CN110812497A - Bionic Janus magnetic-mesoporous silica nanoparticle for CTCs specific capture, and preparation method and application thereof - Google Patents
Bionic Janus magnetic-mesoporous silica nanoparticle for CTCs specific capture, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bionic Janus magnetic-mesoporous silica nanoparticle for CTCs specific capture, and a preparation method and application thereof. The preparation method comprises the following steps: 1) preparing carboxylated Janus magnetic-mesoporous silica nanoparticles; 2) preparing a fused cell membrane; 3) and coating the prepared fused cell membrane on the surface of the Janus magnetic-mesoporous silica nanoparticle to obtain the bionic Janus magnetic-mesoporous silica nanoparticle. The core Janus magnetic-mesoporous silica nanoparticle has an asymmetric structure and a rod-like shape, and has a large specific surface area; by coating the fused cell membrane on the surface, the characteristic that leucocytes cannot be aggregated in a circulating system can be used, the nonspecific adsorption to blood cells can be reduced, and the method can be well applied to the detection of CTCs.
Description
Technical Field
The invention relates to the field of nano materials, in particular to a bionic Janus magnetic-mesoporous silica nanoparticle for CTCs specific capture and a preparation method and application thereof.
Background
Cancer has become a serious disease that impairs human life and health, with over 90% of cancer patients dying from metastases. During tumor metastasis, cancer cells are shed from the primary tumor foci and enter the blood or lymphatic circulation system, becoming Circulating Tumor Cells (CTCs). These CTCs survive in the circulatory system and further develop metastatic tumors in distant organs. Thus, CTCs are a gold entry point for studying the metastatic progression of tumors, monitoring the efficacy and assessing the prognosis of patients as an important vehicle between primary and metastatic foci of tumors. However, the abundance of CTCs in peripheral blood is extremely low, and isolating and detecting CTCs is very challenging.
In recent years, the CTCs capture technology based on nano materials shows bright application prospect in CTCs detection due to the characteristics of simple and convenient operation, rich functions, high recovery rate, small cell damage and the like. The immunofluorescence magnetic ball not only can be specifically combined with CTCs and capture the CTCs through magnetic separation, but also can be used for carrying out fluorescence signal analysis on the captured CTCs, so that the immunofluorescence magnetic ball has great conversion potential in CTCs detection application. In order to further improve the fluorescence signal intensity of the magnetic nanoparticles and enhance the binding capacity of the magnetic nanoparticles with the CTCs, the applicant discloses a nano drug-loading system, and preparation and application thereof in the prior patent 201611270207.6, wherein the magnetic-mesoporous silica nanoparticles with precise and controllable size and asymmetric properties, namely Janus-type magnetic-mesoporous silica nanoparticles, are specifically disclosed to be preparedNanoparticles (Janus Fe)3O4-mSiO2). In the composite component, the mesoporous silica functional unit not only improves the biocompatibility of the nano particles, but also greatly increases the specific surface area of the nano particles, thereby not only improving the adsorption capacity of the nano particles on CTCs, but also providing a favorable space for the combination of fluorescent probes and the modification of functional groups. Furthermore, this Janus Fe3O4-mSiO2The magnetic ball has the advantages of obvious asymmetric structure, the magnetic ball element is exposed outside and does not interfere with the mesoporous silicon dioxide element functionally, the original magnetic performance of the magnetic ball can be well maintained, and convenience is provided for magnetic separation of CTCs. However, clinical detection techniques for CTCs are more demanding. The nanoparticles are required to have sufficient sensitivity to the CTCs, to separate extremely rare CTCs from hundreds of millions of background cells as much as possible, and have extremely high specificity, to capture relatively pure CTCs and to reduce the interference of background cells as much as possible. Thus, Janus Fe3O4-mSiO2The construction needs to be further optimized to meet the urgent clinical requirements on the CTCs detection technology with high specificity and high sensitivity.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a bionic Janus magnetic-mesoporous silica nanoparticle for CTCs specific capture, and a preparation method and application thereof, aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: the bionic Janus magnetic-mesoporous silica nanoparticle for the CTCs specific capture is provided, and comprises the Janus magnetic-mesoporous silica nanoparticle and a fusion cell membrane coated on the surface of the Janus magnetic-mesoporous silica nanoparticle.
The invention also provides a preparation method of the bionic Janus magnetic-mesoporous silica nanoparticle, which comprises the following steps:
1) preparing carboxylated Janus magnetic-mesoporous silica nanoparticles;
2) preparing a fused cell membrane;
3) and coating the prepared fused cell membrane on the surface of the Janus magnetic-mesoporous silica nanoparticle to obtain the bionic Janus magnetic-mesoporous silica nanoparticle.
Preferably, the step 1) specifically includes:
firstly, preparing a magnetic precursor and polyacrylic acid into polyacrylic acid modified magnetic nanospheres by a high-temperature hydrolysis method, and purifying;
then, taking the magnetic nano particles as a substrate, tetraethoxysilane as a silicon source and hexadecyl trimethyl ammonium bromide as a surfactant, and preparing the Janus type magnetic-mesoporous silica nano particles by a sol-gel method;
and then reacting the obtained Janus type magnetic-mesoporous silica nanoparticles with a silane coupling agent to prepare aminated nanoparticles, and performing carboxylation modification on the aminated nanoparticles to prepare negatively charged nanoparticles, namely the needed carboxylated Janus magnetic-mesoporous silica nanoparticles.
Preferably, the step 2) specifically includes:
a mouse 4T1 breast cancer tumor model is constructed in advance, 4T1 cells and white blood cells are collected from tumor tissues and serum of the model, then lysate is added to break the cells by a homogenizer, gradient centrifugation is carried out to collect 4T1 cell membranes, neutrophil granulosa cells, lymphocyte cell membranes and macrophage membrane materials, the cell membrane materials are mixed, and then a fused cell membrane is prepared by a liposome extrusion instrument.
Preferably, the step 3) specifically includes:
uniformly mixing the prepared carboxylated Janus magnetic-mesoporous silica nanoparticles with the fused cell membrane under physiological conditions, and performing multiple extrusion by using a liposome extruder to prepare the bionic Janus magnetic-mesoporous silica nanoparticles.
The invention also provides application of the bionic Janus magnetic-mesoporous silica nanoparticles in specific detection of CTCs.
The invention has the beneficial effects that: the core Janus magnetic-mesoporous silica nanoparticle has an asymmetric structure and a rod-like shape, and has a large specific surface area; by coating the fused cell membrane on the surface, the non-specific adsorption of the bionic Janus magnetic-mesoporous silica nanoparticles to blood cells can be reduced by means of the characteristic that leukocytes cannot be aggregated in a circulating system, and the method can be well applied to the detection of CTCs.
Drawings
Fig. 1 is a transmission electron microscope picture of a biomimetic Janus magnetic-mesoporous silica nanoparticle in example 3 of the present invention;
FIG. 2 shows the results of zeta potential detection analysis in example 3 of the present invention;
FIG. 3 shows the results of biosafety evaluation in example 3 of the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description. The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The reagents involved in the following examples are all commercially available products, and reagents of different manufacturers and models do not cause significant differences in the results.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
The bionic Janus magnetic-mesoporous silica nanoparticle for the CTCs specific capture is provided, and comprises the Janus magnetic-mesoporous silica nanoparticle and a fusion cell membrane coated on the surface of the Janus magnetic-mesoporous silica nanoparticle.
Example 2
The preparation method of the biomimetic Janus magnetic-mesoporous silica nanoparticle described in embodiment 1 is provided, and includes the following steps:
1) preparation of carboxylated Janus magnetic-mesoporous silica nanoparticles (Janus Fe)3O4-mSiO2-COOH)：
Firstly, preparing a magnetic precursor and polyacrylic acid (PAA) by a high-temperature hydrolysis method to obtain polyacrylic acid modified magnetic nanospheres, and purifying;
then, the Janus type magnetic-mesoporous silica nanoparticles are prepared by a sol-gel method by taking the magnetic nanoparticles as a substrate, tetraethyl orthosilicate (TEOS) as a silicon source and Cetyl Trimethyl Ammonium Bromide (CTAB) as a surfactant and a template;
and then reacting the obtained Janus type magnetic-mesoporous silica nanoparticles with a silane coupling Agent (APS) to prepare aminated nanoparticles, and performing carboxylation modification on the aminated nanoparticles to prepare negatively charged nanoparticles, namely the needed carboxylated Janus magnetic-mesoporous silica nanoparticles.
In a further preferred embodiment, step 1) specifically comprises:
1-1) magnetic precursor FeCl30.13g, 0.576g of polyacrylic acid PAA (9000) and 34ml of diethylene glycol DEG, stirring for 30min at 37 ℃ under the protection of nitrogen, rotating at 550rpm, heating to 260 ℃, and continuing stirring for 30min at 550rpm to prepare a first reaction solution;
3.8ml of a diethylene glycol solution of NaOH (20 wt%) at 67.5 ℃ is injected into the first reaction solution, the mixture is continuously stirred and reacted at the rotation speed of 550rpm for 1h, and then separation, water washing and drying (purification) are carried out to obtain magnetic particles;
1-2) preparing the prepared magnetic particles into an aqueous solution of 8.6mg/ml, adding 1ml of the aqueous solution of the magnetic particles into an aqueous solution (5mg/ml, 10ml) containing cetyl trimethyl ammonium bromide serving as a surfactant, fully dispersing, adding 500ml of weak alkaline reagent ammonia water (25% -28%), then slowly adding 0.03ml of ethyl orthosilicate, stirring for 30min, and washing away the surfactant by using ethanol to prepare a magnetic-mesoporous silica nanorod carrier;
1-2) dissolving the prepared magnetic-mesoporous silica nanorod carrier in 10ml of ethanol, adding 1ml of silane coupling agent after ultrasonic dispersion, refluxing reaction liquid at 105 ℃ for 4 hours, separating, washing and drying to prepare the aminated magnetic-mesoporous silica nanorod carrier; adding the aminated magnetic-mesoporous silica nanorod carrier into an ethanol solution (50mg/ml, 10ml) of succinic anhydride, stirring for 24h at 25 ℃, and magnetically separating and washing a reaction product to prepare a carboxylated carrier, namely JanusFe3O4-mSiO2-COOH；
2) Preparation of fused cell membranes:
a mouse 4T1 breast cancer tumor model is constructed in advance, 4T1 cells and white blood cells are collected from tumor tissues and serum of the model, then lysate is added to break the cells by a homogenizer, gradient centrifugation is carried out to collect 4T1 cell membranes, neutrophil granulosa cells, lymphocyte cell membranes and macrophage membrane materials, the cell membrane materials are mixed according to different proportions, and then fused cell membranes with relatively uniform particle size are prepared by a liposome extrusion instrument.
3) Coating the prepared fused cell membrane on the surface of the Janus magnetic-mesoporous silica nanoparticle to obtain the bionic Janus magnetic-mesoporous silica nanoparticle:
uniformly mixing the prepared carboxylated Janus magnetic-mesoporous silica nanoparticles with the fused cell membrane under physiological conditions, and performing multiple extrusion by using a liposome extruder to prepare the bionic Janus magnetic-mesoporous silica nanoparticles. Wherein, the optimal component distribution ratio can be obtained by adjusting the mass ratio of the carboxylated Janus magnetic-mesoporous silica nanoparticles to the fused cell membrane.
Example 3
Performance evaluation of the biomimetic Janus magnetic-mesoporous silica nanoparticles obtained in example 2
1. Referring to fig. 1, a transmission electron microscope picture of the bionic Janus magnetic-mesoporous silica nanoparticle shows that the bionic Janus magnetic-mesoporous silica nanoparticle is in a rod-like shape.
2. Carboxylated Janus magnetic-mesoporous silicaRice grains (Janus Fe)3O4-mSiO2-COOH), fused cell membrane (TWM), biomimetic Janus magnetic-mesoporous silica nanoparticles (TWM-Janus Fe)3O4-mSiO2) Zeta potential detection analysis of (1): referring to fig. 2, the detection result is shown, and the zeta potential of the material is characterized to prove that the fused cell membrane is successfully coated on the surface of the nanoparticle.
3. Evaluation of biological safety:
bionic Janus magnetic-mesoporous silica nanoparticles (TWM-Janus Fe) with different dosages (1.5625,3.125, 6.25,12.5 and 50 mu g/mL) are adopted3O4-mSiO2) And incubating with 4T1 cells for 24 h. And setting comparative examples which are Janus magnetic-mesoporous silica nanoparticles (Janus Fe) with different dosages (1.5625,3.125, 6.25,12.5,50 mu g/mL)3O4-mSiO2) And incubating with 4T1 cells for 24 h. Wherein the control group (CON) did not contain nanoparticles. The bionic Janus magnetic-mesoporous silica nanoparticles obtained by the SRB method and the Janus magnetic-mesoporous silica nanoparticles have the cell survival rate of over 90 percent, and the bionic nanoparticles are proved to have excellent biological safety on the cell level. Refer to fig. 3.
4. Specific capture capacity for CTCs:
adopts bionic Janus magnetic-mesoporous silica nano particles with different concentrations (6.25,12.5,50,100,150 mu g/mL) and 1 x 105A mixture of 4T1 cells. After incubation for 5 min, 15 min, 20 min, 25 min, 30min, the cells in the sample were magnetically separated. The isolated cells were then resuspended and counted using a cell counter. Test results show that the optimal use concentration of the bionic Janus magnetic-mesoporous silica nanoparticles is 50 mug/mL, the reaction time is 20 minutes, the capture efficiency of the bionic Janus magnetic-mesoporous silica nanoparticles to 4T1 cells is 75%, and the bionic Janus magnetic-mesoporous silica nanoparticles have good selectivity to tumor cells.
5. Non-specific adsorption to non-CTCs:
bionic Janus magnetic-mesoporous silica nanoparticles with different concentrations (6.25,12.5,50,100,150 mu g/mL) and 1 library are adopted105Mixing the white blood cells. The cells in the sample were also magnetically separated, resuspended and cell counted after 5 min, 15 min, 20 min, 25 min, 30min of incubation. As a result of the experiment, it was found that the optimal concentration of nanoparticles to be used was 50. mu.g/mL, the reaction time was 20 minutes, and the leukocyte trapping efficiency was 11%. The non-specific adsorption of the bionic Janus magnetic-mesoporous silica nano particles and white blood cells is low.
In this embodiment, the core Janus magnetic-mesoporous silica nanoparticle of the biomimetic Janus magnetic-mesoporous silica nanoparticle has an asymmetric structure and a rod-like morphology, and has a large specific surface area. By coating the fused cell membrane on the surface, the non-specific adsorption of the bionic Janus magnetic-mesoporous silica nanoparticles to blood cells can be reduced by means of the characteristic that leukocytes cannot be aggregated in a circulating system.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (6)
1. A bionic Janus magnetic-mesoporous silica nanoparticle for specifically capturing CTCs is characterized by comprising a Janus magnetic-mesoporous silica nanoparticle and a fused cell membrane coated on the surface of the Janus magnetic-mesoporous silica nanoparticle.
2. The preparation method of the biomimetic Janus magnetic-mesoporous silica nanoparticle as recited in claim 1, comprising the following steps:
1) preparing carboxylated Janus magnetic-mesoporous silica nanoparticles;
2) preparing a fused cell membrane;
3) and coating the prepared fused cell membrane on the surface of the Janus magnetic-mesoporous silica nanoparticle to obtain the bionic Janus magnetic-mesoporous silica nanoparticle.
3. The preparation method of the biomimetic Janus magnetic-mesoporous silica nanoparticle as recited in claim 2, wherein the step 1) specifically comprises:
firstly, preparing a magnetic precursor and polyacrylic acid into polyacrylic acid modified magnetic nanospheres by a high-temperature hydrolysis method, and purifying;
then, taking the magnetic nano particles as a substrate, tetraethoxysilane as a silicon source and hexadecyl trimethyl ammonium bromide as a surfactant, and preparing the Janus type magnetic-mesoporous silica nano particles by a sol-gel method;
and then reacting the obtained Janus type magnetic-mesoporous silica nanoparticles with a silane coupling agent to prepare aminated nanoparticles, and performing carboxylation modification on the aminated nanoparticles to prepare negatively charged nanoparticles, namely the needed carboxylated Janus magnetic-mesoporous silica nanoparticles.
4. The preparation method of the biomimetic Janus magnetic-mesoporous silica nanoparticle as recited in claim 3, wherein the step 2) specifically comprises:
a mouse 4T1 breast cancer tumor model is constructed in advance, 4T1 cells and white blood cells are collected from tumor tissues and serum of the model, then lysate is added to break the cells by a homogenizer, gradient centrifugation is carried out to collect 4T1 cell membranes, neutrophil granulosa cells, lymphocyte cell membranes and macrophage membrane materials, the cell membrane materials are mixed, and then a fused cell membrane is prepared by a liposome extrusion instrument.
5. The preparation method of the biomimetic Janus magnetic-mesoporous silica nanoparticle as recited in claim 4, wherein the step 3) specifically comprises:
uniformly mixing the prepared carboxylated Janus magnetic-mesoporous silica nanoparticles with the fused cell membrane under physiological conditions, and performing multiple extrusion by using a liposome extruder to prepare the bionic Janus magnetic-mesoporous silica nanoparticles.
6. The use of the biomimetic Janus magneto-mesoporous silica nanoparticle according to any of claims 1-5 in the specific detection of CTCs.
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| CN113975244A (en) * | 2021-09-18 | 2022-01-28 | 上海交通大学医学院附属第九人民医院 | Bionic magnetic targeting cationic liposome and preparation method and application thereof |
| CN114259477A (en) * | 2022-01-28 | 2022-04-01 | 中国人民解放军海军军医大学第一附属医院 | Nano delivery system capable of promoting penetration, relieving tumor hypoxia and targeting tumor cells, and preparation method and application thereof |
| CN115245500A (en) * | 2022-06-10 | 2022-10-28 | 温州医科大学 | Phagocyte membrane-coated drug delivery system prepared by low-temperature quick-freezing method |
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| CN109507418A (en) * | 2018-10-26 | 2019-03-22 | 四川大学 | With imitating cyto-architectural magnetic nano-particle, immune magnetic nano particle and the preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113975244A (en) * | 2021-09-18 | 2022-01-28 | 上海交通大学医学院附属第九人民医院 | Bionic magnetic targeting cationic liposome and preparation method and application thereof |
| CN114259477A (en) * | 2022-01-28 | 2022-04-01 | 中国人民解放军海军军医大学第一附属医院 | Nano delivery system capable of promoting penetration, relieving tumor hypoxia and targeting tumor cells, and preparation method and application thereof |
| CN114259477B (en) * | 2022-01-28 | 2023-03-28 | 中国人民解放军海军军医大学第一附属医院 | Nano delivery system capable of promoting penetration, relieving tumor hypoxia and targeting tumor cells, and preparation method and application thereof |
| CN115245500A (en) * | 2022-06-10 | 2022-10-28 | 温州医科大学 | Phagocyte membrane-coated drug delivery system prepared by low-temperature quick-freezing method |
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