CN113304269A - Bioactive preparation based on tumor cell membrane and preparation method and application thereof - Google Patents

Bioactive preparation based on tumor cell membrane and preparation method and application thereof Download PDF

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CN113304269A
CN113304269A CN202110541241.7A CN202110541241A CN113304269A CN 113304269 A CN113304269 A CN 113304269A CN 202110541241 A CN202110541241 A CN 202110541241A CN 113304269 A CN113304269 A CN 113304269A
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cell membrane
tumor cell
inhibitor
exosome
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CN113304269B (en
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栾玉霞
李倩
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Shandong University
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    • 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
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Abstract

The invention provides a bioactive preparation based on tumor cell membranes, and a preparation method and application thereof, and belongs to the technical field of medicines. According to the invention, tumor tissues of tumor-bearing mice are extracted and processed to form tumor cell membrane vesicles, the gel factor capable of forming gel in situ in the tumor tissues is formed after chemical modification, and after the gel factor is loaded with a cyclin-dependent kinase 5(CDK5) inhibitor and an exosome inhibitor, tumor cells and tumor exosomes PD-L1 can be effectively regulated and controlled, so that local and systemic immunosuppression is relieved at the same time, and therefore, the gel factor has a good practical application value.

Description

Bioactive preparation based on tumor cell membrane and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a bioactive preparation based on tumor cell membranes, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Programmed death 1 ligand (PD-L1) is generally thought to play a role on the surface of tumor cells, however the failure rate in clinical cases of immunotherapy based on PD-L1 blockade is still high. Current approaches to enhance the response efficiency of PD-L1-based therapies are limited to locally modulating the tumor site, such as increasing the immunogenicity of the tumor and remodeling the immune microenvironment of the tumor, while ignoring systemic immunosuppressive factors. Notably, PD-L1 is also expressed on the surface of tumor cell-derived exosomes, which circulate throughout the body with exosomes, binding and depleting circulating T cells, producing a systemically immunosuppressive body, and thus, for immune checkpoint therapy, regulation of circulating exosomes PD-L1 is as important as regulation of tumor cells PD-L1.
However, the current PD-L1 blocking antibody is not effective against the exosome PD-L1, one reason being that the unique expression pattern of PD-L1 on exosomes makes it less reactive towards antibodies. Another reason is that exosomes can circulate to some sites that can mask antibody action. To date, there is no drug in clinical practice that can directly target the exosome PD-L1, thus presenting a challenge to precisely regulate exosome PD-L1. Direct inhibition of tumor cell exosome production provides another strategy for reducing total exosome PD-L1. However, considering that exosomes secreted by normal cells are crucial to maintaining normal physiology, how to selectively inhibit tumor-specific exosomes so as to reduce potential systemic toxicity is another challenge to be solved.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a bioactive preparation based on tumor cell membranes, and a preparation method and application thereof. The invention extracts tumor tissues of tumor-bearing mice, processes and forms tumor cell membrane vesicles, forms gel factors which can form gel in situ in the tumor tissues after chemical modification, and can effectively regulate and control tumor cells and tumor exosomes PD-L1 after carrying CDK5 inhibitors and exosome inhibitors together, thereby relieving local and systemic immunosuppression simultaneously, and having good value of practical application.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention there is provided the use of a CDK5 inhibitor in combination with an exosome-inhibitory drug in the preparation of an anti-tumour drug.
The CDK5 inhibitor may be selected from siRNA, shRNA, antibodies, small molecule compounds, and peptide fragments;
preferably, the CDK5 inhibitor is selected from: roscovitine (Rosco), olomoucine (olomoucine) and its derivatives, Dinaciclib (MK-7965, SCH727965), AT7519, CIP, p5, and Cpd1, among others;
more preferably, the CDK5 inhibitor is Roscovitine;
the exosome inhibitory drugs include, but are not limited to, calcium ion channel inhibitors, proton pump inhibitors, sphingomyelin-ribonuclease inhibitors, endothelin receptor a interference drugs;
preferably, the exosome-inhibiting drug may be selected from: dimethyl amiloride (DMA), omeprazole, sulfisoxazole, sphingomyelinase inhibitor GW4869 and the like;
more preferably, the exosome-inhibiting drug is dimethyl amiloride;
further, the CDK5 inhibitor is Roscovitine, the exosome inhibitory drug is dimethyl amiloride, and the mass ratio of the two is 1-10: 1; preferably, the mass ratio of the two is 5: 1.
the anti-tumor medicine at least has one or more of the following purposes:
1) relieving immunosuppression caused by tumor exosomes;
2) relieving immunosuppression brought by PD-L1 on the surface of the tumor cells;
3) improving the anti-tumor efficacy;
wherein the applications 1) and 2) are related to PD-L1.
In a second aspect of the present invention, there is provided a tumor cell membrane-based bioactive agent, wherein the pharmaceutically active ingredient of said cell membrane-based bioactive agent comprises the CDK5 inhibitor and an exosome inhibitory drug as described above.
Further, the bioactive agent is a hydrogel.
In a third aspect of the present invention, there is provided a method for preparing the above hydrogel based on tumor cell membranes, comprising:
the sodium alginate oxide modified tumor cell membrane vesicles form gel factors, CDK5 inhibitors and exosome inhibitory drugs are respectively dissolved and then uniformly mixed, and the gel factors are added into an aqueous solution of the gel factors and stirred to obtain a gelling solution.
In a fourth aspect of the invention, there is provided the use of the above cell membrane-based bioactive agent in the preparation of an anti-tumor medicament.
The beneficial technical effects of one or more technical schemes are as follows:
(1) according to the invention, the CDK5 inhibitor and the exosome inhibitor are jointly applied to the anti-tumor effect, and meanwhile, the immunosuppression brought by tumor exosomes and PD-L1 on the surface of tumor cells is relieved, so that the anti-tumor effect is more excellent;
(2) the invention prepares the sodium alginate oxide modified tumor cell membrane vesicle for the first time, the structure has the function of a gel factor and can quickly form gel in the presence of calcium ions;
(3) the invention synthesizes the cell membrane-based gelator for the first time, the structure of the gelator is different from the prior gelator, and the gelator has higher biological activity;
(4) the hydrogel based on the cell membrane prepared by the invention has better biocompatibility to normal cells, has small toxic and side effects, and provides possibility for designing safer local administration carriers;
(5) the cell membrane-based bioactive preparation inhibits the generation of tumor exosomes by utilizing immunotherapy, down-regulates the expression of tumor cell PD-L1 genes, improves the in-vivo anti-tumor effect and has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a transmission electron microscope picture of the tumor cell membrane vesicle nanostructure before and after modification by sodium alginate oxide in example 2 of the present invention;
FIG. 2 is a gel-forming photograph of O-TMV gel of example 4 of the present invention;
FIG. 3 is a scanning electron micrograph of O-TMV and O-TMV @ DR gels of example 4 of the present invention;
FIG. 4 is a graph showing the experimental characterization of the in vitro cytotoxicity of O-TMV @ hydrogel of example 5 of the present invention;
FIG. 5 is a graph showing the experimental characterization of the in vivo antitumor activity of the O-TMV @ DR hydrogel of example 6 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As mentioned previously, simultaneous regulation of the number of tumor cells and their exosomes PD-L1 is important for increasing PD-L1-based immunotherapy.
In view of the above, the inventors found that CDK5 is highly active in various tumor cells, and the study shows that by inhibiting CDK5, PD-L1 can be effectively down-regulated. CDK5 inhibitors may exert a more thorough effect compared to standard PD-L1 blocking antibodies. Meanwhile, DMA as an exosome inhibitor can effectively inhibit the number of exosomes secreted by tumor cells. Thus, the present invention reduces the number of exosomes PD-L1 by combining an exosome inhibitor (DMA) with the CDK5 inhibitor roscovitine (rosco), in one aspect, inhibiting tumor cell production of exosomes by DMA; on the other hand, through the Rosco down-regulation of PD-L1, IFN-gamma related immune drug resistance is relieved, and the combination of the two can regulate and control the expression of PD-L1 of tumors and exosomes. Meanwhile, the injectable hydrogel based on the tumor cell membrane is used as a carrier, so that on one hand, systemic side effects can be effectively reduced; on the other hand, the tumor cell membrane provides a large amount of endogenous antigens, which is beneficial to enhancing the immunogenicity of the tumor and is more beneficial to the blocking treatment of PD-L1 to exert curative effect.
Accordingly, in an exemplary embodiment of the invention, there is provided the use of a CDK5 inhibitor in combination with an exosome inhibitor in the preparation of an anti-neoplastic medicament.
The CDK5 inhibitor may be selected from siRNA, shRNA, antibodies, small molecule compounds, and peptide fragments;
preferably, the CdkK5 inhibitor is selected from: roscovitine (Rosco), olomoucine (olomoucine) and its derivatives, Dinaciclib (MK-7965, SCH727965), AT7519, CIP, p5, and Cpd1, among others;
more preferably, the CDK5 inhibitor is Roscovitine;
the exosome inhibitory drugs include, but are not limited to, calcium ion channel inhibitors, proton pump inhibitors, sphingomyelin-ribonuclease inhibitors, endothelin receptor a interference drugs;
preferably, the exosome-inhibiting drug may be selected from: dimethyl amiloride (DMA), omeprazole, sulfisoxazole, sphingomyelinase inhibitor GW4869 and the like;
more preferably, the exosome-inhibiting drug is dimethyl amiloride;
when the CDK5 inhibitor is Roscovitine and the exosome inhibitory drug is dimethyl amiloride, the mass ratio of the two is 1-10: 1; when the mass ratio of Roscovitine to dimethyl amiloride is 5:1, the anti-tumor effect is best;
the anti-tumor medicine at least has any one or more of the following purposes:
1) relieving immunosuppression caused by tumor exosomes;
2) relieving immunosuppression brought by PD-L1 on the surface of the tumor cells;
3) improving the anti-tumor efficacy;
wherein the applications 1) and 2) are related to PD-L1.
In a further embodiment of the present invention, there is provided a tumor cell membrane-based biologically active agent, the pharmaceutically active ingredient of said agent comprising the CDK5 inhibitor and an exosome inhibitory drug as defined above.
In yet another embodiment of the present invention, the cell membrane-based bioactive agent is a hydrogel.
In yet another embodiment of the invention, the hydrogel formulation is made of CDK5 inhibitor, exosome inhibitory drug and tumor cell membrane vesicles.
In another embodiment of the present invention, the tumor cell membrane vesicle is a sodium alginate oxide-modified tumor cell membrane vesicle.
In another specific embodiment of the invention, the CDK5 inhibitor is Roscovitine, the exosome inhibitory drug is dimethyl amiloride, and the mass ratio of the two is 1-10: 1; when the mass ratio of Roscovitine to dimethyl amiloride is 5:1, the antitumor effect is best.
In another embodiment of the present invention, the size of the tumor cell membrane vesicle is 150-220 nm.
In another embodiment of the present invention, there is provided a method for preparing the above cell membrane-based bioactive agent, comprising:
the sodium alginate oxide modified tumor cell membrane forms a gelator, CDK5 inhibitor and exosome inhibitory drug are respectively dissolved and then uniformly mixed to form a drug solution, and the drug solution is added into the water solution of the gelator to be stirred and processed, so that a gelator solution is obtained.
In another embodiment of the invention, the solvent for dissolving the CDK5 inhibitor and the exosome inhibitor is deionized water, which has better dissolving effect, and the method for adding the drug solution is dropwise adding, so that the components of the antitumor drug are dispersed more uniformly;
in another embodiment of the present invention, the cell membrane vesicle is prepared by the following method:
(1) collecting mouse tumor tissue, and obtaining tumor single cell suspension by a homogenization method;
(2) ultrasonically treating with a probe to obtain broken tumor cells, removing impurities by ultrafiltration and centrifugation, and extracting tumor cell membrane by gradient centrifugation;
(3) obtaining the tumor cell membrane vesicles by adopting an ultrasonic mode.
In still another embodiment of the present invention, in the above step (1), the tumor single cell suspension is obtained by adding a homogenization buffer, which is a PBS buffer containing sucrose, EDTA, HEPES-NaOH and protease inhibitor, followed by grinding;
further, the concentration of sucrose is 0.1-1 mM, preferably 0.25 mM; the concentration of EDTA is 0.5-5 mM, preferably 1 mM; the concentration of HEPES-NaOH is 5-30 mM, preferably 20 mM; the concentration of the protease inhibitor is 6.5-8.5 mM, and the pH is 7.4.
In another embodiment of the present invention, in the step (2), the specific steps include:
carrying out probe ultrasonic treatment on the collected tumor single cell suspension at a low temperature to break tumor cells to obtain broken tumor cells;
then carrying out medium-speed centrifugation on the broken tumor cells at low temperature, removing melanin and purifying;
carrying out high-speed centrifugation on the purified broken cell suspension at low temperature to obtain tumor cell membrane sediment;
dispersing the tumor cell membrane precipitate again, and freezing and storing;
further, the low temperature is 0-6 ℃, the power amplitude of probe ultrasound is 20-60%, preferably 40%, the probe ultrasound time is 5-15 min, preferably 10min, and the probe ultrasound time interval is as follows: 2-6 s; turning off: 1-4 s, preferably on: 3 s; turning off: 7 s; the rotating speed of the medium-speed centrifugation is 2000-4000 g, preferably 3000g, and the centrifugation time is 5-20 min, preferably 10 min; the rotating speed of the high-speed centrifugation is 10000-20000 g, preferably 15000g, and the centrifugation time is 30-90 min, preferably 60 min; the solvent for redispersing the cell membrane precipitate was PBS;
in another embodiment of the present invention, in the step (3), membrane fusion is performed on the tumor cell membrane by means of low-temperature ultrasound, preferably 0 to 6 ℃, for 30 to 120 seconds, preferably 60 seconds, to obtain the tumor cell membrane vesicle.
In another embodiment of the invention, the sodium alginate oxide modified tumor cell membrane vesicle is prepared by the following method:
dissolving sodium alginate oxide, mixing with tumor cell membrane vesicle uniformly, reacting at room temperature for a period of time, and removing impurities to obtain sodium alginate oxide modified tumor cell membrane vesicle;
preferably, the room temperature is 18-30 ℃;
preferably, the solvent for dissolving the sodium alginate oxide is deionized water, so that the dissolving effect is better;
preferably, the sodium alginate oxide and the tumor cell membrane vesicle are mixed in a stirring mode, so that the mixing uniformity and the reaction speed of the sodium alginate oxide and the tumor cell membrane vesicle are improved;
preferably, the reaction time is 4-12 h, and more preferably 6 h.
In another embodiment of the present invention, the sodium alginate oxide is a product of sodium alginate oxidized with sodium periodate;
in another embodiment of the present invention, the sodium alginate oxide is specifically prepared by the following method:
dissolving sodium alginate in deionized water, adding sodium periodate, reacting under stirring at room temperature, adding ethylene glycol, continuously stirring for reacting, adding NaCl, extracting with ethanol, dissolving again, dialyzing in water, and freeze-drying to obtain sodium alginate oxide.
In another embodiment of the present invention, there is provided a use of the above cell membrane-based bioactive agent for the preparation of an antitumor drug;
the tumor comprises a benign tumor and/or a malignant tumor; the malignant tumor comprises a solid tumor and a blood tumor, wherein the solid tumor comprises melanoma;
the above cell membrane-based bioactive agent has at least one or more of the following uses:
1) relieving immunosuppression caused by tumor exosomes;
2) relieving immunosuppression brought by PD-L1 on the surface of the tumor cells;
3) improving the anti-tumor efficacy.
Wherein the applications 1) and 2) are related to PD-L1.
Also, it is noted that tumors are used in the present invention as known to those skilled in the art, which include benign tumors and/or malignant tumors. Benign tumors are defined as cellular hyperproliferation that fails to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs).
In yet another embodiment of the invention, the medicament of the invention is useful for treating malignant tumors. Examples of malignant tumors that can be treated with the drug of the present invention include solid tumors and hematological tumors. Solid tumors may be, for example, tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as the thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin (such as melanoma), ureter, vagina and vulva. Malignant tumors include hereditary cancers such as retinoblastoma and Wilmstumor. Furthermore, malignant tumors include primary tumors in the organs and corresponding secondary tumors in distant organs (tumor metastases). Hematological tumors can be, for example, aggressive and indolent forms of leukemia and lymphoma, i.e., non-hodgkin's disease, chronic and acute myeloid leukemia (CML/AML), Acute Lymphocytic Leukemia (ALL), hodgkin's disease, multiple myeloma, and T-cell type lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, carcinoid syndromes, and cancers of unknown primary site and AIDS-related malignancies.
The technical solution of the present invention will be described below with specific examples. The raw materials used in the following examples are commercially available and all the equipment used is conventional.
Example 1: preparation of sodium alginate oxide modified tumor cell membrane vesicle
1) Preparation of tumor single cell suspension: after mouse melanoma tissues were collected, they were obtained by adding a homogenization buffer, which is PBS buffer containing 0.25mM sucrose, 1mM EDTA, 20mM HEPES-NaOH and protease inhibitor, followed by grinding.
2) Extracting tumor cell membranes: carrying out probe ultrasonic disruption on the collected tumor single cell suspension at 4 ℃ to obtain disrupted tumor cells, wherein the power amplitude of probe ultrasonic is 40%, the time is 10min, and the interval is as follows: 4s, off: 2 s; centrifuging the crushed tumor cells at 4 deg.C for 10min at 3000g, removing melanin, and purifying; centrifuging the purified broken cell suspension at 4 deg.C for 60min at 15000g to obtain tumor cell membrane precipitate; the cell membrane pellet was redispersed in PBS and stored frozen.
3) Preparing tumor cell membrane vesicles: carrying out ultrasonic treatment on the tumor cell membrane at 4 ℃ for 1min to finally obtain the tumor cell membrane vesicle.
4) Preparing sodium alginate oxide: dissolving Sargassum resin in deionized water, adding sodium periodate, stirring at room temperature in the dark for 4 hr, adding ethylene glycol, stirring for 1 hr, adding NaCl, and extracting with ethanol. The extract was then redissolved and dialyzed against water for 3 days. And freeze-drying to obtain the oxidized sodium alginate.
5) Preparing the sodium alginate oxide modified tumor cell membrane vesicles: and (3) mixing an excessive oxidized sodium alginate solution with the tumor cell membrane vesicle in deionized water, fully stirring, reacting at room temperature for 6 hours, and removing excessive sodium alginate oxide through ultrafiltration and centrifugation to obtain the modified tumor cell membrane vesicle.
Example 2: transmission Electron Microscope (TEM) identification of nanostructure of membrane vesicles of tumor cells before and after modification
And respectively sucking 10 mu L of the tumor cell membrane vesicle solution before and after modification by using a pipette, sucking the redundant liquid on a carbon film copper net and filter paper, drying at room temperature, and then placing under a transmission electron microscope for observation. The results are shown in fig. 1, and transmission electron microscopy pictures can confirm that the shapes of the tumor cell membrane vesicles before and after modification are obviously different, the sizes are 160-200 nm respectively, and the tumor cell membrane vesicles are uniform in size and good in dispersibility.
Example 3: preparation of medicine-carrying O-TMV glue solution
The embodiment provides a hydrogel preparation based on the cell membrane vesicle, which comprises the modified tumor cell membrane vesicle, a CDK5 inhibitor Roscovitine and an exosome inhibitor dimethyl amiloride.
The preparation method comprises the following steps: the CDK5 inhibitor Roscovitine and the exosome inhibitor dimethyl amiloride are precisely weighed and respectively dissolved to 7.82 mg/mL and 1.18 mg/mL by using deionized water-1. And (3) uniformly mixing 100 mu L of the mixture under the ultrasonic condition, slowly dripping the mixture into 800mL of modified tumor cell membrane vesicle aqueous solution in stirring, and stirring for 20min to obtain O-TMV @ DR glue solution.
Example 4: gelling characteristics and microscopic morphology of O-TMV and O-TMV @ DR hydrogels
For more visual observation and comparison, the gelling property of the O-TMV gelator prepared in example 3 was observed by photographing. In figure 2, the O-TMV gelator can rapidly form gel in the presence of calcium ions, and the microstructure of the formed gel is observed, so that the O-TMV and the O-TMV @ DR hydrogel formed after drug loading are both porous structures, and have huge drug loading potential.
Example 5: biocompatibility of O-TMV hydrogels
1. Culture of cells
Human umbilical vein epithelial cells HUVEC were selected as the study subjects. The frozen cells were harvested and cultured at 37 ℃ in 5% CO2Culturing under the conditionCulturing, when the cells grow to high density, carrying out passage, proportionally transferring to a culture flask, continuously culturing and counting the cells.
2. Cytotoxicity test
HUVEC cells were used to assess biocompatibility at various concentrations of O-TMV. By collecting HUVEC cells in logarithmic growth phase at 2X 104The concentration of each well was added to a 48-well plate, and after overnight incubation, O-TMV hydrogel (membrane protein concentration: 50. mu.g. mL) was added at various concentrations-1-500μg·mL-1) 3 multiple holes are arranged. Culturing cells at 37 ℃ for 24h, washing, replacing fresh culture medium, adding 0.5% MTT solution into each hole, continuing to incubate for 4h, then removing liquid in the holes, adding DMSO into each hole for dissolving, measuring absorbance at 490nm by using an enzyme-labeling instrument, and calculating the cell inhibition rate by using the following formula:
Figure BDA0003071645390000091
the results of the cytostatic experiments of several samples at different concentrations are shown in fig. 4. As can be seen from FIG. 4, the O-TMV hydrogel had a small effect on the survival rate of HUVEC cells, and even at high concentrations, the O-TMV hydrogel did not affect the cell morphology and survival status of HUVEC cells.
Therefore, the conclusion is drawn that the O-TMV hydrogel has high biocompatibility and good biological safety to human umbilical vein epithelial cell HUVEC cells.
Example 6O-TMV @ DR hydrogel in vivo tumor suppression study
1. Establishment of animal model
Female C57BL/6 mice, 6 to 8 weeks old, were used to establish an in vivo anti-tumor bilateral model. A suspension of B16F10 cells (8X 10 cells per mouse)5Individual cells) was subcutaneously inoculated into the right forelimb of mice to establish in situ tumors, and 3 days later, a suspension of B16F10 cells (8 × 10 cells per mouse) was added5Individual cells) were inoculated subcutaneously into the left forelimb of mice to establish distal tumors.
2. Tumor inhibition experiment in animal body
The in situ tumor reaches 60mm3After that, the mice were weighed and randomly divided into 6 groups: the normal saline is used asAs a control, ROSCO, DMA, O-TMV, ROSCO + DMA, O-TM @ DR, the last 5 groups were used as experimental groups, each group was administered with a dose of either DMA or ROSCO: DMA: 295 ug kg-1，ROSCO：1.96mg·kg-1. Tumor volumes were measured every two days after once intratumoral administration in situ and were calculated according to the following equation:
Figure BDA0003071645390000101
l represents the maximum diameter (mm), and W represents the minimum diameter (mm).
After each treatment, mice were sacrificed to remove tumors and weighed. And the inhibition rate of the tumor of each group of mice was calculated according to the following formula:
Figure BDA0003071645390000102
Wcmean tumor weight in the saline group, and WtRepresenting the final tumor weight of the other groups.
The results of in vivo tumor suppression experiments are shown in figure 5.
The modified tumor cell membrane vesicle, ROSOCO and DMA are mixed to form O-TMV @ DR glue solution, and the O-TMV @ DR glue solution can quickly form gel in the physiological environment of tumor and play a role in long-acting anti-tumor.
As can be seen from FIG. 5, in both orthotopic tumor and distal tumor, compared with the group administered with DMA or Roscovitine alone, the tumor inhibition rate of the group administered with two drugs in combination is higher than that of the group administered with two drugs alone, because the two drugs in combination can simultaneously reduce the expression level of PD-L1 on the surface of tumor cells and secreted exosomes thereof, and inhibit the local microenvironment and systemic immunosuppression of tumor, thereby achieving better antitumor effect. Particularly, for distant tumors, the tumor inhibition rates of DMA or ROSCO used alone are respectively 27.77% and 24.97%, and the tumor inhibition rate after the two drugs are combined can reach 68.23%, which is obviously higher than the sum of the tumor inhibition rates of the two drugs used alone, so that the DMA and ROSCO are proved to play a synergistic inhibition role in tumor metastasis, and the CDK5 inhibitor and exosome inhibitor are proved to have great potential in the aspect of anti-tumor metastasis treatment by combined application.
The experimental group administered O-TMV alone also showed excellent tumor suppression effect, because the tumor cell membrane provided a large amount of endogenous antigen, contributing to enhancement of tumor immunogenicity. The O-TMV @ DR experimental group shows the optimal effect of inhibiting tumor growth and metastasis, and shows that the drug-loaded bioactive preparation based on the tumor cell membrane is more beneficial to the blocking treatment of PD-L1 to exert curative effect.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

  1. Use of a CDK5 inhibitor in combination with an exosome inhibitor in the preparation of an anti-tumour medicament.
  2. 2. The use according to claim 1, wherein the inhibitor of CDK5 is selected from the group consisting of siRNA, shRNA, antibodies, small molecule compounds and peptide fragments;
    preferably, the CDK5 inhibitor is selected from: roscovitine, olomoucine and its derivatives, Dinaciclib, AT7519, CIP, p5 and Cpd 1;
    more preferably, the CDK5 inhibitor is Roscovitine;
    the exosome inhibitory drugs comprise calcium ion channel inhibitors, proton pump inhibitors, sphingomyelin-ribonuclease inhibitors and endothelin receptor A interference drugs;
    preferably, the exosome-inhibiting drug may be selected from: dimethyl amiloride (DMA), omeprazole, sulfisoxazole, sphingomyelinase inhibitor GW4869 and the like;
    more preferably, the exosome-inhibiting drug is dimethyl amiloride;
    further, the CDK5 inhibitor is Roscovitine, the exosome inhibitory drug is dimethyl amiloride, and the mass ratio of the two is 1-10: 1; preferably, the mass ratio of the two is 5: 1.
  3. 3. the use according to claim 1, wherein the antineoplastic drug has at least one or more of the following uses:
    1) relieving immunosuppression caused by tumor exosomes;
    2) relieving immunosuppression brought by PD-L1 on the surface of the tumor cells;
    3) improving the anti-tumor efficacy;
    wherein the applications 1) and 2) are related to PD-L1.
  4. 4. A tumor cell membrane-based biologically active agent, wherein the pharmaceutically active ingredient of said cell membrane-based biologically active agent comprises a CDK5 inhibitor and an exosome inhibitory drug;
    preferably, the bioactive agent is a hydrogel;
    preferably, the biologically active agent is made from a CDK5 inhibitor, an exosome inhibitory drug and a tumor cell membrane vesicle; more preferably, the tumor cell membrane vesicle is a sodium alginate oxide modified tumor cell membrane vesicle;
    preferably, the size of the tumor cell membrane vesicle is 150-220 nm;
    preferably, the CDK5 inhibitor is Roscovitine and the exosome inhibitory drug is dimethyl amiloride; preferably, the mass ratio of Roscovitine to dimethyl amiloride is 1-10: 1; more preferably, the mass ratio of the two is 5: 1.
  5. 5. A method of preparing a biologically active agent based on tumor cell membranes according to claim 4, comprising:
    forming a gel factor by using the sodium alginate oxide modified tumor cell membrane vesicles, respectively dissolving a CDK5 inhibitor and an exosome inhibitory drug, uniformly mixing to form a drug solution, adding the drug solution into a water solution of the gel factor, and stirring to obtain a gelling solution;
    preferably, the solvent in which the CDK5 inhibitor and exosome inhibitor are dissolved is deionized water; the method of adding the drug solution is dropwise.
  6. 6. The method according to claim 5, wherein the tumor cell membrane vesicles are prepared by:
    (1) collecting mouse tumor tissue, and obtaining tumor single cell suspension by a homogenization method;
    (2) ultrasonically treating with a probe to obtain broken tumor cells, removing impurities by ultrafiltration and centrifugation, and extracting tumor cell membrane by gradient centrifugation;
    (3) obtaining the tumor cell membrane vesicles by adopting an ultrasonic mode.
  7. 7. The preparation method according to claim 6, wherein in the step (1), the single tumor cell suspension is obtained by adding a homogenization buffer, which is a PBS buffer containing sucrose, EDTA, HEPES-NaOH and protease inhibitor, followed by grinding;
    further, the concentration of sucrose is 0.1-1 mM, preferably 0.25 mM; the concentration of EDTA is 0.5-5 mM, preferably 1 mM; the concentration of HEPES-NaOH is 5-30 mM, preferably 20 mM; the concentration of the protease inhibitor is 6.5-8.5 mM, and the pH value is 7.4;
    in the step (2), performing probe ultrasonic treatment on the collected tumor single cell suspension at a low temperature to break tumor cells to obtain broken tumor cells; then carrying out medium-speed centrifugation on the broken tumor cells at low temperature to remove melanin for purification, and carrying out high-speed centrifugation on the purified broken cell suspension at low temperature to obtain tumor cell membrane sediment; dispersing the tumor cell membrane precipitate again, and freezing and storing;
    further, the low temperature is 0-6 ℃, the power amplitude of the probe ultrasonic is 20-60%, and the preferred power is 40%; the ultrasonic time of the probe is 5-15 min, preferably 10min, and the ultrasonic time interval of the probe is as follows: 2-6 s; turning off: 1-4 s, preferably on: 3 s; turning off: 7 s; the rotating speed of the medium-speed centrifugation is 2000-4000 g, preferably 3000g, and the centrifugation time is 5-20 min, preferably 10 min; the rotating speed of the high-speed centrifugation is 10000-20000 g, preferably 15000g, and the centrifugation time is 30-90 min, preferably 60 min; the solvent for redispersing the cell membrane precipitate was PBS;
    in the step (3), membrane fusion is carried out on the tumor cell membrane in a low-temperature head ultrasound mode to obtain the tumor cell membrane vesicle, preferably, the low temperature is 0-6 ℃, and the ultrasound time is 30-120 s, preferably 60 s.
  8. 8. The preparation method of claim 5, wherein the sodium alginate oxide modified tumor cell membrane vesicle is prepared by the following method:
    dissolving sodium alginate oxide, mixing with tumor cell membrane vesicle uniformly, reacting at room temperature for a period of time, and removing impurities to obtain sodium alginate oxide modified tumor cell membrane vesicle;
    preferably, the room temperature is 18-30 ℃; preferably, the solvent for dissolving the sodium alginate oxide is deionized water; preferably, the mixing is carried out in a stirring manner; preferably, the reaction time is 4-12 h, preferably 6 h;
    preferably, the sodium alginate oxide is a product of sodium alginate oxidized by sodium periodate; more preferably, the sodium alginate oxide is prepared by the following method:
    dissolving sodium alginate in deionized water, adding sodium periodate, reacting under stirring at room temperature, adding ethylene glycol, continuously stirring for reacting, adding NaCl, extracting with ethanol, dissolving again, dialyzing in water, and freeze-drying to obtain sodium alginate oxide.
  9. 9. Use of the tumor cell membrane-based bioactive preparation according to claim 4 or the tumor cell membrane-based bioactive preparation prepared by the preparation method according to any one of claims 5 to 8 in the preparation of an antitumor drug.
  10. 10. The use of claim 9, wherein the tumor comprises a benign tumor and/or a malignant tumor; the malignant tumors include solid tumors and hematological tumors; among the solid tumors are melanoma.
CN202110541241.7A 2021-05-18 2021-05-18 Bioactive preparation based on tumor cell membrane as well as preparation method and application thereof Active CN113304269B (en)

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US20170196818A1 (en) * 2014-06-30 2017-07-13 President And Fellows Of Harvard College Hydrogel compositions comprising encapsulated cells and methods of use thereof
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US20170196818A1 (en) * 2014-06-30 2017-07-13 President And Fellows Of Harvard College Hydrogel compositions comprising encapsulated cells and methods of use thereof
CN112040955A (en) * 2018-03-14 2020-12-04 加利福尼亚大学董事会 Inhibitory exosomes in cancer and for immunosuppression

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