CN117582415A - Tumor extracellular vesicle for encapsulating traditional Chinese medicine and preparation method thereof - Google Patents
Tumor extracellular vesicle for encapsulating traditional Chinese medicine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a tumor extracellular vesicle for encapsulating traditional Chinese medicine and a preparation method thereof, and the method comprises the following steps: 1. culturing an adherent lung cancer cell line; 2. collecting tumor extracellular vesicles by adopting a gradient centrifugation method; 3. preparing an electroporation system by using the tumor extracellular vesicles and berberine according to a mass ratio of 1:2, and collecting the berberine-entrapped tumor extracellular vesicles by adopting a centrifugal method. Since the direct incubation method of indirect drug loading is not suitable for berberine with extremely low solubility, the electroporation method is selected to encapsulate berberine into tumor extracellular vesicles. Meanwhile, the medicine carrying efficiency of the vesicle/berberine under different mass ratio conditions is determined, the new technical route is proved to be capable of synthesizing qualified EV-Ber, and the vesicle/berberine mass ratio with highest medicine carrying efficiency is determined.
Description
Technical Field
The invention relates to the field of preparation of targeted drug carriers, in particular to a tumor extracellular vesicle for encapsulating traditional Chinese medicines and a preparation method thereof.
Background
In recent years, the purification of traditional Chinese medicine components, the analysis of traditional Chinese medicine structures and the exploration of traditional Chinese medicine targets by modern scientific technology have become global research hot spots. Berberine (berberbererine) is an active compound isolated from the traditional chinese medicine coptis, and has been used in china as an over the counter drug (OTC) for decades. It has remarkable antibacterial effect on various bacteria such as dysentery bacillus, tubercle bacillus, pneumococcus, typhoid bacillus, diphtheria bacillus, etc., and has the strongest effect on dysentery bacillus. Modern medical research shows that berberine has therapeutic effect on type 2 diabetes, atherosclerosis, parkinson's disease, rheumatoid arthritis, etc. In addition, since berberine has therapeutic potential in a variety of cancers, including liver cancer, ovarian cancer, colon cancer, stomach cancer and lung cancer. Although a large number of in vitro studies and a small number of in vivo studies confirmed the antitumor effect of berberine, the effect of the related animal study as a whole was poor.
Poor water solubility and low bioavailability of berberine are one of the main causes of poor anti-tumor effect in vivo. Therefore, improving the in vivo tumor targeting and bioavailability of berberine is a key to berberine anti-tumor research. Extracellular vesicles (Extracelluar vesicles, EVs) with a diameter of 100-1000nm are vesicle-shaped structures of various cells of an organism, which wrap cell contents to secrete to the outside under physiological and pathological conditions, carry biological information of relevant cells, contain various messenger molecules (RNA, DNA, metabolites and proteins) therein and can transmit bioactive substances among cells through endocytic fusion, ligand-receptor and other modes. Early-stage researches of the team show that TMPs as a novel targeted biological chemotherapeutic drug carrier have the effects of targeted killing tumor and anti-tumor immune activation and mechanism after being used for wrapping chemotherapeutic drugs. Therefore, synthesizing berberine-entrapped drug-loaded vesicles (EV-Ber) is an effective strategy for improving berberine bioavailability and releasing the effective anti-tumor effect in vivo.
There are different methods provided in the literature to achieve extracellular vesicle drug delivery, and these methods can be broadly divided into two main categories: indirect drug delivery and direct drug delivery.
The indirect medicine carrying is realized by treating extracellular vesicle blast cells, the medicine carrying efficiency of the extracellular vesicle is lower, and the indirect medicine carrying is mainly divided into two types:
1) Direct incubation: the medicine to be loaded is directly incubated with the parent cell, so that the secreted extracellular vesicles naturally contain the medicine, and the method is suitable for small-molecule chemical medicines with small cytotoxicity.
2) Transfection or transduction: the medicine to be loaded enters into the parent cell in a transfection mode, so that the extracellular vesicles secreted by the parent cell are also provided with the medicine, and the medicine is mainly applicable to nucleic acid, viral proteins and the like.
Direct drug loading is another more common strategy, and the drug to be loaded is directly loaded into extracellular vesicles, so that the drug loading efficiency is higher, and the method mainly comprises the following steps:
1) Direct incubation: the medicine to be loaded is directly incubated with the extracellular vesicles, and the method is suitable for small molecular chemical medicines.
2) Electroporation: the high-strength electric field is acted on the extracellular vesicles to form transient gaps on the membranes, so that the drugs to be loaded can be promoted to enter the extracellular vesicles through the lipid membranes, and the method is suitable for nucleic acids, chemical drugs and the like.
Other drug-loading strategies include room temperature incubation, saponin permeabilization, freeze-thawing cycles, ultrasound, extrusion, and the like.
Disclosure of Invention
The invention aims to solve the technical problems and provide a tumor extracellular vesicle for encapsulating traditional Chinese medicines and a preparation method thereof.
A preparation method of tumor extracellular vesicles coated with traditional Chinese medicines comprises the following steps:
step 1, when the adherent lung cancer cell line is cultured to 90% density, the supernatant is changed into a serum-free basic culture medium, cells are irradiated for 30 minutes by ultraviolet, and then the cells are placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 24 hours;
step 2, collecting tumor extracellular vesicles by adopting a gradient centrifugation method;
step 3, preparing an electroporation system by using tumor extracellular vesicles and berberine according to a mass ratio of 1:4, wherein berberine is a 25mM dimethyl sulfoxide solution, mixing the tumor extracellular vesicles and berberine, fixing the volume by using cold PBS, blowing by using a pipetting gun, transferring to an electric shock cup, and pre-cooling the whole system on ice for 5 minutes; after one electric shock, the electric shock cup was placed in an incubator at 37 ℃ for 30 minutes to restore the integrity of the vesicle membrane, the system solution was completely sucked into the EP tube by a pipette, and the tumor extracellular vesicles encapsulating berberine were collected by a gradient centrifugation method.
Further, the gradient centrifugation method in the step 2 includes the following steps:
step 2.1, taking 600xg of cell supernatant, 10min, discarding cell sediment, leaving 2000xg of supernatant, 30min, discarding cell debris sediment;
step 2.2, centrifuging the obtained supernatant at 16000xg for 60min to obtain precipitate, namely tumor extracellular vesicles;
step 2.3, washing the tumor extracellular vesicles obtained in the previous step with cold PBS for 2 times, and re-suspending with 100-300ul of cold PBS.
Further, before the tumor extracellular vesicles in step 3 are mixed with berberine, berberine is diluted with cold PBS to prevent the lipid layer of vesicles from being destroyed by DMSO at too high concentration in the system.
The tumor extracellular vesicles coated with the traditional Chinese medicine are prepared by the method.
The beneficial effects of the invention are as follows: the direct incubation method of indirect drug loading is not suitable for berberine with extremely low solubility, so that the invention selects electroporation method to load berberine into tumor extracellular vesicles. However, for drugs of different molecular weights, the mass ratio of vesicles to drug in the electroporation system is unknown. Therefore, the invention successfully realizes EV-Ber synthesis by setting the mass ratio of the tumor extracellular vesicles to berberine and searching the entrapment efficiency under the conditions of different mass ratios.
The applicant characterizes the synthesized tumor extracellular vesicles (EV-Ber) by a multi-dimensional method such as a nanoparticle tracing technology (NTA), a projection electron microscope (TEM), a Western-blot, a nano-flow method and the like; and the content of berberine in EV-Ber is accurately quantified by using High Performance Liquid Chromatography (HPLC), and the drug loading efficiency of vesicles/berberine under different mass ratio conditions is determined. Proved by a new technical route, the qualified EV-Ber can be synthesized, and the vesicle/berberine mass ratio with highest drug carrying efficiency is determined.
The biocompatibility of the EV-Ber is detected at the cellular level, and the biodistribution of the EV-Ber is detected at the animal level, so that the synthesized EV-Ber is proved to have excellent tumor targeting.
Drawings
FIG. 1 is a scheme for synthesizing EV-Ber;
FIG. 2 is an external view of EV (left-hand image) and EV-Ber (right-hand image);
FIG. 3 is a graph of the shape and size of extracellular vesicles identified by nanoparticle tracking system (NTA) and Transmission Electron Microscopy (TEM), wherein A is the result of NTA detection for EV and EV-Ber; panel B is TEM image of EV and EV-Ber;
FIG. 4 is a schematic diagram of the detection results of EV-Ber membrane markers;
fig. 5 shows a HPLC standard curve of berberine, and fig. B shows a statistical diagram of berberine concentration detected by HPLC method under different ratios;
FIG. 6 is a graph of the biocompatibility and biodistribution of EV-Ber, wherein A is the fluorescence of EV-Ber after 1 hour incubation with cells; panel B is the fluorescence of EV-Ber after 24 hours incubation with cells; panel C shows the detection of EV-Ber uptake rates by flow cytometry at 1 hour, 6 hours, and 24 hours of incubation with cells; panel D shows in vivo imaging after 1 hour, 6 hours, and 24 hours of intraperitoneal injection of 60ug DiO-labeled EV-Ber; e is a live imaging image of each viscera after intraperitoneal injection for 60ug of DiO labeled EV-Ber for 1 hour, 6 hours, and 24 hours; panel F is a statistical plot of fluorescence values of panel E.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
1 Synthesis of berberine-loaded tumor extracellular vesicles (EV-Ber)
(1) When an adherent lung cancer cell line (such as H1975, LLC cells) was grown in 150mm cell culture dishes to 90% density, the supernatant was changed to serum-free basal medium, and ultraviolet (300 Jm) -2 ) The cells were irradiated for 30 minutes and then placed at 37℃with 5% CO 2 The incubator continues to incubate for 24 hours.
(2) EV was harvested by gradient centrifugation, and the rotational speed of gradient centrifugation was published in Science Translational Medicine with reference to our previous study. Specifically, 600xg of cell supernatant, 10min, cell pellet was discarded, 2000xg of supernatant was left, 30min, and cell pellet was discarded. The obtained supernatant was centrifuged at 16000Xg for 60min to obtain a precipitate as EV. Finally, after EV was washed 2 times with cold PBS (20000 Xg,45 min), resuspended in 100-300ul cold PBS and either as soon as possible or kept at-80℃for no more than 3 days.
(3) At different W EV :W Ber The electroporation system was prepared in the ratio (EV to berberine mass ratio) of 50ug to 25ug, 50ug to 50ug, 50ug to 100ug, and berberine 25mM DMSO solution, and before mixing, berberine was diluted with cold PBS as much as possible to prevent the lipid layer of vesicles from being damaged by too high concentration DMSO in the system. The vesicles were mixed with berberine, fixed to 400ul with cold PBS, gently blown with a pipette, and transferred to a shock cup (2 mm, BTX, 45-0135), and pre-cooled on ice for 5 min. Electroporation conditions: attenuated wave mode 250v,75uf,2mm. After one shock, the cuvette was placed in an incubator at 37 ℃ for 30 minutes to restore vesicle membrane integrity. The system solution was completely aspirated into a 1.5ml EP tube with a pipette, the cup wall was rinsed with PBS and the wash solution was also transferred to the same EP tube. 20000Xg,30min, and then discarding the supernatant, 1mlThe sediment is resuspended by PBS, the free medicine is removed by centrifugation once under the same condition, and the supernatant is discarded to obtain the sediment, namely EV-Ber. The pellet was resuspended in 100ul cold PBS for later use, or stored at-80℃for no more than 3 days.
Note that: in an 800ul electric rotating system, EV quality is improved to 100ug, the corresponding berberine quality can be 50ug/100ug/200ug, the electric shock cup specification is 4mm (BTX, 45-0136), and electroporation conditions are: attenuation wave mode 400v,150uf,4mm. Other steps and conditions were unchanged.
2 characterization of EV-Ber and detection of the packing fraction
2.1EV-Ber size
The extracted EV-Ber is subjected to nanoparticle tracking system (NTA) and Transmission Electron Microscope (TEM) technology to identify the shape and size of extracellular vesicles. NTA results show that the particle size peaks of EV and EV-Ber are 150.1nm and 176.6nm respectively; TEM images show that the dimensions of EV and EV-Ber are substantially consistent with NTA results, between 100-200nm, in a tea tray shape (FIG. 3).
2.2EV-Ber Membrane markers
Epithelial cell adhesion molecule (EPCAM), tumor susceptibility gene 101 protein (TSG 101), CD9 and CD63 are all classical tumor extracellular vesicle marker proteins, and immunoblotting experiments suggest that EV-beer expresses the above proteins; positive rates of cd63+, epcam+ev-bor were identified as 28.4% and 30.0%, respectively, using nanofluidic techniques (fig. 4).
2.3 packing fraction study
Next, the berberine content in EV-beer was detected by High Performance Liquid Chromatography (HPLC) and the entrapment rate was studied. Two indicators were introduced here to evaluate entrapment efficiency:
encapsulation efficiency (encapsulation efficiency,%) =w/W Ber *100％
Drug loading rate (1 oading efficiency,%) =w/W EV *100％
Huang Liansu mass in the drug-loaded vesicle, W Ber Berberine original mass, W in synthesis system EV Original mass of vesicle in synthetic system, unit: ug.
HPLC results indicate that when W EV ：W Ber Is 50: encapsulation efficiency and at 100The drug loading rate is greater than W EV ：W Ber Is 50:25 and 50: at 50, 50ug of drug-loaded vesicles contained 1.983ug berberine on average (FIG. 5 and Table 1). This shows that EV-Ber synthesized by the improved technical route contains considerable berberine, and the experience obtained by the inventor is that in a system with fixed EV quality, the berberine quality is larger than the EV quality, and the encapsulation efficiency is highest.
Table 1 encapsulation and drug loading calculated based on HPLC method to detect berberine concentration at different ratios
| W EV ：W Ber (ua) | 60：25 | 50：50 | 50：100 |
| Encapsulation efficiency (%) | 0.745±0.2701 | 0.64±0.1127 | 0.992±0.044 |
| Drug loading rate (%) | 0.373±0.135 | 0.64±0.113 | 1.983±0.087 |
3 evaluation of biocompatibility and biodistribution of EV-Ber
Next, we studied the ability of EV-beer to deliver drugs to target cells. LLC cells were labeled with DAPI and PKH26, nuclei and cell membranes were labeled, respectively, and then incubated with 60ug Di0 labeled EV-ber. After 1 hour incubation, only a small amount of green granular EVs were observed around LLC cells (fig. 6A). However, after 24h incubation, the cell membranes were completely stained with green fluorescence (FIG. 6B), indicating that LLC cells had all fused with EV-ber. DiO marked EV-ber green fluorescence staining positive cells are detected by adopting flow cytometry, and the uptake rate is accurately determined. As shown in panel C of FIG. 6, the average positive rates for 1 hour, 6 hours and 24 hours were 1.81%, 52.7% and 95.9%, respectively. These in vitro results demonstrate that EVs can efficiently bind to cell membranes and deliver drugs into cells.
60ug DiO-labeled EV-ber was intraperitoneally injected into C57 mice, and the fluorescence aggregation at different sites of the mice was observed using a small animal imaging system. The results show that EV-ber accumulated very little in vivo at 1 h. After 6 hours fluorescence was widely distributed at various sites including subcutaneous tumors. For 24 hours, fluorescence remained only in subcutaneous tumors and bladder (fig. 6D). The main organs of the heart, lung, liver, spleen, double kidney, subcutaneous tumor and the like of the mice are dissected at different time points, and fluorescence images are obtained. As shown in the E-F plot of FIG. 6, initially, fluorescence is concentrated primarily in the liver, indicating the presence of a first pass effect, as observed in many oral medications. Subsequently, the intrahepatic fluorescence was reduced, whereas the intratumoral fluorescence was gradually increased after 6 hours, with fluorescence lasting within the tumor to 24 hours after injection.
The foregoing is illustrative of the best mode of carrying out the invention, and is not presented in any detail as is known to those of ordinary skill in the art. The protection scope of the invention is defined by the claims, and any equivalent transformation based on the technical teaching of the invention is also within the protection scope of the invention.
Claims (4)
1. The preparation method of the tumor extracellular vesicles coated with the traditional Chinese medicines is characterized by comprising the following steps:
step 1, when the adherent lung cancer cell line is cultured to 90% density, the supernatant is changed into a serum-free basic culture medium, cells are irradiated for 30 minutes by ultraviolet, and then the cells are placed in CO at 37 DEG C 2 Culturing in an incubator for 24 hours;
step 2, collecting tumor extracellular vesicles by adopting a gradient centrifugation method;
step 3, preparing an electroporation system by using tumor extracellular vesicles and berberine according to a mass ratio of 1:2, wherein berberine is a 25mM dimethyl sulfoxide solution, mixing the tumor extracellular vesicles and berberine, fixing the volume by using cold PBS, blowing and uniformly mixing by using a pipetting gun, transferring to an electric shock cup, and precooling the whole system on ice for 5 minutes; after one electric shock, the electric shock cup was incubated in an incubator at 37 ℃ for 30 minutes to restore the integrity of the vesicle membrane, the system solution was completely sucked into the EP tube by a pipette, and the tumor extracellular vesicles encapsulating berberine were collected by centrifugation.
2. The method for preparing tumor extracellular vesicles coated with traditional Chinese medicine according to claim 1, wherein the gradient centrifugation method in step 2 comprises the following steps:
step 2.1, taking 600xg of cell supernatant, 10min, discarding cell sediment, leaving 2000xg of supernatant, 30min, discarding cell debris sediment;
step 2.2, centrifuging the obtained supernatant at 16000xg for 60min to obtain precipitate, namely tumor extracellular vesicles;
step 2.3, washing the tumor extracellular vesicles obtained in the previous step with cold PBS for 2 times, and re-suspending with 100-300ul of cold PBS.
3. The tumor extracellular vesicles coated with the traditional Chinese medicine according to claim 1 and the preparation method thereof, wherein before the tumor extracellular vesicles in the step 3 are mixed with berberine, the berberine is diluted by cold PBS to prevent the lipid layer of the vesicles from being damaged by the DMSO in the system in too high concentration.
4. A tumor extracellular vesicle encapsulating a traditional Chinese medicine, characterized in that it is prepared by the method of any one of claims 1-3.
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| CN115125238A (en) * | 2022-07-23 | 2022-09-30 | 新乡学院 | Method for separating and purifying tumor extracellular vesicle DNA |
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