CN111494639A

CN111494639A - Cell-like structure nano material and preparation method and application thereof

Info

Publication number: CN111494639A
Application number: CN202010195491.5A
Authority: CN
Inventors: 刘中民; 乐文俊; 陈炳地; 吴敏靓; 梅天笑; 李会一; 张一帆
Original assignee: Shanghai East Hospital Tongji University Affiliated East Hospital
Current assignee: Zhihui Medical Technology Shanghai Co ltd
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-08-07

Abstract

The invention discloses a cell-like structure nano material and a preparation method and application thereof, wherein the method comprises the steps of firstly carrying out surface ion modification on a mesoporous nano material to form hydrophilic surface modification; and then carrying out purification treatment such as cracking, discontinuous sucrose-chitosan density gradient extraction and the like on the target cells to obtain target membrane protein, and then rapidly assembling the target membrane protein on the surface of the mesoporous nano particles under the ultrasonic guidance through an electrostatic adsorption principle to form a cell-like nano structure consisting of the target membrane protein, a cavity and a nano material wall. The invention overcomes the random uncertainty and low purity of the existing extracted cell membrane targeting membrane protein, completely retains the biological activity of the original membrane protein, and improves the preparation efficiency of the cell-like structure nano material. The invention can be applied to the biomedical fields of separation and purification of membrane proteins of various cells, drug delivery carriers for resisting tumors, viruses and bacteria, preventive and therapeutic nano vaccine drugs, diagnosis and treatment integrated nano materials and the like.

Description

Cell-like structure nano material and preparation method and application thereof

Technical Field

The invention belongs to the field of nanotechnology, and relates to a nano material in the field of biomedical application, in particular to a cell-like structure nano material, and a preparation method and application thereof.

Background

During the development and progression of tumors, antigens presented on the surface of tumor cells enable the immune system to recognize and strike the tumor cells; on the other hand, however, they also mediate the phenomenon of homologous binding and immune evasion by tumor cells. For example, the level of galectin-3 in blood of a cancer patient is obviously increased, and the galectin-3 interacts with transmembrane mucin MUC1 related to cancer, so that the homologous aggregation phenomenon of circulating tumor cells is improved, anoikis is reduced, and tumor metastasis is promoted. Research shows that the artificially synthesized nano material modified by cell membrane surface antigen can increase the effect of tumor vaccine. In addition, research has been successfully carried out to use cell membranes such as erythrocytes and leukocytes to coat functional nanomaterials (such as gold nanoparticles and silicon nanoparticles), and these membrane-coated nanomaterials can exhibit target cell-specific targeting binding. In addition, the purposes of activating the immune system, improving anti-tumor immune response, prolonging the retention time in circulation, effectively presenting medicaments and the like are achieved. However, the existing technology for extracting membrane protein and coating the membrane protein with the nano material has the problems of complicated steps, low purity of the extracted membrane, easy protein denaturation and the like.

Chinese patent application CN201711360290.0 discloses a breast cancer targeting nanoparticle based on macrophage membrane coating and a preparation method thereof, and the method for realizing macrophage membrane extraction and coating breast cancer targeting nanoparticle comprises the following steps: culturing macrophages by a mouse, repeatedly freezing and thawing the macrophages in liquid nitrogen for 3-5 times, cleaning, centrifuging and extracting cell membranes; then mixing the cell membrane with the nanoparticles, and repeatedly extruding for 10-50 times by a high-pressure nanometer extruder with 400nm, 200nm or 100nm filter membranes to obtain the macrophage-coated nanoparticle preparation loaded with paclitaxel. The substep of preparing the membrane-coated nanoparticles by adopting the physical method is complicated, the conditions are harsh, the yield is not high, and membrane protein deformation and inactivation are easily caused by repeated mechanical extrusion.

Disclosure of Invention

The technical problem to be solved is as follows: in order to overcome the defects of the prior art, the invention extracts high-purity cell membranes by a gradient centrifugation method and forms a membrane-coated nano material to solve the problems of limited cell membrane types, low cell membrane purity, poor coating rate and the like of the coating nano material in the prior art, and provides the cell-structure-imitated nano material for extracting high-purity membrane protein and coating the nano material, and a preparation method and application thereof.

The technical scheme is as follows: a method for extracting high purity membrane proteins and forming membrane-coated nanoparticles, the method comprising the steps of:

(1) culturing target cells in a cell state basically requiring cell viability higher than 60%, cell purity higher than 80%, and cell number of 1-25 × 10⁷One time per time for standby; (2) centrifuging the cells prepared in the step (1) at room temperature of 2000rmp, discarding the supernatant, resuspending the precipitate by using a cell membrane extraction lysate, homogenizing the precipitate on ice, centrifuging at 2000rmp, collecting the supernatant, and resuspending the precipitate by using the cell membrane extraction lysate;

(3) repeating the centrifugation and resuspension steps in step (2) until there are no intact cells;

(4) mixing the supernatants collected in the step (2) and the step (3), and centrifuging by adopting sucrose-chitosan density gradient to obtain a purified cell membrane;

(5) taking each layer of the samples centrifuged in the step (4), analyzing protein components in the samples, diluting the membrane enrichment area by using physiological saline, centrifuging for 0.5 hour at the temperature of 4 ℃ and 20000g, lyophilizing, weighing, adding PBS, and storing at the temperature of 4 ℃;

(6) performing protein quantitative analysis on the cell membranes stored in the step (5), wherein the ratio of the mass concentration of membrane lipid to the mass concentration of membrane protein is 1:4-3: 1; adding a mesoporous nano material which has the same concentration as membrane lipid and is subjected to hydrophilization modification through a surface plasmon resonance technology or a multifunctional mesoporous nano material loaded with a target substance into a cell membrane solution, reacting for 1-12 hours under the conditions of ultrasound and 0-4 ℃, centrifuging at 9000rpm to remove unbound cell membranes, and preparing the nano material with the simulated cell structure.

Preferably, the source of the target cells in step (1) includes, but is not limited to, tumor cells, blood cells obtained by in vitro culture, or various microorganisms such as bacteria and fungi.

Preferably, the cell taking amount in the step (2) is 1 to 25 × 10⁷One time per time, and the time length of two times of centrifugation is 10 min.

Preferably, the cell membrane lysate of step (2) is formulated with 20mM sucrose, 1mM chitosan, 1mM NaOH, 1mM Tris/HCl, 0.1mM PMSF (DMSO solvent), 100. mu.g/m L5. mu.g/m L5. mu.g/m L trypsin inhibitor, final concentration, pH 7.4.

Preferably, the specific method of sucrose-chitosan density gradient centrifugation adopted in step (4) is as follows: pouring the mixed supernatant into discontinuous sucrose-chitosan density gradient liquid, wherein the concentrations w/v of the sucrose-chitosan density gradient liquid are 60%, 45% and 25% respectively; then centrifuging at 20000g for 30min at 4 deg.C, and collecting the sample at the boundary of 45%/25% of sucrose-chitosan density gradient.

Preferably, the mesoporous nano material in the step (6) is: at least one of mesoporous silica, mesoporous silicon, mesoporous carbon, metal oxide, metal sulfide, organic polymer, organic-inorganic hybrid and metal; the mesoporous nano material loaded target substances are as follows: at least one of an anti-tumor drug, an anti-viral drug, an anti-bacterial drug, a biologically active substance (such as an antigen, cytokine, antibody or inhibitor).

The cell-like structure nano material prepared by any one of the methods.

The application of the cell-like structure nano material in preparing a delivery carrier of antitumor, antiviral or antibacterial drugs.

The principle of the preparation method of the cell-like structure nano material is that ① the cracking and discontinuous sucrose-chitosan gradient combined technology adopted by the invention is used for purifying to obtain high-purity target membrane protein, the key defects of random uncertainty, low purity and the like of the existing extracted cell membrane target membrane protein are overcome, ② the surface of the nano material is subjected to hydrophilic modification by adopting the surface plasma resonance technology, and the mesoporous nano material and the target membrane protein are controlled to form the cell-like structure nano material consisting of the target membrane protein, a cavity and a nano material wall through self-assembly under the conditions of low-power ultrasonic guidance and low temperature.

Has the advantages that: (1) the method provided by the invention comprises the steps of sequentially cracking cells, discontinuously extracting sucrose-chitosan with density gradient, and coating cell membranes on corresponding nano materials by ultrasound, so that the purity of membrane protein is improved, and the yield of membrane-coated nano materials is increased, wherein the coating yield of tumor cell membranes is 90-95%, the coating yield of leukemia cells is 94-99%, the coating yield of leukemia cells is 96-99%, and the coating yield of microbial cells is 85-90%; (2) the method has mild reaction conditions, strong repeatability, no repeated freeze thawing operation such as liquid nitrogen and the like, and is suitable for being popularized and applied to large-scale production; (3) the method disclosed by the invention is wide in application range, can be used for obtaining membrane proteins of various target cells, and forming a membrane coating structure with various mesoporous nano materials, provides a foundation for improving the targeting property of different nano materials and realizing diagnosis and treatment integration by subsequently modifying antibodies and carrying drugs, and has wide application potential in the biomedical fields of anti-tumor, anti-virus, anti-bacterial drug delivery, nano vaccine or diagnosis and treatment integration and the like.

Drawings

FIG. 1 is a layer diagram generated by centrifugation using sucrose-chitosan density gradient in example 1;

FIG. 2 is a graph showing the result of Western blot on cell membranes obtained by extraction in example 1; wherein, I is a whole cell lysate, and II is an extracted cell membrane;

FIG. 3 is a transmission electron microscope image of the nano-material with simulated cell structure of example 1;

FIG. 4 is a transmission electron microscope image of the nano-material with simulated cell structure of example 2;

FIG. 5 is a scanning electron micrograph of the nano-material with a simulated cell structure of example 3.

FIG. 6 is a graph showing the antitumor effect of the nano-material with the simulated cell structure in example 4.

FIG. 7 is a graph showing the antibacterial effect of the cell-like nanomaterial of example 5.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

Taking an A549 cell membrane protein coated mesoporous copper sulfide test of a target cell cultured in a laboratory as an example, the specific method is as follows:

(1) culturing target cell A549 in T75 culture flask, observing cell state, and culturing until cell state grows to cell viability higher than 95%, and cell number is 20 × 10⁷When the seeds are planted for one time or several times, the seeds are ready for use;

(2) centrifuging the prepared cell in the step (1) at room temperature of 2000rmp for 5min, discarding the supernatant, resuspending the precipitate in a cell membrane extraction lysate of 2m L, homogenizing the cell membrane extraction lysate on ice, centrifuging at 2000rmp for 5min, collecting the supernatant in a centrifuge tube of 15m L, and resuspending the precipitate by using the cell membrane extraction lysate, wherein the cell membrane extraction lysate comprises 20mM sucrose, 1mM chitosan, 1mM NaOH, 1mM Tris/HCl, 0.1mM PMSF (DMSO solvent), 100 mu g/m L5 mu g/m L5 mu g/m L final concentration of trypsin inhibitor and pH 7.4.

(4) mixing the supernatants collected in the step (2) and the step (3), and centrifuging by adopting sucrose-chitosan density gradient to obtain a purified cell membrane; the specific method adopting sucrose-chitosan density gradient centrifugation comprises the following steps: pouring the mixed supernatant into discontinuous sucrose-chitosan density gradient liquid, wherein the concentrations w/v of the sucrose-chitosan density gradient liquid are 60%, 45% and 25% respectively; then centrifuging at 20000g for 30min at 4 deg.C, and collecting the sample at the boundary of 45%/25% of sucrose-chitosan density gradient.

(5) Taking each layer of the centrifuged sample in the step (4), analyzing protein components in the sample, diluting a membrane enrichment area by using physiological saline, centrifuging for 30min at 20000g and 4 ℃, lyophilizing, weighing, adding PBS, and storing at 4 ℃;

(6) performing protein quantitative analysis on the cell membranes stored in the step (5), wherein the ratio of the mass concentration of membrane lipid to the mass concentration of membrane protein is 1:4-3: 1; adding mesoporous copper sulfide which has the same concentration as membrane lipid and is subjected to hydrophilization modification by a surface plasmon resonance technology into a cell membrane solution, uniformly mixing at 4 ℃, carrying out ultrasonic treatment reaction for 2 hours, and centrifuging at 9000rpm to remove unbound cell membranes to obtain the copper sulfide nano material with the simulated cell structure.

As shown in FIG. 1, from the layered image after gradient centrifugation, a clear white film structure is observed at the 30%/40% interface; as shown in FIG. 2, it can be seen from the Western blot result chart of the extracted cell membrane that the removal of the structure of the cell nucleus and the cell cytoplasm is relatively complete, and the purity of the obtained cell membrane is higher; as can be seen in FIG. 3, the surface of the mesoporous copper sulfide nanomaterial has an obvious film structure as seen in a transmission electron microscope.

Example 2

Taking a membrane protein coated mesoporous silica test of a breast cancer tumor cell from a target cell as an example, the specific method comprises the following steps:

(1) culturing breast cancer tumor cells, and counting the number and the activity of the cells by using placental blue; by H&E, the proportion of each cell is measured by staining, when the cell activity is higher than 60%, the cell purity is higher than 80%, and the cell number is 1-20 × 10⁷One time per time for standby;

(2) - (5) same as in example 1;

(6) performing protein quantitative analysis on the cell membranes stored in the step (5), wherein the ratio of the mass concentration of membrane lipid to the mass concentration of membrane protein is 1:4-3: 1; adding mesoporous silica which has the same concentration as membrane lipid and is subjected to hydrophilization modification by a surface plasmon resonance technology into a cell membrane solution, uniformly mixing at 4 ℃, carrying out ultrasonic treatment reaction for 6 hours, and centrifuging at 9000rpm to remove unbound cell membranes to obtain the multifunctional silicon dioxide nano material with the simulated cell structure.

As shown in FIG. 4, the surface of the mesoporous silica nanomaterial has an obvious film structure as seen from a transmission electron microscope.

Example 3

Taking the membrane protein coating of the target cell derived from the leukocyte as an example to load the porous organosilane polymers (OVA @ POPs) of the egg albumin, the specific method is as follows:

(1) culturing white blood cells by adopting a cell culture medium MEM, and counting the number and the activity of the cells by using placenta blue; by H&E, the proportion of each cell is measured by staining, when the cell activity is higher than 80%, the cell purity is higher than 90%, and the cell number is 1-20 × 10⁷One time per time for standby;

(2) - (5) same as in example 1;

(6) performing protein quantitative analysis on the cell membranes stored in the step (5), wherein the ratio of the mass concentration of membrane lipid to the mass concentration of membrane protein is 1:4-3: 1; adding porous OVA @ POPs which have the same concentration as membrane lipids and are subjected to hydrophilization modification by a surface plasmon resonance technology into a cell membrane solution, uniformly mixing at 4 ℃, carrying out ultrasonic treatment reaction for 6 hours, and centrifuging at 9000rpm to remove unbound cell membranes to obtain the multifunctional cell-structure-simulated porous OVA @ POPs nano material.

As can be seen in FIG. 5, the nano material is in a cell-like spherical shape after being coated by the membrane protein as seen in a scanning electron microscope image.

Example 4

Taking the experiment of the application of the mesoporous silica nano material of the chemotherapy drug wrapped by the tumor cell membrane prepared in the example 2 in the anti-tumor aspect as an example, the specific method is as follows:

(1) referring to example 2, a DOX mesoporous silica nanomaterial (S180@ DOX-SiO) which is a broad spectrum antitumor drug wrapped with S180 cell membrane was prepared₂) And dispersed in PBS for use.

(2) Applying for obtaining ethical batches and establishing a tumor-bearing model, namely resuspending S180 cells by PBS and placing the cells in a wet ice box for low-temperature storage, anesthetizing the mice by 2-5% isoflurane before inoculation, inoculating the cells into 10 mice by intraperitoneal injection, and inoculating 1 × 10 to each mouse⁶s180 cells, the inoculation volume is 200 mu L, and the inoculation part is an abdominal cavity;

(3)15 male BA L B/c mice at 6-8 weeks will be randomized into 3 groups of 5 mice per group based on body weight, the day of cell inoculation is defined as day 0.

The grouping and dosing schedule are shown in table 1:

TABLE 1 grouping and dosing regimens

(3) Morphological changes were observed daily for each mouse, continuously from the start of the cohort to the end of the experiment. All abnormal appearance and behavioral activities were recorded.

(4) In the experimental process, when the weight of the mice is reduced by more than or equal to 15 percent, the administration is stopped, and the drug stopping period is long enough to recover the weight of the mice. Only one mouse is stopped taking the medicine, and the other mice are normally taken; the experiment will continue with reference to the following criteria for weight recovery in mice at drug withdrawal: the weight loss is less than or equal to 10 percent.

(5) During the experiment, animals should be euthanized if any one or more of the following occurs:

a, abnormal movement or paralysis of the animal occurs;

b, animal body weight once decreased more than 20% of body weight at the time of starting drug treatment.

(6) At the end of the experiment, all animals will be sacrificed by inhalation of excess CO2 and cervical dislocation.

As can be seen from fig. 6, from the morphology of the mice 10 days after treatment, the membrane protein-coated chemotherapeutic drug nanomaterial can improve the therapeutic effect of the anti-tumor drug compared to the original drug and placebo treatment groups.

Example 5

Taking the experiment of the application of the porous OVA @ POPs material with the antibiotic wrapped by the white cell membrane prepared in the example 3 in the antibacterial aspect as an example, the specific method is as follows:

(1) reference example 3 porous OVA @ POPs materials (PC-OVA @ POPs) of Penicillin (PC) encapsulated in leukocyte membrane were prepared and dispersed in PBS for use.

(2) Respectively inoculating escherichia coli (E.coli), PC + E.coli or PC-OVA @ POPs + E.coli to L B solid culture medium;

the grouping and dosing schedule are shown in table 2:

TABLE 2 grouping and dosing regimens

(3) The inoculated culture dish was placed upside down in a 37 ℃ incubator and cultured, and the colony was observed after about 12 hours.

As can be seen from fig. 7, the nano material of membrane protein coated antibacterial drug can improve the antibacterial effect of the drug compared with the original drug and placebo treatment group.

Claims

1. The preparation method of the cell-like structure nano material is characterized by comprising the following steps:

(1) culturing target cells in a cell state basically requiring cell viability higher than 60%, cell purity higher than 80%, and cell number of 1-25 × 10⁷One time per time for standby;

(2) centrifuging the target cells in the step (1) at room temperature of 2000rmp, discarding supernatant, resuspending the precipitate by using cell membrane extraction lysate, homogenizing the precipitate on ice, centrifuging at 2000rmp, collecting supernatant, and resuspending the precipitate by using cell membrane extraction lysate;

(6) and (3) carrying out protein quantitative analysis on the cell membranes stored in the step (5), wherein when the ratio of the mass concentration of membrane lipid to the mass concentration of membrane protein is 1:4-3:1, adding a mesoporous nano material which is subjected to hydrophilization modification by a surface plasmon resonance technology and is equal to the concentration of membrane lipid or a multifunctional mesoporous nano material loaded with a target substance into a cell membrane solution, reacting for 1-12 hours under the conditions of ultrasound and 0-4 ℃, and centrifuging at 9000rpm to remove unbound cell membranes to obtain the nano material with the cell-like structure.

2. The method for preparing the cell-like structured nanomaterial according to claim 1, wherein the target cell in the step (1) is a tumor cell, a blood cell, a bacterium, or a fungus.

3. The method for preparing the cell-like structured nanomaterial of claim 1, wherein the number of the cells in the step (2) is 1-25 × 10⁷One time per time, and the time length of two times of centrifugation is 5 min.

4. The method of claim 1, wherein the cell membrane lysate of step (2) is selected from the group consisting of 20mM sucrose, 1mM chitosan, 1mM NaOH, 1mM Tris/HCl, 0.1mM PMSF, trypsin inhibitor 100 μ g/m L5 μ g/m L5 μ g/m L, and pH 7.4.

5. The method for preparing the cell-like structure nano material according to claim 1, wherein the sucrose-chitosan density gradient centrifugation adopted in the step (4) is as follows: pouring the mixed supernatant into discontinuous sucrose-chitosan density gradient liquid, wherein the concentrations w/v of the sucrose-chitosan density gradient liquid are 60%, 45% and 25% respectively; then centrifuging at 20000g for 30min at 4 deg.C, and collecting the sample at the boundary of 45%/25% of sucrose-chitosan density gradient.

6. The method for preparing the cell-like structure nanomaterial according to claim 1, wherein the mesoporous nanomaterial in step (6) is: at least one of mesoporous silica, mesoporous silicon, mesoporous carbon, metal oxide, metal sulfide, organic polymer, organic-inorganic hybrid and metal; the mesoporous nano material loaded target substances are as follows: at least one of antineoplastic drug, antiviral drug, antibacterial drug, and bioactive substance.

7. The cell-like structured nanomaterial prepared by the method of any one of claims 1 to 6.

8. Use of the biomimetic cellular structured nanomaterial of claim 7 in the preparation of a delivery vehicle for an anti-tumor, anti-viral, or anti-bacterial drug.