CN114181284B

CN114181284B - Application of nano short peptide DRF3 in medicine, NK cell carrier and biomedicine

Info

Publication number: CN114181284B
Application number: CN202111358985.1A
Authority: CN
Inventors: 罗忠礼; 万源; 罗茹月; 张宇
Original assignee: Chengdu Saienbei Academy Of External Sciences
Current assignee: Chengdu Saienbei Academy Of External Sciences
Priority date: 2021-11-17
Filing date: 2021-11-17
Publication date: 2024-07-19
Anticipated expiration: 2041-11-17
Also published as: CN114181284A

Abstract

The invention discloses application of nano short peptide DRF3 in medicines, NK cell carriers and biomedicine, relates to the field of self-assembled short peptides, and solves the problems of how to produce hyaluronic acid products with stronger degradation resistance, weaker toxicity, in-situ activation of collagen regeneration and the like, and the amino acid sequence of the hyaluronic acid products is as follows: arg Leu Asp Ile Lys Val Glu Phe Arg Leu Asp Ile Lys Val Glu Phe, the self-assembled short peptide activates and enhances the purity and killing ability of NK cells; but also shows good capacity of promoting DC cell maturation; the mechanical strength of the traditional short peptide nanometer three-dimensional scaffold is improved, the short peptide nanometer three-dimensional scaffold can be used as a carrier and an adjuvant of medicines, biological macromolecules and proteins, and can be respectively combined with hyaluronic acid to prepare a combined system which has better effect, stability, safety and resistance to degradation and lower toxicity compared with the single use of hyaluronic acid, and the short peptide can also be used as a single filler.

Description

Application of nano short peptide DRF3 in medicine, NK cell carrier and biomedicine

Technical Field

The invention relates to the technical field of self-assembled short peptides, in particular to application of self-assembled short peptides in biology, nano medicine, cosmetics and health care products.

Background

Short peptides are ubiquitous in nature. They are found as hormones, pheromones, antibiotics, antifungals, the innate immune system, toxins and pesticides. However, no one has recognized that peptides may be useful as scaffold hydrogel materials. Significant changes have occurred since 1990 when a very interesting repeat was found in yeast proteins. It is now recognized that self-assembled peptides made from 20 natural amino acids have true material properties. Currently, many different applications have evolved from these simple and designed self-assembled peptide scaffold hydrogels and are commercially available. Examples include: (1) the actual 3D tissue cell culture of different tissue cells and various stem cells, (2) repair and regeneration medicine and tissue engineering, (3) 3D tissue printing, (4) sustained release of small molecules, growth factors and monoclonal antibodies, (5) acceleration of wound healing skin and diabetic ulcers and immediate hemostasis applications.

Molecular self-assembly refers to the fact that molecules can self-organize and self-aggregate to form a regular structure under the condition that the molecules are not intervened by external force, namely, the molecules can be converted from a disordered state to an ordered state. In recent years, chiral self-assembled short peptides have been developed as an emerging nano-biomaterial. The biological scaffold nano material imitates the biological functions of ECM, so as to influence biological behaviors such as cell migration, proliferation, differentiation and the like, can be used as a matrix material for three-dimensional cell culture, and has certain effects on wound repair, tissue injury repair and the like.

Natural killer cells (Natural KILLER CELLS, NK) are important immune cells of the innate immune system, are the first line of defense of the human body against cancer cells and virus infection, act as regulators of immune response, have strong immunoregulatory function, can have anti-fibrosis activity by directly killing hepatic stellate cells, when the organism is affected by exogenous infection and tumor cells, the NK cells are activated, have proliferation and killing activity, can well clear necrotic cells and tumor cells affected by inflammation, identify and kill stressed, transformed or virus-infected cells by expressing balance of activating and inhibiting receptor signals, and secrete various effector molecules, and after the NK cells are activated, the NK cells secrete active factors promoting migration, and under factors such as individual differences, cell subpopulations differences, whether the NK cells are activated or not, the types and levels of chemokine receptors are greatly different. How to create a good three-dimensional microenvironment for NK cells and activate the NK cells, reduce the death rate of the NK cells and increase the activity of the NK cells, and prepare the NK vaccine with stronger activity, stronger immunogenicity and longer release time is one of key problems to be solved.

In vivo DCs are classified into lymphoid system DC (lymphoid DC) and myeloid system DC (myeloid DC), the latter being the source of most DCs. Lymphoid DCs develop from precursor cells in the thymus, which release substantial amounts of type I interferon (IFN 1) after activation, and are involved in viral immune responses; myeloid DCs are primarily involved in the induction and initiation of immune responses. Under normal conditions in humans, most DCs are in an immature state, located in the epithelium of non-lymphoid tissues and in various solid organs, and have strong antigen uptake, processing and handling capacity, but they express low levels of costimulatory molecules and major histocompatibility complex class ii molecules (maior histocompatib ility complex class ii molecules, MHC ii) and intercellular adhesion molecules (intercellular cell adhesion molecules, ICAM). How to activate DC cells and activate intercellular signaling, and the preparation of DC vaccine with stronger immunogenicity and longer release time is the second problem to be solved.

With the popularization of cosmetics in recent years, the cosmetics are deeply loved and focused by the young people, the development of whitening, anti-aging, anti-wrinkle and the like is increasingly rapid, and the cosmetics are always highly valued and widely focused in the relevant subject fields, in particular the fields of medical cosmetology, cosmetic science, skin care and health care, skin anti-aging and the like.

It is well known that hyaluronic acid is one of the basic components of connective tissue in humans and other mammals. It is a substance that is widely found in human epidermis, epithelium and nervous tissue. Hyaluronic acid imparts unique resistance and shape retention to the skin. The lack of hyaluronic acid can lead to skin weakening, promoting the formation of wrinkles and blemishes. With age, the concentration of hyaluronic acid in human tissue tends to decrease, thereby impairing its tissue repair function. With progressive aging and repeated exposure to ultraviolet light, the epidermal cells reduce the production of hyaluronic acid, and the aging rate increases. For this reason, HA-based formulations are still considered to be the best epidermal fillers on the market today, since they do not have the risk of skin allergy. Initially, the first hyaluronic acid-based formulation was prepared in the form of particles or microspheres suspended in a gel. However, these gel microsphere-based fillers suffer from the disadvantage of poor stability over time, tending to degrade chemically several months after injection into the skin. Thus, frequent re-injection of filler is required over time to maintain constant repair and epidermal growth. Recently, it has been found that the advantage of subjecting hyaluronic acid to a suitable crosslinking step by using a specific crosslinking agent, so that fillers based on crosslinked hyaluronic acid are used in cosmetic treatments of the face. Frequent injections are still required, and the effects, stability and safety are generally good.

How to construct a three-dimensional cell microenvironment with better biocompatibility, truly simulating cell growth, larger mechanical strength and faster gel formation, how to obtain products with little or no toxicity, stronger degradation resistance, new in-situ activated collagen and the like, which are similar to the application of crosslinked and non-crosslinked hyaluronic acid, and how to construct a maturation-promoting agent with good biocompatibility, little toxicity, capability of independently enhancing in-vivo immunity and good inclusion, and is a great problem to be solved urgently for preparing vaccines with better effects.

Disclosure of Invention

The invention aims at: in order to solve the technical problems, the invention provides application of the nano short peptide DRF3 in medicines, NK cell carriers and biomedicine.

The invention adopts the following technical scheme for realizing the purposes: a self-assembled short peptide having the amino acid sequence:

DRF3：Arg Leu Asp Ile Lys Val Glu Phe Arg Leu Asp Ile Lys Val Glu Phe。

In the technical scheme of the application, a novel self-assembled short peptide is provided, the novel self-assembled short peptide can be mixed with PBS and then self-assembled into injectable hydrogel to serve as a three-dimensional culture bracket of cells, the bracket simulates an extracellular mechanism, not only serves as a physical bracket, but also promotes maturation and differentiation of the cells, and a degradation product is amino acid and has no toxicity; the recombinant DNA can also be used as a maturation promoting agent of DC cells, can increase the killing capacity of NK, and can be used for preparing NK vaccines; the application uses a new self-assembled short peptide as a new thinking of preparing cosmetics and an adjuvant respectively, changes the content of the cosmetics from the source, and the degradation products are amino acids, thus the self-assembled short peptide can be used as a humectant, a slow release agent, a lubricant, an antioxidant, a film forming agent and an emollient, has an auxiliary effect on skin repair, is equivalent to skin nourishment, and has good biocompatibility because the degradation products are amino acids; the novel self-assembled short peptide combines the physical properties and the microstructure of the self-assembled short peptide, is respectively combined with hyaluronic acid to prepare a combined system which has better effect, more stability, better safety, stronger resistance to degradation and smaller toxicity than the single use of hyaluronic acid, and can also be used as a single filler.

Further, the amino acid in the self-assembled short peptide is one or more of L-form, D-form or DL-form.

Furthermore, the amino acids in the self-assembled short peptide are all L-shaped.

Further, the carbon end of the self-assembled short peptide is amidated.

Further, the self-assembled short peptide forms a secondary structure comprising one or more of an alpha helix, a beta sheet, a beta coil, and a random coil.

Use of a self-assembled short peptide as an antigen.

Use of self-assembled short peptides in the preparation of a pharmaceutical carrier material.

The application of self-assembled short peptide in preparing macromolecular carrier material includes one or several of protein medicine, immunoglobulin, serum albumin, P53 protein, P21 protein, igG, sugar, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide.

Application of self-assembled short peptide in preparing antitumor medicine is provided.

Application of self-assembled short peptide in preparation of cell or organoid three-dimensional culture nano scaffold material

An application of self-assembled short peptide in three-dimensional nanometer physical scaffold cultured cells for preparing NK vaccine.

An application of self-assembled short peptide in preparing DC vaccine in culturing cells in three-dimensional nanometer physical scaffold.

A self-assembled short peptide is used as main component for preparing medical and cosmetic products or cosmetics.

Use of a self-assembled short peptide as a vaccine adjuvant.

A self-assembled short peptide hydrogel.

The preparation method of the hydrogel comprises the following steps:

step 1, adding short peptide into water to prepare mother solution;

And step 2, adding ions or PBS into the mother solution to prepare the self-assembled short peptide hydrogel.

Further, the ions include one or more of Na⁺、Mg²⁺、K⁺、Al³⁺、Ca²⁺、Zn²⁺、Fe³⁺、Fe²⁺、H⁺、NH₄ ⁺、Cl^-、SO₄ ^2-、NO₃ ^-、CO₃ ^2-、CH₃COO^-、HCO₃ ^-、OH^-、PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-、HSO₄ ^-.

Further, the concentration of the prepared self-assembled short peptide hydrogel is 1ppM or more.

Furthermore, the concentration of the prepared self-assembled short peptide hydrogel is 1-10mg/ml.

Specific: adding 10mg of self-assembled short peptide into 1ml of water to prepare mother liquor, adding 3ml of PBS to prepare self-assembled short peptide hydrogel with the concentration of 2.5mg/ml, and preparing the hydrogel according to the method, wherein the concentration can be adjusted to be 1-10mg/ml.

The application of hydrogel containing self-assembled short peptide in preparing antitumor targeting medicine.

Further, a biomedical material comprising said hydrogel comprising a self-assembling short peptide.

In the technical scheme of the application, the self-assembly of the short peptide is triggered under the environment of salt ions, wherein the salt ions comprise, but are not limited to ：Na⁺、Mg²⁺、K⁺、Al³⁺、Ca²⁺、Zn²⁺、Fe³⁺、Fe²⁺、H⁺、NH₄ ⁺、Cl^-、SO₄ ^2-、NO₃ ^-、CO₃ ^2-、CH₃COO^-、HCO₃ ^-、OH^-、PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-、HSO₄ ^- and the like.

Provides a novel self-assembled short peptide which has the functions of improving the immunity of organisms, resisting tumors and infections and is used as an adjuvant of medicines and vaccines.

The novel self-assembled short peptide is loaded with drugs, tumor vaccines, cells, antibodies and proteins, and is continuously and stably released in vivo, so that the half life of the drugs is increased.

Provides a novel self-assembled short peptide which can be used as one of the components in cosmetics and health care products.

The short peptide can be self-assembled to form nano fibers in the environments of metal salt ions, cell culture media and the like.

The nanofiber network structure formed under the atomic force microscope and the freeze scanning electron microscope can be applied to the field of cosmetics and used as a slow release agent, an antibacterial agent, a lasting humectant, a lubricant, a rheology regulator, an antioxidant, a film forming agent, an emollient, a stabilizer, a buffer and the like.

The self-assembled short peptide of the invention forms nano fiber, and cells can be subjected to three-dimensional strong attachment and growth, so the self-assembled short peptide can be applied to three-dimensional culture of human, animal and plant cells.

In the application, the application of the compound as a medicament, a protein, a macromolecular adjuvant and a carrier can be used as a carrier and an adjuvant of any compound collection, wherein the collection comprises the connection interaction of various compounds with different structures through Van der Waals force, hydrogen bond, hydrophobic bond and the like. Wherein said compound specifically comprises and is not limited to naturally occurring molecules such as: such as sugars, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, receptors, nucleic acids, nucleotides, oligonucleotides, polynucleotides, including DNA and DNA fragments, RNA and RNA fragments, etc., lipids, steroids, glycopeptides, glycoproteins, proteoglycans, etc., or naturally occurring molecular analogs and derivatives, or small molecule compounds produced by chemical synthesis techniques. The self-assembled short peptide can be used as a carrier and an adjuvant of medicines, macromolecules, proteins and the like which are composed of any compounds. The term "nucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, cDNA or DNA. The term generally refers to a polymeric form of nucleotides (either ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide) that are at least 10 bases in length. The term includes both single-stranded and double-stranded forms of DNA.

"Macromolecule" in the context of the present application refers to biological substances having a relative molecular mass of more than 5000, even more than millions, such as macromolecules including one or more of protein drugs, immunoglobulins, serum albumin, P53 protein, P21 protein, igG, SIgA, saccharides, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, receptors, nucleic acids, nucleotides, oligonucleotides, polynucleotides, and the like. It has a very close relationship with vital movements and consists of simple molecular units which are considered to be monomers. In the solution there is a gel-forming substance. Compounds having a relative molecular mass exceeding ten thousand are generally referred to as macromolecular compounds or macromolecular compounds. It is composed of a number of repeating structural units, typically having a linear structure, and sometimes having a dendritic structure. Many substances having important biological functions, such as proteins and nucleic acids, belong to this class of compounds.

In the present application, the "drug" can only affect physiological, biochemical and pathological processes of the organism, and is used for preventing, diagnosing, treating diseases and chemical substances of fertility, wherein the self-assembled short peptide can be used as a carrier of the drug, and the action type of the "drug" specifically comprises and is not limited to: drugs acting on the efferent nervous system (efferent nervous system drugs, parasympathetic drugs, cholinergic blockers, adrenoceptor agonists, adrenoceptor blockers), drugs acting on the central nervous system (sedative-hypnotics, antiepileptics and anticonvulsants, antiparkinsonism and drugs for the treatment of Alzheimer's disease, antipsychotics, analgesics, antipyretic-analgesic anti-inflammatory drugs, anesthetics), drugs acting on the circulatory and blood systems (antiarrhythmic drugs, antihypertensives, anti-chronic congestive heart failure drugs, anti-atherosclerosis drugs, anti-angina drugs, drugs for the blood and hematopoietic systems), drugs acting on the visceral system (diuretics and dehydrates) antitussive, expectorant, antiasthmatic, anti-peptic ulcer and digestive tract function regulating, uterine smooth muscle acting), autogenous active substances acting on endocrine system agents (adrenocortical hormone, thyroid hormone and antithyroid, insulin and oral hypoglycemic agents, sex hormone agents and contraceptives), chemotherapeutic agents (beta-lactam antibiotics, aminoglycosides and polymetacins, macrolides, lincomycin, vancomycin, tetracyclines, chloramphenicol, synthetic antibacterial agents, antitubercular agents, antileprosy agents, antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, antitumor agents), immune system agents (immunosuppressants, immunopotentiators).

In the present application, the self-assembled short peptide is used as a carrier of a "drug", wherein the drug specifically includes, but is not limited to (the drug name and chemical name both have consistency): chicken Ovalbumin (OVA), botulinum toxin, tyramine, reserpine, cocaine tricyclic, carbachol, pilocarpine, nicotine, neostigmine, norepinephrine, phenylephrine, clonidine, epinephrine, isoprenaline, dobutamine, salbutamol, acetylcholine, clofibrate choline, methacholine, betulin, cholinesterase, penicillin, oxacillin, ampicillin, amoxicillin potassium clavulanate, piperacillin sodium tazobactam sodium, Cefradine, ceftriaxone, gentamicin, erythromycin, azithromycin, clindamycin, compound sulfadiazine, norfloxacin, moxifloxacin, metronidazole, nitrofurantoin, pyrazinamide, ethambutol, streptomycin, sodium p-aminosalicylate, dapsone, fluconazole, itraconazole, amphotericin B, acyclovir, ganciclovir, oseltamivir, ribavirin, sofosbu Wei Weipa tavir, tenofovir dipivoxil, AIDS drugs, chloroquine, hydroxychloroquine, artemisinin drugs, antimonous sodium gluconate, praziquantel, albendazole, lidocaine, bupivacaine, ropivacaine, ketamine, propofol, remifentanil, sevoflurane, Rocuronium bromide, succinylcholine chloride, vecuronium bromide, fentanyl, pethidine, morphine, pregabalin, acetaminophen, aspirin, ibuprofen, sodium diclofenac, indomethacin, mesalamine (oxazine), penicillamine, allopurinol, colchicine, benzbromarone, amantadine, benzhaline, dopa-hydrazine, pramipexole, bromocriptine, neostigmine, carbamazepine, sodium valproate, phenobarbital, lamotrigine, nimodipine, betahistine, mannitol, citicoline sodium, nicolazine, huperzine A, perphenazine, chlorpromazine, haloperidol, sulpiride, amisulpride, olanzapine, risperidone, paliperidone, aripiprazole, paroxetine, fluoxetine, amitriptyline, clomipramine, diazepam, clonazepam, eszomib, tandospirone, lithium carbonate, midazolam, isosorbide dinitrate, nifedipine, mexiletine, metoprolol, digoxin, captopril, valsartan, nitrendipine, urapidil, prazosin, phentolamine, dopamine, simvastatin, bromhexine, carbocisteine, compound liquorice, aminophylline, compound aluminum hydroxide, lactase, anisodamine, domperidone, enema, bifendate, bacillus licheniformis viable, ursodeoxycholic acid, sulfasalazine, furosemide, hydrochlorothiazide, glycerinum fructose, tamsulosin (tamsulosin), Finasteride, ferrous sulfate, ferric dextran, vitamin B12, adenosylcobalamin, mecobalamin, aspirin, ticagrelor, thrombin, vitamin K1, protamine, heparin, warfarin, urokinase, dabigatran etexilate, recombinant human tissue type plasminogen kinase derivative, hydroxyethyl starch, chorionic acid, recombinant human growth hormone, hydrocortisone, methylprednisolone, insulin, metformin, glibenclamide, glipizide, glimepiride, gliquidone, acarbose, liraglutide, sitagliptin, thyroslice, levothyroxine sodium, methimazole, propylthiouracil, cinacalcet, testosterone propionate, testosterone undecanoate, Progesterone, medroxyprogesterone, diethylstilbestrol, nielestrol, vitamin D2, chlorpheniramine, diphenhydramine, promethazine, triptolide, azathioprine, mycophenolate mofetil, semustine, ifosfamide, methotrexate, mercaptopurine, hydroxyurea, fluorouracil, etoposide, daunorubicin, hydroxycamptothecin, vincristine, paclitaxel, cisplatin, arsenite (arsenic trioxide), tretinoin, capecitabine, tamoxifen, letrozole, ondansetron, gefitinib, rituximab, pemetrexed, vitamin series, calcium gluconate, compound amino acid 18AA, fatty milk amino acid glucose, oral rehydration salts, Glucose sodium chloride, compound sodium chloride, sodium lactate ringer, glucose, sodium thiosulfate, clodroxyphosdine, iodoxyphosdine, penehyclidine, methylene blue, naloxone, acetamide, penicillamine, tetanus antitoxin, anti-rabies serum, tetanus human immunoglobulin, mitomycin, anti-snake venom serum, national immune programming vaccine, diatrizosamine, iodized oil, iohexol, tuberculin pure protein derivative, erythromycin, imi Kangqu Annelideconazole, mupirocin, ichthyol, salicylic acid, mometasone furoate, tretinoin, chloramphenicol, levofloxacin, atropine, oxymetazoline, posterior pituitary injection, contraceptive, caffeine, and the like.

In the present application, as a "maturation-promoting agent" of a cell, the meaning of the maturation-promoting agent shall be expressed as: the cell is specialized, so that the cell generates corresponding protein and has related functions, such as the DC cell in the application, the DC cell is cultured after the short peptide is added, and the expression quantity of CD86 protein of the DC cell is increased, namely the DC cell is considered to be mature. Similarly, self-assembled short peptide continuously activates NK cells to prepare NK and DC vaccines with better effect.

In the present application, DRF3 three-dimensionally cultures cells, wherein "cells" include, but are not limited to: DC cells (dendritic cells), NK cells (natural killer cells), lymphocytes, monocytes/macrophages, granulocytes, mast cells, leukocytes, phagocytic cells, T lymphocytes, B lymphocytes, K lymphocytes (K lymphocytes), embryonic stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, hepatic stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, etc., and further includes primary and passaged cells not limited to one or more of tonsils, adrenals, bile ducts, bladder, bones, bone marrow, brain, chest, cervix, colorectal, esophagus, eyes, head and neck, kidney, liver, lung, lymph node, nervous system, ovary, pancreas, prostate, skin, soft tissue, stomach, testis, thymus, thyroid, uterus, etc.

The species of all the above cells include, but are not limited to: including unicellular eukaryotes such as yeast and fungi and multicellular eukaryotes such as animals, non-limiting examples include invertebrates (e.g., insects, coelenterates, echinoderms, nematodes, etc.); eukaryotic parasites, e.g., malaria parasites, such as plasmodium falciparum (Plasmodium falciparum), worms, etc.; vertebrates (e.g., fish, amphibian, reptile, bird, mammal); and mammals (e.g., rodents, primates such as humans and non-human primates).

In the application, the term "three-dimensional culture" refers to the co-culture of carriers with three-dimensional structures and various different kinds of cells in vitro, so that the cells can migrate and grow in the three-dimensional space structure of the carriers to form a three-dimensional cell-carrier compound, the characteristics of the cells on the wall of the culture process are changed or reduced, the cells can obtain more living space in space, the cell contact inhibition is reduced, and the cells are attached to self-assembled short peptides and generally present a round shape under a microscope.

In the present application, the term "organoid" means that the organoid belongs to a three-dimensional (3D) cell culture, comprising some key properties that represent the organ. Such in vitro culture systems comprise a self-renewing stem cell population that can differentiate into a plurality of organ-specific cell types, possess similar spatial organization to the corresponding organ and are capable of reproducing part of the function of the corresponding organ, thereby providing a highly physiologically relevant system. Tissue samples containing adult stem cells, single adult stem cells, or induced differentiation by the orientation of pluripotent stem cells can all produce organoids wherein organoid species include, but are not limited to: tonsil, adrenal gland, bile duct, bladder, bone marrow, brain, chest, cervix, colorectal, esophagus, eye, head and neck, kidney, liver, lung, lymph node, nervous system, ovary, pancreas, prostate, skin, soft tissue, stomach, testis, thymus, thyroid, uterus, etc. The tumor organoids included are mainly and not limited to: colon cancer, adrenal gland cancer, soft tissue sarcoma, lymphoma, nerve cancer, brain cancer, skin cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, breast cancer, bone cancer intraocular melanoma, retinoblastoma, fallopian tube cancer, gall bladder cancer, stomach cancer, soft tissue sarcoma, central nervous system bacterial cell tumor (brain cancer), pediatric extracranial bacterial cell tumor, ectopic bacterial cell tumor, ovarian bacterial cell tumor, testicular cancer, heart tumor, liver cell cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumors, renal cell cancer, laryngeal cancer, leukemia lip cancer and oral cancer, liver cancer, lung cancer (non-small cell, alveolar tumor and tracheobronchial tumor), lymphoma, male breast cancer, melanoma, skin cancer, mesothelioma, metastatic squamous neck cancer (head and neck cancer), oral cancer, multiple myeloma/plasma cell tumor, myeloproliferative tumor, paranasal sinus cancer, neuroblastoma, oral cancer, lip cancer, pharyngeal cancer, pancreatic cancer, islet cell tumor, nasal cavity cancer, parathyroid cancer, pharyngeal cancer, pituitary tumor, primary peritoneal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, T-cell lymphoma testicular cancer, nasopharyngeal cancer, esophageal cancer, hypopharyngeal cancer, thymoma, thyroid cancer, renal cancer, transitional cell carcinoma of urinary tract cancer, cancer of uterus, endometrial cancer, vaginal cancer, vascular tumor, and the like.

In the present application, the term "antigen" and grammatical equivalents thereof (e.g., "antigenic") refers to a compound that can specifically bind by a specific humoral or cellular immune product (e.g., an antibody molecule or T cell receptor). The antigen may be any type of molecule, including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids and hormones, and macromolecules such as complex sugars (e.g., polysaccharides), phospholipids and proteins. Common classes of antigens include, but are not limited to, at least one of viral antigens, bacterial antigens, fungal antigens, protozoal and other parasitic antigens, tumor antigens, antigens involved in autoimmune diseases, allergies and transplant rejection, toxins and other miscellaneous antigens.

The invention can be used as a collagen promoter in combination with a collagen promoter, such as, but not limited to: ascorbyl phosphate salts such as ascorbic acid, sodium ascorbyl phosphate, and magnesium ascorbyl phosphate; ascorbyl fatty acid esters such as ascorbyl monostearate, ascorbyl monopalmitate, ascorbyl dipalmitate, and ascorbyl tetraisopalmitate; ascorbyl ethers such as 3-O-ethyl ascorbate, 2-O-ethyl ascorbate, cetyl ascorbate, glycerin ascorbate, and hexyl glyceryl ascorbate 12; ascorbyl glucoside such as ascorbyl-2-glucoside and fatty acid esters thereof; ascorbic acid derivatives such as ascorbyl sulfate and tocopheryl phosphate; retinoids such as retinol, retinol acetate, retinol palmitate, hydrogenated retinol, and the like; nicotinamide, glutathione, cysteine, crocetin, sericin, geraniol (geraniol), glyceroglycosides, lactoferrin, procyanidins, pantothenic acid, panthenol, soyasaponin, resveratrol, isoflavones, coenzyme Q10, chondroitin sulfate, acetylglucosamine, glycerophosphorylcholine, hydrolyzed hyaluronic acid, collagen peptides, shell matrix hydrolysates, 5' adenosine monophosphate, proline, glycine, arginine, aspartic acid, alanine; and at least one of extracts of plants having collagen production promoting effect, such as hibiscus, licorice leaves, roman chamomile, sweet tea, hawthorn, impatiens balsamina, lotus leaves, kudzu root, sparassis crispa, chlorella, bear's bamboo, burdock, sophorae radix, cabbage heart, loquat leaves, perilla, pea, mulberry leaf, thyme, spruce, basil, dayflower, platycladi seed, cassia seed, snakegourd seed, garden burnet, kuh-seng, persimmon, peony, perilla, dried orange peel, mallow, ginger, chamomile, strawberry seed, swertia, soybean, wheat, ginseng, coix seed, fangku, cardamom, rosemary, sage, etc.

The present invention may be used in combination with hyaluronic acid as a dermal filler, anti-wrinkle, in particular in combination with a pro-agent such as, but not limited to: ascorbyl phosphate salts such as hyaluronic acid, collagen, ascorbic acid, sodium ascorbyl phosphate, and magnesium ascorbyl phosphate; ascorbyl fatty acid esters such as ascorbyl monostearate, ascorbyl monopalmitate, ascorbyl dipalmitate, and ascorbyl tetraisopalmitate; ascorbyl ethers such as 3-O-ethyl ascorbate, 2-O-ethyl ascorbate, cetyl ascorbate, glycerin ascorbate, and hexylglycerin ascorbate; ascorbyl glucoside such as ascorbyl-2-glucoside and fatty acid esters thereof; ascorbic acid derivatives such as ascorbyl sulfate and tocopheryl phosphate; retinoids such as retinol, retinol acetate, retinol palmitate, hydrogenated retinol, and the like; nicotinamide, carotenes, tocopherols, tocotrienols, chondroitin sulfate, acetylglucosamine, glycerophosphorylcholine, glyceroglycosides, hydrolyzed hyaluronic acid, collagen peptides, sitosterol, carnosine, creatine, phytic acid, N-methylserine, 3-methylcyclopentadecanone, saponins, genistein, soy flavone, phytol; and at least one plant extract having hyaluronic acid production promoting effect, such as herba Cynomorii, mentha piperita, herba Mali Pumilae, perillae herba, fructus Citri Limoniae, meng Sang, fructus Chebulae, broussonetia papyrifera, fructus fici, ulva, kochiae fructus, herba Houttuyniae, achillea millefolium, semen Armeniacae amarum, fructus crataegi, semen Lini, fructus Gardeniae, herba Urticae Cannabinae, strawberry seed, fructus Avenae Fatuae, fructus Foeniculi, stigma croci Sativi, herba Camelliae Japonicae, fructus Cucurbitae Moschatae, luffae, germinatus Phragmitis, herba Gynostemmatis, radix Sophorae Flavescentis, pericarpium Citri Tangerinae, radix Paeoniae, fructus kaki, saviae Miltiorrhizae radix centella, pu' er tea, maitake place in water shield, herba Equiseti hiemalis, fructus Pyri, and flos Matricariae.

The invention can be used for preparing anti-aging agents by combining with the anti-aging agents, such as, but not limited to: tocopherol (vitamin E), disodium vitamin E phosphate, tocopheryl acetate, fullerene, ubiquinone, grape seed extract, tea extract, retinol acetate, ginkgo extract, phytosterol, resveratrol, ceramide, ginseng root extract, puerarin, soybean isoflavone, etc.

All the self-assembled short peptide containing words and functions of the self-assembled short peptide are macroscopic in hydrogel.

The 16 sequences of the invention are polypeptides, which are referred to herein as "self-assembling short peptides".

The beneficial effects of the invention are as follows:

1. A novel self-assembled short peptide is provided, increasing the type of self-assembled short peptide.

2. The novel self-assembled short peptide materials can form stable nanofibers, and the nanofibers can be applied to three-dimensional culture of stem cells and organoids to simulate living environments in cells and provide three-dimensional microenvironments outside the cells.

3. The self-assembled material with the medicine carrying function can carry medicines and macromolecules, has obvious anti-tumor effect and opens up a new path for vaccine adjuvants.

4. The self-assembled material with the novel vaccine adjuvant function can enhance the immunity in vivo, and can be used for preparing health-care products and medicines.

5. The self-assembled material can be used as a slow release agent, an antibacterial agent, a lasting humectant, a lubricant, a rheology regulator, an antioxidant, a film forming agent, an emollient, a stabilizer and a buffer agent in cosmetics, and provides a novel hydrogel with good biocompatibility and beneficial degradation products for cosmetic raw materials.

6. Provided is a novel self-assembled short peptide which is useful as a maturation promoting agent for DC, an activator for NK cells, or the like.

Drawings

FIG. 1 is a circular dichroism spectrum of self-assembled short peptide DRF3 of the invention;

FIG. 2 is a transmission electron microscope image of the self-assembled short peptide DRF3 of the present invention;

FIG. 3 is an atomic force microscope image of the self-assembled short peptide DRF3 of the present invention;

FIG. 4 is a cryo-scanning electron microscope image of the self-assembled short peptide DRF3 of the present invention;

FIG. 5 is a Congo red staining chart of the self-assembled short peptide DRF3 of the invention;

FIG. 6 is an aniline blue staining chart of the self-assembled short peptide DRF3 of the invention;

FIG. 7 is a three-dimensional culture of the self-assembled short peptide DRF3 of the invention on the third day of tonsillar cells;

FIG. 8 is a three-dimensional culture of the self-assembled short peptide DRF3 of the invention on the third day of tonsillar organoids;

FIG. 9 is a graph showing the toxicity test of CCK8 of three-dimensionally cultured fibroblasts after mixing the self-assembled oligopeptide DRF3 with hyaluronic acid according to the invention;

FIG. 10 is a graph showing CCK8 toxicity attack experiment of self-assembled short peptide DRF3 on colon cancer cells;

FIG. 11 is a graph of the controlled release of the self-assembled short peptide DRF3 versus OVA of the present invention;

FIG. 12 is a flow-through sorting graph of the self-assembled short peptide DRF3 of the invention promoting DC cell maturation;

FIG. 13 is a flow apoptosis graph of the self-assembled short peptide DRF3 of the present invention promoting NK cell maturation.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The concentrations of the examples according to the invention were all 2.5mg/ml, unless specific concentrations were stated.

Example 1

This example provides the preparation of self-assembled short peptide DRF3 consisting of L-amino acids

Materials:

The raw materials are prepared according to the amino acid sequence as follows: fmoc-L-Arg-OH (9-fluorenylmethoxycarbonyl-L-arginine-. Gamma. -t-butoxycarbonyl), fmoc-L-Lys-OH (9-fluorenylmethoxycarbonyl-L-lysine-. Epsilon. -t-butoxycarbonyl), fmoc-L-Asp (OtBu) -OH (fluorenylmethoxycarbonyl-L-aspartic acid-. Epsilon. -t-butoxycarbonyl), fmoc-L-Val-OH (9-fluorenylmethoxycarbonyl-L-valine), fmoc-L-Phe-OH (9-fluorenylmethoxycarbonyl-L-phenylalanine-. Gamma. -t-butoxycarbonyl), fmoc-L-Glu (OtBu) -OH (9-fluorenylmethoxycarbonyl-L-glutamic acid-. Epsilon. -t-butoxycarbonyl), fmoc-L-Leu-OH (9-fluorenylmethoxycarbonyl-L-leucine), fmoc-L-Ile-OH (9-fluorenylmethoxycarbonyl-L-isoleucine), fmoc-L-Phe-OH (9-fluorenylmethoxycarbonyl-L-isobutoxycarbonyl-L-valine), fmoc-L-phenyloxycarbonyl-gamma. -t-butoxycarbonyl), N, N ', N' -tetramethyluronium tetrafluoroborate), HBTU (O-benzotriazol-1-yl-N, N, N, N-tetramethyluronium hexafluorophosphate) and HOBT (1-hydroxybenzotriazole), piperidine, acetic anhydride, dichloromethane; solvent: DMF (N, N-dimethylformamide), TFA (trifluoroacetic acid), ACN (acetonitrile), glacial ethyl ether, NMM (N-methylmorpholine).

The Fmoc (fluorenylmethoxycarbonyl) protected solid phase synthesis method is adopted, and the process steps are briefly described as follows:

(1) 0.5mmol/G RINK AMIDE RESIN g was weighed into a peptide synthesizer, after soaking the resin in 200ml DCM (dichloromethane) for 30min, the resin was washed three times with 400: 400inIDMF for 3min each time, and the washing solution was suction filtered. After 10-20 minutes, 100ml of 20% piperidine/DMF is used for oscillation reaction for 30 minutes, after the reaction is finished, the dry cleaning liquid is pumped and filtered, the resin is cleaned for 5 times by 400mIDMF times, each time for 3 minutes, a little resin is taken to be checked as the previous trione after the cleaning is finished, the resin is positive, and then the raw materials are added into a reactor:

Fomc-L-Leu-OH 14.312g

HBTU 14.15g

HOBT 8.77g

NMM 8.27ml

DMF 243ml

After the above raw materials are added, the reaction is carried out for 40min, the filtration is carried out, the resin is washed by 30ml DMF for 4 times, each time for 3 min, a little resin is taken as the previous trione for examination, and the resin is negative.

(2) The resin was then washed 4 more times with 40ml dmf and checked for positive resin before taking a few resin as the trione, and the following raw materials were added to the reaction vessel:

(a)Fomc-L-Arg-OH 15.122g

(b)HOBT 26.30g

(c)NMM 7.36ml

(d)DMF 247ml

After the raw materials are added, the reaction is carried out for 40 minutes by shaking, after the reaction is finished, the raw materials are washed with 30ml of DMF for 4 times for 3 minutes each time, a little resin is taken as the previous trione for examination, and the resin is negative.

(3) Changing the raw materials (a) and (b) (c) (d) in the step ⑵, and repeating the operation of the step ⑵: in the step (2), the raw material (a) is replaced by the amino acid of the corresponding primary structure sequence.

Repeating ⑴⑵⑴ steps again, synthesizing the raw materials and the dosage of each step according to the DRF3 sequence without changing; after the last beam-attachment, fmoc-protecting group was removed, 20% piperidine/DMF (volume concentration) was reacted for 30 minutes, the resin was washed, 160ml 50% acetic anhydride/DMF (volume concentration of acetic anhydride) was added for 30 minutes, the resin was washed with 40ml DMF, and then the resin was washed 4 times with methanol, suction-filtered and dried under vacuum for 8 hours. 50ml of 90% TFA/DCM (volume concentration of TFA) is added into a container containing peptide resin, the reaction is carried out for 3 hours, suction filtration is carried out, filtrate is concentrated, diethyl ether is added into residual liquid, white solid is separated out, the solid is suction filtered, crude peptide is obtained, purification is carried out through HPLC (high performance liquid chromatography), and freeze drying is carried out, thus obtaining the short peptide DRF3 of the invention, the amino acid sequence of which is described in a sequence table.

Example 2

As shown in FIG. 1, the self-assembled short peptide DRF3 self-assembled 24h round dichroism spectrum (CD)

The self-assembled short peptide after self-assembly for 24 hours at 37 ℃ is displayed in a circular dichroscope, after 24 hours of assembly, the short peptide DRF3 can be self-assembled to form a nanofiber interweaved membranous structure, the secondary junctions of the two structures are in a mirror image related beta-sheet structure, as shown in figure 1, and figure 1 is a circular dichroscope of the self-assembled short peptide DRF3 when assembled for 24 hours; (Wavelength Wavelength; CD circular dichroism)

Example 3

Self-assembled short peptide DRF3 self-assembled 24h Transmission Electron Microscope (TEM)

1. Experimental materials

DRF3, main solutions were: deionized water H ₂ O; PBS solution (Na ⁺、K⁺、PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-, etc.).

2. Main laboratory instrument

Transmission electron microscope (TEM, H-200, hitachi)

3. Experimental method

(1) Preparing a working solution with a final concentration of 100 mu M of DRF3 by deionized water or PBS (phosphate buffered saline), and observing by a transmission electron microscope; (2) taking a small amount of working solution and carrying out negative dyeing by using 1% phosphotungstic acid: sucking a drop of about (10-30 mu l) working solution on the surface of a clean glass slide by using a clean suction head, carefully clamping a copper mesh covered with a Formvar film by using forceps, dipping the working solution on the glass slide by using the copper mesh, standing for a plurality of seconds, dipping a small amount of 1% phosphotungstic acid by using the copper mesh after the working solution is fully combined with the copper mesh, and carrying out negative dyeing on the adsorbed working solution; (3) The excess liquid on the copper mesh is sucked up by filter paper, and the copper mesh is left to stand in the air for a few minutes until the copper mesh is dried.

And scanning the copper net by using TEM, and directly observing the self-assembled structure of the short peptide on the copper net.

4. Experimental results

The self-assembled short peptide after self-assembly for 1 hour under the environment of 37 ℃ is displayed in a transmission electron microscope, after the self-assembled short peptide DRF3 is assembled for 1 hour, the short peptide DRF3 can be self-assembled to form a nanofiber interweaved membranous structure, and the two secondary junctions are in a mirror image related beta-sheet structure, so that proteins, medicines, macromolecules, cells and the like can be effectively loaded, a physical support is provided for cell growth, and the application of the self-assembled short peptide in preparation of a medicine carrier material is supported; application of self-assembled short peptide in preparation of macromolecular carrier material, wherein the macromolecules comprise one or more of protein drugs, immunoglobulin, serum albumin, P53 protein, P21 protein, igG, sugar, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide; application of self-assembled short peptide in preparing three-dimensional culture nanometer bracket material for cells or organoids; application of self-assembled short peptide in three-dimensional nanometer physical scaffold culture cells as preparation of DC vaccine; an application of self-assembled short peptide in three-dimensional nanometer physical scaffold cultured cells as preparation of NK vaccine; a self-assembled short peptide hydrogel has a concentration of 1ppM or more. As shown in FIG. 2 (200 μm, 500 μm, 2 μm, respectively), FIG. 2 is a transmission electron micrograph of the self-assembled short peptide DRF3 for 24 hours, which shows that the short peptide is now in a network structure.

Example 4

As shown in FIG. 3, the self-assembled short peptides are shown in atomic force microscope images of 400 μm, 1 μm and 2 μm respectively, and the short peptides are shown as net structures.

1. Experimental materials

DRF3, main solution: sterile deionized water H ₂ O; millipore Milli-Q system, autoclaved at 4℃for further use.

2. Main instrument

Atomic force microscope AFM (multimode 8)

3. Experimental method

Preparing a working solution of DRF3 by deionized water, wherein the final concentration is 100 mu M; respectively dripping 5 mu l of configured working solution of DRF3 on the surface of a newly stripped mica sheet; about 30s after the coating was completed, the unattached short peptides were removed by rinsing with 1000 μl deionized water; air-drying each short peptide working solution smear at room temperature; AFM scanning is carried out on the mica sheet in a gas phase, and an AFM image is collected by using a recording mode of SPI 4000; using a 20 μm scanner (400), an Olympus Si-DF20 microcantilever, and a needle with a spring constant of 12N/M (Si, radius 10nm, rectangular substrate 200 μm); the free resonance frequency of the cantilever is 127KHz; the phase map is recorded at a pixel resolution of 512 x 512; to show the self-assembled short peptide nanofiber structure, scans were performed in the 600nm by 600nm and 200nm by 200nm ranges.

4. Experimental results

The self-assembled short peptide after 24 hours of self-assembly at 37 ℃ is shown in an atomic force microscope, and fig. 3 is an atomic force microscope image of the self-assembled short peptide DRF3 after 24 hours of assembly, the short peptide forms a dense regular nanorod-like fiber structure, and the rod-like nanofibers are mutually integrated into a dense nanofiber mesh scaffold. Further, the self-assembled short peptide can be widely applied to the biomedical field, provides physical nano-brackets, drug carriers, cell maturation promoting agents and the like for preparing medical products, cosmetics or health-care products, provides theoretical basis for loading drugs, cells, proteins and macromolecules, and supports the application of the self-assembled short peptide in preparing drug carrier materials; application of self-assembled short peptide in preparation of macromolecular carrier material, wherein the macromolecules comprise one or more of protein drugs, immunoglobulin, serum albumin, P53 protein, P21 protein, igG, SIgA, sugar, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide; application of self-assembled short peptide in preparing three-dimensional culture nanometer bracket material for cells or organoids; application of self-assembled short peptide in three-dimensional nanometer physical scaffold culture cells as preparation of DC vaccine; an application of self-assembled short peptide in three-dimensional nanometer physical scaffold cultured cells as preparation of NK vaccine; a self-assembled short peptide hydrogel has a concentration of 1ppM or more.

Example 5

As shown in FIG. 4, the self-assembled short peptide DRF3 is respectively frozen at 10 μm, 5 μm and 20 μm for scanning electron microscope, and the short peptide DRF3 is shown as a fiber network structure.

The self-assembled short peptide after self-assembly for 24 hours at 37 ℃ is shown in a frozen scanning electron microscope, and fig. 4 is a frozen scanning electron microscope image of the self-assembled short peptide DRF3 after assembly for 24 hours, wherein the short peptide forms a dense regular nano rod-shaped fiber structure, and rod-shaped nano fibers are mutually assembled to form a dense nano fiber net-shaped bracket. Further shows that the preparation method can be widely applied to the biomedical field, and provides physical nano-brackets, drug carriers, cell maturation promoting agents, cell vaccines and the like for preparing medical products, cosmetics or health care products.

Example 6

As shown in FIG. 5, the self-assembled short peptide DRF3 was self-assembled for 24h Congo red staining

1. Experimental materials

Short peptide: DRF3

The mixed solution comprises the following components: 1) Preparing a mixed liquid with a fixed concentration (containing chicken ovalbumin, immunoglobulin, insulin, thiotepa, carmustine, mitomycin, collagen, hydroxycamptothecin, taxol, docetaxel, cephalotaxine, interleukin-2, hyaluronic acid, metronidazole, puromycin, CD40 and PD-L1), wherein the lowest concentration in the components is 1ppM; 2) Dyeing liquid: congo red staining.

2. Experimental procedure

10Mg/ml of short peptide solution mother liquor is diluted to 2.5mg/ml by PBS solution, and is placed in 37 ℃ environment to be self-assembled for 0 hour, 4 hours, 12 hours and 24 hours respectively, and then Congo red staining detection is carried out. 15 μl of the short peptide solution was pipetted onto a slide glass, stained with Congo red staining solution for about 30s, and observed under an optical microscope and photographed.

3. Experimental results

Adding a mixed liquid (containing chicken ovalbumin, immunoglobulin, insulin, thiotepa, carmustine, mitomycin, collagen, hydroxycamptothecin, taxol, docetaxel, cephalotaxine, interleukin-2, hyaluronic acid, metronidazole, puromycin, CD40 and PD-L1) with a predetermined concentration, and the Congo red staining result shows that DRF3 presents fiber gel under a microscope, the assembly is basically completed in 12 hours, the complete assembly is successful in 24 hours, the assembly is stable in 48 hours, and the self-assembled short peptide DRF3 is subjected to the Congo red staining in 24 hours in FIG. 5. Further shows that the self-assembled short peptide can be used for preparing medical and aesthetic products, cosmetics or health care products, provides a theoretical basis for loading medicines, cells, proteins and macromolecules, and supports the application of the self-assembled short peptide in preparing medicine carrier materials; application of self-assembled short peptide in preparation of macromolecular carrier material, wherein the macromolecules comprise one or more of protein drugs, immunoglobulin, serum albumin, P53 protein, P21 protein, igG, SIgA, sugar, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide; application of self-assembled short peptide in preparing three-dimensional culture nanometer bracket material for cells or organoids; application of self-assembled short peptide in three-dimensional nanometer physical scaffold culture cells as preparation of DC vaccine; an application of self-assembled short peptide in three-dimensional nanometer physical scaffold cultured cells as preparation of NK vaccine; a self-assembled short peptide as main component for preparing medical and cosmetic products or cosmetics; a self-assembled short peptide hydrogel has a concentration of 1ppM or more.

Example 7

As shown in FIG. 6, the self-assembled short peptide DRF3 was self-assembled for 24h aniline blue staining

The aniline blue staining solution is one of the components of the Masson trichromatic staining kit, mainly comprises aniline blue, weak acid and the like, is acidic, is often used together with ponceau staining solution and the like to stain the collagen fibers, and is mainly used for distinguishing the collagen fibers from the muscle fibers, wherein the dyed muscle fibers are red and the collagen fibers are blue.

1. Experimental materials

Short peptide: DRF3

The mixed solution comprises the following components: 1) Preparing a mixed liquid with a fixed concentration (such as chicken ovalbumin, collagen, hyaluronic acid, glutathione, thyroxine, acetylcholine, bevacizumab, arsenite, gefitinib, chlorambucil, CD40, CD19, CD21, PD-L1 and vitamin B ₁₂), wherein the lowest concentration of the components is 1ppM; 2) Dyeing liquid: aniline blue staining solution, 95% ethanol, xylene and phosphomolybdic acid.

2. Experimental method

Conventionally dewaxing slices to water; dyeing with the prepared Weigert iron hematoxylin staining solution for 5-10min; differentiation of the acidic ethanol differentiation solution for 1-2s, and washing with distilled water; returning Masson bluing liquid to blue for 1min, and washing with distilled water; dyeing the ponceau dyeing liquid for 5-10min, washing with weak acid working solution for 3-5s, and differentiating with phosphomolybdic acid solution for 1-2min; pouring out the differentiation liquid, directly putting into aniline blue staining solution for staining for 1-2min, and washing with weak acid working solution for 1min; rapidly dehydrating 95% ethanol, and dehydrating absolute ethanol for 3 times, each time for 5-10s; the xylene is transparent for 3 times, each time for 1-2min, and the neutral gum is sealed.

3. Experimental results

FIG. 6 shows aniline blue staining of self-assembled short peptide DRF3 for 24h, mixed solution (such as chicken ovalbumin, collagen, hyaluronic acid, glutathione, thyroxine, acetylcholine, bevacizumab, arsenite, gefitinib, chlorambucil, CD40, CD19, CD21, PD-L1 and vitamin B12) with certain concentration, aniline blue staining shows that DRF3 shows fiber gel under a microscope, and is basically assembled for 12h, and is completely assembled for 24h and is assembled successfully for 48 h. Further shows that the self-assembled short peptide can be used in certain biomedical fields, can also be used for preparing medical products, cosmetics or health care products, and the like, provides a theoretical basis for loading medicines, cells, proteins and macromolecules, and supports the application of the self-assembled short peptide in the preparation of a medicine carrier material; application of self-assembled short peptide in preparation of macromolecular carrier material, wherein the macromolecules comprise one or more of protein drugs, immunoglobulin, serum albumin, P53 protein, P21 protein, sugar, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide; application of self-assembled short peptide in preparing three-dimensional culture nanometer bracket material for cells or organoids; application of self-assembled short peptide in three-dimensional nanometer physical scaffold culture cells as preparation of DC vaccine; an application of self-assembled short peptide in three-dimensional nanometer physical scaffold cultured cells as preparation of NK vaccine; a self-assembled short peptide as main component for preparing medical and cosmetic products or cosmetics; a self-assembled short peptide hydrogel has a concentration of 1ppM or more.

Example 8

Tonsil cells were selected as a model or representative of general cell culture, and cultured in a three-dimensional environment constructed with DRF 3.

Respectively placing tonsil cells in 37 ℃ water bath to dissolve rapidly; then adding RPMI-1640 (Gibco company) culture solution, suspending the centrifugally precipitated cells; then inoculating into a 25cm culture flask, and adding a complete culture medium with the culture solution of RPMI-1640.

The main components of the kit are 1% double-antibody solution (green streptomycin-streptomycin) and 8-10% (volume concentration) fetal bovine serum (Gibco company), and the culture flask is placed in a 37 ℃ incubator for culture with the volume fraction of 5% carbon dioxide;

Changing the liquid once every 2 days, and dividing the cells into bottles for passage after the growth condition of the cells is good;

During passage, firstly sucking out the culture solution in the culture flask by using a suction pipe in an ultra-clean bench, adding 1ml of 0.25% pancreatin into the culture flask to enable cells to be dissociated, and properly oscillating; transferring the liquid in the culture flask into a centrifuge tube, centrifuging at 1000 rpm for 8 minutes to precipitate cells, discarding supernatant, suspending cells in a complete culture medium culture solution of RPMI-1640, and inoculating in separate bottles; when the growth state of the inoculated cells is good, the inoculated cells are reserved for later use;

the three-dimensional culture steps are as follows: (1) performing three-dimensional culture after the cell growth state is good; (2) centrifugation at 1000rpm, counting; (4) Adding short peptide solution and the like, and uniformly mixing to form three-dimensional suspension cell liquid; placing the cells in a 96-well plate in a constant temperature incubator (37 ℃,5% CO ₂₎ for culture, observation and analysis, wherein part of tonsil cells are in an adherent growth state and are in a long fusiform shape in a two-dimensional environment;

When the cells are cultured in a three-dimensional environment constructed by DRF3, the cells are spherically embedded in the short peptide hydrogel; cells were clear and boundaries were clearly visible, representing a multi-layer growth.

Cell growth and proliferation are inhibited, while in the three-dimensional culture environment constructed by DRF3, cells are clear and cell numbers still increase. The tonsil cells can be used as a representative or model of cell culture to grow and proliferate in a three-dimensional culture environment constructed by the short peptide hydrogel, and the tonsil cells have good conditions. Furthermore, the self-assembled short peptide can be used for the treatment in the biomedical field such as cell culture and stem cell field, can also provide support for preparing certain special personalized medical and aesthetic products, and supports the application of the self-assembled short peptide in preparing a cell or organoid three-dimensional culture nano scaffold material; application of self-assembled short peptide in three-dimensional nanometer physical scaffold culture cells as preparation of DC vaccine; an application of self-assembled short peptide in three-dimensional nanometer physical scaffold cultured cells as preparation of NK vaccine; a self-assembled short peptide hydrogel has a concentration of 1ppM or more. As shown in fig. 7, tonsil cells were rounded in morphology after three days of three-dimensional culture.

Example 9

Self-assembled short peptide three-dimensional culture tonsillar organoids

1. Experimental materials

The (human) tonsil tissue is derived from medical waste disposal of a first hospital affiliated to Chongqing medical university, and accords with corresponding national and ethical regulations.

2. Experimental method

Immersing freshly removed tonsil tissue in 1640 medium containing 10% diabody for 1h; taking out tonsil tissue, placing into a culture dish, and cutting tonsil tissue into tissue blocks with the size of about 1mm multiplied by 1 mm; the cells were digested with digestive enzymes in an oven at 37℃for 2h.

Preparing the digested cells into tonsil cell suspension; the cell suspension is adjusted to a concentration of 1X 10 ⁷ cells/ml, transferred into 24-well plates, and 1ml per well; 200 μl of self-assembled short peptide at a concentration of 5mg/ml was added to each well to construct a three-dimensional culture system.

Transferring into a cell culture box for culture.

3. Experimental results

As shown in FIG. 8, cells are observed to aggregate and grow into a cell cluster under a microscope under the system, which indicates that the three-dimensional nano physical scaffold formed by the short peptide supports tissue and organ culture.

The tonsil is an organoid model, which shows that the self-assembled short peptide can be applied to the field of three-dimensional culture organoid culture, and supports the application of the self-assembled short peptide in preparing a cell or organoid three-dimensional culture nano scaffold material; application of self-assembled short peptide in three-dimensional nanometer physical scaffold culture cells as preparation of DC vaccine; an application of self-assembled short peptide in three-dimensional nanometer physical scaffold cultured cells for preparing NK vaccine.

Example 10

CCK8 experiment of three-dimensional culture of fibroblast after self-assembled short peptide and hyaluronic acid are mixed

The experimental steps are as follows:

The self-assembled short peptide DRF3 (concentration 5 mg/ml) was mixed with hyaluronic acid (concentration, volume ratio 1:1) and then mixed with fibroblasts to perform the three-dimensional culture procedure as shown in example 8.

1. Preparing a cell suspension: cell count

2. Inoculated into 96-well plates: the same sample can be replicated 3 times per well with about 100ul of cell suspension per well, depending on the appropriate number of plated cells.

3. Culturing in a 37 ℃ incubator: the cell attachment requires about 2-4 hours of incubation after inoculation, which can be omitted if attachment is not required.

4. Adding toxic substances with different concentrations

5. Culturing in a 37 ℃ incubator: the culture time for adding the toxic substance is determined according to the nature of the toxic substance, the cell sensitivity and the cell cycle. It generally takes more than one generation of time to culture.

6. 10Ul CCK8 was added: since there is a small amount of CCK8 added per well, there is a potential for error due to the reagent sticking to the walls of the wells, it is recommended to gently tap the plate after the reagent addition is completed to aid in homogenization.

7. Culturing for 1-4 hours: the amount of Formazan formed varies from cell type to cell type. If the color development is insufficient, the culture may be continued to confirm the optimal conditions. In particular, the amount of Formazan formed in blood cells is small, and a long development time (5 to 6 hours) is required.

8. Measurement of absorbance at 450 nm: the dual wavelength is adopted for measurement, the detection wavelength is 450-490nm, and the reference wavelength is 600-650nm.

Experimental results: as shown in fig. 9 (HA hyaluronic acid; OD absorbance) shows that after the three-dimensional culture of fibroblasts by combining DRF3 with hyaluronic acid, the toxicity is almost nontoxic or weaker than that of hyaluronic acid alone, which indicates that the short peptide DRF3 can synergistically reduce the toxicity of hyaluronic acid, further, the fiber network structure of DRF3 is similar to that of hyaluronic acid (but HAs smaller toxicity), almost no toxicity or extremely low cytotoxicity provides theoretical support for substituting hyaluronic acid with short peptide to become an independent filler, and further indicates that hyaluronic acid and other main compounds of cosmetics have great application value in the medical and cosmetic fields in combination with self-assembled short peptide, which supports a self-assembled short peptide as a main component for preparing medical and cosmetic products or cosmetics; a self-assembled short peptide hydrogel has a concentration of 1ppM or more.

Example 11

CCK8 toxicity attack experiment of self-assembled short peptide DRF3 on colon cancer cells

CCK8 experiments are shown in example 10 (concentration; absorbance value).

The toxicity of colon cancer cells at different concentrations of short peptides was tested here and grouped into: 1ug/ml, 2.5mg/ml, 5mg/ml

Experimental results: as shown in fig. 10, the higher the concentration of the short peptide at 24 and 48 hours, the greater the toxicity of colon cancer cells, further showing that the application prospect of the short peptide DRF3 as the preparation field of anti-tumor drugs supports the application of a self-assembled short peptide in the preparation of anti-tumor drugs; a self-assembled short peptide hydrogel has a concentration of 1ppM or more.

Example 12

As shown in FIG. 11 (Days of Days; OVA release), DRF3 released antigen OVA in vitro

The release of OVA from the DRF3 hydrogel was examined, 200. Mu. lOVA containing the solution was placed in 200. Mu.l of the hydrogel, and 200. Mu.l of PBS solution was added to the top of the gel.

100 Μl PBS was aspirated daily and an equal volume of PBS solution was supplemented. The OVA concentration in the samples was extracted by BCA assay and the cumulative OVA release was calculated and plotted.

The results of fig. 11 show that the duration of time for which DRF3 was able to release OVA was around 5 days and the duration of time for which DRF3 was able to release OVA was around 10 days. Further, the DRF3 can continuously release the medicine (protein) OVA, provides a great rising space for the half life of the medicine, further provides a real possibility for the self-assembled short peptide to be applied to the clinical medicine field, especially the medicine, macromolecule, vaccine and cell field multi-scene application, and supports the application of the self-assembled short peptide in preparing medicine carrier materials; the application of self-assembled short peptide in preparing macromolecular carrier material includes one or several of protein medicine, immunoglobulin, serum albumin, P53 protein, P21 protein, igG, SIgA, sugar, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide. Here we demonstrate that DRF3 is capable of carrying protein drugs using OVA protein as a model, and further DRF3 is also capable of carrying other macromolecular proteins (such as immunoglobulins, serum albumin, P53 protein, P21 protein, etc. compounds mentioned in the specification).

Example 13

Flow sorting experiment for promoting DC cell maturation by self-assembled short peptide DRF3

After mixing short peptides (2.5 mg/ml) with DC cells for 7 days, flow-sorting experiments were performed.

1 Seeding cells

3X 104/well of drug stimulation was typically applied for 24 hours and on-board.

2 Machine-on method

In cellquest environment, using control cell to calibrate loading condition, storing needed loading file closing cellqest software under preset file folder (INS file and SET file), opening MPM software, performing a series of settings under MPM software, firstly performing SETTINGINITIAL and FINALWASHING operations under Autosample, cleaning the whole pipeline, performing loading sample setting, mainly setting loading volume (100 ul in general), mixing times, cleaning times, etc., setting current plate under Acquisition menu (353263 as the plate number used now), dateStorageFolder (i.e. data storage file folder), acquisitionDocument and InstrumentSettingsFile (loading conditions selected from stored INS and SET files), selecting loading sample range of 96-well plate under Acquisition, loading sample, exiting loading sample plate, cleaning pipeline (adding water on 96-well plate, cleaning pipeline in the above mode)

It should be noted that: if PBS is used as the sheath liquid for loading, water is used as the sheath liquid after loading is finished, and the pipeline is repeatedly washed to prevent PBS salt crystals from blocking the pipeline.

Experimental results: FIG. 12 is a flow-through sorting graph of DC cells three-dimensionally cultured with short peptides, showing that CD86 expression of DC cells in the short peptide group is significantly increased compared with PBS group, showing that the short peptides promote maturation of DC cells, showing that the DC cells three-dimensionally cultured with short peptides can effectively activate DC cells, promote maturation of DC cells, prepare DC vaccine, further verify that DRF3 can enter the body as an antigen component, activate DC cells, prepare DC vaccine, and lay a theoretical foundation for self-assembled short peptides applied to vaccines, drugs, macromolecules, protein carriers and adjuvants, and this embodiment supports the application of a self-assembled short peptide in preparing drug carrier materials; application of self-assembled short peptide in preparation of macromolecular carrier material, wherein the macromolecules comprise one or more of protein drugs, immunoglobulin, serum albumin, P53 protein, P21 protein, igG, SIgA, sugar, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide; a self-assembled short peptide hydrogel has a concentration of 1ppM or more.

Preparation of DC vaccine: 1. the cultured cells were prepared in DC solution by adding 1ml of medium at a concentration of 5X 10 ⁵ cells/ml.

2. 200. Mu.l of DRF3 was added to 200. Mu.l of PBS (phosphate solution) to prepare solution 1.

3. Solution 1 was rapidly added to the DC solution to formulate a DC vaccine.

Example 14

NK cell apoptosis experiment for detecting short peptide culture by flow separation

After mixing short peptides (2.5 mg/ml) with NK cells for 7 days, a flow apoptosis experiment was performed.

1. Culturing cells by using a 6-hole plate, sucking out old culture medium when the growth of the cells reaches 60% -70%, and processing according to experimental requirements to continue culturing.

2. According to the experimental treatment time, the cell culture solution is sucked into a proper centrifuge tube, the adherent cells are washed once by PBS, and a proper amount of pancreatin cell digestion solution is added to digest the cells. Incubating at room temperature until the cells are gently blown off, and sucking out pancreatin cell digestive juice. Excessive digestion of pancreatin should be avoided. ( Note that: suspension cells do not need pancreatin digestion and can be directly collected into a centrifuge tube )

3. Adding the cell culture solution collected in the step (2), slightly mixing, transferring into a centrifuge tube, centrifuging for 5min at 1000g, discarding the supernatant, collecting cells, and lightly suspending the cells with PBS and counting. Note that: the cell culture solution added in the step (2) can collect suspended cells undergoing apoptosis or necrosis on one hand, and serum in the cell culture solution can effectively inhibit or neutralize residual pancreatin on the other hand; residual pancreatin can digest and degrade subsequently added Annexin V-FITC resulting in staining failure.

4. 5-10 Ten thousand resuspended cells were taken, centrifuged at 200g for 5min, the supernatant was discarded, and 195. Mu.l Annexin V-FITC conjugate was added to gently resuspend the cells.

5. Mu.l Annexin V-FITC was added and gently mixed.

6. Incubate at room temperature (20-25 ℃) for 10min in the dark. Light protection can be performed using aluminum foil.

7.200G was centrifuged for 5min, the supernatant was discarded, and 190. Mu.l Annexin V-FITC conjugate was added to resuspend the cells.

8. Add 10 μl propidium iodide staining solution, mix gently, and place in ice bath in the dark.

9. And (3) detecting by using a flow cytometer, wherein Annexin V-FITC is green fluorescence, and PI is red fluorescence.

Note that: FITC is green fluorescent and this detection method is not applicable to cells that have been labeled with green fluorescent.

Experimental results: FIG. 13 shows that the apoptosis rate of NK cells in the short peptide group is smaller than that of the PBS group, which indicates that the three-dimensional microenvironment constructed by the short peptide has good biocompatibility with the NK cells, so that the NK cells are effectively activated, and further, the embodiment has great application value in the field of preparing NK vaccines, and supports the application of the self-assembled short peptide in preparing NK vaccines in three-dimensional nanometer physical scaffold cultured cells; a self-assembled short peptide hydrogel has a concentration of 1ppM or more.

Preparation of NK vaccine: 1. the cultured cells were prepared into NK solution by adding 1ml of medium at a concentration of 5X 105 cells/ml.

3. Solution 1 was rapidly added to NK solution to formulate NK vaccine.

Further indicated by the above examples:

1. The round secondary chromatography of the short peptide is tested to detect that the short peptide can form a stable secondary structure, and a theoretical basis is laid for the short peptide DRF3 to form a stable fiber network structure.

2. The short peptide DRF3 can form a stable fiber network structure through testing a transmission electron microscope image, an atomic force microscope image and a frozen scanning electron microscope image of the short peptide, and can provide a stable three-dimensional nano physical bracket for cells, so that a theoretical basis is further laid for the DRF3 to load and wrap medicines, proteins, macromolecules, vaccines and the like.

3. The Congo red and aniline blue staining of the short peptide (containing the drug) shows that the short peptide DRF3 can be mixed with the drug and the protein to form a fiber network structure, and direct evidence is provided for loading the drug, the protein and the macromolecule on the DRF 3.

4. Further, the in vitro controlled release capability of the short peptide DRF3 on chicken Ovalbumin (OVA) is tested, so that the short peptide can be directly verified to be used as a long-term carrier and an adjuvant of a drug, protein and macromolecules, and the half life of the drug is increased.

5. By testing the three-dimensional culture capacity of the short peptide DRF3, the graph shows that the short peptide can provide a stable and durable three-dimensional nanometer physical scaffold for tonsil cells and organoids, and further according to the model, confidence is provided for preparing DC and NK vaccines by wrapping DC cells and NK cells in subsequent culture.

6. By testing the short peptide, three-dimensionally culturing DC and NK cells, using a flow separation technology to identify the maturation of the DC cells and the death rate of the NK cells, direct evidence is provided for the short peptide DRF3 to promote the maturation of the DC cells and activate the NK cells, evidence is provided for the DRF3 as an antigen component, and evidence is provided for the DRF3 to wrap the NK and the DC and prepare the DC and NK vaccine.

7. The experiment of testing the toxicity of the short peptide DRF3 and CCK8 cultured by colon cells shows that the short peptide has toxicity to colon cancer cells, and provides direct evidence for the short peptide DRF3 serving as a vaccine and a drug adjuvant and also serving as a drug for killing tumors.

The three-dimensional culture of fibroblasts after the short peptide DRF3 and hyaluronic acid are mixed shows that the toxicity of hyaluronic acid can be reduced after hyaluronic acid is combined, further shows that the hyaluronic acid and other main cosmetic compounds are combined to self-assemble the short peptide, has great application value in the medical and cosmetic fields, and provides theoretical support for the short peptide to replace hyaluronic acid as a filler.

And (3) a sequence table:

SEQUENCE LISTING

<110> Chengdu Saint Engineer academy of surgery

<120> A self-assembled short peptide, its use in carrier materials and biomedical applications

<130>

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> PRT

<213> Artificial sequence

<400> 1

Arg Leu Asp Ile Lys Val Glu Phe Arg Leu Asp Ile Lys Val Glu Phe

1 5 10 15

Claims

1. A self-assembling short peptide, characterized in that it has the amino acid sequence:

DRF3：ArgLeuAspIleLysValGluPheArgLeuAspIleLysValGluPhe。

2. Use of a self-assembling short peptide according to claim 1 for the preparation of a pharmaceutical carrier material.