CN112680397B - Vesicle centrifugal protective agent and application thereof, and method for centrifugally extracting vesicles - Google Patents
Vesicle centrifugal protective agent and application thereof, and method for centrifugally extracting vesicles Download PDFInfo
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Abstract
The invention provides a vesicle centrifugal protective agent, application thereof and a method for centrifugally extracting vesicles, and belongs to the technical field of biology. The vesicle centrifugal protective agent comprises glycerol and sodium carboxymethyl cellulose. According to the invention, on the basis of differential centrifugation, glycerin and sodium carboxymethyl cellulose are used as a centrifugal protective agent, the glycerin can penetrate into cells to improve the solute concentration of an intracellular solution, and the sodium carboxymethyl cellulose is outside the cells to improve the solute concentration of an extracellular solution, so that the osmotic pressure balance inside and outside the vesicle is realized, the extrusion deformation resistance of the vesicle can be improved, the vesicle can be protected from being damaged in the ultra-high speed centrifugation process to the maximum extent, and the yield of the vesicle is improved. In addition, the addition of the sodium carboxymethyl cellulose can change the surface charge property of the vesicles, destroy the electrostatic repulsion force of the vesicles, cause adjacent vesicles to aggregate and precipitate, improve the vesicle sedimentation speed, shorten the extraction time and further improve the vesicle yield and the separation efficiency.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a vesicle centrifugal protective agent and application thereof in centrifugal extraction of vesicles, and further relates to a method for centrifugal extraction of vesicles.
Background
The cytoskeleton is an important structure by which eukaryotic cells maintain their basic morphology, and cells, after being stimulated exogenously or endogenously, cause rearrangement of the cytoskeleton, resulting in uneven local stress on the cell membrane. The abnormally stressed cytoplasm expands outwards, and is released to the outside of the cell in the form of vesicles after randomly wrapping part of the cell contents, and the special subcellular structure with the diameter of about 0.1-1 mu m is called as 'cell vesicles'. Since the cell vesicles carry the biomarker molecules of the original cells, the cell vesicles can be specifically identified by target cells, and more researchers begin to adopt the cell vesicles derived from tumor cells as a new generation of drug delivery system to achieve the advantages of targeted drug delivery, reduction of biological barriers, improvement of biocompatibility and the like, so as to enhance the treatment effect of chemotherapeutic drugs. Therefore, the cell vesicle has wide application prospect in the field of drug carriers. Obtaining cell vesicles with stable properties and high purity is a prerequisite for carrying out the research work.
At present, the extraction and purification of the cell vesicles mainly adopt an ultracentrifugation method or a gradient density centrifugation method, a dialysis method, an ultrafiltration method, a gel exclusion chromatography method and an immunomagnetic bead method. The centrifugation method is based on the difference of sedimentation rates of cell vesicles and other biological component densities for separation, and is simple to operate, but the cell vesicles are small in size and light in weight, so that the cell vesicles can be settled by high centrifugal force and long centrifugal time, and the influence of long-time high-speed centrifugation on the number of the vesicles is large, so that the cell vesicles are pressed, broken and agglomerated easily, the loss of the cell vesicles is caused, the yield is low, and the subsequent experiment is influenced. Both dialysis and ultrafiltration separate the cell vesicles from the impurities according to molecular weight cut-off, with the difference: the dialysis method is a method for purifying cells by using cell vesicles which cannot permeate a semipermeable membrane, and the time required in the whole separation and extraction process is long, and the vesicle recovery efficiency is low; the ultrafiltration method is a method for removing impurities by using the fact that cell vesicles cannot penetrate through an ultrafiltration membrane by taking pressure difference between two sides of the ultrafiltration membrane as a driving force and the ultrafiltration membrane as a filtering medium, the purification efficiency is greatly improved, but the loss amount of the vesicles is large, the filter membrane is easily blocked by the impurities, a large amount of cell vesicles are lost when the filter membrane is damaged, and the recovery rate is low. Gel exclusion chromatography is based on the size difference between cell vesicles and other biological components in a solution, and the cell vesicles are separated from other components by utilizing the difference of migration speeds of the components with different sizes in a gel chromatographic column to obtain the cell vesicles with higher purity. Immunomagnetic beads are based on the use of specific proteins on the surface of cellular vesicles such as: CD9/CD63/CD81 and the like are combined with immunomagnetic beads coated with corresponding antibodies for magnetic separation, but because the immunomagnetic beads have smaller volume and the particle size is nano-grade and less than or equal to the diameter of cell vesicles, the steric hindrance combined with the cell vesicles is larger, the separation efficiency is not high, and because the price of the antibodies is more expensive, the separation cost of the method is higher; in addition, the cell vesicles obtained by the immunomagnetic bead method are eluted by using an acidic eluent, so that the morphology of the cell vesicles is incomplete, and downstream application of the cell vesicles is affected. In addition, a method based on hydrophilic polymer precipitation is used for extracting the cell vesicles, but the separated cell vesicles are usually doped with a lot of impure protein components, so that the purity and the yield of the cell vesicles are extremely low, and the cell vesicles are not suitable for application scenes with high requirements on purity.
Disclosure of Invention
Aiming at the problems of low cell vesicle separation efficiency and low yield in the prior art, the invention provides a vesicle centrifugal protective agent, application of the vesicle centrifugal protective agent in vesicle centrifugal extraction, and a method for extracting vesicles through centrifugation.
In order to achieve the purpose, the invention is specifically realized by the following technical scheme:
a vesicular centrifugal protectant comprises glycerol and sodium carboxymethylcellulose.
Further, the vesicle cryoprotectant comprises 3-percent wt-7% wt glycerol and 2-percent wt-8% wt sodium carboxymethylcellulose.
Further, the vesicle cryoprotectant comprises 5% wt glycerol and 4% wt sodium carboxymethylcellulose.
Further, the vesicle is a cell vesicle or a drug-loaded vesicle.
In addition, the invention provides application of the vesicle centrifugation protective agent in centrifugal extraction of cell vesicles or drug-loaded vesicles.
In addition, the invention provides a method for centrifugally extracting vesicles, which comprises the following steps:
s1, adding the vesicle centrifugation protective agent into a cell suspension containing vesicles, and uniformly mixing to obtain a suspension;
s2, centrifuging the suspension for 8-13min under a first centrifugal force, then centrifuging the supernatant for 0.5-3min under a second centrifugal force, then centrifuging the supernatant for 30-90min under a third centrifugal force, and obtaining the sediment which is the vesicle;
wherein the first centrifugal force is less than the second centrifugal force and less than or equal to the third centrifugal force.
Further, the cell suspension containing vesicles is a cell suspension containing cell vesicles or a cell suspension containing drug-loaded vesicles.
Further, the preparation method of the cell suspension containing the cell vesicles comprises the following steps: inducing apoptosis of the cells by chemical, physical and/or biological methods to obtain the cell suspension containing the cell vesicles.
Further, the preparation method of the cell suspension containing the drug-loaded vesicle comprises the following steps: inducing the cell to generate apoptosis by chemical, physical and/or biological methods, and incubating the apoptotic cell with an effective amount of drug to obtain the cell suspension containing the drug-loaded vesicle.
Further, the first centrifugal force is 1500-2500g, and the second centrifugal force is 11000-30000g; the third centrifugal force is 11000-30000g.
Further, the first centrifugal force is 2000g, and the second centrifugal force is 14000g; the third centrifugal force was 28000g.
Further, in step S2, the suspension is centrifuged at 2000g for 10min, then the supernatant is centrifuged at 14000g for 2min, and then the supernatant is centrifuged at 28000g for 30min, and the precipitate is the vesicle.
Has the advantages that:
according to the invention, on the basis of differential centrifugation, glycerol and sodium carboxymethyl cellulose are used as a centrifugal protective agent, glycerol can penetrate into cells to improve the solute concentration of an intracellular solution, and sodium carboxymethyl cellulose is arranged outside the cells to improve the solute concentration of an extracellular solution, so that the osmotic pressure balance inside and outside the vesicle is realized, the change degree of the vesicle volume is small, the extrusion deformation resistance of the vesicle is favorably improved, the vesicle is protected from being damaged in the ultra-high speed centrifugation process to the maximum extent, and the vesicle yield is improved. In addition, the addition of the sodium carboxymethyl cellulose can change the surface charge property of the vesicles, destroy the electrostatic repulsion force of the vesicles, cause adjacent vesicles to aggregate and precipitate, improve the vesicle sedimentation speed, shorten the extraction time and further improve the vesicle yield and the separation efficiency.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the number of vesicles obtained from cells using different vesicle centrifugation protectors in example 1 of the present invention;
FIG. 2 is a diagram showing the number of cell vesicles obtained by using sodium carboxymethyl cellulose with different concentrations in example 2 of the present invention;
FIG. 3 is a graph showing the number of vesicles obtained by using glycerol at different concentrations in example 3 of the present invention;
FIG. 4 is a graph showing the number of vesicles obtained by centrifugation under different conditions in example 4 of the present invention;
FIG. 5 is a cell vesicle size distribution graph obtained by using glycerol and sodium carboxymethyl cellulose in combination in example 4 of the present invention;
FIG. 6 is a morphology chart of cell vesicles obtained by using glycerol and sodium carboxymethyl cellulose in combination according to example 4 of the present invention;
FIG. 7 is a cell vesicle size distribution graph obtained without adding a vesicle centrifugation protective agent in example 4 of the present invention;
FIG. 8 is a morphology chart of cell vesicles obtained without addition of a vesicle centrifugation protectant in example 4 of the present invention;
fig. 9 is a detection chart of the drug content of the drug-loaded vesicle obtained in example 5 of the present invention;
fig. 10 is a graph showing the killing effect of the drug-loaded vesicle obtained in example 5 of the present invention on tumor cells.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. In addition, the terms "comprising", "containing", "having" and "having" are intended to be non-limiting, i.e., that other steps and other ingredients can be added which do not affect the results. Materials, equipment and reagents are commercially available unless otherwise specified.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, volume fractions, and other numerical values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Among the prior art techniques for separating extracellular vesicles, ultracentrifugation is most widely used because of its simple operation and high reproducibility. However, methods of ultracentrifugation for separating extracellular vesicles have limitations. The hydrostatic pressure at any point in the centrifuge tube increases along with the increase of the height of the liquid column, and the cell membrane can be cracked and seriously damaged if the generated pressure is too strong under the action of centrifugal shearing force and hydrostatic pressure. And under the action of stronger centrifugal field pressure, extracellular vesicle agglomeration is easily caused, so that the vesicle yield is low.
In order to reduce the damage of centrifugal shearing force to extracellular vesicles in the centrifugal process, the embodiment of the invention provides a vesicle centrifugal protective agent, which comprises glycerol and sodium carboxymethyl cellulose.
Glycerol is often used as a microbial cryopreservation protectant, which can protect cell membranes from mechanical damage under extreme conditions, and sodium carboxymethyl cellulose is a good stabilizer, which can maintain the stability of the solution and prevent the components in the solution from coagulating. According to the invention, on the basis of differential centrifugation, glycerol and sodium carboxymethyl cellulose are used together, on one hand, the glycerol has low molecular weight and good compatibility with membrane lipid and can rapidly pass through a cell membrane, while the sodium carboxymethyl cellulose has high molecular weight and can not enter cells, but the solute concentration of an extracellular solution can be improved, so that through matching, osmotic pressure balance inside and outside the vesicle is realized, water absorption and dehydration balance are maintained, the change degree of the volume of the vesicle is smaller, the extrusion deformation resistance of the vesicle is improved, the vesicle is protected against the damage of centrifugal shear force, and the recovery rate of the vesicle in the separation process is improved.
On the other hand, the tumor cell membrane contains more electronegative molecules such as phosphatidylserine and glycosylated mucin, the surface of the cell membrane has stronger negative charges, a layer of oppositely charged ions (an electric double layer) is shielded around the cell membrane in a solution, and electrostatic repulsion between cells is generated, so that when the electric double layer is broken, the electrostatic repulsion is also reduced to promote cell aggregation. Sodium ions generated by ionization of sodium carboxymethyl cellulose can specifically neutralize negative charges on the surface of a cell membrane, so that the surface charge property of the cell membrane is changed, adjacent vesicles tend to aggregate and precipitate, and the sodium carboxymethyl cellulose can reduce the surface tension between membrane lipid and a solution and reduce the sedimentation resistance, so that the vesicle sedimentation speed is increased, the centrifugation time is greatly shortened, and the aggregation and agglomeration of the vesicles or agglomeration caused by long-time centrifugation are prevented. Therefore, the centrifugal protective agent provided by the invention can protect the vesicles from being damaged to the maximum extent in the ultra-high speed centrifugation process, and the time required by vesicle extraction is shortened to a certain extent while the yield of the vesicles is improved.
If the concentration of the glycerol is high or the concentration of the sodium carboxymethyl cellulose is low, the osmotic pressure in the vesicle is increased, the cells can continuously absorb water when exposed to the surrounding low-concentration environment, and the cell membrane can be ruptured when the expansion exceeds the tolerable limit; correspondingly, if the concentration of glycerol is low or the concentration of sodium carboxymethylcellulose is high, the osmotic pressure outside the vesicle is increased, cells can continuously lose water when exposed to the surrounding high-concentration environment, and cell membranes can be ruptured when the cells shrink beyond tolerable limits; that is, an imbalance in the concentration of glycerol and sodium carboxymethylcellulose will cause the vesicles to be excessively dehydrated or to absorb water to cause rupture, reducing the yield. Suitable concentrations of glycerol and sodium carboxymethylcellulose maintain the osmotic pressure within a range that facilitates vesicle morphology preservation. Moreover, the concentration of sodium carboxymethyl cellulose can also affect the vesicle sedimentation speed, and proper size and charge distribution can destroy the inherent electrostatic repulsion among vesicles to cause agglutination phenomenon, wherein the agglutination can increase along with the increase of the concentration of sodium carboxymethyl cellulose and can cause saturation phenomenon, if the concentration of sodium carboxymethyl cellulose is too high, the sedimentation speed is too high, some cell fragments or other impurities can be nonspecifically captured into the vesicle clot, and if the concentration of sodium carboxymethyl cellulose is too low, the sedimentation speed is too low, the centrifugal time can be prolonged or the centrifugal speed needs to be increased to cause vesicle agglomeration, so that the yield is reduced. Thus, preferably, the vesicle cryoprotectant comprises 3-wt% wt-7% wt glycerol and 2-8% wt sodium carboxymethylcellulose; more preferably comprises 5% wt glycerol and 4% wt sodium carboxymethyl cellulose. The above measurement is in terms of mass percent.
Another embodiment of the present invention provides an application of the vesicle centrifugation protective agent as described above, specifically, an application of the vesicle centrifugation protective agent in centrifugal extraction of cell vesicles or drug-loaded vesicles. The application of the vesicle centrifugal protection agent is the same as the advantages of the vesicle centrifugal protection agent relative to the prior art, and the details are not repeated.
In addition, the invention provides an application method of the vesicle centrifugation protective agent, specifically a method for centrifugally extracting cell vesicles or a method for centrifugally extracting drug-loaded vesicles.
A method of centrifuging vesicles, comprising the steps of:
s1, adding the vesicle centrifugation protective agent into a cell suspension containing vesicles, and uniformly mixing to obtain a suspension;
s2, subjecting the suspension to a first centrifugal force CF 1 Centrifuging for 8-13min, and collecting supernatant under second centrifugal force CF 2 Centrifuging for 0.5-3min, and collecting supernatant under third centrifugal force CF 3 Centrifuging for 30-90min, and precipitating to obtain vesicle;
wherein the first centrifugal force CF 1 < second centrifugal force CF 2 Third centrifugal force CF not more than 3 。
The suspension is firstly centrifuged at low speed to remove dead cell sediment and large fragments, the obtained supernatant contains vesicles and cell fragments, the cell fragments and other precipitates are removed by high-speed instantaneous centrifugation, the obtained supernatant is mainly vesicles, the high-purity vesicle sediment is obtained by ultra-high speed centrifugation for a long time, and the sediment is resuspended to obtain the standby vesicles. The vesicle centrifugation protective agent is added in the centrifugation process, the vesicle structure is retained, the mechanical damage of ultra-high speed centrifugation is reduced, the separation efficiency is high, and the yield is obviously improved.
The vesicle-containing cell suspension can be a variety of vesicle-containing media, and can be, for example, extracellular fluid, plant juice, and the like, in general. The extracellular fluid may be plasma, excreta (e.g., urine), secretions (e.g., milk, saliva), or cell culture fluid, etc., and the plant juice may be typically the cellular fluid of plant cells, etc. Wherein, the vesicle can be a cell vesicle or a medicine-carrying vesicle.
In some embodiments, the cell suspension containing cell vesicles is prepared by: inducing the cell to generate apoptosis by chemical, physical and/or biological methods, wherein in the process of apoptosis, the skeleton of the cell membrane of the apoptotic cell is changed, the cell membrane bulges outwards locally, randomly wraps the cell content and is released outside the cell in the form of vesicle to generate the cell vesicle, and a cell suspension containing the cell vesicle is obtained, wherein the cell suspension specifically comprises cells, cell fragments and the cell vesicle.
In some embodiments, the cell suspension containing drug-loaded vesicles is prepared by: inducing the cell to apoptosis by chemical, physical and/or biological methods to generate cell vesicles, incubating the apoptotic cell with an effective amount of a drug to enable the drug to be encapsulated into the cell vesicles, wherein the drug-encapsulated cell vesicles are drug-loaded vesicles to obtain a cell suspension containing the drug-loaded vesicles, and the cell suspension specifically comprises cells, cell fragments and the drug-loaded vesicles.
The chemical method can be used for inducing apoptosis by co-incubation of chemotherapeutic drugs and tumor cells or co-incubation of high-concentration calcium ions and calcium ion carriers and tumor cells, the physical method can be used for inducing apoptosis by ultraviolet irradiation or dehydration and apoptosis by hypertonic solution, and the biological method can be used for cracking cell membranes by various lytic enzymes and the like.
Optionally, the CF 1 From 1500 to 2500g, said CF 2 11000 to 30000g; the CF 3 11000 to 30000g. Speed and time of centrifugation to vesicle harvestingThe amount of aggregates and their morphology have a large impact. First centrifugal force CF 1 And a second centrifugal force CF 2 When the centrifugal force is lower than 1500-2500g and 11000-30000g, cells and cell fragments are difficult to completely remove, and when the centrifugal force is too high, part of vesicles are wrapped by the cells and the cell fragments during sedimentation, so that the yield of the vesicles is reduced. Third centrifugal force CF 3 In 11000-14000g, although vesicles can be collected, the amount of vesicles is small, the yield can be improved to some extent by prolonging the centrifugation time, and when the centrifugation force is increased to 14000-28000g, the amount of vesicles collected is more desirable, and when the amount of vesicles is further increased to 28000g or more, aggregation and agglomeration are easily caused.
In order to better maintain vesicle morphology and improve vesicle yield, preferably, the CF is 1 2000g of said CF 2 14000g; the CF 3 Was 28000g.
More preferably, in step S2, the suspension is centrifuged at 2000g for 10min, then the supernatant is centrifuged at 14000g for 2min, and then the supernatant is centrifuged at 28000g for 30min, and the precipitate is the vesicle.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are examples of experimental procedures not specified under specific conditions, generally according to the conditions recommended by the manufacturer.
In the following examples, unless otherwise specified, A549 lung adenocarcinoma cell line was used as tumor cells, and apoptosis of tumor cells was induced by irradiation with ultraviolet rays.
Example 1 Glycerol and sodium carboxymethylcellulose in combination as vesicular centrifugation protectants
Preparation of cell suspensions containing cell vesicles: inoculating the purified tumor cells into RPMI 1640 medium containing 10% fetal bovine serum (v/v), 100U/mL penicillin, and 100mg/mL streptomycin, placing the tumor cells-inoculated medium in a carbon dioxide incubator at 37 deg.C under gas atmosphere of 5% 2 Under the conditions of (1) until the cell concentration reaches 2X 10 7 one/mL, then collecting tumor cells and transferring them to a culture dish containing fresh medium, and culturingIrradiating the dish under ultraviolet rays for 1h to induce apoptosis, and then culturing in a carbon dioxide incubator for 16-20h to form stable cell vesicles, thereby obtaining cell suspension containing the cell vesicles.
Differential centrifugation: the differential centrifugation of the cell suspension containing the cell vesicles is used for separating the cell vesicles, and the method specifically comprises the following steps:
s1, transferring cell suspension containing cell vesicles to a plurality of centrifuge tubes and dividing the cell suspension into a plurality of groups, adding different vesicle centrifugal protective agents into each group respectively, and uniformly mixing to obtain suspension; specific grouping information is shown in table 1, wherein the mass concentration shown in table 1 is the final concentration of the components of the vesicle centrifugation protective agent in the cell suspension, and in the following examples, all mass concentrations are also the final concentration of the components of the vesicle centrifugation protective agent in the cell suspension unless otherwise specified;
s2, centrifuging the suspension for 10min under 2000g of centrifugal force, then centrifuging the supernatant for 2min under 14000g of centrifugal force, then centrifuging the supernatant for 60min under 14000g of centrifugal force, and obtaining the sediment which is the cell vesicle. The number of cell vesicles was characterized using a Malvern NS300 particle tracking analyzer and the results are shown in figure 1.
Table 1 grouping information of embodiment 1
As can be seen from fig. 1, when sodium carboxymethylcellulose (CMC) is used alone, the protective agent has a stronger protective effect on cell vesicles than other protective agents, and when the protective agent is used in combination with a plurality of commercially available common protective agents, glycerol has a stronger synergistic effect on the cell vesicles than propylene glycol and dextran, indicating that glycerol and sodium carboxymethylcellulose are used in combination as a stronger protective agent.
Example 2 optimization of sodium carboxymethylcellulose concentration
Preparing cell suspensions by the method of example 1, dividing the cell suspensions into different groups, adding 5% by weight of glycerol, respectively, adding 0.25%, 0.5%, 1%, 2%, 4%, 8% by weight of sodium carboxymethylcellulose, respectively, to each group, and mixing to obtain suspension; the suspension was subjected to differential centrifugation according to step S2 of example 1, and the number of vesicles obtained by differential centrifugation was characterized using a Malvern NS300 particle tracking analyzer, the results of which are shown in fig. 2.
As can be seen from fig. 2, the vesicle yield depends on the concentration of sodium carboxymethyl cellulose within a certain range, and as the concentration of sodium carboxymethyl cellulose increases, the number of cell vesicles significantly increases, because the increase of sodium carboxymethyl cellulose is beneficial to reducing the mechanical damage of cell vesicles, and the influence on the cell membrane charge accelerates the sedimentation rate of cell vesicles, but as the concentration of sodium carboxymethyl cellulose further increases, the viscosity of the cell suspension starts to increase rapidly, which leads to significant increase of the cell sedimentation resistance, and the influence of the cell suspension viscosity on the cell sedimentation rate is greater than the influence of the cell membrane surface charge change, which causes coagulation, and the combined effect reduces the vesicle yield. Therefore, the mass concentration of the sodium carboxymethylcellulose is preferably 2 to 8%, more preferably 4%.
Example 3 optimization of Glycerol concentration
Preparing a cell suspension by the method of example 1, and then dividing the cell suspension into two groups, each group being divided into different groups, wherein the two groups are added with 0.5% wt and 4% wt of sodium carboxymethyl cellulose, respectively, and then the groups are added with 0, 1%, 2%, 3%, 4%, 5%, 6%, and 7% wt of glycerol, respectively, and mixed uniformly to obtain a suspension; the suspension was subjected to differential centrifugation according to step S2 of example 1, and the number of vesicles obtained by differential centrifugation was characterized using a Malvern NS300 particle tracking analyzer, and the results are shown in fig. 3.
As can be seen from fig. 3, glycerol in a certain concentration range can improve the protection effect of sodium carboxymethylcellulose on cell vesicles, and during high-speed centrifugation, appropriate concentrations of glycerol and sodium carboxymethylcellulose can maintain the osmotic pressure balance inside and outside the cell vesicles, which is of great significance for the cell vesicles to resist centrifugal shear force. Preferably, the glycerol has a mass concentration of 3-7%, more preferably 5%.
Example 4 Glycerol in combination with sodium carboxymethylcellulose protects vesicles during ultracentrifugation
Preparing cell suspensions by the method of example 1, then dividing the cell suspensions into different groups, adding 5% wt of glycerol and 4% wt of sodium carboxymethylcellulose into each group, and mixing to obtain suspensions; the cell suspension was centrifuged at 2000g for 10min, then the supernatant was centrifuged at 14000g for 2min, then the supernatant was centrifuged at 14000g and 28000g for 30min, 60min and 90min respectively, while differential centrifugation was performed under the same conditions without addition of glycerol and sodium carboxymethyl cellulose as a control. The number of the cell vesicles obtained by differential centrifugation was characterized by using a Malvern NS300 particle tracking analyzer, and the characterization results are shown in FIG. 4.
As can be seen from fig. 4, at a lower centrifugal force of 14000g, the number of the cell vesicles significantly increases with the increase of the centrifugal time, and after 60min, the number of the cell vesicles does not have statistical difference with the increase of the centrifugal time, and at this time, the cell vesicles can be observed to be squeezed and deformed to different degrees under an electron microscope, but the cell membrane structure is basically intact. Under the condition of a high 28000g centrifugal force, the number of the cell vesicles is remarkably increased along with the prolonging of the centrifugal time, and after 60min, the number of the cell vesicles is greatly reduced, because the cell vesicles are compressed and deformed due to the high centrifugal rotating speed and the long centrifugal time, partial cell membranes are extruded and broken, the number of the complete vesicles is reduced, and the cell vesicle yield is reduced. However, after the vesicle centrifugation protective agent is added, the yield of the cell vesicles is obviously increased, which shows that the vesicle centrifugation protective agent well protects the cell vesicles and improves the compression and deformation resistance of the cell vesicles, so that the vesicle centrifugation protective agent can ensure that the cell vesicles are quickly centrifuged at a higher speed in a shorter time, greatly shortens the centrifugation time, well preserves the ultrastructure of the cell vesicles, is beneficial to obtaining the high-quality cell vesicles, and the obtained cell vesicles have good dispersibility in a solution after being resuspended and do not have a large amount of aggregation and agglomeration. Compared with high-speed long-time centrifugation, ultrahigh-speed short-time centrifugation is more beneficial to increase of the yield of the cell vesicles.
For example, drug-loaded vesicles obtained by centrifuging at 28000g for 30min were subjected to Malvern NS300 particle tracking analyzer to characterize the particle size of the cell vesicles, and Hitachi HT7800 120kV transmission electron microscopy was used to characterize the morphology of the cell vesicles, with the characterization results shown in FIGS. 5-6, respectively. As can be seen from fig. 5, the cell vesicles have narrow and single peak-shaped particle size distribution, uniform particle size, and average particle size of 206.3nm, while the control group has multiple peaks and wide particle size distribution range, and cell membranes are ruptured and recombined due to ultra-high speed centrifugation, thereby forming cell vesicles with different sizes (see fig. 7). The experimental group has a narrow particle size distribution range compared with the control group, and is easier to pass through the barrier of tumor cells when used for treatment. Further, it can be seen from the electron microscope that the purified vesicles extracted from the experimental group (see fig. 6) have a classical morphology, a complete structure, uniform dispersion, high background purity and fewer impurities than the control group (see fig. 8).
Example 5 Effect of Glycerol in combination with sodium carboxymethylcellulose on vesicle drug Loading
Preparation of cell suspension containing drug-loaded vesicles: inoculating the purified tumor cells into RPMI 1640 medium containing 10% fetal bovine serum (v/v), 100U/mL penicillin, and 100mg/mL streptomycin, placing the tumor cells-inoculated medium in a carbon dioxide incubator, and reacting at 37 deg.C under 5% CO in gas atmosphere 2 Under the conditions of (1) until the cell concentration reaches 2X 10 7 Then, the tumor cells were collected and transferred to a culture dish containing a fresh medium, the culture dish was irradiated with ultraviolet rays for 1 hour to induce apoptosis, and then different kinds of chemotherapeutic drugs (methotrexate (MTX): final concentration 2mg/mL, doxorubicin hydrochloride (DOX): final concentration 200. Mu.g/mL, cisplatin (CDDP): final concentration 200. Mu.g/mL, hydroxycamptothecin (HCPT): final concentration 500. Mu.g/mL) were added, respectively, at a temperature of 37 ℃ under a gas atmosphere of 5 CO 2 The cell suspension containing the medicine-carrying vesicles is obtained after 16-24h of incubation under the condition of (1).
Differential centrifugation: differential centrifugation is carried out on the cell suspension containing the medicine-carrying vesicles to separate the medicine-carrying vesicles, and the method specifically comprises the following steps:
s1, transferring the cell suspension containing the drug-loaded vesicles to a plurality of centrifuge tubes, adding 5-wt% of glycerol and 4-wt% of sodium carboxymethyl cellulose into the centrifuge tubes, and uniformly mixing to obtain a suspension;
s2, centrifuging the suspension for 10min under 2000g of centrifugal force, then centrifuging the supernatant for 2min under 14000g of centrifugal force, then centrifuging the supernatant for 30min under 28000g of centrifugal force, and obtaining the precipitate, namely the drug-loaded vesicle.
Detecting the drug loading amount of the drug-loaded vesicle: high Performance Liquid Chromatography (HPLC) for unit drug loaded vesicles (10) 10 individuals/mL), wherein the liquid phase detection method of Methotrexate (MTX), doxorubicin hydrochloride (DOX) and Cisplatin (CDDP) refers to pharmacopoeia 2015 edition of the people's republic of China (column C18250X 4.6 mm). The HPLC detection conditions of Hydroxycamptothecin (HCPT) are as follows: mobile phase methanol: water =50 (v/v), column: thermo Acclaim TM 120, column temperature 30 ℃, flow rate: 1mL/min, detection wavelength: 266nm. The results are shown in FIG. 9. At the same time, differential centrifugation was performed under the same conditions without addition of glycerol and sodium carboxymethylcellulose as a control group.
Anti-tumor experiment of the drug-loaded vesicle: staining the prepared methotrexate-loaded vesicles by using a cell membrane staining kit (PKH 26 fluorescent probe), and then respectively inoculating an A549 lung adenocarcinoma cell line, an OVCAR-3 ovarian cancer cell line, a HepG2 liver cancer cell line, an MCF-7 breast cancer cell line and an HCT-8 colon cancer cell line into a 24-well plate with the cell size of 1 × 10 per well 5 Cell, at 37 ℃ and 5% CO 2 And (3) incubating in an incubator overnight, adding the three PKH 26-labeled drug-loaded vesicles into each hole in equal amount, incubating for 3 hours, digesting the cells in the holes, transferring the cells to a flow tube, measuring the apoptosis rate by using a BDFACSCAnto II flow cytometer, and performing in-vitro tumor cell killing comparison, wherein the measurement result is shown in figure 10.
As can be seen from fig. 9-10, the unit drug loading of the drug-loaded vesicles obtained by adding the vesicle centrifugation protective agent of the present invention during the centrifugation extraction process is higher than that of the control group, which indicates that the vesicle centrifugation protective agent is helpful for improving the stability of the drug-loaded vesicles and directly improving the productivity of the drug-loaded vesicles, and the existence of the vesicle centrifugation protective agent does not affect the loading effect of the cell vesicles on the chemotherapeutic drugs. In addition, the killing effect of the obtained medicine-carrying vesicle on several tumor cells is not lower than that of the medicine-carrying vesicle obtained by the conventional extraction method, and the medicine-carrying vesicle has a more excellent in-vitro anti-tumor effect, so that the vesicle centrifugal protective agent can be used under the condition of not removing, namely the purified medicine-carrying vesicle does not need to remove glycerol and sodium carboxymethyl cellulose.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present disclosure, and these changes and modifications are intended to be within the scope of the present disclosure.
Claims (7)
1. The application of the vesicle centrifugal protective agent in centrifugal extraction of cell vesicles or drug-loaded vesicles is characterized in that the vesicle centrifugal protective agent consists of glycerol and sodium carboxymethyl cellulose; wherein the ratio of glycerol added to the cell suspension containing cell vesicles or drug-loaded vesicles is 3-7% by weight, and the ratio of sodium carboxymethylcellulose added is 2-8% by weight.
2. The use according to claim 1, wherein the glycerol is added to the cell suspension containing the cell vesicles or the drug-loaded vesicles at a ratio of 5% to wt, and the sodium carboxymethylcellulose is added at a ratio of 4% to wt.
3. A method for centrifugally extracting vesicles, comprising the steps of:
s1, adding the vesicle centrifugation protective agent in the application of the vesicle centrifugation protective agent in any one of claims 1-2 in centrifugal extraction of cell vesicles or drug-loaded vesicles into cell suspension containing vesicles, and uniformly mixing to obtain suspension;
s2, centrifuging the suspension for 8-13min under a first centrifugal force, then centrifuging the supernatant for 0.5-3min under a second centrifugal force, then centrifuging the supernatant for 30-90min under a third centrifugal force, and obtaining the sediment which is the vesicle;
wherein the first centrifugal force is less than the second centrifugal force and less than or equal to the third centrifugal force.
4. The method for centrifugally extracting vesicles according to claim 3, wherein the cell suspension containing vesicles is a cell suspension containing cell vesicles or a cell suspension containing drug-loaded vesicles.
5. The method for centrifugally extracting vesicles according to claim 4, wherein the cell suspension containing the cell vesicles is prepared by: inducing apoptosis of cells by chemical, physical and/or biological methods to obtain the cell suspension containing the cell vesicles;
the preparation method of the cell suspension containing the drug-loaded vesicles comprises the following steps: inducing the cell to generate apoptosis by chemical, physical and/or biological methods, and incubating the apoptotic cell with an effective amount of drug to obtain the cell suspension containing the drug-loaded vesicle.
6. The method for centrifugally extracting vesicles according to any one of claims 3 to 5, wherein the first centrifugal force is 1500-2500g, and the second centrifugal force is 11000-30000g; the third centrifugal force is 11000-30000g.
7. The method for centrifugally extracting vesicles according to claim 6, wherein in step S2, the suspension is centrifuged at 2000g for 10min, then the supernatant is centrifuged at 14000g for 2min, and then the supernatant is centrifuged at 28000g for 30min, so that the precipitate is vesicles.
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