CN112553162B - Apoptosis inducer, drug-loaded vesicle and application thereof - Google Patents

Apoptosis inducer, drug-loaded vesicle and application thereof Download PDF

Info

Publication number
CN112553162B
CN112553162B CN202011477409.4A CN202011477409A CN112553162B CN 112553162 B CN112553162 B CN 112553162B CN 202011477409 A CN202011477409 A CN 202011477409A CN 112553162 B CN112553162 B CN 112553162B
Authority
CN
China
Prior art keywords
cell
apoptosis
drug
vesicles
vesicle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202011477409.4A
Other languages
Chinese (zh)
Other versions
CN112553162A (en
Inventor
童雅琪
陈彬
张一�
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hubei Soundny Bio Tech Co ltd
Original Assignee
Hubei Soundny Bio Tech Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hubei Soundny Bio Tech Co ltd filed Critical Hubei Soundny Bio Tech Co ltd
Priority to CN202011477409.4A priority Critical patent/CN112553162B/en
Publication of CN112553162A publication Critical patent/CN112553162A/en
Application granted granted Critical
Publication of CN112553162B publication Critical patent/CN112553162B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides

Abstract

The invention provides an apoptosis inducer, a medicine carrying vesicle and application thereof, belonging to the technical field of biology. The apoptosis inducer comprises an effective amount of yeast mannan and an effective amount of calcium ion. According to the invention, yeast mannan is used as a calcium ion carrier, so that the permeability of a cell membrane to calcium ions can be improved, high-concentration free calcium ions can be gathered in cells in a short time, and further, an apoptosis program is triggered to generate cell vesicles, and the obtained cell vesicles have the advantages of high yield, good uniformity and small average cell vesicle diameter. The cell vesicle coated with the drug can efficiently penetrate through the barrier of the tumor cell and be endocytosed by the tumor cell, thereby improving the drug delivery efficiency.

Description

Apoptosis inducer, drug-loaded vesicle and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to an apoptosis inducer, and further relates to a method for preparing a drug-loaded vesicle by using the apoptosis inducer to induce apoptosis, the drug-loaded vesicle prepared by the method, and a pharmaceutical composition containing the drug-loaded vesicle.
Background
In recent years, the incidence of tumors has increased year by year. Although scientists have been exploring to combat tumors in various ways, clinical cure rates for tumors have not yet shown significant improvements. The cancer treatment means mainly comprises surgical treatment, radiation treatment, drug treatment and the like, however, the traditional cancer treatment means has certain limitations because the targeting property is poor and the toxicity is strong, and the cancer treatment means can also damage normal cells and generate adverse reactions while killing tumor cells. In order to overcome the toxic and side effects of traditional cancer treatment methods, more and more scientists begin to use carriers such as specific liposomes to encapsulate chemotherapeutic drugs, so that the chemotherapeutic drugs are selectively released at tumor sites and even selectively enter tumor cells, thereby achieving the effect of selectively killing the tumor cells. However, the exogenous carrier also has certain toxic and side effects on organisms, and the particle size of the clinically used carrier particles also enables the particles to easily penetrate through cell membranes of normal cells, so that the toxic and side effects of the carrier and the chemotherapeutic drugs wrapped by the carrier on the organisms are increased.
Based on this, some scientists begin to adopt the cell vesicles derived from tumor cells as a new generation of drug delivery system, and the cell vesicles carry bioactive molecules of original tumor cells, show molecular biological imprints of the original tumor cells, have the advantages of strong targeting, long circulation time, accessible biological barriers and the like, and can also effectively activate immune responses of organisms and enhance the treatment effect of chemotherapeutic drugs. Therefore, cell vesicles derived from tumor cells are receiving increasing attention.
Currently, the cell vesicles derived from tumor cells are usually prepared by inducing apoptosis of tumor cells by ultraviolet irradiation or by administering an effective amount of chemotherapeutic drugs. When the method of inducing tumor cell apoptosis by ultraviolet irradiation is adopted, the cell apoptosis degree near a light source is too high and the cell apoptosis degree far from the light source is not enough due to weak ultraviolet penetrating power, so that the obtained cell vesicle has low yield and poor uniformity. When the method for inducing apoptosis of tumor cells is adopted, because different chemotherapeutic drugs induce apoptosis in different mechanisms, for example, anthracyclines and platinum chemotherapeutic drugs directly destroy the double helix structure of DNA so that the DNA can not be copied, and antimetabolites chemotherapeutic drugs are combined with enzymes necessary for synthesizing normal metabolites so as to interfere the synthesis of nucleic acids. Therefore, not all chemotherapeutic drugs can induce apoptosis or release cell vesicles, such as taxanes and vinca chemotherapeutic drugs, and the uniformity of cell vesicles obtained by inducing apoptosis with chemotherapeutic drugs is poor, limiting the application of this method.
Disclosure of Invention
Aiming at the problems of low yield and poor uniformity of the cell vesicles in the prior art, the invention provides a cell apoptosis inducer, a method for preparing drug-loaded vesicles by inducing apoptosis of cells by using the cell apoptosis inducer, the drug-loaded vesicles prepared by using the method and a pharmaceutical composition containing the drug-loaded vesicles.
In order to achieve the purpose, the invention is realized by the following technology:
an apoptosis-inducing agent comprising an effective amount of yeast mannan and an effective amount of calcium ions.
Further, the apoptosis inducer comprises 5-20mg/L of yeast mannan and 50-800. Mu. Mol/L of calcium ion.
Further, the apoptosis inducer comprises 10mg/L of yeast mannan and 100-150. Mu. Mol/L of calcium ion.
Further, the yeast mannan is one or more of candida utilis mannan, pichia pastoris mannan, baker's yeast mannan, beer yeast mannan and saccharomyces boulardii mannan.
In addition, the invention provides application of the apoptosis inducer in inducing apoptosis or preparing medicine-carrying vesicles.
In addition, the invention provides a preparation method of the medicine carrying vesicle, which comprises the following steps: adding the apoptosis inducer into a tumor cell suspension in a logarithmic growth phase, co-culturing for a period of time to induce apoptosis to generate cell vesicles, collecting the cell vesicles, and incubating the cell vesicles with an effective amount of a drug to obtain drug-loaded vesicles.
Further, the co-cultivation time was 60min.
In addition, the invention provides a medicine carrying vesicle, which is prepared by the method for preparing the medicine carrying vesicle.
In addition, the invention provides application of the medicine carrying vesicle in preparation of a medicine for treating tumors.
In addition, the invention provides a pharmaceutical composition, which comprises the medicine-carrying vesicle and pharmaceutically acceptable auxiliary agents.
Has the advantages that:
1. the invention utilizes the yeast mannan as a calcium ion carrier, the yeast mannan is easy to penetrate through cell membranes, the permeability of the cell membranes to calcium ions can be improved, high-concentration free calcium ions can be gathered in cells in a short time, and then an apoptosis program is triggered, so that the cell membranes are locally expanded outwards and released to the outside of the cells in the form of vesicles, and the cell vesicles are generated. In a uniform culture solution, the degrees of calcium ion stress of all tumor cells are the same, the degrees of apoptosis are basically the same, and the obtained cell vesicles have high yield, good uniformity and small average cell vesicle diameter. The cell vesicle coated with the drug can efficiently penetrate through the barrier of the tumor cell and be endocytosed by the tumor cell, thereby improving the drug delivery efficiency.
2. The drug-loaded vesicle obtained by inducing apoptosis by the apoptosis inducer of the invention contains a small amount of yeast mannan besides the encapsulated drug, the yeast mannan can enhance the release of cytokine and the activity of interferon, activate macrophage, enhance the innate immunity of organisms, is matched with the action of the drug in the drug-loaded vesicle, is beneficial to improving the killing effect on tumor cells and enhancing the treatment effect.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a relative fluorescence intensity detection chart of Fluo 3-AM of example 1 of the present invention;
FIG. 2 is a photograph of the apoptosis assay of example 1 of the present invention;
FIG. 3 is a graph showing the detection of apoptosis after induction by different concentrations of mannans from Saccharomyces boulardii in example 2 of the present invention;
FIG. 4 is a graph showing the detection of apoptosis after the induction of calcium ions at different concentrations in example 3 of the present invention;
FIG. 5 is a graph showing the distribution of cell vesicles obtained by the synergy between yeast mannan and calcium ion to induce apoptosis in example 3 of the present invention;
FIG. 6 is a diagram of the morphology of cell vesicles obtained by the synergy of yeast mannan and calcium ion to induce apoptosis in example 3 of the present invention;
FIG. 7 is a graph showing the distribution of the sizes of vesicles obtained by the synergy of the calcium ionophore A23187 with calcium ions to induce apoptosis in example 3 of the present invention;
FIG. 8 is a graph showing the distribution of the size of vesicles obtained by inducing apoptosis by UV irradiation in example 3 of the present invention;
FIG. 9 is a graph showing apoptosis detection after co-culture induction at different times in example 4 of the present invention;
FIG. 10 is a number detection chart of drug-loaded vesicles obtained by different induction methods in example 5 of the present invention;
fig. 11 is a drug content detection graph of drug-loaded vesicles obtained by different induction methods in example 5 of the present invention;
FIG. 12 is a graph showing in vitro tumor cell uptake detection of drug-loaded vesicles obtained by different induction methods in example 6 of the present invention;
fig. 13 is a graph of the killing effect of the drug-loaded vesicles obtained by different induction methods in example 6 of the present invention on tumor cells.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. In addition, the terms "comprising," "including," and "having" are intended to be non-limiting, i.e., other steps and other ingredients can be added that do not affect the results. Materials, equipment and reagents are commercially available unless otherwise specified.
For a better understanding of the invention, without limiting its scope, all numbers expressing quantities, volume fractions, and other numerical values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
The cell is composed of cell membrane wrapped with cell content, the cell membrane is phospholipid bilayer, has permeability, and its spherical structure is maintained by centripetal pull force formed by protein fiber filament called cytoskeleton in the cell. When cells are stimulated (such as medicines, ultraviolet rays and high temperature) to die, part of protein fiber filaments at the cell membrane attaching part of a cytoskeleton are broken or lose attachment, and centripetal pulling force suddenly disappears, so that a local cell membrane structure expands outwards and protrudes under the action of the outward pulling force, and cell contents are wrapped and released to a sublayer structure between cells and molecules outside the cells in a vesicle form, and the size of the sublayer structure is basically between 100 and 1000nm.
Apoptosis is a programmed cell death process initiated by apoptosis-related genes and involved by various physiological and pathological factors, and scientists find and summarize that four signal paths causing apoptosis of mammalian cells are respectively as follows: external pathways (death receptor pathways), internal pathways (mitochondrial pathways), caspase-2 dependent pathways, and Caspase-2 independent pathways (GrA mediated pathways).
Death receptor pathway: the mechanism by which Fas, a death receptor, triggers apoptosis is achieved by raising calcium ion concentrations. After calcium binding protein with strong sensitivity to calcium ion change in endoplasmic reticulum cavity is combined with Fas, calcium ion is made to flow inwards to start apoptosis and activate Caspase-8.
Mitochondrial pathways: excessive calcium ions in cells are taken up by mitochondria to cause calcium ion overload, so that the mitochondria are damaged, cytochrome C is released, and apoptotic bodies are induced to form, thereby activating Caspase-9, activating a direct executor Caspase-3 of apoptosis and inducing apoptosis to occur.
Caspase-2 dependent pathways: the DNA damage causes the activation of Caspase-2, and the Caspase-2 can stimulate the release of mitochondrial cytochrome C and the activation of multiple effect caspases, thereby triggering the apoptosis, and the process needs the participation of calcium ions.
Caspase-2 independent pathways: serine protease GrA can enter cytoplasm of target cells only by depending on calcium ions and perforin, cuts proteins on endoplasmic reticulum membrane such as SET, HMG2, ape1 and the like, and causes release and activation of DNA endonuclease GAAD, and the activated GAAD is transferred into nucleus, thereby triggering DNA fragmentation and inducing apoptosis.
Calcium ions, which play an important role in many vital activities, are closely related to the four apoptotic pathways described above. Based on this, the present invention aims to provide an apoptosis inducer using yeast mannan to assist calcium ion (Ca) 2+ ) Enter the cells, thereby inducing apoptosis of tumor cells to extract vesicles.
The embodiment of the invention provides an apoptosis inducer, which comprises effective amount of yeast mannan and effective amount of calcium ions.
The term "effective amount" refers to any amount of a compound, agent, formulation, or composition sufficient to achieve a desired biological or medical response in its intended target, e.g., a cell, tissue, or subject. For example, in certain embodiments of the invention, the desired goal may be: promoting or inducing apoptosis, such as apoptosis associated with tumor cells. In other embodiments of the invention, the intended target may be: ameliorating and/or treating a disease. The term "treatment" and similar terms encompass any treatment of a disease in a mammal, particularly a human, and includes: (a) prevention of disease: (ii) in an individual predisposed to, but not yet diagnosed with, said disease; (b) inhibition of disease: i.e., arrest disease progression; and (c) relieving the disease, i.e., causing regression of the disease.
Yeast mannan is a layer of polysaccharide polymer with biological activity coated on yeast cell wall, its main chain is formed from sugar chain formed from mannose by alpha- (1, 6) glycosidic bond, and on the main chain there are rich branched chains connected by alpha-1, 2 or alpha-1, 3 glycosidic bond, and it is a lipophilic water-soluble material, can easily pass through cell membrane, so that it utilizes yeast mannan as raw materialIs calcium ionophore, and can promote calcium ion permeability, increase calcium ion flow rate into cell, and accumulate Ca for short time in cell 2+ The concentration and high concentration of free calcium ions trigger the apoptosis process, and in the apoptosis process, the skeleton of the cell membrane of an apoptotic cell changes, the cell membrane bulges outwards locally, randomly wraps the cell content, and is released to the outside of the cell in the form of vesicles, so that the cell vesicles are generated. In a uniform culture solution, the liquid environment of each tumor cell is the same and the contact chance with yeast mannan and calcium ions is equal, namely the degrees stressed by the calcium ions are the same, so the apoptosis degrees of the tumor cells are basically the same, the yield of the obtained cell vesicles is high, the uniformity is good, the average cell vesicle diameter is small and is 185.8nm, and the cell vesicles can efficiently penetrate through the cell membrane of the tumor cells and are endocytosed by the tumor cells, so the drug delivery efficiency is improved.
Because cell contents can be randomly wrapped in the process of forming the cell vesicle, the drug-loaded vesicle obtained by inducing apoptosis by the apoptosis inducer can wrap a certain amount of yeast mannan and calcium ions besides the drug, the yeast mannan has a certain immunoregulation function, can enhance the release of cytokines and the activity of interferon, promote or coordinate the activities of different immune cells, enhance the migration of leucocytes and the like to infected parts, activate macrophages, thereby enhancing the innate immunity of an organism, further being beneficial to improving the killing effect on tumor cells of the organism and enhancing the treatment effect. Many reactions closely related to life activities can not leave calcium ions, a large amount of calcium ions are stored in endoplasmic reticulum in cells, and the calcium ions do not have toxic or side effects on organisms within a certain concentration range, namely, the calcium ions do not have effects on the functions of the medicine-carrying vesicles.
Calcium ion regulates four main apoptosis signal pathways in the body, regulates Ca in cells 2+ The concentration can directly influence the apoptosis degree of cells, the apoptosis strength is in positive correlation with the concentration of intracellular free calcium ions, and the damage degree of cell membranes is increased along with the increase of the concentration of the intracellular free calcium ions. The concentration of intracellular free calcium ions is too low, the apoptosis rate is low, the damage degree of cell membranes is small, and the yield of cell vesicles is low. However, if the concentration of intracellular free calcium ions is too high, cell apoptosis is sufficient, and cell membranes are completely broken, but cell membranes are fragmented, and the fragmented cell membranes cannot be bubbled to generate stable cell vesicles, which also results in low cell vesicle yield. By studying the effect of different concentrations of yeast mannan and calcium ion on the induction of apoptosis to produce cell vesicles, preferably, the apoptosis inducer comprises 5-20mg/L yeast mannan and 50-800. Mu. Mol/L calcium ion. More preferably, it comprises 10-15mg/L of yeast mannan and 100-400. Mu. Mol/L of calcium ion. Further preferably, the yeast mannan is 10mg/L and the calcium ion is 100-150 mu mol/L, and the apoptosis rate of tumor cells and the yield of cell vesicles are high and the uniformity is good.
The yeast mannan is not particularly limited in kind, and can effectively promote calcium ion inflow, and in some preferred embodiments, the yeast mannan is one or more of candida utilis mannan, pichia pastoris mannan, saccharomyces cerevisiae mannan, brewers yeast mannan, and saccharomyces boulardii mannan.
Another embodiment of the present invention provides an application of the apoptosis inducer, specifically, an application of the apoptosis inducer in inducing apoptosis or preparing drug-loaded vesicles. The application of the apoptosis inducer is the same as the advantages of the apoptosis inducer relative to the prior art, and the detailed description is omitted here.
In addition, the invention provides an application method of the apoptosis inducer, and specifically relates to a method for inducing apoptosis or a method for preparing a medicine carrying vesicle by using the apoptosis inducer.
The method for inducing apoptosis comprises the following steps: the apoptosis-inducing agent as described above is added to a tumor cell suspension in the logarithmic growth phase, and co-cultured for a period of time to induce apoptosis.
The method for preparing the medicine-carrying vesicle comprises the following steps: adding the apoptosis inducer into a tumor cell suspension in a logarithmic growth phase, co-culturing for a period of time to induce apoptosis to generate cell vesicles, collecting the cell vesicles, and incubating the cell vesicles with an effective amount of a drug to obtain drug-loaded vesicles.
The tumor cells and the apoptosis inducer are co-cultured, the activity of the tumor cells in the logarithmic growth phase is strong, the metabolism is active, calcium ions can rapidly enter cells under the concentration gradient of the calcium ions and the cooperative transportation action of the yeast mannan, the apoptosis is further triggered to stably release the cell vesicles, and the cell vesicle yield and the particle size of the produced cell vesicles are uniform. And then, the cell vesicles are used as carriers to wrap the medicine, and the cell membrane is a phospholipid bilayer and has permeability, so that the medicine can enter the cell vesicles along the concentration gradient to form uniform solute in the cell vesicles, and the medicine-carrying vesicles with the average diameter of about 185.8nm are obtained. The medicine-carrying vesicle is derived from tumor cells, can be easily fused with cell membranes of target tumor cells to be treated, is enriched at diseased positions, has stronger targeting property, can not cause any damage to normal tissues, and avoids toxic action on organisms caused by using exogenous materials as carriers. Moreover, the obtained drug-loaded vesicle also contains a certain amount of yeast mannan, which is beneficial to enhancing the innate immunity of organisms, and can significantly improve the anti-tumor effect by matching with drugs.
Preferably, the co-cultivation conditions are: temperature 37. + -. 3 ℃ C., gas atmosphere 5% CO 2 . It will be appreciated that each cell has its specific optimal growth temperature and gas atmosphere, and the present invention selects these parameters according to the type of tumor cells to be cultured. When the skilled person selects other cell sources, the temperature and gas atmosphere, etc. can be adapted.
In the early stage, the apoptosis rate of tumor cells and the yield of cell vesicles increase with the extension of the induction time, and after 60min, the increase of the apoptosis rate is slowed down, but the yield of cell vesicles is reduced, which is closely related to the fragmentation of cell membranes. Therefore, preferably, the co-cultivation time is 60min.
The method for collecting the cell vesicles adopts the conventional technology in the field, and separates the cell vesicles from impurities such as cell membrane fragments and inclusion bodies by collection, such as differential centrifugal separation, density gradient centrifugal separation and other technical means, and the invention is not repeated.
Among them, the cell vesicles for encapsulating the drug are preferably derived from the same type of tumor cells as the tumor cells to be treated. Such tumor cells include, but are not limited to: ovarian cancer, breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer, bladder cancer, leukemia and/or glioma. Accordingly, the drugs include, but are not limited to: a medicine for treating ovarian cancer, breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer, bladder cancer, leukemia or glioma. Specifically, the drugs include, but are not limited to: methotrexate, doxorubicin, cisplatin, paclitaxel, 5-fluorouracil, gemcitabine, and/or 10-hydroxycamptothecin. The cell vesicle prepared by the invention is suitable for various drugs on the market, and can cross the property limitation of the drugs.
In another embodiment of the present invention, a drug-loaded vesicle is provided, which comprises a cell vesicle derived from an apoptotic tumor cell and an effective amount of a drug encapsulated in the cell vesicle, wherein the drug-loaded vesicle is prepared according to the method for preparing a drug-loaded vesicle described above. Compared with the prior art, the medicine carrying vesicle has the same advantages as the method for preparing the medicine carrying vesicle, and the details are not repeated.
The invention further provides application of the medicine-carrying vesicle in preparation of a medicine for treating tumors.
In addition, the invention provides a pharmaceutical composition, which comprises the medicine-carrying vesicle and a pharmaceutically acceptable auxiliary agent. The advantages of the pharmaceutical composition and the drug-loaded vesicle are the same as those of the prior art, and are not repeated herein.
The term "pharmaceutically acceptable adjuvant" refers to a component that does not interfere with the efficacy of the biological activity of the active ingredient and that is not significantly toxic to the subject at the concentrations at which it is administered, and includes solvents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such components for pharmaceutically active substances is well known in the art.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are examples of experimental procedures not specified under specific conditions, generally according to the conditions recommended by the manufacturer.
In the following examples, the yeast mannan was Saccharomyces boulardii mannan, and the tumor cells were A549 lung adenocarcinoma cell line, unless otherwise specified.
Example 1 synergy of Saccharomyces boulardii mannan with calcium ions induces apoptosis
Tumor cell culture: inoculating the purified tumor cells into RPMI 1640 medium containing 10% fetal bovine serum (v/v), 100U/mL penicillin, and 100mg/mL streptomycin, and adjusting the temperature at 37 deg.C and gas atmosphere to 5% CO 2 The culture was performed under the conditions of (1), and the digestion was performed when the tumor cells grew to the logarithmic phase, after which the dispersed tumor cells were inoculated in a 24-well plate and incubated overnight for use.
Tumor cell apoptosis: dividing tumor cells in a 24-well plate into 2 groups, namely an experimental group and a control group, dividing the experimental group into 6 groups, respectively adding 10mg/L of Saccharomyces boulardii mannan, and respectively adding 0, 50, 100, 200, 400 and 800 mu mol/L of CaCl into each group 2 . The control group was also divided into 6 groups, and 0, 50, 100, 200, 400, 800. Mu. Mol/L CaCl was added to each group 2 And no Bladdia mannan was added. Placing the tumor cells of the experimental group and the control group in a gas atmosphere at 37 ℃ to 5% CO 2 Culturing for 1h, sampling and detecting the calcium ion concentration in each group of tumor cells, replacing a fresh RPMI 1640 culture medium after culturing for 1h, performing apoptosis detection after culturing overnight, and calculating the apoptosis rate.
The calcium ion concentration detection method comprises the following steps: cells are incubated by Hanks solution containing calcium ion probe Fluo 3-AM, the relative fluorescence intensity of Fluo 3-AM in each group of cells is detected under a laser confocal microscope to indicate the calcium ion concentration, the detection result is shown in figure 1, and the higher the relative fluorescence intensity is, the higher the calcium ion concentration is.
The apoptosis detection method comprises the following steps: and (3) resuspending the cultured overnight tumor cells in Annexin V binding buffer, adding an apoptosis detection reagent Annexin V-FITC and PI, incubating for 30min, and performing apoptosis detection by using a BD FACSCANTO II flow cytometer, wherein Annexin V positive groups are apoptotic cells, and the detection result is shown in figure 2.
As can be seen from fig. 1, compared to the control group, after the experiment group is added with the blattella manihot mannan for co-culture, the concentration of free calcium ions in the tumor cells is greatly increased, which indicates that the blattella manihot mannan can be used as a calcium ion carrier, so as to improve the permeability of cell membranes to calcium ions, promote the calcium ion inflow, further significantly increase the intracellular calcium ion concentration, and induce the occurrence of apoptosis program, therefore, the apoptosis rate of the tumor cells in the experiment group is also significantly higher than that in the control group (see fig. 2).
As can be seen from FIGS. 1-2, the apoptosis effect of the saccharomyces boulardii mannan and calcium ion level cells is in a dose-dependent manner, under the condition of 10mg/L saccharomyces boulardii mannan, the apoptosis rate of the tumor cells increases along with the increase of calcium ion concentration, when the calcium ion stimulation concentration reaches 200 mu mol/L, the apoptosis rate of the tumor cells reaches more than 90%, but when the calcium ion concentration exceeds 200 mu mol/L, the apoptosis degree of the tumor cells does not change obviously, and preferably, the calcium ion concentration is 100-400 mu mol/L.
Example 2 optimization of the concentration of mannans in Saccharomyces boulardii
Tumor cell cultures were performed as described in example 1, after which the tumor cells in the 24-well plates were divided into different groups and 100. Mu. Mol/L and 400. Mu. Mol/L CaCl were added 2 And gradient concentration of Saccharomyces boulardii mannan (0, 5, 10, 15, 20 mg/L), placing the tumor cells in a gas atmosphere at 37 deg.C for 5% CO 2 Culturing for 1h, replacing fresh RPMI 1640 culture medium after culturing for 1h, performing apoptosis detection after culturing overnight, and calculating apoptosis rateThe detection result is shown in FIG. 3.
It can be known from fig. 3 that the efficiency of the saccharomyces boulardii mannan cooperating with calcium ions entering tumor cells depends on the concentration of the saccharomyces boulardii mannan within a certain range, and the apoptosis rate of the tumor cells is remarkably improved along with the increase of the concentration of the saccharomyces boulardii mannan, because the increase of calcium ion carriers accelerates the accumulation speed of the concentration of intracellular free calcium ions, the increase of cell stress damage causes the increase of the release speed of the cell vesicles, but the increase of the apoptosis rate of the tumor cells is not obvious along with the further increase of the concentration of the saccharomyces boulardii mannan, but the excessive damage of cell membranes is easily caused, a large amount of fragmented cell membranes are formed, and the yield of the cell vesicles is reduced. Therefore, the mannans of the Saccharomyces boulardii are preferably 10-15mg/L, and more preferably 10mg/L.
Example 3 optimization of calcium ion concentration
Tumor cell cultures were performed by the method of example 1, after which the tumor cells in the 24-well plates were divided into different groups, which were treated as follows:
experimental groups: adding 10mg/L Saccharomyces boulardii mannan and CaCl with gradient concentration into tumor cells 2 (0, 25, 50, 100, 150, 200. Mu. Mol/L), placing the tumor cells at a temperature of 37 ℃ in a gaseous atmosphere, 5% 2 Culturing for 1h, replacing a fresh RPMI 1640 culture medium after culturing for 1h, culturing overnight, and centrifuging with density gradient to obtain the cell vesicle without drug loading. The number and the particle size of the cell vesicles are characterized by using a Malvern NS300 particle tracking analyzer, and the morphology of the cell vesicles is characterized by using a Hitachi HT7800 120kV transmission electron microscope, wherein the characterization results are respectively shown in figures 4-6.
Control group 1: the common calcium ionophore A23187 in the prior art is used for equivalently replacing the saccharomyces boulardii mannan of the invention, a tumor apoptosis experiment is carried out under the same condition as that of an experimental group, the cell vesicle particle size of a control group 1 is characterized by using a Malvern NS300 particle tracking analyzer, and the result is shown in figure 7.
Control group 2: the cell vesicle is prepared by adopting the common ultraviolet irradiation technology in the prior art, and the specific preparation process refers to Chinese patentThe cell vesicle particle size of control 2 was characterized using a Malvern NS300 particle tracking analyzer according to CN102302784B, and the results are shown in fig. 8. In FIGS. 5 and 7 to 8, the abscissa Size represents the particle Size of the cell vesicle in units of: nm, ordinate Concentration represents Concentration in units of: 10 8 One per mL.
As can be seen from FIG. 4, the induction of apoptosis by the saccharomyces boulardii mannan, which cooperates with calcium ions entering tumor cells, can promote the release of vesicles from the tumor cells, and the production of vesicles in a certain range depends on the calcium ion concentration. The appropriate calcium ion concentration and the concentration of mannans in Saccharomyces boulardii can ensure the apoptosis rate to release cell vesicles, and simultaneously, does not cause cell membrane fragmentation to ensure the cell vesicle yield. Therefore, preferably, the apoptosis inducer comprises 10mg/L of yeast mannan and 100-150. Mu. Mol/L of calcium ion. More preferably, 10mg/L of yeast mannan and 100. Mu. Mol/L of calcium ion are included, and the cell vesicles obtained under these conditions are subjected to performance characterization. As can be seen from FIGS. 5-6, the cell vesicles have narrow and single peak-shaped particle size distribution, uniform particle size, and average particle size of 185.8nm, which is greatly reduced compared to the average particle size of control group 1 and control group 2 (see FIGS. 7-8), and has a narrower distribution range, and thus can more easily cross the barrier of tumor cells when used for treatment. And the obtained cell vesicle has obvious liposome characteristics and high stability.
Example 4 optimization of Co-culture time of tumor cells with apoptosis-inducing Agents
Tumor cell culture was performed by the method of example 1, after which the tumor cells in the 24-well plate were divided into different groups, and 10mg/L Saccharomyces boulardii mannan and 100. Mu. Mol/L CaCl were added to the tumor cells, respectively 2 Thereafter, subjecting the tumor cells to a temperature of 37 ℃ and a gas atmosphere of 5% 2 Culturing for different time (0, 15, 30, 45, 60, 75 and 90 min), replacing fresh RPMI 1640 culture medium, culturing overnight, and centrifuging with density gradient to obtain cell vesicle without drug. The number of cellular vesicles was characterized using a Malvern NS300 particle tracking analyzer and the results are shown in figure 9.
As can be seen from fig. 9, the cell vesicle yield at the initial stage of co-culture showed an exponential increase trend with the increase of the induction time, and the cell vesicle yield decreased after the co-culture time exceeded 60min, which is closely related to the cell membrane fragmentation. Therefore, preferably, the co-cultivation time is 60min.
Example 5 preparation of drug-loaded vesicles
Tumor cell culture: inoculating the purified tumor cells into RPMI 1640 medium containing 10% fetal bovine serum (v/v), 100U/mL penicillin, and 100mg/mL streptomycin, and adjusting the temperature at 37 deg.C and gas atmosphere to 5% CO 2 The culture was performed under the conditions of (1), and the digestion was performed when the tumor cells grew to the logarithmic phase, after which the dispersed tumor cells were inoculated in a 24-well plate and incubated overnight for use.
Preparing a drug-loaded vesicle: adding 10mg/L Saccharomyces boulardii mannan and 100. Mu. Mol/L CaCl into tumor cells 2 Thereafter, the tumor cells were exposed to a gas atmosphere at 37 ℃ to 5% CO 2 After culturing for 1 hour, the RPMI 1640 medium was replaced with fresh medium, and various chemotherapeutic agents (methotrexate (MTX): final concentration 2mg/mL, doxorubicin hydrochloride (DOX): final concentration 200. Mu.g/mL, cisplatin (CDDP): final concentration 200. Mu.g/mL, hydroxycamptothecin (HCPT): final concentration 500. Mu.g/mL) were added thereto, respectively, and the mixture was incubated at 37 ℃ under a gas atmosphere of 5% CO 2 Incubating for 16-24h under the condition of (1), and then performing density gradient centrifugation to obtain the medicine-carrying vesicle. Meanwhile, a calcium ionophore A23187 is used for equivalently replacing the saccharomyces boulardii mannan and the medicine carrying vesicles prepared under the same condition as the invention are used as a control group 1; the drug-loaded vesicle prepared by ultraviolet-induced apoptosis in Chinese patent CN102302784B was used as control group 2. Characterization of the number of drug-loaded vesicles was performed using a Malvern NS300 particle tracking analyzer and the results are shown in FIG. 10, using High Performance Liquid Chromatography (HPLC) for unit drug-loaded vesicles (10) 10 individuals/mL) of the drug content, wherein the liquid phase detection methods of Methotrexate (MTX), doxorubicin hydrochloride (DOX) and Cisplatin (CDDP) refer to pharmacopoeia 2015 edition of the people's republic of china (column C18250 × 4.6 mm). HPLC detection conditions of Hydroxycamptothecin (HCPT) are as follows: mobile phase methanol: water =500 (v/v), column: thermo Acclaim TM 120, column temperature 30 ℃, flow rate: 1mL/min, detection wavelength: 266nm. The results are shown in FIG. 11.
From fig. 10 to 11, it can be seen that the number of the drug-loaded vesicles prepared by inducing tumor cell apoptosis with the apoptosis inducer of the present invention is significantly higher than that of the group induced by calcium ionophore a23187 and the group induced by ultraviolet irradiation, and the drug-loaded vesicles per unit is also significantly higher than that of the other 2 groups.
Example 6 verification of antitumor function of drug-loaded vesicles
The apoptosis inducer of the invention in example 5, the MTX-loaded vesicles prepared by the prior art in which apoptosis was induced by calcium ion vector A23187 and ultraviolet irradiation, were stained with a cell membrane staining kit (PKH 26 fluorescent probe), and then an A549 lung adenocarcinoma cell line, an OVCAR-3 ovarian cancer cell line, a HepG2 liver cancer cell line, an MCF-7 breast cancer cell line, and an HCT-8 colon cancer cell line were selected and inoculated in 24-well plates, 1X 10 per well 5 Cell, at 37 ℃ and 5% CO 2 And (3) incubating in an incubator overnight, adding the three drug-loaded vesicles marked by the PKH26 in the same amount into each hole, after incubating for 3 hours, digesting cells in the holes, transferring the cells into a flow tube, carrying out drug-loaded vesicle uptake detection by using a BDFACSCANTO II flow cytometer, detecting the PKH26 by using a PE channel, wherein the result shows the percentage of PE positive cells, the detection result is shown in figure 12, meanwhile, the apoptosis rate of the cells is determined, and the killing comparison of in vitro tumor cells is carried out, and the detection result is shown in figure 13.
The in vitro drug-loaded vesicle ingestion result shows that the drug-loaded vesicle prepared by the apoptosis inducer inducing apoptosis can be ingested by tumor cells, has high ingestion rate and good tumor cell killing effect, has more excellent in vitro anti-tumor effect, and can be used as a potential tumor pharmaceutical preparation.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present disclosure, and these changes and modifications are intended to be within the scope of the present disclosure.

Claims (8)

1. An apoptosis inducer comprising 10mg/L of yeast mannan and 100 to 150. Mu. Mol/L of calcium ion.
2. The apoptosis inducer of claim 1, wherein the yeast mannan is one or more of candida utilis mannan, pichia pastoris mannan, baker's yeast mannan, brewer's yeast mannan, and blakeda mannan.
3. Use of an apoptosis-inducing agent according to any one of claims 1 to 2 in the preparation of a medicament for inducing apoptosis or in the preparation of a drug-loaded vesicle.
4. The preparation method of the medicine-carrying vesicle is characterized by comprising the following steps: adding the apoptosis-inducing agent of any one of claims 1-2 to a suspension of tumor cells in logarithmic growth phase, co-culturing for a period of time to induce apoptosis to produce cellular vesicles, then collecting the cellular vesicles, and incubating the cellular vesicles with an effective amount of a drug to obtain drug-loaded vesicles.
5. The method for preparing drug-loaded vesicles according to claim 4, wherein the co-culturing time is 60min.
6. A drug-loaded vesicle, which is prepared by the preparation method of the drug-loaded vesicle of claim 4 or 5.
7. Use of the drug-loaded vesicle of claim 6 for the preparation of a medicament for the treatment of tumors.
8. A pharmaceutical composition comprising the drug-loaded vesicle of claim 6 and a pharmaceutically acceptable adjuvant.
CN202011477409.4A 2020-12-15 2020-12-15 Apoptosis inducer, drug-loaded vesicle and application thereof Active CN112553162B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011477409.4A CN112553162B (en) 2020-12-15 2020-12-15 Apoptosis inducer, drug-loaded vesicle and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011477409.4A CN112553162B (en) 2020-12-15 2020-12-15 Apoptosis inducer, drug-loaded vesicle and application thereof

Publications (2)

Publication Number Publication Date
CN112553162A CN112553162A (en) 2021-03-26
CN112553162B true CN112553162B (en) 2023-01-13

Family

ID=75064751

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011477409.4A Active CN112553162B (en) 2020-12-15 2020-12-15 Apoptosis inducer, drug-loaded vesicle and application thereof

Country Status (1)

Country Link
CN (1) CN112553162B (en)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63202380A (en) * 1987-02-19 1988-08-22 Snow Brand Milk Prod Co Ltd Production of plasminogen activation factor by cell culture
CA2243543A1 (en) * 1996-01-26 1997-07-31 Research Institute For Glycotechnology Apoptosis inducers
FR2862649B1 (en) * 2003-11-26 2006-01-27 Centre Nat Rech Scient NOVEL CYCLODEXTRIN DIMERS AND DERIVATIVES THEREOF, PROCESSES FOR THEIR PREPARATION AND THEIR USE, IN PARTICULAR FOR THE SOLUBILIZATION OF PHARMACOLOGICALLY ACTIVE SUBSTANCES
CN103446580B (en) * 2012-05-31 2016-08-03 湖北盛齐安生物科技有限公司 A kind of tumor vaccine and preparation method thereof
CN111067868B (en) * 2019-12-20 2021-12-21 湖北盛齐安生物科技股份有限公司 Medicine carrying vesicle

Also Published As

Publication number Publication date
CN112553162A (en) 2021-03-26

Similar Documents

Publication Publication Date Title
US11717480B2 (en) Extracellular vesicles and methods and uses thereof
CN109837306A (en) Contain the excretion body and its preparation method and application of miRNA-204-5p
CN106309369A (en) DC cell membrane bionic liposome drug carrier as well as preparation method and application thereof
CN106139148B (en) A kind of tumor chemotherapeutic medicinal preparation combination
CN104140953B (en) Breast cancer multidrug-resistant cell strain constructed by virtue of epirubicin induction as well as construction method and application thereof
CN106344924B (en) It is a kind of combine metabolic block nano-formulation and its drug resistance inversion application
Sun et al. Activation of macrophages by the ophiopogon polysaccharide liposome from the root tuber of Ophiopogon japonicus
CN110124058A (en) It is a kind of from the preparation of mesenchymal stem cell excretion body-adriamycin nano targeted drug and the research of external anti-osteosarcoma
Hu et al. Microparticles: biogenesis, characteristics and intervention therapy for cancers in preclinical and clinical research
CN108178803A (en) A kind of preparation of cinnamic acid-dextran polymer self-assembled nanometer grain for carrying medicine and its antitumor application thereof
CN114376986B (en) Bionic nanoparticle for homologous recombination exosome multi-drug delivery and preparation method and application thereof
CN112553162B (en) Apoptosis inducer, drug-loaded vesicle and application thereof
CN110711249B (en) Preparation method of lysosome membrane-coated nanoparticles
CN111617053A (en) Method for preparing vesicle by inducing apoptosis with high osmotic pressure
CN110934944A (en) Method for extracting total saponins of Trillium japonicum and pharmaceutical application
CN112891381B (en) Preparation method and application of bacterial wall modified liposome-carried adriamycin
CN109276570A (en) The Nano medication and preparation method thereof of biological source macrocycle molecule
CN105521497B (en) Embedded cationic liposomal gene carrier system of sucrose fatty ester and its preparation method and application
Zhang et al. Regulating the surface topography of CpG nanoadjuvants via coordination-driven self-assembly for enhanced tumor immunotherapy
CN116139251A (en) Polypeptide with immunity improving, anti-tumor and life prolonging functions and application thereof
CN102363044A (en) Application of three anthraquinone substances of targeted mitochondria as nasopharyngeal darcinoma radiosensitizers
Niemelä Nanoparticles as Targeting System for Cancer Treatment: From idea towards reality
CN110898035B (en) Tumor chemotherapy pharmaceutical preparation and preparation method thereof
CN114181985B (en) Fusarium graminearum extracellular polysaccharide, preparation method thereof and application thereof in preparing medicines for treating gastric cancer
CN110938152B (en) Polysaccharide RPP1 prepared from shellfish fermented by bacillus natto, and purification method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant