CN110269946B - Preparation and application of natural cell membrane derived ultrasonic contrast agent - Google Patents
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Abstract
The invention relates to a novel natural cell membrane derived ultrasonic contrast agent, a preparation method of the ultrasonic contrast agent and application of the ultrasonic contrast agent in diagnosis and treatment of various diseases such as cardiovascular diseases, cancers and the like. Such ultrasound contrast agents are characterized by membrane components that are entirely derived from natural cell membranes and then internally entrap a gas or liquid that substantially maintains the structural integrity and functional activity of the natural cell membranes used. The novel contrast agent can maintain the original functional activity of a cell membrane, so that the novel contrast agent has high-efficiency immune clearance avoiding capability and blood long-circulating capability in vivo and has good active targeting performance.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to preparation of a novel natural cell membrane-derived ultrasonic contrast agent and application of the novel natural cell membrane-derived ultrasonic contrast agent in diagnosis and treatment of various diseases such as cardiovascular diseases, cancers and the like.
Background
Non-invasive ultrasound imaging has been widely used in the diagnosis of various diseases such as cancer and cardiovascular diseases because of its incomparable advantages such as convenience, low price and real-time imaging. Compared with other imaging methods, the ultrasonic examination has no pain and radioactivity, does not need special environmental conditions, provides accurate, rich and visual information, and is widely loved by patients and doctors. Because of the economical and practical properties of ultrasonic examination, the popularization of ultrasonic diagnostic equipment in China is far higher than that of expensive imaging technologies such as CT, magnetic resonance and the like.
The ultrasound microbubble contrast agent enables echo signals of the part to be obviously enhanced by changing the ultrasound characteristics (such as backscattering coefficient, attenuation coefficient, sound velocity, nonlinear effect and the like) of the tissue, and can obtain more abundant information to assist diagnosis. The development of ultrasound contrast agents has gone through two generations: the first generation of contrast agent is formed by wrapping a layer of albumin, lipid, polymer or surfactant on the periphery of air bubbles to serve as a membrane shell, has good stability and small size, so that microbubbles can reach the left ventricle and peripheral blood vessels through pulmonary circulation after intravenous injection so as to be developed, but has weak enhancement effect, short duration and low diagnostic value; the second generation is a microbubble ultrasound contrast agent containing fluorocarbon gas, the molecular weight of the fluorocarbon gas is much larger than that of air, the fluorocarbon gas has lower solubility and dispersivity, the fluorocarbon gas is not easy to diffuse in microbubbles, and simultaneously, the fluorocarbon gas is inert gas and can keep good stability in vivo, so the prepared second generation microbubble contrast agent has obvious enhancement effect, obviously prolongs the duration time, and has definite diagnostic value for various diseases.
The important direction for the development of the ultrasonic contrast agent is the improvement of a film-forming material, and the purpose is to ensure that the ultrasonic contrast agent is safer and has stronger functionality, thereby playing a more important role in the diagnosis and treatment of various diseases. The current film forming materials mainly comprise the following materials: proteins, phospholipids, high molecular weight polymers, and surfactants. The application of the ultrasound contrast agent based on the above membrane materials is still limited to contrast enhancement of ultrasound signals, and is slowly progressing in functional imaging reflecting pathological information. For example: the targeted ultrasound microbubble contrast agent can be used for carrying out molecular level imaging on microscopic lesions of tissues and organs in vivo, and has very important significance for diagnosis and treatment of diseases. The preparation of targeted microbubble contrast agents is generally carried out by linking target molecules such as antibodies or ligands to the contrast agents, and the targeted microbubble contrast agents are combined with antigens or receptors expressed by specific cells in vivo to realize targeted imaging. However, there are still many problems to be solved: the coupling technology of the ultrasonic contrast agent and the targeting molecule is immature, the preparation process is complicated, and the repeatability is poor; the sensitivity of the combination of the target molecules and the receptors is low, and the efficiency of the targeted combination needs to be improved; the targeted ultrasound contrast agent has short circulation time in vivo, is easy to be cleared by immunity, and the like.
In fields similar to the development of ultrasound contrast agents, such as the field of nanomaterials, problems of poor safety and insufficient functionality are also encountered. In the research process of novel nano materials, people try to camouflage the nano materials by using the novel bionic materials, so that the nano materials become self substances to avoid the recognition of an immune system. Among the numerous biomimetic materials, cell membranes are a material that can confer unique biological properties to nanoparticles. By fusing cell membranes on the surfaces of the nanoparticles, the nanoparticles can have complex and unique surface physicochemical properties of original cells, and simultaneously approximately maintain the original natural structural integrity and functional activity of the cell membranes. The method for directly combining the cell membrane with the nanoparticles not only can reduce the adsorption of in vivo nonspecific protein to the nanoparticles, but also has relatively simple preparation process. A number of preclinical studies have demonstrated that cell membrane camouflaged nanomaterials can be used for specific molecular imaging of cancer, atherosclerosis, and the like.
Based on the above consideration, we have developed a novel ultrasound contrast agent prepared by using natural cell membranes instead of artificially synthesized lipids, which can participate in physiological and pathological processes in patients due to its ability to substantially maintain the original natural structural integrity and functional activity of cell membranes, and directly reflect the critical information of the location, progress and degree of disease occurrence; in addition, the natural cell membrane is taken from the patient, so the natural cell membrane is absolutely safe, has innate immune compatibility and can efficiently avoid immune clearance.
Disclosure of Invention
The invention aims to provide a novel ultrasonic contrast agent derived from natural cell membranes and a preparation method of the novel contrast agent.
Another object of the present invention is to provide the use of the above novel ultrasound contrast agents derived from natural cell membranes in various diseases such as cardiovascular diseases and cancer.
The novel ultrasonic contrast agent is characterized in that the membrane components are completely derived from natural cell membranes, the cell membranes include but are not limited to red blood cells, white blood cells, platelets, tumor cells, macrophages, stem cells and a combination of two or more of the red blood cells, the white blood cells, the platelets, the tumor cells, the macrophages and the stem cells, and then inert gas or liquid is encapsulated inside the novel ultrasonic contrast agent.
The invention provides a preparation method of a novel ultrasonic contrast agent derived from natural cell membranes, which comprises the following steps:
1) the natural cells were collected and washed 3 times with buffer to obtain a pure suspension of natural cells.
2) And (3) treating the natural cell suspension by using a freeze-drying buffer solution containing a freeze-drying protective agent to ensure that cell membranes are fully combined with the freeze-drying protective agent to obtain the natural cell freeze-drying suspension.
3) Packaging the natural cell freeze-dried suspension in a penicillin bottle, freeze-drying according to a corresponding freeze-drying process, removing water in a cell structure, and backfilling with inert gas to obtain the natural cell freeze-dried powder.
4) And adding a buffer solution into a penicillin bottle containing the natural cell freeze-dried powder, and slightly shaking to obtain the uniformly dispersed natural cell ultrasonic contrast agent.
The novel ultrasonic contrast agent can approximately maintain the structural integrity and the functional activity of the used natural cell membrane, so that the novel ultrasonic contrast agent has high-efficiency immune clearance avoiding capability and blood long-circulating capability in vivo and has excellent active targeting performance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a microscopic observation result of a platelet ultrasonic contrast agent prepared in specific example 1;
FIG. 2 shows the results of particle size distribution of platelet ultrasound contrast agent prepared in example 1;
FIG. 3 is an in vitro ultrasound contrast image of platelet ultrasound contrast agent of specific example 4;
fig. 4 is an ultrasound contrast image of platelet ultrasound contrast agent at animal heart tissue in specific example 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The following detailed description will help to understand the present invention, but does not limit the contents of the present invention.
Example 1
Platelet hypercompetencePreparation of acoustic contrast agent: draw a volume of peripheral anticoagulated venous blood into a sterile centrifuge tube and record the initial volume (V)0mL). The high-concentration platelet plasma is prepared by a two-step centrifugation method. The first time, centrifugal force 200 Xg is adopted, and the centrifugation is carried out for 10 min. Carefully aspirate the upper plasma into another centrifuge tube and discard the lower red blood cells. The second centrifugation was performed for 15min at 1390 Xg. The platelet plasma volume required to be preserved was calculated as 1/10 of the initial volume retained. And removing the redundant plasma on the upper layer, and uniformly blowing the platelet sediment on the lower layer to obtain the high-concentration platelet plasma.
The freshly prepared platelet plasma was centrifuged by 12000 Xg for one second by centrifugal force to separate platelets from the upper plasma. Recording plasma partial volume (V)1mL). The lower platelet pellet was then resuspended and washed twice with Buffer A (100mM NaCl, 10mM KCl, 10mM EGTA, 10mM imidazole, 10mg/mL prostaglandin-E1, pH 6.8). The platelet concentration was then adjusted to 1X 10 with Buffer A solution containing 33mM trehalose9mL, incubation at 37 ℃ for 4 h. After the incubation was complete, centrifugation at 12000 Xg for one second was carried out using lyophilization buffer (9.5mM HEPES, 142.5mM NaCl, 4.8mM KCl, 1mM MgCl)230mM trehalose) to obtain a platelet-containing frozen stock solution. Packaging, freeze-drying, and using perfluoropropane inert gas (C)3F8) Backfilling, and storing in a sealed manner to obtain the platelet freeze-dried powder. After addition of PBS to restore the aqueous state, a suspension of evenly dispersed platelet ultrasound contrast agent was obtained by gentle shaking, with the platelet structure remaining intact (FIG. 1) and the particle size distribution centered at 3.1 μm (FIG. 2).
Example 2
Preparing an erythrocyte ultrasonic contrast agent: drawing a certain amount of peripheral anticoagulated venous blood into a sterile centrifuge tube, centrifuging the blood for 5min at 2500r/min, discarding plasma and leucocyte, centrifuging erythrocytes for 5min at 2500r/min by using a phosphate buffer solution with the isotonic pH of 7.4, repeatedly washing for 3 times, discarding supernatant, and obtaining erythrocytes; the obtained washed red blood cells and 800mM trehalose solution are uniformly mixed in a ratio of 1:1.5, and trehalose is loaded into the cells by an electric pulse method to enable the intracellular trehalose concentration to reach about 60mM, so as to obtain the trehalose red blood cell concentrated solution. Mixing the erythrocyte concentrated solution and a freeze-drying buffer solution (15% PVP, 4% trehalose, 3% glycerol, 5% BSA and 0.05% HSI) according to a volume ratio of 1:4, subpackaging, freeze-drying, backfilling with nitrogen, sealing and storing to obtain the erythrocyte ultrasonic contrast agent freeze-dried powder.
Example 3
Preparation of prostate cancer cell (PC-3) ultrasonic contrast agent: mammalian sample cells suspended in albumin at 4 ℃, 1% phosphate buffer were collected and the cell suspension was centrifuged to obtain a cell pellet. Cells were resuspended using trehalose isotonic solution and incubated for 1 h. The cell suspension was added to the vessel, cooled to 4 ℃ and the vessel was shaken to allow the cells to spread evenly and frozen at about-70 ℃ for 1 h. Transferring the container into a freeze-drying device for freeze-drying, and using inert gas SF6And backfilling, and sealing and storing to obtain the freeze-dried powder of the prostate cancer cell ultrasonic contrast agent.
Example 4
The biofilm ultrasound contrast agent obtained in examples 1 to 3 was prepared with physiological saline in a volume ratio of 1:3, and then injected into a latex tube, 500ml of ultrasound degassed water was placed in a water tank, the above latex tube was placed in the middle of the liquid, and the ultrasonic diagnostic apparatus contract mode was used, MI:0.04 (mechanical index), probe frequency: 3-12MHz, and the in vitro ultrasonic contrast effect of the biomembrane ultrasonic contrast agent is researched. The in vitro ultrasound contrast image is shown in fig. 3, the silica gel tube wall has a high echo effect in an ultrasound field due to its high density, and the inner cavity of the silica gel tube is in an anechoic state (PBS, fresh platelets) when no biomembrane ultrasound contrast agent exists. After the biomembrane ultrasonic contrast agent is injected, remarkable echo signal enhancement is observed in the inner cavity of the silicone tube, so that the biomembrane ultrasonic contrast agent is proved to have good capacity of in-vitro ultrasonic development enhancement.
Example 5
In order to evaluate the in vivo ultrasound imaging enhancing performance of the biofilm ultrasound contrast agents obtained in examples 1-3, SD rats were subjected to cardiac tissue ultrasound imaging. Concentration of microbubbles 1 x 108mL, tail vein injection at a concentration of 1mL/kg, followed by injection of 100. mu.L of physiologicalBrine. Using the ultrasonic diagnostic apparatus contract mode, MI:0.04 (mechanical index), probe frequency: 3-12 MHz. The in vivo ultrasound contrast image is shown in figure 4, and the heart ultrasound signal is obviously enhanced 10s after injection, which shows that the biomembrane ultrasound contrast agent can successfully pass through the pulmonary circulation and enter the systemic circulation system. Therefore, the method can predict the huge application prospect of the platelet ultrasonic contrast agent in the diagnosis of blood-related diseases.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (1)
1. A preparation method of an ultrasonic contrast agent derived from natural cell membranes is characterized in that membrane components of the ultrasonic contrast agent are completely derived from the natural cell membranes, and gas is encapsulated in the components;
the contrast agent can maintain the structural integrity and the functional activity of the used natural cell membrane;
the natural cell is derived from a multicellular organism or a unicellular organism; the multicellular organism is an animal or a plant selected from the group consisting of red blood cells, white blood cells, platelets, tumor cells, macrophages, stem cells; the unicellular organism is a bacterium or a fungus;
the natural cell membrane is a mixture of one or more different kinds of natural cell membranes; the gas is inert gas;
the ultrasonic contrast agent is formed by wrapping gas by a film-forming material, and the particle size range of the ultrasonic contrast agent is 10nm-100 mu m;
the preparation method of the natural cell membrane derived ultrasonic contrast agent comprises the following steps: 1) collecting natural cells, washing for 3 times by using buffer solution to obtain pure natural cell suspension; 2) treating the natural cell suspension with a freeze-drying buffer solution containing a freeze-drying protective agent to fully combine cell membranes with the freeze-drying protective agent to obtain a natural cell freeze-drying suspension; 3) subpackaging the natural cell freeze-dried suspension in a penicillin bottle, freeze-drying according to a corresponding freeze-drying process, removing water in a cell structure, and backfilling with inert gas to obtain natural cell freeze-dried powder; 4) and adding a buffer solution into a penicillin bottle containing the natural cell freeze-dried powder, and slightly shaking to obtain the uniformly dispersed natural cell ultrasonic contrast agent.
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| CN105536000B (en) * | 2015-12-10 | 2018-11-27 | 北京大学 | A kind of acoustic contrast agent and its preparation method and application based on pentaerythritol ester |
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