polypeptide, and has the effect of inhibiting cancer cell proliferation. CN 112494427a discloses a polylactic acid-polypeptide micelle and application thereof, which is formed by self-assembly of amphiphilic polylactic acid-polypeptide polymers. The hydrophilic polypeptide forms an outer shell of the polylactic acid-polypeptide micelle, and the hydrophobic polylactic acid forms an inner core of the polylactic acid-polypeptide micelle; the amphiphilic polylactic acid-polypeptide polymer is also connected with an aptamer targeting tumor cells, and has important significance in the field of combined treatment of tumors. The polypeptide nano-micelle provided by the patent is used as a medicine, but has a few reports on the cosmetic application.
In view of the above, there is a need to develop a method for preparing anti-wrinkle polypeptide products with good transdermal properties and high safety.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: overcomes the problems of polypeptide transdermal property and efficacy and ensures the safety, and provides a botulinum-like polypeptide nano micelle and a preparation method thereof, which are applied to the technical field of cosmetics.
The technical scheme adopted for solving the technical problems comprises the following steps:
a method for preparing a botulinum-like polypeptide nano-micelle, comprising the steps of:
(1) Design of a botulinum-like polypeptide: using Discovery studio software to obtain a polypeptide sequence capable of blocking interaction between SNAP-25 and Rabpylin-3A based on a simulated design; modifying the nitrogen end of the polypeptide sequence by a hydrophobic group;
(2) Preparation of a botulinum-like polypeptide: synthesizing a crude polypeptide sequence modified by a hydrophobic group, purifying and freeze-drying to obtain a purified botulinum-like polypeptide;
(3) Preparation of botulinum-like polypeptide nano-micelles: the botulinum-like polypeptide is prepared into a solution with the concentration of more than or equal to 0.2mg/mL, and the botulinum-like polypeptide is self-assembled into nano-micelles. Characterizing the polypeptide nano micelle solution by using a transmission electron microscope, and performing negative dyeing treatment on a sample by using 3% phosphotungstic acid; the particle size of the sample was tested using dynamic light scattering and was less than 50nm. The botulinum-like polypeptide micelle has obvious anti-wrinkle effect, and can be used as a carrier to load other hydrophilic active ingredients.
In the step (1), the hydrophobic group is modified by myristoyl or palmitoyl.
In the step (2), resin is used as a carrier, a polypeptide solid-phase organic synthesis technology is used, and in an N, N-dimethylformamide system, a polypeptide sequence modified by a hydrophobic group is synthesized by an Fmoc synthesis strategy, purified and freeze-dried to obtain the purified polypeptide. Specific:
rink Aminde MBHA Resin is selected as a carrier, and polypeptide solid-phase organic synthesis technology is used for synthesizing Myristyl-Glu-Asn-Arg-Ser-Phe-Met-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala-NH in an N, N-dimethylformamide system by using Fmoc synthesis strategy 2 Removing the protecting group by using 20% piperidine/DMF solution, and performing final nitrogen end modification by using myristoyl chloride or palmitoyl chloride to obtain full-protection peptide resin; cracking the full-protection peptide resin under the action of trifluoroacetic acid, and simultaneously removing a polypeptide side chain protecting group to obtain a polypeptide crude product; using counterPurifying the polypeptide by phase preparation liquid chromatography, and filling: c18, phase A0.1% TFA/H 2 Phase O, phase B0.1% TFA/MeOH, phase A equilibrated loading, elution procedure: 25% -85% of 60min, preparing and purifying, and freeze-drying to obtain purified polypeptide; or the synthetic sequence is Palmitoyl-ENRSFMELEEMQRRA-NH 2 Is a polypeptide of the genus.
(4) The efficacy of the botulinum-like polypeptide nano micelle prepared by the invention is verified:
permeability test: the distribution of the samples in the pig skin was examined using fluorescent-labeled botulinum-like polypeptide nanomicelle, fluorescent-labeled pentadecapeptide (pentadecapeptide disclosed in CN113512092 a), and the permeability was evaluated. The permeability of the polypeptide is obviously enhanced under the form of nano micelle.
Anti-wrinkle efficacy: detecting the change condition of cell proliferation and cell migration after the action of the botulinum-like polypeptide nano micelle sample based on fibroblasts; the UVA is adopted to irradiate the fibroblasts, and the change condition of the content of the Collagen I (Collagen I) and the content of the matrix metalloproteinase 1 (MMP-1) is detected, so that the botulinum-like polypeptide nano micelle has obvious anti-wrinkle effect; based on Ex vivo skin tissues, the detection of the change conditions of Collagen density and Collagen IV (Collagen IV) content shows that the botulinum-like polypeptide nano-scale can achieve the anti-wrinkle effect. The neurotransmitter acetylcholine content was tested on a neuronal cell basis at the 2D cell level and 3D skin model level using chromogenic methods. Compared with a control group (hexapeptide), the content of the botulinum-like polypeptide nano micelle acetylcholine is obviously reduced, and a strong anti-wrinkle effect is achieved.
(5) The botulinum-like polypeptide nano micelle is a nano particle formed by self-assembly of amphiphilic molecules, the structure of the botulinum-like polypeptide nano micelle comprises a lipophilic inner core and a hydrophilic outer shell, and the hydrophilic outer shell can be combined with a hydrophilic active ingredient through non-covalent bonds (such as Van der Waals force and intermolecular hydrogen bonds) to form the supramolecular nano particle.
The human collagen I is simulated by computer, a hydrophilic polypeptide containing 21 amino acid sequences is designed, and the sequence is Ac-GP- (Hyp) -GP- (Hyp) -GF- (Hyp) -GERGP- (Hyp) -GP- (Hyp) -GP- (Hyp) -NH 2 Through biological verification, the composition has the effects of tightening and resisting aging. Characterization of binding by Transmission Electron microscopyThe rear nano micelle structure is nano particles, supermolecule nano particles are formed, the particle size is about 100nm, and the sequence of the collagen peptide is as follows: ac-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly-Glu-Arg-Gly-Pro-Hyp-Gly-Pro-Hyp-NH 2 . Verification of efficacy at cellular and tissue level: the botulinum-like polypeptide nano micelle loads collagen peptide, not only can play a role in resisting wrinkle, but also can play a role in tightening and resisting aging.
(6) Selecting a bee venom peptide with hydrophilic property, and the sequence is H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH 2 Has remarkable anti-inflammatory effect, can be combined with hydrophilic shells of the botulinum-like polypeptide nano-micelle, is applied to cosmetics, and can play a role of relieving when being used for resisting wrinkles. The botulinum-like polypeptide nano-micelle hydrophilic shell loads hydrophilic melittin, and supermolecule nano-particles are formed through non-covalent bond self-assembly, and the particle size is about 100nm.
In the above step (2), the substitution degree of Rink Amide MBHA Resin is preferably 0.5 to 0.6mmol/g.
In the step (3), the concentration of the botulinum-like polypeptide nano micelle solution is preferably 0.2mg/mL, and the concentration is less than 0.2mg/mL by the characterization of a transmission electron microscope, so that the nanoparticles cannot be formed.
In the step (4), the carboxyl terminal of the botulinum-like polypeptide is preferably labeled with Cy5, and fluorescent labeling is performed to prepare nano micelle; preferably, the hexapeptide is used as an efficacy verification bid product control group to carry out anti-wrinkle activity comparison experiments with the botulinum-like nano-micelle.
In the step (5), the concentration ratio of the botulinum-like polypeptide nano micelle to the collagen peptide is preferably 2:1.
In the step (6), the concentration ratio of the botulinum-like polypeptide nano micelle to the melittin is preferably 2:1.
Compared with the prior art, the invention has the following beneficial effects:
1. the botulinum-like polypeptide is an amphiphilic molecule, and in an aqueous solution, a lipophilic group is inward, and a hydrophilic group is outward and can be self-assembled into a nano micelle. The nano micelle can rapidly penetrate through the skin barrier, dissociate into polypeptide monomers after transdermal penetration, exert the anti-wrinkle effect, and can be applied to the technical field of cosmetics.
2. The invention develops an application technology of polypeptide nano micelle, which is defined as POLYPP TM Polypeptide hypertonic delivery techniques. The polypeptide with stronger functions is preferably subjected to biological modification to self-assemble into supermolecule spherical microcapsules, so that the supermolecule spherical microcapsules play roles among cells. The botulinum-like polypeptide nano micelle disclosed by the invention has the advantages that the polypeptide sequence contains 15 amino acids, the length of the 15 amino acids is difficult to transdermal, but the nano micelle can be quickly transdermal after the botulinum-like polypeptide nano micelle is formed, the difficult problem of difficult transdermal due to strong long peptide efficacy in cosmetics is perfectly solved, and the innovation of a cosmetic polypeptide delivery mode is realized. The whole preparation process does not contain any exogenous substances, completely avoids the addition of nano gold, has high biocompatibility and high safety, and has doubled anti-wrinkle effect compared with the commercial product of hexapeptide.
3. The general polypeptide nano-micelle comprises a lipophilic inner core and a hydrophilic outer shell, and in the field of pharmaceutical peptides, the polypeptide nano-micelle is mostly applied with the lipophilic inner core, can embed or load a plurality of drug effect molecules with poor water solubility, improves the drug loading capacity, and has a fresh report on the function of the hydrophilic outer shell; the botulinum-like polypeptide nano micelle disclosed by the invention comprises a lipophilic inner core and a hydrophilic outer shell, wherein the hydrophilic outer shell can load hydrophilic polypeptides with other effects, and the botulinum-like anti-wrinkle effects are exerted, and meanwhile, other effects, such as the tightening effects of combining collagen peptide and the anti-inflammatory and soothing effects of combining melittin, are exerted, so that the technical effect of double effects of the double peptides is achieved.
Drawings
FIG. 1 is an HPLC profile of a botulinum-like polypeptide;
fig. 2 is an ESI-MS profile of a botulinum-like polypeptide, m= 2135.3;
FIG. 3 is a transmission electron microscope image (TEM image) of a botulinum-like polypeptide nano-micelle with a (CMC=0.2 mg/mL) particle size of less than 50nm;
FIG. 4 is a transmission electron microscopy image at a concentration of 0.1mg/mL of a botulinum-like polypeptide nano-micelle solution;
FIG. 5 is a permeation experiment of fluorescence intensity of a milk pig skin based botulinum-like polypeptide nano-micelle;
a, fluorescence microscopic observation, B, accumulated permeation quantity;
FIG. 6 is a graph showing the results of cell proliferation experiments of botulinum-like polypeptide nano-micelles based on fibroblasts;
FIG. 7 is a graph showing the results of cell migration experiments of botulinum-like polypeptide nano-micelles based on fibroblasts;
a,4 times mirror image, B, relative mobility average;
FIG. 8 is an experimental result of in vitro anti-wrinkle verification detection of Collage I based on UVA irradiated fibroblasts;
FIG. 9 is an experimental result of in vitro anti-wrinkle verification detection of matrix metalloproteinase MMP-1 based on UVA irradiation of fibroblasts;
FIG. 10 is a graph showing anti-wrinkle efficacy verification-detection of collagen density results based on UVA irradiation of ex vivo skin;
FIG. 11 is a graph showing anti-wrinkle efficacy verification-detection of the content of Collagen IV based on UVA irradiation of the skin ex vivo;
a, staining chart, B, relative IOD/Area average;
FIG. 12 shows measurement results of neurotransmitter acetylcholine content based on HT22 cells;
FIG. 13 is a comparison of the safety of introduction of botulinum-like polypeptide nanomicelles and nanogold polypeptide hybrids;
a, a post-operation photo of polypeptide nanometer hybrid, B, a post-operation photo of the botulinum-like polypeptide micelle;
fig. 14 is ESI-MS of collagen peptide, m= 2055,3;
FIG. 15 is a TEM image of a botulinum-like collagen composite nano-micelle with a particle size of less than 100nm;
FIG. 16 is a graph showing detection of collagen fiber content of a botulinum-like polypeptide nano-micelle loaded collagen peptide based on UV-stimulated ex vivo skin;
a, staining chart, B, relative IOD/Area average;
fig. 17 is ESI-MS diagram of melittin, m= 2846.5;
FIG. 18 is a TEM image of botulinum-like bee venom complex nanomicelle having a particle size of about 100nm;
fig. 19 is a graph showing the results of macrophage-based anti-inflammatory experiments with botulinum-like venenum nanomicelles.
In the drawings of the present invention, for simplicity of labeling, the botulinum-like polypeptide nano-micelles are labeled as tylosin bundles; the nanometer micelle formed by loading the collagen peptide with the botulinum-like polypeptide is used for drawing, and the nanometer micelle of the melittin with the botulinum-like polypeptide is used for drawing.
Detailed Description
In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings.
In the description of the present invention, unless otherwise specified, all reagents are commercially available and methods are conventional in the art.
In the following, a brief description of the specification is given, rink Amide MBHA Resin (Tianjin Nankai and Technical Co., ltd.) with a substitution of 0.5mmol/g; otBu: t-butoxy; trt: a trityl group; pbf:2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl; tBu: a tertiary butyl group; myristonyl-Cl: myristoyl chloride; palmitoyl-Cl: palmitoyl chloride.
The analytical high performance liquid chromatograph is Agilent 1260; the preparation type high performance liquid chromatograph is innovative constant-pass LC3000, the C18 analysis chromatographic column is 4.6mm multiplied by 250mm of Dalian physical chemistry research institute, and the C18 preparation chromatographic column is 10cm multiplied by 65cm of Chengdu science popularization biological limited company; the mass spectrometer was a Waters UPLC-lc.
Example 1
A botulinum-like polypeptide nano-micelle, the botulinum-like polypeptide sequence is: R-Glu-Asn-Arg-Ser-Phe-Met-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala-NH 2 (SEQ ID NO. 1), R is Myr (myristoyl) or Pal (palmitoyl).
Example 2
A method for preparing a botulinum-like polypeptide nano-micelle, comprising:
1. design and preparation of botulinum-like polypeptides
(1) Computer-simulated design of polypeptide sequences:
based on SNAP-25 and Rabpylin-3A crystal structures, natural amino acid mutation is carried out on corresponding amino acid sites by using Discovery studio software, and then roots are obtainedAccording to the energy minimization principle, energy minimization simulation was performed on the mutant complex proteins. According to computational chemistry, calculating the polypeptide hydrophilicity and hydrophobicity and charge distribution to obtain an optimal sequence: ENRSFMELEEMQRRA. Modifying the polypeptide nitrogen end with hydrophobic group, preferably myristoyl modification, to obtain Myr-ENRSFMELEEMQRRA-NH 2 ；
(2) Synthesis of botulinum-like polypeptides:
(2.1) Synthesis of Fmoc-Ala-Rink Amide MBHA Resin
10g Rink Amide MBHA Resin (SD=0.5 mmol/g) was weighed and put into a synthesis column, the mixture was swelled with 80mL of N, N-dimethylformamide for 4 hours, the solvent was removed by suction filtration, 80mL of a 20% by volume solution of piperidine in N, N-dimethylformamide was used to remove Fmoc protecting group for 40 minutes, suction filtration was performed, the filter cake was washed twice with isopropyl alcohol and N, N-dimethylformamide in this order, 80mL each time, ninhydrin detection resin was black, 100mL of N, N-dimethylformamide, 3.1g (10 mmol) of Fmoc-Ala-OH, 2g (15 mmol) of 1-hydroxybenzotriazole, 2.3mL (15 mmol) of N, N' -diisopropylcarbodiimide were added to the synthesis column, and the mixture was stirred at room temperature for 2 hours under nitrogen protection, and the ninhydrin detection resin was washed with isopropyl alcohol and N, N-dimethylformamide in this order to obtain Fmoc-Ala-Rink Amide MBHA Resin.
(2.2) Fmoc protected fully protected peptide resin Synthesis
6.4g (10 mmol) of Fmoc-Arg (Pbf) -OH, 6.1g (10 mmol) of Fmoc-Gln (Trt) -OH, 3.7g (10 mmol) of Fmoc-Met-OH, 4.2g (10 mmol) of Fmoc-Glu (OtBu) -OH, 3.5g of Fmoc-Leu-OH, 4.2g (10 mmol) of Fmoc-Glu (OtBu) -OH, 3.7g (10 mmol) of Fmoc-Met-OH, 3.8g of Fmoc-Phe-OH, 3.8g of Fmoc-Ser (tBu) -OH, 6.4g (10 mmol) of Fmoc-Glu (OtBu) -OH, 4.2g (10 mmol) of Fmoc-Glu (OtBu) -OH, synthesis of Fmoc-Glu (OtBu) -Asn (Trt) -Arg (Pbf) -Ser (tBu) -Phe-Met-Glu (OtBu) -Leu-Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Rink Amide MBHA Resin.
(2.3) removing the protecting group by using a piperidine/DMF solution with the volume fraction of 20%, and carrying out final nitrogen end modification by using myristoyl chloride to obtain the full-protection peptide resin. The method comprises the following steps:
80mL of 20% piperidine N, N-dimethylformamide solution is added to the synthesis column to remove Fmoc protecting groups for 40 minutes, suction filtration is carried out, filter cakes are washed twice with isopropanol and N, N-dimethylformamide in sequence, 80mL of ninhydrin detection resin is black, 100mL of dichloromethane, 7mL of myristoyl chloride, 15mLN and N-diisopropylethylamine are added to the synthesis column, the ninhydrin detection resin is reacted for 1 hour and is colorless and transparent, and Myr-Glu (OtBu) -Asn (Trt) -Arg (Pbf) -Ser (tBu) -Phe-Met-Glu (OtBu) -Leu-Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Rink Amide MBHA Resin is washed with isopropanol, N-dimethylformamide and methanol in sequence to dry the resin.
(2.4) cracking the full-protection peptide resin under the action of trifluoroacetic acid, and simultaneously removing the side chain protecting group of the polypeptide to obtain a crude polypeptide. The method comprises the following steps: adding 132mL of trifluoroacetic acid, 7.5mL of triisopropylsilane, 6mL of phenylsulfide and 4.5mL of water into the dried resin, carrying out suction filtration after 2.5 hours, concentrating the filtrate, decompressing and removing TFA, adding 10 times of cold diethyl ether into the concentrated solution, crystallizing and stirring for 1 hour, filtering, collecting a filter cake, and carrying out vacuum drying to obtain a crude product of the botulinum-like polypeptide (trifluoroacetate);
(2.5) purification:
adding 50% acetonitrile water solution into crude product of botulinum-like polypeptide, adding small amount of trifluoroacetic acid to assist dissolution, filtering, fixing volume, purifying by reverse phase chromatography (filler: C18), and performing chromatography: phase A0.1% TFA/H 2 O (v/v), phase B0.1% TFA/ACN (v/v), phase A equilibrated loading, elution procedure: 25% -85% of 60min, and collecting the eluent. The elution procedure was followed using 50mmol/L ammonium acetate buffer for salt transfer: 55% -85%60min, and lyophilizing to obtain pure botulinum-like polypeptide acetate (hereinafter called botulinum-like polypeptide lyophilized powder) 5.1g, i.e. Myr-Glu-Asn-Arg-Ser-Phe-Met-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala-NH 2 The yield thereof was found to be 48%.
High performance liquid chromatography and mass spectrometry were used to characterize the structure and purity of the polypeptides, respectively. High performance liquid chromatography testing; purity greater than 90% (as shown in figure 1). Electrospray mass spectrometry ESI-MS was used for structural characterization: (M+3H/3,100) (as shown in FIG. 2).
Supplementary explanation: when R in the amino acid sequence of a botulinum-like polypeptide is palmitoyl modified, reference may be made to the preparation of a myristoyl-modified polypeptide, but replacement of the myristoyl-modified moiety is required. Other chemical methods may also be used to synthesize the amino acid sequence of the botulinum-like polypeptide.
2. Preparation of polypeptide nano micelle
Respectively weighing 10mg, 25mg and 50mg of myristoyl modified botulinum-like polypeptide freeze-dried powder, respectively adding the powder into 50mL of double distilled water to prepare 0.2mg/mL, 0.5mg/mL and 1mg/mL solutions, and carrying out ultrasonic oscillation for 5-10 min to obtain colorless solution of the botulinum-like polypeptide nano-micelle.
3. Structural characterization of polypeptide nanomicelle
Characterizing the polypeptide nano micelle solution by using a transmission electron microscope, and performing negative dyeing treatment on a sample by using 3% phosphotungstic acid; particle morphology at different concentrations was examined: the samples showed nanoparticle morphology at concentrations of 1mg/mL, 0.5mg/mL, and 0.2mg/mL (as shown in FIG. 3). The particle size is measured by dynamic light scattering and is less than 50nm.
As measured by experiment: at a concentration of 0.2mg/mL, the polypeptide solution can form nano-micelles, and less than 0.2mg/mL cannot form nano-micelles. As shown in FIG. 4, at a concentration of 0.1mg/mL, almost no nanoparticles could be observed.
4. Permeability test of polypeptide nanomicelle
Based on a milk pigskin-Franz cell system, the distribution of fluorescence-labeled botulinum-like polypeptide nano-micelles and fluorescence-labeled pentadecapeptides (the polypeptide nano-micelles are the pentadecapeptides disclosed in CN113512092A for permeability test experiments) in pigskin at different time points is observed through a fluorescence microscope, and the skin permeation behaviors of different samples are evaluated. The method comprises the following steps:
(1) Fluorescent labeling of polypeptides: dissolving the botulinum-like polypeptide in an aqueous solution, adding 1.2 equivalents of Cy5 fluorescent dye, carrying out light-shielding reaction for 24 hours, carrying out dialysis treatment, and freeze-drying to obtain red freeze-dried powder, and sequentially preparing fluorescent-labeled botulinum-like polypeptide nano-micelles; as a control group, fifteen peptides were also treated with Cy5 fluorescent label.
(2) Based on the milk pigskin-Franz cell system, the skin permeation behavior of different samples was evaluated by fluorescence microscopy of the distribution of fluorescent marker substances in the pigskin at different time points.
(3) Experimental results: the fluorescence intensity values at the time points of 2h and 4h show that the accumulated permeation amounts of the botulinum-like polypeptide nano micelle fluorescent marker and the pentadecapeptide fluorescent marker are larger than that at the previous time point, namely, the sample permeation amount is continuously increased along with the time; the cumulative permeation amounts were ordered at 2h, 4 h: botulinum-like polypeptide nanomicelle fluorescent label >15 peptide fluorescent label (as shown in fig. 5). The final results showed that: the transdermal permeability of the botulinum-like polypeptide nano micelle is far higher than that of pentadecapeptide.
5. Anti-wrinkle activity verification of polypeptide nano-micelle
(1) Cell proliferation assay based on fibroblasts:
detecting the change of cell proliferation and cell migration after the action of a sample based on fibroblasts; and (3) irradiating the fibroblasts by adopting UVA, and evaluating the anti-wrinkle effect of the sample to be tested by detecting the change condition of the content of Collagen I (Collagen I) protein and matrix metalloproteinase 1 (MMP-1) after the sample acts.
Sample working solutions were prepared according to table 1 and incubated overnight in an incubator.
TABLE 1 fibroblast-based cell proliferation test sample formulation
Note that: "/" indicates no related operations.
Dosing was performed according to the test protocol of Table 1, with culture in an incubator for 24h, 48h, 72h, respectively, after incubation of the cells, the supernatant was discarded, MTT working solution (0.5 mg/mL, as prepared) was added, and the results were expressed as mean+ -SD, calculated according to the cell viability equation and plotted using GraphPad Prism. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences, indicating that: compared with a blank control group (hexapeptide), the botulinum-like polypeptide nano micelle has obviously increased cell viability after 24 hours, 48 hours and 72 hours of administration, and the lifting rates are respectively 12.92%, 42.77% and 60.98%, as shown in fig. 6.
(2) Cell scratch test based on fibroblasts (cell migration experiment based on fibroblasts): cell inoculation, liquid preparation and administration, and scratching after incubation of the sample is finished. Two passes were drawn longitudinally in a six-well plate with a 5mL gun head, with vertical marks as the base line (gun head vertical ruler edge), with mark spacing maintained at 2cm; after the vertical scratches are made, the horizontal scratches are made at the vicinity of the central axis of the six-hole plate perpendicular to the base line. The gun head is used for scratching, so that the force is uniform as much as possible, and the width of the scratch is kept consistent as much as possible. After the scratch was completed, the cells were washed 3 times with PBS, and then added with a normal cell culture solution, incubator (37 ℃,5% CO) 2 ) Culturing for 24h. After scratch, photographing under a 4-fold mirror for 0h, and after scratch, washing with PBS once for 24h, photographing under the 4-fold mirror. The relative mobility of each group was calculated by normalizing the Blank (BC) group mobility. The results show that: compared with a blank control group, the botulinum-like polypeptide nano micelle and the hexapeptide have remarkably increased cell mobility after 24 hours of administration, and the increase rates are 56% and 37% respectively. The mobility of botulinum-like polypeptide nanomicelle cells was increased compared to the hexapeptide group as shown in fig. 7.
(3) Anti-wrinkle efficacy test based on UVA-stimulated fibroblasts: cell seeding, dispensing according to the following table protocol.
Table 2 anti-wrinkle efficacy test sample formulation based on UVA stimulated fibroblasts
Note that: "/" indicates no related operations.
According to the test protocol of Table 2, the group administration was performed in an incubator (37 ℃ C., 5% CO) 2 ) And incubated for 24h. According to the test scheme, UVA irradiation is carried out on the groups needing to be irradiated, and the irradiation dose is 30J/cm 2 . After the irradiation, the mixture was placed in an incubator (37 ℃ C., 5% CO) 2 ) The culture was continued for 24 hours. And (3) sample collection: after 24h of incubation, the cell culture supernatant was collected in an EP tube and stored in a freezer at-80 ℃. After 24h incubation, the cell culture supernatants were collected in EP tubes, stored frozen in a refrigerator at-80℃and tested according to the instructions of ELISA kit, plotted using GraphPad Prism, and expressed as mean.+ -. SD. The results show that: compared with a negative control group, the content of the Collagen I of the botulinum-like polypeptide nano micelle and the hexapeptide is obviously increased, and the increase rate is 107.73% and 21.73%.
The Collagen I content of the tenascus was significantly increased compared to the hexapeptide group (as shown in fig. 8); compared with a negative control group, the content of the matrix metalloproteinase MMP-1 of the polypeptide nano-micelle and the hexapeptide is obviously reduced, and the reduction rate is 28.05% and 19.44%. Compared to the hexapeptide group, the MMP-1 content of the botulinum-like polypeptide nano-micelle group was significantly reduced (as shown in FIG. 9).
(4) Anti-wrinkle efficacy test based on ex vivo skin tissue: sample working solutions were prepared according to the following table.
TABLE 3 anti-wrinkle efficacy test sample formulation based on ex vivo skin tissue
Note that: "/" indicates no related operations.
Immersing freshly obtained skin tissue in 75% alcohol, washing for 30s, and then buffering with sterile PBSWashing with the flushing liquid for three times; after the completion, the skin was cut into pieces of 24.+ -. 2mm 2 Is placed into a culture mold, then the culture mold is transferred into a 6-well plate, 3.7mL of culture solution is added into each well, and the temperature is 37 ℃ and the concentration of CO is 5% 2 Culturing in incubator, and changing liquid every day; after 2 days of in vitro skin tissue culture, irradiation and administration were started with reference to the test protocols and corresponding treatment conditions in table 3; the irradiation dose was UVA (30J/cm) 2 ) And UVB (50 mJ/cm) 2 ) Continuously irradiating for 4 days, changing fresh culture solution after each irradiation, and carrying out drug administration treatment, wherein a sample to be tested is carried out in a surface drug administration mode; after 4 days of continuous irradiation, the isolated skin tissue was cultured for 3 further days, during which time no irradiation was performed and only sample administration was performed. The detection was performed according to the immunohistochemical procedure, the detection was performed according to the collagen density specific procedure using a skin sonicator, and GraphPad Prism was used, and the result was expressed as mean±sd. The results show that: the collagen density content of the botulinum-like polypeptide nano-micelle group was increased compared to the botulinum injection (as shown in fig. 10); the Collagen IV content of the botulinum-like polypeptide nano-micelle group was increased compared to the botulinum injection (as shown in fig. 11).
(5) Acetylcholine content detection on HT22 cells: based on mouse hippocampal nerve cells, the content of neurotransmitter acetylcholine is tested by using a chromogenic method at the level of 2D cells and the level of a 3D skin model, and hexapeptide is selected as a bid product control. The results show that: compared with a blank control group, the content of acetylcholine in the hexapeptide and botulinum-like polypeptide nano-micelle group is obviously reduced, and the reduction rates are 23.29% and 49.60% respectively. Compared with hexapeptide, the content of acetylcholine in the botulinum-like polypeptide nano-micelle group is significantly reduced (as shown in figure 12).
(6) Human body safety verification of botulinum-like polypeptide nano micelle
The polypeptide nanometer hybrid containing the nanometer gold generates a series of adverse reactions when being applied to leading-in products: the botulinum-like polypeptide micelle is used as an introduced product, and has no allergic stimulus response. As shown in fig. 13.
Using the nanogold-containing polypeptide nanohybrids (the polypeptide nanohybrid structure disclosed in example 1 of CN 113512092A), a plating hand needle was used for periorbital and frontal treatment with a total dose of 0.4ml with slight swelling immediately after surgery, accompanied by a skin dome (fig. 13A). Whereas, with botulinum-like polypeptide micelles injected, after seven days of use, the macroscopic true wrinkles became shallow and the numerous fine lines disappeared, the VISIA data indicated 76% improvement in ocular wrinkles with no skin irritation response (fig. 13B).
Example 3
A preparation method of a botulinum-like polypeptide nano micelle loaded collagen peptide comprises the following steps:
(1) Preparation of collagen peptide: rink Amide MBHA Resin is selected, and a solid-phase polypeptide synthesis technology is adopted to synthesize collagen peptide in a manner of synthesizing botulinum-like polypeptide, so as to obtain a crude collagen peptide; purifying collagen peptide crude product by reversed phase liquid phase preparative chromatography, and freeze drying to obtain collagen peptide pure product, namely Ac-GP- (Hyp) -GF- (Hyp) -GERGP- (Hyp) -GP- (Hyp) -NH 2 . Molecular weight was determined using electrospray ionization mass spectrometry (ESI-MS), and the ESI-MS of the collagen peptide is shown in FIG. 14.
(2) Preparation of botulinum-like polypeptide nano-micelle loaded collagen peptide: 3mg of collagen peptide is weighed and dissolved in 10mL of pure water, ultrasonic is carried out to dissolve completely, 6mg of botulinum-like polypeptide is weighed and dissolved in 20mL of pure water, and the collagen peptide-containing solution and the botulinum-like polypeptide-containing solution are mixed and stirred uniformly, thus completing the preparation of the botulinum-like collagen nanocomposite (composite nano micelle). The structural morphology was examined using a transmission electron microscope, and the sample was negatively stained with 3% phosphotungstic acid and the structural morphology was nanoparticle (as shown in fig. 15).
(3) Based on the tightening and anti-wrinkle efficacy test of the isolated skin tissue: sample working solutions were prepared according to the following table
TABLE 4 preparation of test samples for anti-wrinkle efficacy based on tightening of ex vivo skin tissue
Note that: "/" indicates no related operations.
Tissue-treated and then administered, ex vivo skin, according to the method of Table 4After tissue culture for 2 days, carrying out irradiation and drug administration according to conditions, continuously irradiating for 4 days, changing fresh culture solution after each irradiation, carrying out drug administration treatment, carrying out surface drug administration on a sample to be tested, fixing a model of skin tissue after drug administration by adopting 4% paraformaldehyde, embedding the tissue, respectively carrying out Masson staining after slicing, recovering slicing results, photographing by using a microscope, and using
Plus image processing software performs the analysis. The results show that: the collagen fiber content of the tenascin peptide group was increased compared to commercially available palmitoyl pentapeptide-4 (as shown in fig. 16).
Example 4
A preparation method of a botulinum-like polypeptide nano micelle loaded melittin comprises the following steps:
(1) Preparation of melittin: rink Amide MBHA Resin is selected, and a solid-phase polypeptide synthesis technology is adopted to synthesize the melittin in a manner of synthesizing the botulinum-like polypeptide, so as to obtain a crude melittin product; purifying the crude product with reversed phase liquid phase preparative chromatography, and lyophilizing to obtain pure product of melittin, namely H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH 2 . Molecular weight was determined using electrospray ionization mass spectrometry (ESI-MS), which is shown in FIG. 17.
(2) Preparation of a botulinum-like bee venom composite nano micelle: weighing 3mg of melittin, dissolving in 10mL of pure water, completely dissolving by ultrasonic, weighing 6mg of botulinum-like polypeptide, dissolving in 20mL of pure water, mixing the solution containing the melittin and the solution containing the botulinum-like polypeptide, and stirring and uniformly mixing to obtain the botulinum-like bee venom nanocomposite. The structural morphology was examined using a transmission electron microscope, and the sample was negatively stained with 3% phosphotungstic acid and the structural morphology was nanoparticle (as shown in fig. 18).
(3) Macrophage-based anti-inflammatory assay: the samples were tested under the following conditions.
TABLE 5 preparation of test samples for anti-wrinkle efficacy based on tightening of ex vivo skin tissue
Note that: "/" indicates no related operations.
The results show that: the sample tylosin can obviously reduce the content of IL-6, and the inhibition rates can respectively reach 67.84 percent, as shown in figure 19.
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. The preparation method of the botulinum-like polypeptide nano micelle is characterized by comprising the following steps of:
design of a botulinum-like polypeptide: based on SNAP-25 and Rabpylin-3A crystal structures, a polypeptide sequence capable of blocking interaction between the SNAP-25 and Rabpylin-3A crystal structures is obtained through simulation design, and a hydrophobic group modification is carried out on the nitrogen end of the polypeptide sequence;
preparation of a botulinum-like polypeptide: synthesizing a crude polypeptide sequence modified by a hydrophobic group, purifying and freeze-drying to obtain a purified botulinum-like polypeptide;
preparation of botulinum-like polypeptide nano-micelles: the purified botulinum-like polypeptide is prepared into a solution with the concentration of more than or equal to 0.2mg/mL, and the botulinum-like polypeptide is self-assembled into nano-micelles.
2. The method of claim 1, wherein the hydrophobic group is modified to be myristoyl or palmitoyl.
3. The method for preparing a botulinum-like polypeptide according to claim 1, wherein the polypeptide sequence modified by hydrophobic groups is synthesized in an N, N-dimethylformamide system by Fmoc synthesis strategy using a solid-phase organic synthesis technique of the polypeptide using a resin as a carrier, purified and lyophilized to obtain the purified polypeptide.
4. The botulinum-like polypeptide produced by the method of claim 2, wherein the amino acid sequence of the botulinum-like polypeptide is:
R-Glu-Asn-Arg-Ser-Phe-Met-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala-NH 2 r is myristoyl or palmitoyl.
5. The botulinum-like polypeptide nano-micelle prepared by the method of claim 2, wherein the critical micelle concentration of the botulinum-like polypeptide nano-micelle is 0.2mg/mL.
6. The use of a botulinum-like polypeptide nano-micelle according to claim 5, wherein the botulinum-like polypeptide nano-micelle is used for the preparation of an anti-wrinkle, anti-aging product.
7. The use of a botulinum-like polypeptide nano-micelle according to claim 5, wherein the nano-micelle structure comprises a lipophilic inner core and a hydrophilic outer shell, which is capable of loading a hydrophilic active ingredient through a non-covalent bond, and has the efficacy of the active ingredient while preventing wrinkles.
8. The use of a botulinum-like polypeptide nano-micelle according to claim 5, wherein the botulinum-like polypeptide nano-micelle hydrophilic shell is loaded with a hydrophilic collagen peptide or melittin to form supramolecular nanoparticles for the preparation of cosmetic products.
9. The use of a botulinum-like polypeptide nano-micelle according to claim 8, wherein when the botulinum-like polypeptide nano-micelle is loaded with collagen peptide, it is in the form of a botulinum-like-collagen composite nano-micelle for preparing an anti-wrinkle, compact product.
10. The use of a botulinum-like polypeptide nano-micelle according to claim 8, wherein the botulinum-like polypeptide and the melittin form supramolecular nanoparticles having the structure of a botulinum-like-melittin composite nano-micelle for the preparation of anti-wrinkle, soothing products.
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