CN110628719B - Method for inducing cells to rapidly produce vesicles and application thereof - Google Patents
Method for inducing cells to rapidly produce vesicles and application thereof Download PDFInfo
- Publication number
- CN110628719B CN110628719B CN201910900505.6A CN201910900505A CN110628719B CN 110628719 B CN110628719 B CN 110628719B CN 201910900505 A CN201910900505 A CN 201910900505A CN 110628719 B CN110628719 B CN 110628719B
- Authority
- CN
- China
- Prior art keywords
- vesicles
- cells
- protein
- uio
- 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
Links
Images
Classifications
-
- 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
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- 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
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary cells
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
-
- 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
- C12N2510/00—Genetically modified cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Abstract
The invention provides a method for inducing cells to rapidly generate vesicles and application thereof, and the invention obtains the vesicles by using a novel nanoparticle 2I-Bodipy-5C-UiO-Hf, co-culturing with nucleated cells and illuminating. Meanwhile, the high-efficiency expression of the target protein is obtained in the vesicle, and the protein can be widely applied to various nucleated cells. Provides a universal, efficient and feasible method in the aspects of exploring the interaction among proteins, the molecular function of cell membrane protein, immunotherapy and the like, and has good practical application value.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a method for inducing cells to rapidly produce vesicles 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.
Extracellular vesicles are the focus of research in the medical and health industry at present, and the research currently considers that: according to the size of the extracellular vesicles, the extracellular vesicles can be divided into apoptotic bodies, exosomes (60-100nm), small vesicles (100-1000nm) and large vesicles (more than 1 mu M).
Vesicles play an important role:
therapeutic effect of vesicles. Loading the drug with vesicles to transport the drug with severe side effects to the target cells will reduce the patient's pain and inconvenience during treatment. In SCID mice, Cisplatin-loaded vesicles significantly inhibited the growth of ovarian cancer cells. In liver cancer H22 tumor-bearing mice, MTX-loaded vesicles significantly increased the survival rate of the mice. In the clinical treatment of patients with advanced lung cancer malignant pleural effusion, the pleural perfusion methotrexate vesicle can kill tumor cells in a targeted manner, effectively treat recurrent malignant pleural effusion and show good safety. If vesicles derived from allogeneic cells are used as the therapeutic substance, the patient will experience immune rejection due to differences in MHC; if vesicles derived from autologous cells are used, the patient will not develop an immune response and may be used in combination with an immunosuppressive agent. In addition, no cell nucleus exists in the vesicle, so that the transformation effect of the nuclear substance in the tumor cell can be avoided, and the vesicle has a wide application prospect in clinic.
2, research on the functions of the vesicles in the biological membrane protein molecules. Plasma membrane proteins account for 70% of all known drug targets, including ion channel proteins, G protein-coupled receptors, transporters, immunoglobulins, adhesion molecules, etc., and the plasma membrane proteome is very important for elucidating the biological functions of proteins, deeply understanding physiological processes, immune responses, disease mechanisms and discovering new drug targets.
3, other uses of vesicles in the medical field. The vesicle produced by tumor cell contains a large amount of tumor-associated antigen substance, and can be used as tumor vaccine after being processed by antigen presenting cell. The vesicles can be used for diagnosis of diseases by loading biologically active substances such as primers, probes, contrast agents, antisense nucleic acids, fluorescent molecules or antibodies into the vesicles. Upon loading of the vesicles with biologically active substances, such as Cas9 protein and grnas, vesicles are produced that can have gene editing functions.
At present, the research on the vesicle forming agent is numerous, and a chemical reagent method (a sulfhydryl blocking agent method), a cytotoxin method (such as cytochalasin or melittin) and the like are available, but the inventor finds that the efficiency of vesicle formation, the time required by vesicle formation and the subsequent treatment required by vesicle collection are all defects, and the problems of long cycle, low yield, complex flow and the like still exist in the current process of inducing cells to generate vesicles, so that the application research of the vesicles is restricted.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a method for inducing cells to rapidly generate vesicles and application thereof, and the invention obtains the vesicles by using a novel nanoparticle 2I-Bodipy-5C-UiO-Hf, co-culturing the nanoparticle with nucleated cells and illuminating. Meanwhile, the high-efficiency expression of the target protein can be obtained in the vesicle, and the protein can be widely applied to various nucleated cells, so that the protein has good practical application value.
In one aspect of the present invention, there is provided a method of inducing rapid vesicle production by a cell, the method comprising: the nano particles and cells are incubated and cultured together, and the cells are induced to generate vesicles rapidly and efficiently under the action of illumination.
Wherein the nano-particles are 2I-Bodipy-5C-UiO-Hf.
In a second aspect of the invention, vesicles produced by the above method are provided. The diameter of the vesicle prepared by the method is not less than 100nm after the vesicle is generated, and the liquid vesicle with the diameter of 100-10000nm is further preferable. The method for producing vesicles according to the present invention is not produced by inducing apoptosis, and therefore the vesicles do not contain apoptotic bodies.
In a third aspect of the invention, there is provided the use of a method as described above and/or a vesicle as described above in any one or more of:
1) studying protein-protein interactions;
2) studying the molecular function of membrane proteins;
3) drug delivery;
4) disease diagnosis or preparation of disease diagnosis drugs and/or reagents;
5) disease treatment or preparation of disease treatment drugs and/or agents.
Further, the disease is cancer, including, but not limited to, leukemia, soft tissue tumors, malignant melanoma, bone tumors, skin cancer, lung cancer, thyroid cancer, liver cancer, endometrial cancer, prostate cancer, colon cancer, esophageal cancer, stomach cancer, and breast cancer.
By adopting the technical scheme of the invention, cells can be efficiently induced to generate vesicles, and compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. the invention does not need expensive instrument and equipment, has short consumed time and high efficiency, and can obtain a large amount of vesicles within 1 hour;
2. by a transgenic technology, the EGFP protein expressed in cytoplasm can be obtained in the vesicle, and the membrane protein can also be efficiently expressed in the vesicle;
3. the method can be used for obtaining the nucleated cell vesicles of different species and different tissue sources of mice, rats and humans, so the method has universal applicability. Therefore, the invention provides a more optimized technical scheme in the research fields of comparing proteomes in different cell lines, comparing protein expression profiles and protein interaction in target cells and clinically tumor vaccines and medicines, thereby having 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 diagram of the steps of the nanoparticles 2I-Bodipy-5C-UiO-Hf combined with light to induce MCF-7 to generate vesicles; wherein the content of the first and second substances,
a: when the cells are in the logarithmic growth phase, adding the nanoparticles 2I-Bodipy-5C-UiO-Hf into a cell culture dish to make the final concentration of the nanoparticles 1 mu M;
b: light protection, at 37 ℃ 5% CO2Under the condition, co-incubation and culture are carried out for 30 min;
c: washing the cells 2 times with PBS to remove residual nanoparticles in the culture medium;
d: irradiating green excitation light for 2-3min to induce MCF-7 cells to generate vesicles, and shaking the vesicles through agate balls to enable the vesicles to fall off and be dissociated in supernatant;
e: adding 2M/L sucrose solution into the cell supernatant solution, centrifuging at 1500 rpm for 20min to allow cell supernatant to be layered, depositing cell debris in the sucrose layer, and allowing secreted vesicles to exist in the vesicle layer.
FIG. 2 is a graph showing the test results of fluorescent microscope detection of nanoparticles 2I-Bodipy-5C-UiO-Hf after incubation for 30min, phagocytosis by MCF-7 cells and 2min irradiation by green light; wherein the content of the first and second substances,
FIG. 2A is a diagram showing vesicle-free production of MCF-7 cells after irradiation with green light;
FIG. 2B is a graph of vesicle-free production of MCF-7 cells after co-incubation with nanoparticle 2I-Bodipy-5C-UiO-Hf;
FIG. 2C is a diagram of vesicles produced by MCF-7 cells incubated with the nanoparticle 2I-Bodipy-5C-UiO-Hf under green light illumination;
FIG. 2D is a graph of shed vesicles produced by MCF-7 cells after co-incubation with the nanoparticle 2I-Bodipy-5C-UiO-Hf under green light illumination;
FIG. 3 is a graph relating to the induction of MCF-7 cells by the nanoparticle 2I-Bodipy-5C-UiO-Hf to produce vesicles encapsulating a protein of interest; wherein the content of the first and second substances,
FIG. 3A is a diagram of vesicles produced by MCF-7 cells incubated with the nanoparticle 2I-Bodipy-5C-UiO-Hf under green light illumination;
FIG. 3B is a vesicle map generated by MCF-7-EGFP-stabilized cells incubated with nanoparticle 2I-Bodipy-5C-UiO-Hf under green light irradiation;
FIG. 3C is a fluorescent microscopic picture of the green-free fluorescence of vesicles produced by MCF-7 cells incubated with the nanoparticle 2I-Bodipy-5C-UiO-Hf under green light irradiation;
FIG. 3D is a green fluorescence image of vesicles produced by MCF-7-EGFP-stabilized cells incubated with nanoparticle 2I-Bodipy-5C-UiO-Hf under green light irradiation.
FIG. 4 is a diagram of flow cytometry to detect that the nanoparticle 2I-Bodipy-5C-UiO-Hf is phagocytosed by MCF-7 cells within 30min and generates vesicles after light treatment; wherein the content of the first and second substances,
FIG. 4A is a flow chart of MCF-7 cells as a negative control;
FIG. 4B is a flow chart of vesicles that produced no green fluorescence under green illumination after co-incubation of MCF-7 cells with nanoparticle 2I-Bodipy-5C-UiO-Hf, showing no significant clustering at SSC-, FITC +;
FIG. 4C is a flow chart of MCF-7-EGFP-transfected cells incubated with nanoparticles 2I-Bodipy-5C-UiO-Hf and under green light irradiation, which shows significant clustering at the SSC-, FITC + sites, with a number of 5% of the total cell number.
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 example embodiments in accordance with the present application. 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.
As described above, many studies on vesicle-forming agents currently exist, including chemical reagent methods (thiol blocking agent methods), cytotoxin methods (such as cytochalasin or melittin), etc., but the inventors found that the efficiency of vesicle formation, the time required for vesicle formation, the subsequent treatment required for vesicle collection are all deficient, and all processes for inducing cells to produce vesicles still have problems of long cycle, low yield, complex flow, etc.
In view of the above, in one embodiment of the present invention, there is provided a method for inducing rapid vesicle production in a cell, the method comprising: the nano particles and cells are incubated and cultured together, and the cells are induced to generate vesicles rapidly and efficiently under the action of illumination.
Wherein the nano-particles are 2I-Bodipy-5C-UiO-Hf.
The nanoparticles were used at a concentration of 1-2 μ M (based on BODIPY).
In another embodiment of the present invention, the incubation time of the nanocarrier and the cell is 30-60min, and the illumination time is 1-30 min.
In yet another embodiment of the present invention, the wavelength of the light is distributed in the range of 1-580nm, preferably in the range of 450-570 nm; by controlling the illumination wavelength, the vesicle generation efficiency can be effectively improved.
In another embodiment of the present invention, the cells are nucleated cells, and the origin of the species from which the nucleated cells are derived and the origin of the tissues are not limited, as long as the cells capable of producing vesicles are within the scope of the present invention, such as various human tumor cells (including human breast cancer cells MCF-7).
The vesicles produced at the same time may be loaded with at least one of the following: target proteins, microvesicles, nucleic acids, drugs and nanocarriers; further, the loaded substances are two or more different substances, such as cas9 protein and nucleic acid gRNA.
In another embodiment of the present invention, the target protein at least comprises any one of the following proteins: cytokines, antibodies, targeting proteins, bioactive peptides, toxins, and fusion proteins.
In another embodiment of the present invention, the method for preparing a target protein comprises: transient or stable cell lines are obtained by constructing plasmids and utilizing transgenic technologies such as liposome, lentiviral vector, adenoviral vector and the like, so that target proteins are effectively expressed in cytoplasm or cell membrane and are wrapped in vesicles in the process of producing the vesicles.
The nucleic acid is any one or more of antisense nucleic acid, DNA, mRNA, lncRNA, circRNA, PNA and gRNA.
In yet another embodiment of the present invention, the method further comprises the step of collecting the vesicles, said step comprising collecting the vesicles using density gradient centrifugation.
In yet another embodiment of the present invention, vesicles produced by the above method are provided. The diameter of the vesicle prepared by the method is not less than 100nm after the vesicle is generated, and the liquid vesicle with the diameter of 100-10000nm is further preferable. The method for producing vesicles according to the present invention is not produced by inducing apoptosis, and therefore the vesicles do not contain apoptotic bodies.
In a further embodiment of the invention, there is provided the use of a vesicle as described above and/or in any one or more of the following:
1) studying protein-protein interactions;
2) studying the molecular function of membrane proteins;
3) drug delivery;
4) disease diagnosis or preparation of disease diagnosis drugs and/or reagents;
5) disease treatment or preparation of disease treatment drugs and/or agents.
In yet another embodiment of the present invention, the disease is cancer, including but not limited to leukemia, soft tissue tumors, malignant melanoma, bone tumors, skin cancers, lung cancers, thyroid cancers, liver cancers, endometrial cancers, prostate cancers, colon cancers, esophageal cancers, stomach cancers, and breast cancers.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 Synthesis of nanoparticles 2I-Bodipy-5C-UiO-Hf
1. Synthesis of UiO-66-OH (Hf) (1)
Reacting HfCl4(336mg,1.05mmol), 2-hydroxyterephthalic acid (182mg,1.0mmol) and acetic acid (4.0mL) in water (6.0mL) heated to 100 ℃ for 48 h; and (3) carrying out centrifugal separation (12000rpm, 30min), washing by using ethanol, and drying at 40 ℃ to obtain the nanometer UiO-66-OH (Hf) particles. FT-IR (ATR, cm)-1):3253(b),2977(m),1699(w),1583(s),1500(s),1424(vs),1245(m),1158(w),1045(w),963(w),798(w),768(m),680(m),578(w),475(w).
2. Synthesis of BODIPYC5Br
Under a nitrogen atmosphere, 6-bromohexanoyl chloride (5.6g,26mmol) and 2, 4-dimethyl-1H-pyrrole (5.0g,52.5mmol) were refluxed in dichloromethane (200mL) for 1H, cooled to room temperature, and then N, N-diisopropylethylamine (15mL) was added thereto and heated under reflux for 1H. Then BF is added3·Et2O (15mL) was heated under reflux for an additional 3 h. Stirring overnight, purifying with neutral alumina column (dichloromethane as eluent) to obtain BODIPYC5Br. yield: 4.7g (46%).1H NMR(400MHz,Chloroform-d)δ6.06(s,2H),3.44(t,J＝5.1Hz,2H),2.99-2.94(m,2H),2.52(s,6H),2.42(s,6H),1.95-1.89(m,2H),1.69-1.63(m,4H).13C NMR(101MHz,Chloroform-d)δ153.90,145.90,140.25,131.36,121.66,33.38,32.18,30.88,28.55,28.19,16.39,14.44.MALDI-TOF MS,Calcd.For[M],396.118,Found,396.386.UV-vis(EtOH),497nm.FT-IR(ATR,cm-1):2927(m),2868(w),1550(vs),1509(vs),1475(s),1440(m),1409(m),1372(m),1340(w),1307(m),1270(m),1252(w),1224(m),1200(vs),1157(m),1107(m),1080(s),1070(s),1028(m),986(s),837(w),819(w),798(w),716(w),644(vw),626(w),583(w),554(vw),482(w).Anal.Calcd.For C18H24BBrF2N2(％):C,54.44；H,6.09；N,7.05,Found:C,54.14；H,5.91；N,7.32.
3. Synthesis of 2I-BODIPYC5Br(2)
Mixing BODIPYC5Br (1.0g,2.5mmol) and iodine (1.6g,6.25mmol), and an aqueous solution of iodic acid (1.0mL,0.9g/mL) was added to ethanol (400mL) and treated at reflux for 30 min. Cooling to room temperature, purifying with neutral alumina column (eluent, dichloromethane/n-hexane, v/v ═ 1:1) to obtain 2I-BODIPYC5Br is added. Yield 1.1g (69%).1H NMR(400MHz,Chloroform-d)δ3.44(t,J＝6.5Hz,2H),3.07-2.99(m,2H),2.62(s,6H),2.49(s,6H),1.93(p,J＝6.5Hz,2H),1.73-1.60(m,4H).13C NMR(101MHz,Chloroform-d)δ155.41,145.54,142.15,131.31,86.56,33.21,32.07,30.64,29.03,28.43,19.00,16.13.MALDI-TOF MS,Calcd.For[M],647.912,Found,647.557.UV-vis(EtOH),527nm.FT-IR(ATR,cm-1):2925(m),2859(w),1538(vs),1462(m),1394(m),1347(m),1306(w),1272(w),1252(w),1207(w),1186(s),1146(m),1077(m),994(m),753(w),719(w),587(w),527(w).Anal.Calcd.For C18H22BBrF2I2N2(％):C,33.32；H,3.42；N,4.32,Found:C,33.48；H,3.21；N,4.55.
4. Synthesis of 2I-Bodipy-5C-UiO-Hf
Mixing UiO-66-OH (Hf) (5.0mg) and 2I-BODIPYC5Br (5.0mg, 7.7. mu. mol) and KI (1.0mg, 6.0. mu. mol) were dissolved in DMF (5.0mL) and treated with stirring at room temperature for 72 h. The 2I-Bodipy-5C-UiO-Hf is obtained by centrifugal separation (12000rpm, 30min) and sequential washing with DMF and water. FT-IR (ATR, cm)-1):3233(b),2976(m),1700(w),1584(s),1500(s),1424(vs),1245(m),1158(w),1099(w),1044(w),963(w),833(w),798(w),768(m),592(w),576(w),527(vw),475(w).
Example 2
Vesicle formation (FIG. 1).
Subculturing human breast cancer cells MCF-7, adjusting the concentration of MCF-7 cells to enable the cells to be in a logarithmic growth phase, adding 1 mu M (measured by BODIPY) of nanoparticles 2I-Bodipy-5C-UiO-Hf into a culture medium, co-incubating and culturing for 30min with the MCF-7 cells, observing the cell morphology through a fluorescence microscope after co-incubation is finished, and enabling the cells phagocytosing the nanoparticles 2I-Bodipy-5C-UiO-Hf to show green fluorescence under the fluorescence microscope. And (3) rinsing the cells for 2 times by using PBS to remove the nano particles, and then irradiating the cells by using green excitation light for 2-3min to induce the cells to generate vesicles. The cells can be seen to generate colorless vesicles under an inverted microscope, the volume of the vesicles is increased along with the prolonging of the illumination time, the cells are in a semi-adherent state, and the vesicles can fall into a supernatant solution (figure 2).
2, collecting vesicles:
when about 50% of cells generate vesicles, agate balls are added, and the cell culture flask or the culture dish is shaken gently to break off the vesicles and free in the supernatant. And (3) collecting the vesicles by a density gradient centrifugation method, carefully and gently adding the vesicle generation liquid into a 2M sucrose solution, rotating at 1500 rpm, centrifuging for 10 minutes to enrich the vesicles in the upper layer, and enrich cell debris in the lower sucrose layer.
3, loading green fluorescent protein in MCF-7 cell vesicles:
extracting plasmid pLVX-EGFP-IRES-PURO, co-transfecting 293T cells with pSPAX2 and pMD2.G through liposome, packaging to generate EGFP lentivirus, infecting breast cancer cells MCF-7, observing the expression of green fluorescent protein in the cells, and stably screening through puromycin to obtain the MCF-7-EGFP stable cell line. Cells are quickly induced to generate vesicles after being subjected to light irradiation through co-incubation culture with the nanoparticle 2I-Bodipy-5C-UiO-Hf. Compared with the colorless vesicles induced by the control MCF-7 cells, the vesicles generated by the MCF-7-EGFP stabilized cells are green vesicles containing EGFP protein (FIG. 3), which shows that the vesicles are loaded with the EGFP protein stably expressed in the cytoplasm. The detection of various parameters of the cytopoietic vesicles by flow cytometry showed that the green vesicles were concentrated in the SSC-, FITC + position and that the number of vesicles produced in this population was about 5% of the total cell number (fig. 4).
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 (4)
1. A method of inducing rapid vesicle production by a cell, the method comprising: co-incubating and culturing the nanoparticles and cells, and inducing the cells to generate vesicles under the action of illumination; the incubation time of the nano particles and the cells is 30-60min, and the illumination time is 1-30 min; the wavelength of the light is distributed at 450-570 nm;
wherein the nano-particles are 2I-Bodipy-5C-UiO-Hf; the nanoparticle has a structure of
2I-Bodipy-5C-UiO-Hf is generated by the reaction of UiO-66-OH (Hf), 2I-BODIPYC5Br and KI,
(ii) a And the vesicles are loaded with the protein of interest by the following method: transient or stable cell lines are obtained using liposomes, lentiviral vectors or adenoviral vectors, allowing efficient expression of the protein of interest in the cytoplasm or on the cell membrane, and encapsulated within vesicles during the production of vesicles as described above.
2. The method of claim 1, further comprising the step of collecting the vesicles, said step comprising collecting the vesicles using density gradient centrifugation.
3. Use of a vesicle loaded with a protein of interest by a method according to claim 1 or 2, comprising: transient or stable cell lines are obtained using liposomes, lentiviral vectors or adenoviral vectors, allowing efficient expression of the protein of interest in the cytoplasm or on the cell membrane and encapsulated within vesicles in a method for producing vesicles as claimed in claim 1.
4. The use of claim 3, further comprising studying the molecular function of the membrane protein or the interaction between the proteins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910900505.6A CN110628719B (en) | 2019-09-23 | 2019-09-23 | Method for inducing cells to rapidly produce vesicles and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910900505.6A CN110628719B (en) | 2019-09-23 | 2019-09-23 | Method for inducing cells to rapidly produce vesicles and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110628719A CN110628719A (en) | 2019-12-31 |
| CN110628719B true CN110628719B (en) | 2021-08-24 |
Family
ID=68974091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910900505.6A Active CN110628719B (en) | 2019-09-23 | 2019-09-23 | Method for inducing cells to rapidly produce vesicles and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110628719B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142646A (en) * | 2013-02-12 | 2015-12-09 | 兰诺龙有限公司 | Method of producing microparticles |
| CN107001031A (en) * | 2014-10-14 | 2017-08-01 | 芝加哥大学 | Nano particle for photodynamic therapy, the photodynamic therapy of X ray induction, radiotherapy, chemotherapy, immunotherapy and its any combination |
| CN107964509A (en) * | 2016-10-20 | 2018-04-27 | 湖北盛齐安生物科技股份有限公司 | Promote the equipment of cell release vesica and its promote the method for cell release vesica |
| CN110129266A (en) * | 2019-05-21 | 2019-08-16 | 上海交通大学 | A kind of method and application promoting cell secretion excretion body |
| CN110172071A (en) * | 2019-06-10 | 2019-08-27 | 山东师范大学 | A kind of bromine amyl replaces BODIPY derivative and the preparation method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040171175A1 (en) * | 2003-02-28 | 2004-09-02 | Swanson Basil I. | Process for conjugating biomolecules to hydrophobic membrane-incorporated molecules |
| KR20120132183A (en) * | 2011-05-27 | 2012-12-05 | 포항공과대학교 산학협력단 | Nanovesicle derived from tumor tissue and tumor vaccine using the same |
| CN102302784B (en) * | 2011-08-22 | 2012-12-05 | 湖北盛齐安生物科技有限公司 | Tumor chemotherapeutic medicinal preparation and preparation method thereof |
| KR101720851B1 (en) * | 2015-01-29 | 2017-03-28 | 포항공과대학교 산학협력단 | Nanovesicles derived from cell membrane and use thereof |
-
2019
- 2019-09-23 CN CN201910900505.6A patent/CN110628719B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142646A (en) * | 2013-02-12 | 2015-12-09 | 兰诺龙有限公司 | Method of producing microparticles |
| CN107001031A (en) * | 2014-10-14 | 2017-08-01 | 芝加哥大学 | Nano particle for photodynamic therapy, the photodynamic therapy of X ray induction, radiotherapy, chemotherapy, immunotherapy and its any combination |
| CN107964509A (en) * | 2016-10-20 | 2018-04-27 | 湖北盛齐安生物科技股份有限公司 | Promote the equipment of cell release vesica and its promote the method for cell release vesica |
| CN110129266A (en) * | 2019-05-21 | 2019-08-16 | 上海交通大学 | A kind of method and application promoting cell secretion excretion body |
| CN110172071A (en) * | 2019-06-10 | 2019-08-27 | 山东师范大学 | A kind of bromine amyl replaces BODIPY derivative and the preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| 《A pH-activatable and aniline-substituted photosensitizer for near-infrared cancer theranostics》;Jiangwei Tian等;《CHEMICAL SCIENCE 》;20151231;第6卷(第10期);全文 * |
| 《Acid Base-Controllable FRET and Self-Assembling Systems Fabricated by Rhodamine B Functionalized Pillar arene-Based Host−Guest Recognition Motifs》;Sun, Jifu等;《ORGANIC LETTERS》;20180119;第20卷(第2期);全文 * |
| 《Aqueous self-assembly of a charged BODIPY amphiphile via nucleation–growth mechanism》;Le Yang等;《PHYSICAL CHEMISTRY CHEMICAL PHYSICS》;20151231;第17卷(第14期);全文 * |
| 《Luminescent metal–organic frameworks based on dipyrromethene metal complexes and BODIPYs》;Baudron, Stephane A;《CRYSTENGCOMM》;20161231;第18卷(第25期);全文 * |
| Chang Yeon Lee等.《Light-Harvesting Metal-Organic Frameworks (MOFs): Efficient Strut-to-Strut Energy Transfer in Bodipy and Porphyrin-Based MOFs》.《American Chemical Society》.2011,第133卷15858–15861. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110628719A (en) | 2019-12-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Extracellular vesicles: A bright star of nanomedicine | |
| Yang et al. | Exosome biochemistry and advanced nanotechnology for next‐generation theranostic platforms | |
| CN112957478B (en) | Method for surface modification of targeting ligand of Extracellular Vesicles (EVs) | |
| Srivastava et al. | Organically derived exosomes as carriers of anticancer drugs and imaging agents for cancer treatment | |
| Ohno et al. | Exosome-mediated targeted delivery of miRNAs | |
| CN102596179A (en) | Liposomal compositions and uses of same | |
| CN103221034A (en) | Nano-vehicle derived from tumor tissue, and cancer vaccine using same | |
| Yang et al. | Magainin II modified polydiacetylene micelles for cancer therapy | |
| CN111346226A (en) | Self-oxygen-generating nanoparticles and application thereof in mediating tumor photodynamic therapy | |
| Thomas et al. | Exosomes: biological pharmaceutical nanovectors for theranostics | |
| CN112195156B (en) | Biological nano-particle and multi-dimensional multi-target-point composite nano-particle from endoplasmic reticulum of cell as well as preparation and application thereof | |
| CN111116904A (en) | Phenylboronic acid modified fluorine-containing high polymer material and application thereof in intracellular delivery of protein | |
| Panigrahi et al. | Exosomes: Insights and therapeutic applications in cancer | |
| CN115252582A (en) | Preparation and application of erythrocyte membrane heterozygosis pH liposome coated oncolytic virus preparation | |
| Yang et al. | GSH/pH Dual Activatable Cross‐linked and Fluorinated PEI for Cancer Gene Therapy Through Endogenous Iron De‐Hijacking and in Situ ROS Amplification | |
| Desai et al. | Tumor-derived systems as novel biomedical tools—turning the enemy into an ally | |
| Zhu et al. | Immuno-affinitive supramolecular magnetic nanoparticles incorporating cucurbit [8] uril-mediated ternary host-guest complexation structures for high-efficient small extracellular vesicle enrichment | |
| Moosavian et al. | Melanoma-derived exosomes: Versatile extracellular vesicles for diagnosis, metastasis, immune modulation, and treatment of melanoma | |
| Yang et al. | Black phosphorus nanosheets assist nanoerythrosomes for efficient mRNA vaccine delivery and Immune Activation | |
| CN110628719B (en) | Method for inducing cells to rapidly produce vesicles and application thereof | |
| Desai et al. | “Bioinspired” membrane-coated nanosystems in cancer theranostics: a comprehensive review | |
| Yan et al. | Reversal of HMGA1-mediated immunosuppression synergizes with immunogenic magnetothermodynamic for improved hepatocellular carcinoma therapy | |
| CN116763938A (en) | Extracellular vesicle nucleic acid nano-drug delivery system, preparation method and application thereof | |
| CN115040643A (en) | Tumor cell-bacterium fusion material and preparation method and application thereof | |
| Hao et al. | The GSH responsive indocyanine green loaded PD-1 inhibitory polypeptide AUNP12 modified MOF nanoparticles for photothermal and immunotherapy of melanoma |
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 |