CN114181284A

CN114181284A - Application of nano short peptide DRF3 in medicines, NK cell carriers and biomedicine

Info

Publication number: CN114181284A
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: 2022-03-15

Abstract

The invention discloses an 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 problem of how to produce hyaluronic acid products with stronger degradation resistance, weaker toxicity, in-situ activated collagen regeneration and the like, and the amino acid sequence of the hyaluronic acid product is as follows: arg Leu Asp Ile Lys Val Glu Phe Arg Leu Asp Ile Lys Val Glu Phe, the self-assembly short peptide activates and enhances the purity and killing ability of NK cells; also shows good ability to promote 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, more stability, better safety, stronger resistance to degradation and less toxicity than the single use of hyaluronic acid, and the short peptide can also be used as a single filling agent.

Description

Application of nano short peptide DRF3 in medicines, NK cell carriers 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, antifungal agents, innate immune systems, toxins and pesticides. No one has considered peptides to be useful as scaffold hydrogel materials. Significant changes have occurred since 1990 after a very interesting repeat was found in the yeast protein. It is now recognized that self-assembling peptides made from 20 natural amino acids have true material properties. Currently, many different applications have been developed from these simple and designed self-assembling peptide scaffold hydrogels and are commercially available. Examples include: (1) true 3D tissue cell culture of different tissue cells and various stem cells, (2) repair and regenerative medicine and tissue engineering, (3)3D tissue printing, (4) sustained release of small molecules, growth factors and monoclonal antibodies, (5) accelerated wound healing skin and diabetic ulcers and immediate hemostatic applications.

Molecular self-assembly refers to the ability of molecules to self-organize and self-assemble into a regular structure, i.e., change from a disordered state to an ordered state, without the intervention of an external force. In recent years, chiral self-assembled short peptides have been developed into an emerging nano biomaterial. The ECM-imitated biological scaffold nano material can imitate partial functions of ECM, thereby influencing 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 (NK) are important immune cells of the innate immune system, are the first line of defense of the human body against cancer cells and viral infection, serve as regulators of immune response, have strong immunoregulatory functions, can have anti-fibrosis activity by directly killing hepatic stellate cells, when an organism is infected by exogenous sources and invaded by tumor cells, NK cells are activated, have proliferation and killing activities, can well clear necrotic cells and tumor cells affected by inflammation, recognize and kill cells infected by stress, transformation or viruses by expressing the balance of activation and inhibition of receptor signals, secrete various effector molecules, and secrete active factors for promoting migration after the NK cells are activated, and under the factors of individual difference, cell subgroup difference, whether the NK cells are activated and the like, the types and levels of chemokine receptors vary widely. How to create a good three-dimensional microenvironment for NK cells and activate the NK cells, so that the death rate of the NK cells is reduced, the activity of the NK cells is increased, and the NK vaccine which has stronger activity, stronger immunogenicity and longer release time is prepared is one of the key problems to be solved.

DC in the human body is classified into lymphoid DC (lymphoid DC) and myeloid DC (myeloid DC), which are the sources of most DC. Lymphoid lineage DC is developed by differentiation of precursor cells in thymus, and after activation, a large amount of type I interferon (interferon 1, IFN1) is mainly released and participates in virus immune response; myeloid DC are mainly involved in the induction and initiation of immune responses. Under normal conditions in humans, most DCs are in an immature state, are located in the epithelium and various parenchymal organs of non-lymphoid tissues, have strong antigen uptake, processing and handling capabilities, but express low levels of costimulatory molecules and major histocompatibility complex ii molecules (MHC ii), intercellular adhesion molecules (ICAM). How to activate DC cells and enable signal transmission among the cells to be active, and the preparation of DC vaccine with stronger immunogenicity and longer release time is the second problem to be solved.

In recent years, along with the popularization of cosmetics, the cosmetics are deeply loved and concerned by young people, and the development of whitening, anti-aging, anti-wrinkle and the like is rapidly increased, so that the cosmetics are highly concerned and generally concerned by related subject fields, particularly the fields of medical cosmetology, cosmetic science, skin care and health care, skin anti-aging and the like.

Hyaluronic acid is well known to be one of the basic components of human and other mammalian connective tissues. It is a substance widely found in human epidermal, epithelial and neural tissues. Hyaluronic acid imparts unique resistance and shape retention to the skin. The lack of hyaluronic acid can cause skin weakness and promote the formation of wrinkles and blemishes. With age, the concentration of hyaluronic acid in human tissue tends to decrease, impairing its tissue repair function. With progressive aging and repeated exposure to ultraviolet light, epidermal cells decrease the production of hyaluronic acid, and the rate of aging increases. For this reason, HA-based formulations are still today considered as the best epidermal filler on the market, since they do not risk skin allergic reactions. Initially, a first formulation based on hyaluronic acid was prepared in the form of particles or microspheres suspended in a gel. However, these fillers based on gelled microspheres have the disadvantage of poor stability over time, tending to chemically degrade months after injection into the skin. Thus, over time, frequent re-injection of filler is required to maintain the repair and epidermal growth constant. Recently, it has been found that hyaluronic acid is subjected to the advantage of a suitable crosslinking step by using a specific crosslinking agent, and therefore fillers based on crosslinked hyaluronic acid are used in the cosmetic treatment of the face. But the injection is required frequently, and the effect, the stability and the safety are moderate.

How to construct a three-dimensional microenvironment of cells with better biocompatibility, truly simulated cell growth, higher mechanical strength and faster gelatinization, how to obtain a product with the similar application of crosslinking hyaluronic acid and non-crosslinking hyaluronic acid, better effect, low toxicity or nontoxicity, stronger degradation resistance, in-situ activated collagen regeneration and the like, how to establish a maturation promoter with good biocompatibility, low toxicity, capability of independently enhancing the immunity in vivo and good encapsulation of DC and NK cells, and the great problem to be solved for preparing the vaccine with better effect.

Disclosure of Invention

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

The invention specifically adopts the following technical scheme for realizing the purpose: a self-assembled short peptide, the amino acid sequence of which is:

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-assembly short peptide is provided, the novel self-assembly short peptide and the novel self-assembly short peptide can be mixed with PBS and then self-assembled into injectable hydrogel to be used as a three-dimensional culture bracket of cells, the bracket simulates an extracellular mechanism, not only is used as a physical bracket, but also promotes the maturation and differentiation of the cells, and the degradation product of the novel self-assembly short peptide is amino acid and has no toxicity; can also be used as maturation-promoting agent of DC cells, increase killing ability of NK, and can be used for preparing NK vaccine; the invention takes a new self-assembly short peptide as a new thought of preparation and adjuvant of cosmetics respectively, changes the content of the cosmetics from the source, the degradation product is amino acid, not only can be used as a humectant, a slow release agent, a lubricant, an antioxidant, a film-forming agent and an emollient, but also can have auxiliary effect on the repair of skin, is equivalent to skin nourishment, and the degradation product is amino acid and has good biocompatibility; the novel self-assembly short peptide is combined with physical properties and a microscopic nano structure, is respectively combined with hyaluronic acid for use, is prepared into a combined system which has better effect, more stability, better safety, stronger resistance to degradation and less toxicity than the single use of hyaluronic acid, and can also be used as a single filling agent.

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

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

Further, the carbon terminal of the self-assembly short peptide is amidated.

Further, self-organizing short peptides form secondary structures, which include one or more of alpha helices, beta sheets, beta coils, and random coils.

An application of self-assembled short peptide as antigen.

An application of self-assembled short peptide in preparing medicine 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.

An application of self-assembled short peptide in preparing antineoplastic medicine.

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 preparing NK vaccine in three-dimensional nano physical scaffold cultured cells.

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

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

An application of self-assembled short peptide as vaccine adjuvant.

A self-assembled short peptide hydrogel.

The preparation method of the hydrogel comprises the following steps:

step 1, adding short peptides into water to prepare mother liquor;

and 2, adding ions or PBS into the mother liquor to prepare the self-assembled short peptide hydrogel.

Further, the ion includes 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 ^-One or more of (a).

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

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

Specifically, the method comprises the following steps: adding 10mg of self-assembly short peptide into 1ml of water to prepare mother solution, adding 3ml of PBS to prepare the self-assembly 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 1-10 mg/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-assembled short peptide.

In the technical scheme of the application, short peptide self-assembly is triggered in a salt ion environment, wherein the salt ions include and are not limited to: na (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-assembly short peptide which has the functions of improving the immunity of organisms, resisting tumors and infection and is used as an adjuvant of medicaments and vaccines.

Provides a novel self-assembly short peptide which is loaded with drugs, tumor vaccines, cells, antibodies and proteins, can be continuously and stably released in vivo and can increase the half-life of the drugs.

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

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

The nano-fiber network structure formed under an atomic force microscope and a cryo-scanning electron microscope can be applied to the field of cosmetics and used as a slow release agent, an antibacterial agent, a lasting moisturizing agent, a lubricant, a rheology regulator, an antioxidant, a film forming agent, an emollient, a stabilizer, a buffering agent and the like.

The nanofiber formed by the self-assembled short peptide can be used for three-dimensional attachment and growth of cells on the nanofiber, so that the self-assembled short peptide can be applied to three-dimensional culture of human, animal and plant cells.

In the present application, the application as a drug, a protein, a macromolecular adjuvant, or a carrier can be used as a carrier or an adjuvant for any compound set, wherein the set comprises a plurality of compounds with different structures which are connected and interacted through van der waals force, hydrogen bonds, hydrophobic bonds, and the like. The compounds described therein specifically include and are 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, and the like, lipids, steroids, glycopeptides, glycoproteins, proteoglycans, and the like or naturally occurring molecular analogs and derivatives or small molecule compounds produced by chemical synthesis techniques. The self-assembly short peptide can be used as a carrier and an adjuvant of drugs, macromolecules, proteins and the like consisting of any compounds. The term "nucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, cDNA, or DNA. The term generally refers to nucleotides (either ribonucleotides or deoxyribonucleotides or either type of nucleotide in modified form) in the form of a polymer of at least 10 bases in length. The term includes both single-stranded and double-stranded forms of DNA.

"macromolecule" in this application refers to a biological substance with a relative molecular mass of 5000 or more, even over a million, such as macromolecules including one or more of protein drugs, immunoglobulins, serum albumin, P53 protein, P21 protein, IgG, SIgA, sugars, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, receptors, nucleic acids, nucleotides, oligonucleotides, polynucleotides, and the like. It is very closely related to life activities and consists of simple molecular units thought to be monomers. There are gel-forming substances in solution. Compounds having a relative molecular mass of more than ten thousand are generally referred to as macromolecular compounds or macromolecular compounds. It is composed of many repeating structural units, generally having a linear structure, and some having a dendritic structure. Many substances with important biological effects, such as proteins and nucleic acids, belong to this class of compounds.

In the present application, the "drug" can only affect the physiological, biochemical and pathological processes of the body, and can be used as a chemical substance for preventing, diagnosing, treating diseases and fertility, wherein the self-assembled short peptide can be used as a carrier of the drug, and the action types of the "drug" specifically include and are not limited to: acting on the drugs of efferent nervous system (efferent nervous system drug, parasympathomimetic drug, cholinergic receptor blocking drug, adrenoceptor agonist, adrenoceptor blocking drug), acting on the central nervous system (sedative hypnotic drug, antiepileptic drug, anticonvulsant drug, anti-Parkinson disease and Alzheimer disease treating drug, anti-psychotic drug, analgesic drug, antipyretic analgesic and anti-inflammatory drug, anesthetic drug), acting on the circulatory system and blood system (antiarrhythmic drug, antihypertensive drug, anti-chronic congestive heart failure drug, anti-atherosclerotic drug, anti-angina drug, drug affecting the blood and hematopoietic system), acting on the visceral system (diuretic drug and dehydratant, antitussive, expectorant and antiasthmatic drug, anti-peptic ulcer and digestive tract function regulating drug, acting on uterine smooth muscle drug), Acting on autologous active substances and endocrine system drugs (adrenocortical hormone, thyroid hormone and antithyroid drug, insulin and oral hypoglycemic drug, sex hormone drug and contraceptive), chemotherapeutic drugs (beta-lactam antibiotics, aminoglycosides and doramectin, macrolides, lincomycins, vancomycin drugs, tetracyclines, chloromycetins, artificially synthesized antibacterial drugs, antituberculosis drugs, antileprosy drugs, antibacterial drugs, antiviral drugs, antifungal drugs, antiparasitic drugs, antitumor drugs), and acting on immune system drugs (immunosuppressive drugs, immunopotentiating drugs).

In the present application, the self-assembled short peptide serves as a carrier of a "drug", wherein the drug specifically includes but is not limited to (the drug name and the chemical name have consistency): egg albumin (OVA), carnitine, tyramine, reserpine, ***e tricyclo, carbachol, pilocarpine, nicotine, neostigmine, norepinephrine, phenylephrine, clonidine, epinephrine, isoproterenol, dobutamine, albuterol, acetylcholine, clobecholine, methacholine, arecoline, cholinesterase, penicillin, oxacillin, ampicillin, amoxicillin clavulanate, piperacillin sodium tazobactam sodium, cephradine, ceftriaxone, gentamicin, erythromycin, azithromycin, clindamycin, sulfamethoxazole, norfloxacin, moxifloxacin, metronidazole, nitrofurantoin, pyrazinamide, ethambutol, streptomycin, sodium p-aminosalicylate, dapsone, fluconazole, itraconazole, amphotericin B, acyclovir, ganciclovir, oseltamivir, stastastaphyl, doxetasone, doxorabicolol, doxorabicine, doxoraline, doxora, Ribavirin, solifosbuvir, valprovir, tenofovir disoproxil, aids, chloroquine, hydroxychloroquine, artemisinin-based drugs, sodium stibogluconate, praziquantel, albendazole, lidocaine, bupivacaine, ropivacaine, ketamine, propofol, remifentanil, sevoflurane, rocuronium, succinylcholine chloride, vecuronium, fentanyl, pethidine, morphine, pregabalin, acetaminophen, aspirin, ibuprofen, diclofenac sodium, indomethacin, mesalamine, penicillamine, allopurinol, colchicine, benzbromarone, amantadine, trihexyphenidyl, plaxozine, bromocriptine, neostigmine, carbamazepine, sodium valproate, phenobarbital, lamotrigine, nimodipine, betahistine, mannitol, sodium phosphatidylcholine, meclizine, methamphetamine, perzine, methamphetazine, and the like, Chlorpromazine, haloperidol, sulpiride, amisulpride, olanzapine, risperidone, paliperidone, aripiprazole, paroxetine, fluoxetine, amitriptyline, clomipramine, diazepam, clonazepam, estazolam, tandospirone, lithium carbonate, midazolam, isosorbide dinitrate, nifedipine, mexiletine, metoprolol, digoxin, captopril, valsartan, nitrendipine, urapidil, prazosin, phentolamine, dopamine, simvastatin, bromhexine, carbocisteine, compound licorice, aminophylline, compound aluminum hydroxide, lactasin, anisodamine, domperidone, ganciclovir, bifendate, viable bacteria of bacillus, ursodesoxycholic acid, sulfasalazine, furosemide, hydrochlorothiazine, fructose, tamsulosin (tamsulosin), finasteride, ferrous sulfate, vitamin B12, Adcobalamine, mecobalamin, aspirin, ticagrelor, thrombin, vitamin K1, protamine, heparin, warfarin, urokinase, dabigatran etexilate, recombinant human tissue plasminogen kinase derivatives, hydroxyethyl starch, chorionic gonadotropin, recombinant human growth hormone, hydrocortisone, methylprednisolone, insulin, metformin, glyburide, glipizide, glimepiride, gliquidone, acarbose, liraglutide, sitagliptin, thyroid tablets, levothyroxine sodium, methimazole, propylthiouracil, cinacalcet, testosterone propionate, testosterone undecanoate, progesterone, medroxyprogesterone, diethylstilbestrol, nileol, vitamin D2, chlorpheniramine, diphenhydramine, promethazine, triptolide, azathioprine, mycophenolate mofetil, semaphorin, ifosfamide, methotrexate, mercaptopurine, hydroxyurea, hydroxyprogesterone, hydroxapridine, and the like, Fluorouracil, etoposide, daunorubicin, hydroxycamptothecin, vincristine, paclitaxel, cisplatin, arsenous acid (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, sodium chloride, compound sodium chloride, sodium lactate, glucose, sodium thiosulfate, pralidoxime, penehyclidine, methylene blue, naloxone, acetamide, penicillamine, tetanus antitoxin, anti-rabies serum, tetanus human immunoglobulin, mitomycin, snake venom antiserum, national vaccine for immune programming, diatrizoate, iodized oil, iohexol, tuberculin protein derivatives, erythromycin, miconazole, triamcinolone, and econazole, Mupirocin, ichthammol, salicylic acid, mometasone furoate, tretinoin, chloramphenicol, levofloxacin, atropine, oxymetazoline, posterior pituitary injection, contraceptive, and caffeine.

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

In the present application, DRF3 is a three-dimensional culture of cells, wherein "cells" includes, but is not limited to: DC cells (dendritic cells), NK cells (natural killer cells), lymphocytes, monocytes/macrophages, granulocytes, mast cells, leukocytes, phagocytes, 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, and the like, also included are, without limitation, one or more of primary and secondary cells in tonsils, adrenal glands, bile ducts, bladder, bone marrow, brain, breast, cervix, colorectal, esophagus, eye, head and neck, kidney, liver, lung, lymph nodes, nervous system, ovary, pancreas, prostate, skin, soft tissue, stomach, testis, thymus, thyroid, uterus, and like organs.

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, and the like); eukaryotic parasites such as malaria parasites, e.g., Plasmodium falciparum (helminth), worms, etc.); vertebrates (e.g., fish, amphibians, reptiles, birds, mammals); and mammals (e.g., rodents, primates such as humans and non-human primates).

In the present application, the term "three-dimensional culture" refers to co-culturing a carrier with different materials having a three-dimensional structure and various types of cells in vitro, so that the cells can migrate and grow in the three-dimensional spatial structure of the carrier to form a three-dimensional cell-carrier complex, and changing or reducing the characteristic of adherence of the cells in the culture process, so that the cells obtain more living space in space, cell contact inhibition is reduced, and the cells are attached to and self-assembled short peptides and generally appear as circles under a microscope.

In the present application, the term "organoid" means that the organoid belongs to a three-dimensional (3D) cell culture, including some key characteristics that represent the organ. Such in vitro culture systems comprise a population of self-renewing stem cells that can differentiate into a plurality of organ-specific cell types, have similar spatial organization as the corresponding organ and are capable of reproducing a portion 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 directed differentiation of pluripotent stem cells can produce organoids wherein the class of organoids includes and is 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, and the like. The tumor organoids involved 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, intraocular melanoma of bone cancer, retinoblastoma, fallopian tube cancer, gall bladder cancer, stomach cancer, soft tissue sarcoma, bacterial cell tumor of the central nervous system (brain cancer), bacterial cell tumor of extracranial children, bacterial cell tumor of external horn, bacterial cell tumor of ovary, testicular cancer, heart tumor, hepatocellular carcinoma, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, renal cell carcinoma, laryngeal cancer, leukemic lip and oral cancer, liver cancer, lung cancer (non-small cell, alveolar tumor and tracheobronchial tumor), lymphoma, male breast cancer, melanoma, skin cancer, mesothelioma, metastatic neck cancer (head and neck cancer), Oral cancer, multiple myeloma/plasmacytic 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 pelvis cancer, transitional cell carcinoma of urethral cancer, uterine cancer, endometrial cancer, vaginal cancer, vascular tumor, and the like.

In the present application, the term "antigen" and grammatical equivalents thereof (e.g., "antigenicity") refers to a compound that can be specifically bound by a product of a specific humoral or cellular immunity, such as an antibody molecule or T cell receptor. The antigen may be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as 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 and be used together with the collagen promoter, such as and not limited to: ascorbic acid phosphate ester salts such as ascorbic acid, sodium ascorbic acid phosphate ester salts and magnesium ascorbic acid phosphate ester salts; ascorbic acid fatty acid esters such as ascorbic acid monostearate, ascorbic acid monopalmitate, ascorbic acid dipalmitate, and ascorbic acid tetraisopalmitate; ascorbic acid ethers such as 3-O-ethyl ascorbic acid, 2-O-ethyl ascorbic acid, cetyl ascorbic acid, glycerol ascorbic acid, and hexylglyceryl ascorbic acid 12; ascorbyl glucoside such as ascorbic acid-2-glucoside and fatty acid esters thereof; ascorbic acid derivatives such as ascorbyl sulfate and ascorbyl tocopherol phosphate; retinol, retinol acetate, retinol palmitate, hydrogenated retinol, and other vitamin A compounds; nicotinamide, glutathione, cysteine, crocetin, sericin, geraniol (geraniol), glycerol glucoside, lactoferrin, procyanidins, pantothenic acid, panthenol, soyasaponin, resveratrol, isoflavones, coenzyme Q10, chondroitin sulfate, acetylglucosamine, glycerophosphatidylcholine, hydrolyzed hyaluronic acid, collagen peptides, shell matrix hydrolysate, 5' -adenosine monophosphate, proline, glycine, arginine, aspartic acid, alanine; and at least one of plant extracts having collagen production promoting effect, such as hibiscus, licorice leaves, roman chamomile, sweet tea, hawthorn, balsamine, lotus leaves, kudzu roots, Sparassis crispa, chlorella, Sasa Veitchii Rehder, burdock, Japanese pagodatree roots, flowering cabbage, loquat leaves, purple perilla, peas, mulberry leaves, thyme, spruce, Lepidium Merrill, Commelina communis, Platycladi seeds, cassia seeds, snakegourd seeds, sanguisorba, sophora flavescens, persimmon, peony, perilla, dried orange peel, mallow, ginger, chamomile, strawberry seeds, swertia herb, soybean, wheat, ginseng, coix seeds, pelargonium prevention, cardamom, rosemary, and sage.

The present invention can promote the production and assembly of hyaluronic acid, and can be used in combination with hyaluronic acid as a dermal filler and anti-wrinkle, specifically, a combinable production promoter such as, but not limited to: ascorbyl phosphate ester salts such as hyaluronic acid, collagen, ascorbic acid, sodium ascorbyl phosphate, and magnesium ascorbyl phosphate; ascorbic acid fatty acid esters such as ascorbic acid monostearate, ascorbic acid monopalmitate, ascorbic acid dipalmitate, and ascorbic acid tetraisopalmitate; ascorbic acid ethers such as 3-O-ethyl ascorbic acid, 2-O-ethyl ascorbic acid, cetyl ascorbic acid, glycerol ascorbic acid, and hexylglycerol ascorbic acid; ascorbyl glucoside such as ascorbic acid-2-glucoside and fatty acid esters thereof; ascorbic acid derivatives such as ascorbyl sulfate and ascorbyl tocopherol phosphate; retinol, retinol acetate, retinol palmitate, hydrogenated retinol, and other vitamin A compounds; nicotinamide, carotene, tocopherol, tocotrienol, chondroitin sulfate, acetylglucosamine, glycerophosphatidylcholine, glyceroglucoside, hydrolyzed hyaluronic acid, collagen peptides, sitosterol, carnosine, creatine, phytic acid, N-methylserine, 3-methylcyclopentadecanone, saponin, genistein, daidzein, phytol; and at least one plant extract having a hyaluronic acid production promoting effect, such as marjoram, peppermint, apple mint, perilla, beefsteak plant, lemon, Mongolian mulberry, breadfruit, paper mulberry, fig, ulva, mallow, waixue grass, phellodendron, houttuynia cordata, yarrow, almond, hawthorn, linseed, gardenia, nettle, strawberry seed, carambola, passion fruit, sea grape, saffron, camellia, pumpkin, luffa, asparagus, gynostemma pentaphylla, sophora flavescens, dried orange peel, peony, persimmon, salvia miltiorrhiza, centella asiatica, pu' er tea, maitake mushroom, water shield, horsetail, pear, chamomile, and the like.

The invention can resist aging and is combined with an anti-aging agent to be used as the preparation of the anti-aging agent, such as and not limited to: at least one of tocopherol (vitamin E), disodium vitamin E phosphate, tocopherol acetate, fullerene, ubiquinone, grape seed extract, tea extract, retinol acetate, ginkgo biloba extract, phytosterols, resveratrol, ceramide, ginseng root extract, puerarin, soybean isoflavone, etc.

All the words comprising the self-assembled short peptides and the functions of the self-assembled short peptides have the macroscopic expression of hydrogel.

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

The invention has the following beneficial effects:

1. provides a novel self-assembly short peptide, and increases the types of the self-assembly short peptide.

2. The novel self-assembled short peptides can form stable nano fibers, and the nano fibers can be used for three-dimensional culture of stem cells and organoids, simulating the living environment in cells and providing a three-dimensional microenvironment in vitro.

3. The novel self-assembly material with the drug loading function is provided, can load drugs and macromolecules, has a remarkable anti-tumor effect, and opens up a new path for vaccine adjuvants.

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

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

6. Disclosed 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 the self-assembled short peptide DRF3 of the present invention;

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

FIG. 5 is a congo red staining pattern of the self-assembled short peptide DRF3 of the present invention;

FIG. 6 is a aniline blue staining pattern of the self-assembled short peptide DRF3 of the present invention;

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

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

FIG. 9 is a CCK8 toxicity test chart of fibroblasts cultured in three dimensions after mixing the self-assembled short peptide DRF3 of the present invention with hyaluronic acid;

FIG. 10 is a diagram of the experiment of CCK8 challenge of colon cancer cells by the self-assembled short peptide DRF3 of the present invention;

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

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

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

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention.

Thus, the following detailed description of the embodiments of the present invention, 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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The concentration of the examples of the present invention is 2.5mg/ml unless specified.

Example 1

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

Materials:

preparing raw materials according to an amino acid sequence as follows: Fmoc-L-Arg-OH (9-fluorenylmethoxycarbonyl-L-arginine-gamma-tert-butoxycarbonyl), Fmoc-L-Lys-OH (9-fluorenylmethoxycarbonyl-L-lysine-epsilon-tert-butoxycarbonyl), Fmoc-L-Asp (OtBu) -OH (fluorenylmethoxycarbonyl-L-aspartic acid-epsilon-tert-butoxycarbonyl), Fmoc-L-Val-OH (9-fluorenylmethoxycarbonyl-L-valine), Fmoc-L-Phe-OH (9-fluorenylmethoxycarbonyl-L-phenylalanine-gamma-tert-butoxycarbonyl), Fmoc-L-Glu (OtBu) -OH (9-fluorenylmethoxycarbonyl-L-glutamic acid-epsilon-tert-butoxycarbonyl), Fmoc-L-Leu-OH (9-fluorenylmethoxycarbonyl-L-leucine), Fmoc-L-Ile-OH (9-fluorenylmethoxycarbonyl-L-isoleucine-gamma-tert-butoxycarbonyl), TBTU (O-benzotriazol-N, 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 solid-phase synthesis method adopting Fmoc (fluorenylmethyloxycarbonyl) protection has the following process steps:

(1) 0.5mmol/g Rink amide resin 20g was weighed into a peptide synthesizer, the resin was soaked with 200ml DCM (dichloromethane) for 30 minutes, then the resin was washed with 400inIDMF for 3min three times, and the washing solution was drained by suction filtration. Shaking and reacting for 30 minutes with 100ml of 20% piperidine/DMF for 10-20 minutes, after the reaction is finished, carrying out suction filtration on dry washing liquid, washing the resin for 5 times with 400m IDMF (intermediate frequency) for 3min each time, taking a little resin after the washing is finished to carry out the first three-ketone test, wherein the resin is positive, and then adding raw materials into a reactor:

Fomc-L-Leu-OH 14.312g

HBTU 14.15g

HOBT 8.77g

NMM 8.27ml

DMF 243ml

after the raw materials are added, the reaction is carried out for 40min, the suction filtration is carried out, the resin is washed by 30ml of DMF for 4 times, each time for 3min, a little resin is taken to be tested for the first three ketones, and the resin is negative.

(2) And then washing the resin by 40ml of DMF for 4 times, taking a little of resin for the first triketone check, and adding the following raw materials into a reaction vessel, wherein the resin is positive:

(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 resin is washed by 30ml of DMF for 4 times, each time is 3 minutes, a little resin is taken to be used as the previous triketone test, and the resin is negative.

(3) The method comprises the following steps of (a) raw materials, (b) (c) (d) raw materials and the addition amount are unchanged, and the steps are repeated: in the step (2), the raw material (a) is replaced by amino acid of a corresponding primary structure sequence.

The operation of the first step and the second step is repeated again, the raw materials and the consumption of the steps are unchanged, and the steps are synthesized according to the DRF3 sequence; after the last beam, the Fmoc-protecting group was removed, 20% piperidine/DMF (vol./vol.) was reacted for 30 minutes, the resin was washed, 160ml of 50% acetic anhydride/DMF (vol./vol. of acetic anhydride) was added and reacted for 30 minutes, the resin was washed with 40ml DMF, and the resin was washed with methanol 4 times, filtered dry and dried under vacuum for 8 hours. Adding 50ml of 90% TFA/DCM (TFA volume concentration) into a container containing peptide resin, reacting for 3 hours, carrying out suction filtration, concentrating filtrate, adding diethyl ether into residual liquid, separating out white solid, carrying out suction filtration on the solid to obtain crude peptide, purifying by HPLC (high performance liquid chromatography), and carrying out freeze drying to obtain the short peptide DRF3 with the amino acid sequence as shown in the sequence table.

Example 2

As shown in FIG. 1, self-assembled short peptide DRF3 self-assembled for 24h Circular Dichroism (CD)

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

Example 3

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

1. Experimental Material

DRF3, the main solutions were: deionized water H₂O; PBS solution (Na)⁺、K⁺、PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-Etc.).

2. Main experimental instrument

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

3. Experimental methods

(1) Preparing a working solution with DRF3 to a final concentration of 100 mu M by using deionized water or PBS for observation by using 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 by using a clean suction head to drop on the surface of a clean glass slide, carefully clamping a copper mesh covered with a Formvar film on a TEM by using tweezers, dipping the working solution on the glass slide by using the copper mesh, standing for several 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) and (4) sucking the redundant liquid on the copper mesh by using filter paper, and standing in the air for a plurality of minutes until the copper mesh is dried.

Scanning the copper mesh by a TEM (transmission electron microscope), and directly observing the short peptide self-assembly structure on the copper mesh.

4. Results of the experiment

The self-assembled short peptide after self-assembly for 1 hour at 37 ℃ is displayed in a transmission electron microscope, after the self-assembled short peptide is assembled for 1 hour, the short peptide DRF3 can be self-assembled to form a nanofiber interwoven membranous structure, secondary junctions of the two structures are mirror image related beta folded structures, protein, medicines, macromolecules, cells and the like can be effectively loaded, a physical scaffold is provided for cell growth, and the application of the self-assembled short peptide in preparation of a medicine carrier material is supported; the application of a self-assembled short peptide in preparing a macromolecular carrier material, wherein 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; the application of self-assembled short peptide in preparing cell or organoid three-dimensional culture nano-scaffold material; the application of the self-assembled short peptide in the preparation of DC vaccine in the three-dimensional nano physical scaffold culture cell; the application of the self-assembled short peptide in preparing NK vaccine in three-dimensional nano physical scaffold cultured cells; a self-assembled short peptide hydrogel with a concentration of 1ppM or more. As shown in FIG. 2 (200 μm, 500 μm, 2 μm, respectively), FIG. 2 is a transmission electron microscope image of the self-assembled short peptide DRF3 assembled for 24h, which shows that the short peptide is now a network structure.

Example 4

As shown in FIG. 3, the atomic force microscopy images of the self-assembled short peptide at 400 μm, 1 μm and 2 μm respectively show that the short peptide is now in a network structure.

1. Experimental Material

DRF3, main solution: sterile deionized water H₂O; millipore Milli-Q system, autoclaved and stored at 4 ℃ until use.

2. Main instrument

Atomic force microscope AFM (multimode8)

3. Experimental methods

Preparing a working solution of DRF3 by using deionized water, wherein the final concentration is 100 mu M; respectively dripping 5 mul of the prepared working solution of DRF3 on the surface of a newly stripped mica sheet; about 30s after the coating is finished, washing with 1000 μ l of deionized water to remove the unattached short peptide; drying the short peptide working solution smears in air at room temperature; performing AFM scanning on the mica sheet in a gas phase, and collecting AFM images by using a SPI4000 recording mode; using a 20 μ M scanner (400), an Olympus Si-DF20 microcantilever, and a pin with a spring constant of 12N/M (Si, radius 10nm, rectangular base 200 μ M); the free resonance frequency of the cantilever is 127 KHz; the phase map is recorded at a pixel resolution of 512 x 512; to show the nanofiber structure of the self-assembled short peptide, scanning was performed in the range of 600nm × 600nm, 200nm × 200 nm.

4. Results of the experiment

The self-assembled short peptide after self-assembling for 24 hours in the environment of 37 ℃ is shown in an atomic force microscope, and figure 3 is an atomic force microscope picture of the self-assembled short peptide DRF3 when assembled for 24 hours, after 24 hours of assembly, the short peptide forms a dense and regular nano rod-shaped fiber structure, and rod-shaped nano fibers are mutually aggregated to form a dense nano fiber mesh scaffold. Further shows that the self-assembly short peptide can be widely applied to the field of biomedicine, provides a physical nano bracket, a drug carrier, a cell maturation promoting agent and the like for preparing medical and cosmetic products, cosmetics or health products, provides a theoretical basis for loading drugs, cells, proteins and macromolecules, and supports the application of the self-assembly short peptide in preparing a drug carrier material; the application of a self-assembled short peptide in preparing a macromolecular carrier material, wherein 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; the application of self-assembled short peptide in preparing cell or organoid three-dimensional culture nano-scaffold material; the application of the self-assembled short peptide in the preparation of DC vaccine in the three-dimensional nano physical scaffold culture cell; the application of the self-assembled short peptide in preparing NK vaccine in three-dimensional nano physical scaffold cultured cells; a self-assembled short peptide hydrogel with a concentration of 1ppM or more.

Example 5

As shown in FIG. 4, the self-assembly short peptide DRF3 is shown to be in a fiber network structure by cryo-scanning electron microscopy at 10 μm, 5 μm and 20 μm, respectively.

The self-assembled short peptide after self-assembly for 24 hours in the environment of 37 ℃ is shown in a frozen scanning electron microscope, and figure 4 is a frozen scanning electron microscope image of the self-assembled short peptide DRF3 when the self-assembled short peptide is assembled for 24 hours, after the self-assembled short peptide is assembled for 24 hours, the short peptide forms a dense and regular nano rod-shaped fiber structure, and rod-shaped nano fibers are mutually aggregated to form a dense nano fiber mesh scaffold. Further shows that the nano-scaffold can be widely applied to the field of biomedicine and provides physical nano-scaffolds, drug carriers, cell maturation promoters, cell vaccines and the like for preparing medical and cosmetic products, cosmetics or health products.

Example 6

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

1. Experimental Material

Short peptides: DRF3

The mixed solution comprises the following components: 1) preparing a mixed liquid with a certain concentration (containing chicken ovalbumin, immunoglobulin, insulin, thiotepa, carmustine, mitomycin, collagen, hydroxycamptothecin, paclitaxel, docetaxel, cephalotaxine, interleukin-2, hyaluronic acid, metronidazole, puromycin, CD40 and PD-L1), wherein the lowest concentration in the components is 1 ppM; 2) dyeing liquid: and (5) dyeing with Congo red.

2. Procedure of experiment

Diluting the stock solution of the short peptide solution with 10mg/ml to 2.5mg/ml by using a PBS solution, and performing Congo red staining detection after self-assembly for 0 hour, 4 hours, 12 hours and 24 hours respectively at 37 ℃. Sucking 15 mul of short peptide solution on a glass slide, dropping Congo red staining solution for staining for about 30s, observing under an optical microscope, and taking a picture.

3. Results of the experiment

Adding mixed liquid (containing chicken ovalbumin, immunoglobulin, insulin, thiotepa, carmustine, mitomycin, collagen, hydroxycamptothecin, paclitaxel, docetaxel, cephalotaxine, interleukin-2, hyaluronic acid, metronidazole, puromycin, CD40 and PD-L1) with set concentration, and the Congo red staining result shows that DRF3 is in fiber gel state under a microscope, the assembly is basically completed within 12h, the complete assembly is successful within 24h and the assembly is stable within 48 h, and the figure 5 shows that the Congo red staining is performed when the self-assembled short peptide DRF3 is assembled for 24 h. Further shows that the polypeptide can be used for preparing medical and cosmetic products, cosmetics or health care products and other fields, provides a theoretical basis for loading drugs, cells, proteins and macromolecules, and supports the application of the self-assembled short peptide in preparing a drug carrier material; the application of a self-assembled short peptide in preparing a macromolecular carrier material, wherein 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; the application of self-assembled short peptide in preparing cell or organoid three-dimensional culture nano-scaffold material; the application of the self-assembled short peptide in the preparation of DC vaccine in the three-dimensional nano physical scaffold culture cell; the application of the self-assembled short peptide in preparing NK vaccine in three-dimensional nano physical scaffold cultured cells; a self-assembled short peptide is used as main ingredient for preparing medicinal and cosmetic products or cosmetics; a self-assembled short peptide hydrogel with a concentration of 1ppM or more.

Example 7

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

The aniline blue staining solution is one of compositions of Masson trichrome staining kits, mainly comprises aniline blue, weak acid and the like, is acidic, is often used in combination with ponceau fuchsin staining solution and the like to dye collagen fibers, and is mainly used for distinguishing collagen fibers from muscle fibers, wherein the dyed muscle fibers are red, and the dyed collagen fibers are blue.

1. Experimental Material

Short peptides: DRF3

The mixed solution comprises the following components: 1) mixing liquid (such as chicken egg albumin, collagen, hyaluronic acid, glutathione, thyroxine, acetylcholine, bevacizumab, arsenous acid, gefitinib, chlorambucil, CD40, CD19, CD21, PD-L1, vitamin B)₁₂) Wherein the lowest concentration of the components is 1 ppM; 2) dyeing liquid: aniline blue staining solution, 95% ethanol, xylene and phosphomolybdic acid.

2. Experimental methods

Slicing and dewaxing to water conventionally; staining for 5-10min by using prepared Weibert iron hematoxylin staining solution; differentiating the acidic ethanol differentiation solution for 1-2s, and washing with distilled water; returning the Masson bluing liquid to blue for 1min, and washing with distilled water; dyeing the fuchsin dyeing solution for 5-10min, washing with weak acid working solution for 3-5s, and differentiating with phosphomolybdic acid solution for 1-2 min; pouring off the differentiation solution, directly placing into aniline blue staining solution for staining for 1-2min, and washing with weak acid working solution for 1 min; dehydrating with 95% ethanol for 5-10s for 3 times; the xylene is transparent for 3 times, each for 1-2min, and sealed with neutral gum.

3. Results of the experiment

FIG. 6 is aniline blue staining of self-assembled short peptide DRF3 after 24h assembly, mixed with a mixed solution containing a certain concentration (such as chicken egg albumin, collagen, hyaluronic acid, glutathione, thyroxine, acetylcholine, bevacizumab, arsenous acid, gefitinib, chlorambucil, CD40, CD19, CD21, PD-L1, vitamin B12), which shows that DRF3 is in a fiber gel state under a microscope, and the assembly is basically completed within 12h, the complete assembly within 24h is successful, and the assembly is stable within 48 h. Further shows that the self-assembly short peptide can be used in certain biomedical fields, can also be used for preparing medical and cosmetic products, cosmetics or health care products and the like, provides a theoretical basis for loading drugs, cells, proteins and macromolecules, and supports the application of the self-assembly short peptide in preparing a drug carrier material; the application of a self-assembled short peptide in preparing a macromolecular carrier material, wherein 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; the application of self-assembled short peptide in preparing cell or organoid three-dimensional culture nano-scaffold material; the application of the self-assembled short peptide in the preparation of DC vaccine in the three-dimensional nano physical scaffold culture cell; the application of the self-assembled short peptide in preparing NK vaccine in three-dimensional nano physical scaffold cultured cells; a self-assembled short peptide is used as main ingredient for preparing medicinal and cosmetic products or cosmetics; a self-assembled short peptide hydrogel with 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.

Placing the tonsil cells in water bath at 37 deg.C respectively for rapid dissolution; then adding RPMI-1640(Gibco company) culture solution, and suspending and centrifuging the precipitated cells; then, the culture medium was inoculated into a 25cm flask, and the complete medium in which the culture medium was RPMI-1640 was added.

The main components of the culture medium are 1 percent double-antibody solution (streptomycin-penicillin) and 8 to 10 percent fetal bovine serum (Gibco company), a culture bottle is placed at 37 ℃ and the volume fraction is 5 percent carbon dioxide, and the culture medium is cultured in an incubator;

changing the liquid every 2 days, and bottling the cells for passage after the growth condition of the cells is good;

when in passage, firstly sucking out the culture solution in the culture bottle by using a suction pipe in a super clean bench, adding 1ml of 0.25 percent pancreatin into the culture bottle to enable the cells to be free, and properly oscillating; transferring the liquid in the culture bottle into a centrifuge tube, centrifuging and precipitating cells at 1000rpm for 8 minutes, removing supernatant, suspending the cells by using complete culture medium culture solution of RPMI-1640, and inoculating in bottles; when the growth state of the inoculated cells is good, the inoculated cells are ready for use;

the three-dimensional culture steps are as follows: (1) after the cell growth state is good, three-dimensional culture is carried out; (2) centrifuging at 1000rpm, and counting; (4) adding solution such as short peptide, and mixing uniformly to form three-dimensional suspension cell sap; placing 96-well plate in constant temperature incubator (37 deg.C, 5% CO)₂₎Culturing, observing and analyzing; in a two-dimensional environment, part of the tonsil cells are in an adherent growth state and are in a long fusiform shape;

when the cells are cultured in a three-dimensional environment constructed by DRF3, the cells are embedded in the short peptide hydrogel in a spherical shape; the cells were clear and the boundaries were clearly visible, appearing as multi-layer growth.

The growth and proliferation of the cells are inhibited, while in the three-dimensional culture environment constructed by DRF3, the cells are transparent and the cell number is still increased. The tonsil cells as a representative or model of cell culture can grow and proliferate in a three-dimensional culture environment constructed by the short peptide hydrogel, and the state is good. Further shows that the self-assembly short peptide can be used for the treatment in the biomedical field such as the cell culture and stem cell field, can also provide support for preparing some special personalized medical and cosmetic products, and supports the application of the self-assembly short peptide in preparing the cell or organoid three-dimensional culture nano stent material; the application of the self-assembled short peptide in the preparation of DC vaccine in the three-dimensional nano physical scaffold culture cell; the application of the self-assembled short peptide in preparing NK vaccine in three-dimensional nano physical scaffold cultured cells; a self-assembled short peptide hydrogel with a concentration of 1ppM or more. As shown in FIG. 7, the morphology of tonsil cells after three days of three-dimensional culture was circular.

Example 9

Self-assembled short peptide three-dimensional culture tonsil organoid

1. Experimental Material

Tonsil tissue is derived from medical waste of the first hospital affiliated to Chongqing medical university, and conforms to corresponding national and ethical regulations.

2. Experimental methods

Soaking the tonsil tissue freshly taken from the operation in a 1640 culture medium containing 10% double antibody for 1 h; taking the tonsil tissue out, putting the tonsil tissue into a culture dish, and cutting the tonsil tissue into tissue blocks with the size of about 1mm multiplied by 1 mm; cells were digested with digestive enzymes in an oven at 37 ℃ for 2 h.

Preparing the digested cells into tonsil cell suspension; the cell suspension was adjusted to a concentration of 1X 10⁷Transferring the cells/ml into a 24-well plate, wherein each well contains 1 ml; 200 mul of self-assembly short peptide with the concentration of 5mg/ml is added into each hole to construct a three-dimensional culture system.

Transferring into a cell culture box for culture.

3. Results of the experiment

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

Tonsil is an organoid model, which indicates that the self-assembly short peptide can be applied to the field of three-dimensional culture of organoids, and supports the application of the self-assembly short peptide in preparing cell or organoid three-dimensional culture nano-scaffold materials; the application of the self-assembled short peptide in the preparation of DC vaccine in the three-dimensional nano physical scaffold culture cell; an application of self-assembled short peptide in preparing NK vaccine in three-dimensional nano physical scaffold cultured cells.

Example 10

CCK8 experiment for three-dimensional culture of fibroblasts after mixing self-assembled short peptide and hyaluronic acid

The experimental steps are as follows:

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

1. Preparing a cell suspension: cell counting

2. Plating into 96-well plates: the same samples were replicated in 3 replicates, with an appropriate number of plated cells, about 100ul of cell suspension per well.

3. Culturing in an incubator at 37 ℃: the cells need to be cultured for about 2-4 hours to adhere after inoculation, and if adhesion is not needed, the step can be omitted.

4. Adding toxic substances at different concentrations

5. Culturing in an incubator at 37 ℃: the incubation 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 culture time.

6. Add 10ul CCK 8: since the amount of CCK8 added to each well was small, and there was a possibility of errors due to the reagents sticking to the walls of the wells, it was recommended to gently tap the plate after the reagents had been added to aid in mixing.

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

8. Measurement of absorbance at 450 nm: the measurement is carried out by adopting double wavelengths, the detection wavelength is 450-490nm, and the reference wavelength is 600-650 nm.

The experimental results are as follows: as shown in fig. 9(HA hyaluronic acid; OD absorbance), it is shown that DRF3 is almost non-toxic after combined three-dimensional culture of fibroblasts with hyaluronic acid, or the toxicity is weaker than that of hyaluronic acid alone in culture, indicating that the short peptide DRF3 can synergistically reduce the toxicity of hyaluronic acid, further indicating that the fiber network structure of DRF3 is similar to (but less toxic than) that of hyaluronic acid, almost non-toxic or extremely low in cytotoxicity, and also providing theoretical support for the short peptide to replace hyaluronic acid as a separate filler, further indicating that hyaluronic acid and other major cosmetic compounds combined self-assembled short peptide have significant application value in the medical and cosmetic fields, and supporting a self-assembled short peptide as a major component for preparing medical and cosmetic products or cosmetics; a self-assembled short peptide hydrogel with a concentration of 1ppM or more.

Example 11

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

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

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

The experimental results are as follows: as shown in fig. 10, the higher the short peptide concentration is at 24 and 48 hours, the greater the toxicity to colon cancer cells, further indicating the application prospect of the short peptide DRF3 as the anti-tumor drug preparation field, supporting the application of a self-assembled short peptide in the preparation of anti-tumor drugs; a self-assembled short peptide hydrogel with a concentration of 1ppM or more.

Example 12

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

The DRF3 hydrogel was tested for OVA release by placing 200. mu.l OVA in 200. mu.l hydrogel and then adding 200. mu.l PBS solution on top of the gel.

Each day 100. mu.l PBS was aspirated and supplemented with an equal volume of PBS solution. OVA concentrations in samples were extracted by BCA assay and cumulative OVA release was calculated and plotted.

The results in FIG. 11 show that DRF3 can release OVA continuously for about 5 days, and DRF3 can release OVA continuously for about 10 days. Further shows that DRF3 can continuously release OVA (ovalbumin) of the drug, provides a great rising space for the half-life period of the drug, further provides real possibility for the self-assembly short peptide to be applied to the clinical medicine field, in particular to multi-scene application in the fields of drugs, macromolecules, vaccines and cells, and supports the application of the self-assembly short peptide in the preparation of a drug 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, SIgA, saccharide, monosaccharide, oligosaccharide, polysaccharide, amino acid, peptide, oligopeptide, polypeptide, protein, receptor, nucleic acid, nucleotide, oligonucleotide and polynucleotide. Here we demonstrated that DRF3 can load protein drugs using OVA protein as a model, and further DRF3 can also load other macromolecular proteins (such as immunoglobulin, serum albumin, P53 protein, P21 protein, etc. and compounds mentioned in the specification).

Example 13

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

After the short peptide (2.5mg/ml) was mixed with DC cells and cultured for 7 days, a flow sorting experiment was performed.

1 seeding of cells

The 3X 104/well drug stimulation was generally applied for 24 hours, and the machine was operated.

2-computer-operating method

Under the cellquest environment, a control cell is utilized to calibrate the loading condition, and under the SET folders (INS file and SET file), the required sample loading file is stored, the cellqest software is closed, the MPM software is opened, a series of settings are carried out under the MPM software, firstly, the SettingInitial and FinaWashing operations are carried out under the Autosample, the sample loading conditions are SET to mainly SET the sample loading volume (generally 100ul), the mixing times, the cleaning times and the like after the whole pipeline is cleaned, setting a current plate (the plate number is 353263 currently used), a DateStorage folder, an acquitionDocument and an Instrument SettingsFile (the sample loading condition is selected from stored INS and SET files) under an Acquisition menu, selecting the sample loading range of a 96-well plate under the Acquisition, selecting Acquire under the Acquisition, withdrawing the sample loading plate after the sample loading is finished, using water, and cleaning a pipeline (adding water on the 96-well plate and cleaning the pipeline in a sample loading manner).

It is necessary to know that: if PBS is used as the sheath fluid for sample loading, after the sample loading is finished, water is used as the sheath fluid, and the pipeline is repeatedly flushed, so that the PBS salt is prevented from crystallizing and blocking the pipeline.

The experimental results are as follows: FIG. 12 is a flow-sorting graph of a DC cell cultured in three-dimensional short peptide, in which the expression of CD86 in the DC cell in the short peptide group is significantly increased compared with that in PBS group, which indicates that the short peptide promotes the maturation of the DC cell, indicates that the DC cell cultured in three-dimensional short peptide can effectively activate the DC cell, promote the maturation of the DC cell, prepare the DC vaccine, and further verifies that DRF3 can enter the body as an antigen component to activate the DC cell, prepare the DC vaccine, and lay a theoretical basis for the application of the self-assembled short peptide in vaccines, drugs, macromolecules, protein carriers and adjuvants, and this example supports the application of the self-assembled short peptide in the preparation of drug carrier materials; the application of a self-assembled short peptide in preparing a macromolecular carrier material, wherein 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 with a concentration of 1ppM or more.

Preparation of DC vaccine: 1. culturing the cultured cells at a concentration of 5X 10⁵1ml of the culture medium was added to each ml to prepare a DC solution.

2. Solution 1 was prepared by adding 200. mu.l of DRF3 to 200. mu.l of PBS (phosphate solution).

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

Example 14

Experiment for detecting short peptide culture NK cell apoptosis by flow sorting

After short peptides (2.5mg/ml) were cultured in a mixed culture with NK cells for 7 days, a flow apoptosis test was conducted.

1. Culturing cells with 6-well plate, sucking out old culture medium when cell growth reaches 60-70%, treating according to experiment requirement, and continuing culturing.

2. According to the experimental treatment time, the cell culture solution is sucked out into a proper centrifugal tube, the adherent cells are washed once by PBS, and a proper amount of pancreatin cell digestive juice is added to digest the cells. And (4) incubating at room temperature until the adherent cells can be blown down by gentle blowing, and sucking the digestive juice of the pancreatin cells. Excessive digestion of pancreatin is to be avoided. (Note: suspension cells need not be digested with pancreatin, and can be collected directly into a centrifuge tube)

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

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

5. Add 5. mu.l Annexin V-FITC and mix gently.

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

7.200g, centrifuge for 5min, discard the supernatant, add 190. mu.l Annexin V-FITC conjugate to resuspend the cells.

8. Add 10. mu.l of propidium iodide staining solution, mix gently, and place in ice bath and in dark.

9. And (5) detecting by using a flow cytometer, wherein Annexin V-FITC is green fluorescence, and PI is red fluorescence.

Note that: FITC is green fluorescence, and the detection method is not suitable for cells with green fluorescence labels.

The experimental results are as follows: fig. 13 shows that the NK cell apoptosis rate in the short peptide group is smaller compared to the PBS group, which indicates that the three-dimensional microenvironment constructed by the short peptide has good biocompatibility with NK cells, effectively activates NK cells, and further has significant application value in the field of NK vaccine preparation, and this example supports the application of a self-assembled short peptide as NK vaccine preparation in three-dimensional nano physical scaffold cultured cells; a self-assembled short peptide hydrogel with a concentration of 1ppM or more.

Preparation of NK vaccine: 1. cultured cells were added to 1ml of a medium at a concentration of 5X 105 cells/ml to prepare an NK solution.

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

Further demonstrated by the above examples:

1. the circular dichroism chromatogram of the short peptide is tested, so 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 transmission electron microscope image, the atomic force microscope image and the cryo-scanning electron microscope image of the short peptide are tested to show that the short peptide DRF3 can form a stable fiber network structure, can provide a stable three-dimensional nano physical scaffold for cells, and further lay a theoretical foundation for loading and wrapping drugs, proteins, macromolecules, vaccines and the like by the DRF 3.

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 protein to form a fiber network structure, and direct evidence is provided for loading the drug, protein and macromolecule to DRF 3.

4. Furthermore, by testing the in-vitro controlled release capacity of the short peptide DRF3 on the Ovalbumin (OVA), the short peptide can be further directly verified to be used as a long-term carrier and an adjuvant of a medicament, a protein and a macromolecule, so that the half-life period of the medicament is increased.

5. By testing the three-dimensional culture capacity of the short peptide DRF3, a chart shows that the short peptide can provide a stable and durable three-dimensional nano physical scaffold for tonsil cells and organoids, and further provides confidence for the subsequent culture and encapsulation of DC cells and NK cells and the preparation of DC and NK vaccines according to the model.

6. The DC and NK cells are cultured in a three-dimensional mode through testing the short peptide, maturation of the DC cells and mortality of the NK cells are identified through a flow sorting method, direct evidence is provided for the fact that the short peptide DRF3 can promote maturation of the DC cells and activate the NK cells, evidence is provided for the fact that DRF3 serves as an antigen component, and evidence is provided for the fact that DRF3 can wrap the NK cells and the DC cells and can be prepared into DC and NK vaccines.

7. The test on the short peptide DRF3 and the CCK8 challenge experiment of colon cell culture shows that the short peptide has toxicity on colon cancer cells, and provides direct evidence for the short peptide serving as a vaccine and a pharmaceutical adjuvant and also serving as a medicament for killing tumors.

Tests show that the toxicity of hyaluronic acid can be reduced after hyaluronic acid is combined by three-dimensional cultured fibroblasts after the short peptide DRF3 is mixed with hyaluronic acid, and further show that hyaluronic acid and other main cosmetic compound combined self-assembled short peptides have great application values in the fields of medicine and beauty and cosmetics, and provide theoretical support for the short peptide to replace hyaluronic acid as a filler.

A sequence table:

SEQUENCE LISTING

<110> Chengdu Saibei scientific research institute

<120> a self-assembling short peptide, its use in carrier material and biomedicine

<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-assembled short peptide, which is characterized in that the amino acid sequence is as follows:

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

2. use of a self-assembling short peptide according to claim 1 as an antigen.

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

4. The use of a self-assembled short peptide according to claim 1 for preparing a macromolecular carrier material, wherein the macromolecule comprises one or more of protein drugs, immunoglobulins, serum albumin, P53 protein, P21 protein, IgG, sugars, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, receptors, nucleic acids, nucleotides, oligonucleotides, and polynucleotides.

5. Use of a self-assembled short peptide according to claim 1 for the preparation of an anti-neoplastic medicament.

6. Use of the self-assembled short peptide of claim 1 in preparing a nano-scaffold material for three-dimensional culture of cells or organoids.

7. The use of the self-assembled short peptide of claim 1 in three-dimensional nano physical scaffold cultured cells for preparing NK vaccine.

8. The use of the self-assembled short peptide of claim 1 in the preparation of DC vaccine in three-dimensional nano physical scaffold cultured cells.

9. A self-assembled short peptide as claimed in claim 1, which is used as a main ingredient for the preparation of a medicinal and cosmetic product or a cosmetic.

10. A hydrogel comprising the self-assembled short peptide of claim 1, wherein the concentration of the self-assembled short peptide hydrogel is 1ppM or more.