CN112029728A - Fluorescent magnetic nano-composite and preparation method and application thereof - Google Patents

Fluorescent magnetic nano-composite and preparation method and application thereof Download PDF

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CN112029728A
CN112029728A CN202010987421.3A CN202010987421A CN112029728A CN 112029728 A CN112029728 A CN 112029728A CN 202010987421 A CN202010987421 A CN 202010987421A CN 112029728 A CN112029728 A CN 112029728A
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liver cancer
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张娜
初琪慧
刘永军
牟伟伟
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Abstract

The invention provides a fluorescent magnetic nano-composite and a preparation method and application thereof, belonging to the technical field of tumor diagnosis and detection and biomedicine. The invention obtains a fluorescent magnetic nano-composite by coating a silicon dioxide layer on the surface of a magnetic nanoparticle, loading fluorescein in a silicon hole and connecting a targeting factor GPC3 specifically combined with liver cancer circulating tumor cells on the surface; the test proves that the nano-particles have uniform particle size, and good stability, magnetic responsiveness and biocompatibility. The fluorescent magnetic nano-composite can realize the specific capture of the circulating tumor cells of the liver cancer, thereby developing application prospects for the detection and analysis of the circulating tumor cells of the liver cancer, and having good practical application values.

Description

Fluorescent magnetic nano-composite and preparation method and application thereof
Technical Field
The invention belongs to the technical field of tumor diagnosis and detection and biomedicine, and particularly relates to a fluorescent magnetic nano-composite as well as a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Primary liver cancer is one of the most common malignant tumors, with hidden onset and rapid progression, and 80% of patients are diagnosed at an advanced stage. Therefore, the five-year survival rate of the liver cancer patient is extremely low, the survival prognosis is poor, and the effective way for improving the survival rate of the liver cancer patient is to realize early diagnosis and early treatment. Circulating Tumor Cells (CTCs) are released from a primary focus into Tumor cells in peripheral blood circulation, the number of CTCs in blood is closely related to the occurrence, development and metastasis of tumors, and when the diameter of the tumors is 1mm, the CTCs enter the circulation system, which is difficult to find by a traditional imaging practice.
Compared with the traditional detection method, the CTC detection has the obvious characteristics of no wound, real time, convenience, high efficiency and the like, and is expected to open up a personalized accurate treatment way for liver cancer patients. Therefore, CTC detection can be used to achieve early diagnosis of liver cancer. However, the CTC content in the blood of a patient is extremely low, and the design of a system for efficiently capturing the CTC of the liver cancer is very important. The conventional CTC detection methods mainly comprise a density gradient centrifugation method, a microfiltration method, a microfluidic method, a dielectrophoresis method, an immunomagnetic separation method based on a magnetic nano-carrier and the like. The magnetic nano-carrier has the advantages of good biocompatibility, easy surface modification, excellent magnetic enrichment function and the like, and is widely applied to the field of magnetic biological separation. Currently, based on immunomagnetic separation
Figure BDA0002689712830000011
The system is the only approved CTC detection means on the market by FDA, and the visible magnetic nano-carrier has heavy weight in CTC separation detectionThe important position.
Figure BDA0002689712830000021
The method is characterized in that CTC is captured based on epithelial specific adhesion molecule (EpCAM) antibody modification, CTC expressing EpCAM in blood is identified through antigen-antibody specific binding, and the CTC is separated from other cells in the blood under the action of an external magnetic field. However, the inventor finds that only 0-20% of patients with liver cancer clinically express EpCAM, so that the invention of a high-sensitivity and high-specificity method for capturing circulating tumor cells of liver cancer is of great importance for clinical guidance of liver cancer.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a fluorescent magnetic nano-composite and a preparation method and application thereof. The fluorescent magnetic nano-composite is prepared, and experiments prove that the fluorescent magnetic nano-composite can efficiently and quickly capture circulating tumor cells of the liver cancer, lays a solid foundation for the CTC diagnosis technology, is favorable for realizing early diagnosis of the liver cancer, and has good practical application value.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
the invention provides a fluorescent magnetic nano-composite, which comprises magnetic nanoparticles, wherein mesoporous silica layers are coated on the surfaces of the magnetic nanoparticles, fluorescein is loaded in mesopores of the mesoporous silica layers, and phosphatidylinositol proteoglycan-3 (GPC3) targeting factors are modified on the surfaces of the mesoporous silica layers.
In a second aspect of the present invention, there is provided a method for preparing the above fluorescent magnetic nanocomposite, the method comprising:
s1, mixing the magnetic nanoparticles, a template agent and a silicon source for reaction, and removing the template to obtain the magnetic mesoporous silica nanoparticles;
s2, loading fluorescein into the mesopores by adopting a passive drug loading method to obtain fluorescent magnetic mesoporous silica nanoparticles;
s3, modifying the connecting molecules on the surface of the silicon layer to provide active groups, and connecting the connecting molecules with GPC3 targeting factors through chemical bonds.
In a third aspect of the present invention, there is provided a use of the fluorescent magnetic nanocomposite as described above in any one or more of:
1) specifically capturing circulating tumor cells of the liver cancer and/or preparing products for specifically capturing the circulating tumor cells of the liver cancer;
2) detecting and analyzing the circulating tumor cells of the liver cancer and/or preparing a product for detecting and analyzing the circulating tumor cells of the liver cancer;
3) products for early diagnosis of liver cancer and/or early diagnosis of liver cancer.
In a fourth aspect of the present invention, a method for specifically capturing circulating tumor cells of liver cancer is provided, wherein the method comprises adding the fluorescent magnetic nanocomposite to a sample to be tested.
One or more of the technical schemes have the following beneficial technical effects:
according to the technical scheme, a silicon dioxide layer is coated on the surface of the magnetic nanoparticle, fluorescein is loaded in a silicon hole, and a targeting factor GPC3 specifically combined with the circulating tumor cells of the liver cancer is connected to the surface of the magnetic nanoparticle to obtain a fluorescent magnetic nanocomposite; the test proves that the nano-particles have uniform particle size, and good stability, magnetic responsiveness and biocompatibility. The fluorescent magnetic nano-composite can realize the specific capture of the circulating tumor cells of the liver cancer, thereby developing application prospects for the detection and analysis of the circulating tumor cells of the liver cancer, and having good practical application values.
Drawings
FIG. 1 is a transmission electron microscope image of the fluorescent magnetic nanocomposite prepared in example 1 of the present invention.
FIG. 2 is a hysteresis curve of the fluorescent magnetic nanocomposite prepared in example 1 of the present invention.
FIG. 3 is a graph showing the safety results of fluorescent magnetic nanocomposites prepared with different concentrations in example 1 of the present invention.
FIG. 4 is a graph showing the results of the capture efficiency of fluorescent magnetic nanocomplexes with different concentrations prepared in example 1 of the present invention to HepG2 and Jurkat T cells.
FIG. 5 is a graph showing the results of the capture efficiency of the fluorescent magnetic nanocomplexes prepared in example 1 of the present invention on HepG2 and Jurkat T cells at different incubation times.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
In a typical embodiment of the present invention, a fluorescent magnetic nanocomposite is provided, where the fluorescent magnetic nanocomposite includes magnetic nanoparticles, the surfaces of the magnetic nanoparticles are coated with mesoporous silica layers, the mesopores of the mesoporous silica layers are loaded with fluorescein, and the surfaces of the mesoporous silica layers are modified with glypican-3 (GPC3) targeting factors.
Wherein the magnetic nanoparticles can be iron, cobalt, nickel and oxides thereof, such as gamma-Fe2O3、Fe3O4、CoFe2O4And Co3O4(ii) a Preferably Fe3O4，Fe3O4The nano particles have the characteristics of good biocompatibility, superparamagnetism, photo-thermal property and the like, so the nano particles have wide application value in the field of nano drug carriers.
Silica is widely used in the field of medical materials because of its low toxicity and good biocompatibility. The nano-sized mesoporous silica has high specific surface area, and the pore channels are orderly arranged, uniform in size and stable in structure. And the surface of the material contains a large number of hydroxyl groups, so that good chemical modification conditions are brought to the material.
The fluorescein can be coumarin-6, Nile Red, Cy5.5, IR780, Dil and other hydrophobic fluorescein; in a specific embodiment of the invention, the fluorescein is coumarin-6 and nile red.
Wherein, the phosphatidylinositol proteoglycan-3 (GPC3) targeting factor is modified on the surface of the mesoporous silica layer, specifically, a connecting molecule is modified on the surface of the mesoporous silica layer to provide an active group; and then the connecting molecule is connected with the GPC3 targeting factor through a chemical bond.
GPC3 targeting factors include, but are not limited to, GC33 mab, YP7 mab, HN3 mab, HS20 mab, GPC3 polypeptides, and combinations thereof.
Wherein the linker molecules include, but are not limited to, carboxyl, amino, hydroxyl, thiol, maleimide, and azide groups.
The fluorescent magnetic nano-composite prepared by the invention has uniform quality and appearance through detection, and the average particle size is about 110 nm.
In another embodiment of the present invention, there is provided a method for preparing the fluorescent magnetic nanocomposite, comprising:
s1, mixing the magnetic nanoparticles, a template agent and a silicon source for reaction, and removing the template to obtain the magnetic mesoporous silica nanoparticles;
s2, loading fluorescein into the mesopores by adopting a passive drug loading method to obtain fluorescent magnetic mesoporous silica nanoparticles;
s3, modifying the connecting molecules on the surface of the silicon layer to provide active groups, and connecting the connecting molecules with GPC3 targeting factors through chemical bonds.
Wherein the magnetic nanoparticles can be iron, cobalt, nickel and oxides thereof, such as gamma-Fe2O3、Fe3O4、CoFe2O4And Co3O4(ii) a Preferably Fe3O4；
The silicon source includes, but is not limited to, ordinary sodium silicate, high modulus ratio sodium silicate, silica sol, tetraethyl orthosilicate (TEOS), methyl orthosilicate (TMOS), and bis- [3- (triethoxysilyl) propyl ] -disulfide;
the template may be cetyltrimethylammonium bromide (CTAB).
The fluorescein can be coumarin-6, Nile Red, Cy5.5, IR780, Dil and other hydrophobic fluorescein; in a specific embodiment of the invention, the fluorescein is coumarin-6 and nile red.
The linking molecules include, but are not limited to, carboxyl, amino, hydroxyl, thiol, maleimide, and azide groups.
GPC3 targeting factors include, but are not limited to, GC33 mab, YP7 mab, HN3 mab, HS20 mab, GPC3 polypeptides, and combinations thereof.
In another embodiment of the present invention, there is provided a use of the fluorescent magnetic nanocomposite as described above in any one or more of:
1) specifically capturing circulating tumor cells of the liver cancer and/or preparing products for specifically capturing the circulating tumor cells of the liver cancer;
2) detecting and analyzing the circulating tumor cells of the liver cancer and/or preparing a product for detecting and analyzing the circulating tumor cells of the liver cancer;
3) products for early diagnosis of liver cancer and/or early diagnosis of liver cancer.
In another embodiment of the present invention, a method for specifically capturing circulating tumor cells of liver cancer is provided, which comprises adding the fluorescent magnetic nanocomposite to a sample to be tested.
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited thereto in any way.
Example 1, a fluorescent magnetic nanocomposite for specifically capturing circulating tumor cells of liver cancer based on targeted GPC3, comprising:
Fe3O4 10mg
0.24g of tetraethoxysilane
Coumarin-61 mg
Carboxy-polyethylene glycol-Maleimide (MAL-PEG-COOH)30mg
GPC3 monoclonal antibody 2mg
The preparation method of the fluorescent magnetic nano-composite comprises the following steps:
(1) dissolving 0.1g of template agent in 20mL of deionized water, and dissolving the magnetic nanoparticles Fe3O4Ultrasonically dispersing for 1h in advance, adding the mixture into the aqueous solution, mechanically stirring for 2h at the temperature of 80 ℃ to uniformly mix the mixture, dropwise adding 0.24g of tetraethoxysilane, and continuously reacting for 2 h; after the reaction is finished, washing the reaction product for 2 to 3 times by using absolute ethyl alcohol and deionized water respectively, and performing magnetic separation on the obtained precipitate to remove a template to obtain Magnetic Mesoporous Silica Nanoparticles (MMSN); re-dispersing the precipitate in 10mL of water, adding 3-Aminopropyltriethoxysilane (APTES) under the heating condition of 80 ℃, mechanically stirring for reaction for 1h, introducing amino functional groups on the surface of the precipitate, and removing the template to obtain the magnetic mesoporous silica nanoparticles (MMSN-NH) with amino2)；
(2) Dissolving fluorescein coumarin-6 and magnetic mesoporous silica nanoparticles in 20mL methanol by passive drug loading method, mechanically stirring at room temperature in dark to volatilize most of solvent, and loading fluorescein into the mesopores after stirring to obtain fluorescent magnetic mesoporous silica nanoparticles (C6/MMSN-NH)2)；
(3) Adding linking molecule MAL-PEG-COOH, 4.2mg EDC and 2.1mg NHS, activating carboxyl under ice bath, and reacting with C6/MMSN-NH2The reaction was overnight and the linkage was by reaction of the carboxyl with the amino group. After the reaction is finished, the reaction solution is centrifuged, washed and the precipitate is collected to obtain the fluorescent magnetic nano-composite (C6/MMSN-PEG-Mal) with the active group maleimide.
(4) Carrying out sulfhydrylation on the GPC3 antibody, and connecting the GPC3 antibody by utilizing the reaction of maleimide and sulfhydrylation to obtain the fluorescent magnetic nano-composite capable of specifically capturing the circulating tumor cells of the liver cancer.
Example 2, a fluorescent magnetic nanocomposite for specifically capturing circulating tumor cells of liver cancer based on targeted GPC3, comprising: gamma-Fe2O37.5mg, tetraethoxysilane 0.24g, coumarin-61 mg, carboxyl-polyethylene glycol-maleimide (MAL-PEG-COOH)30mg, phosphorusGlypican-3 (GPC3) 2 mg.
The preparation method is the same as example 1, but the magnetic nanoparticles are gamma-Fe2O3。
Example 3, a fluorescent magnetic nanocomposite for specifically capturing circulating tumor cells of liver cancer based on targeted GPC3, comprising: fe3O410mg, tetraethoxysilane 0.24g, coumarin-61 mg, carboxyl-polyethylene glycol-maleimide (MAL-PEG-COOH)30mg, and GC33 monoclonal antibody 2 mg.
The preparation method is the same as example 1, but the targeting factor is GC33 monoclonal antibody.
Example 4, a fluorescent magnetic nanocomposite for specifically capturing circulating tumor cells of liver cancer based on targeted GPC3, comprising: fe3O410mg, tetraethoxysilane 0.24g, nile red 1mg, carboxyl-polyethylene glycol-maleimide (MAL-PEG-COOH)30mg, GPC3 monoclonal antibody 2 mg.
The preparation method is the same as example 1, but the hydrophobic fluorescein is nile red.
Example 5, a fluorescent magnetic nanocomposite for specifically capturing circulating tumor cells of liver cancer based on targeted GPC3, comprising: fe3O410mg, methyl orthosilicate 0.17g, nile red 1mg, carboxyl-polyethylene glycol-maleimide (MAL-PEG-COOH)30mg, GPC3 monoclonal antibody 2 mg.
The preparation method is the same as example 1, but the silicon source is methyl orthosilicate.
The fluorescent magnetic nano-composite prepared in example 1 is shown in a transmission electron microscope picture of figure 1, and the nano-particles are round in shape, have a better spherical structure and have an average particle size of 111.74 +/-1.48 nm; as shown in the hysteresis curve chart of fig. 2, the magnetic separator has higher saturation magnetization and can realize quick separation; FIG. 3 is a graph showing the safety results of different concentrations of vectors against HepG2 cells, which indicates that the survival rate of HepG2 cells is still above 80% when the concentration is increased to 400. mu.g/mL, indicating better safety; FIG. 4 is a graph of the effect of fluorescent magnetic nanocomplex concentration on HepG2 capture efficiency, showing that when the vector concentration exceeds 400. mu.g/mL, the capture efficiency does not increase any more, so the optimal capture concentration is selected to be 400. mu.g/mL, and there is no significant change in the capture efficiency for Jurkat T cells as the vector concentration increases; when the incubation time of the carrier and the liver cancer cells is different, the capture efficiency reaches over 90% after the incubation for 30min, and the capture efficiency does not change obviously after the incubation time exceeds 30min, so the capture concentration is selected to be 30min, and the capture efficiency of the Jurkat T cells does not change obviously with the increase of the incubation time (figure 5).
In conclusion, the prepared fluorescent magnetic nano-composite with the core-shell structure has excellent magnetic response capability, can specifically target the liver cancer CTC on the surface based on the target GPC3, and provides possibility for in-vitro identification and detection of the liver cancer CTC.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. The fluorescent magnetic nano-composite is characterized by comprising magnetic nanoparticles, wherein mesoporous silica layers are coated on the surfaces of the magnetic nanoparticles, fluorescein is loaded in mesopores of the mesoporous silica layers, and phosphatidylinositol proteoglycan-3 targeting factors are modified on the surfaces of the mesoporous silica layers.
2. The fluorescent magnetic nanocomposite of claim 1, wherein the magnetic nanoparticles comprise iron, cobalt, nickel and oxides thereof, preferably γ -Fe2O3、Fe3O4、CoFe2O4And Co3O4(ii) a Further preferably Fe3O4。
3. The fluorescent magnetic nanocomplex of claim 1, wherein said fluorescein comprises coumarin-6, nile red, cy5.5, IR780, Dil and other hydrophobic fluorescein; preferably, the fluorescein is coumarin-6 and nile red.
4. The fluorescent magnetic nanocomposite of claim 1, wherein the glypican-3 (GPC3) targeting factor is modified on the surface of the mesoporous silica layer by modifying a linker molecule on the surface of the mesoporous silica layer to provide an active group; then the connecting molecule is connected with a GPC3 targeting factor through a chemical bond;
preferably, the GPC3 targeting factors include GC33 mab, YP7 mab, HN3 mab, HS20 mab, GPC3 polypeptide, and combinations thereof;
the linking molecules include, but are not limited to, carboxyl, amino, hydroxyl, thiol, maleimide, and azide groups.
5. The fluorescent magnetic nanocomposite of claim 1, wherein the fluorescent magnetic nanocomposite has an average particle size of 110 nm.
6. The method for preparing the fluorescent magnetic nanocomposite as claimed in any one of claims 1 to 5, wherein the preparation method comprises:
s1, mixing the magnetic nanoparticles, a template agent and a silicon source for reaction, and removing the template to obtain the magnetic mesoporous silica nanoparticles;
s2, loading fluorescein into the mesopores by adopting a passive drug loading method to obtain fluorescent magnetic mesoporous silica nanoparticles;
s3, modifying the connecting molecules on the surface of the silicon layer to provide active groups, and connecting the connecting molecules with GPC3 targeting factors through chemical bonds.
7. The method of claim 6, wherein the magnetic nanoparticles comprise iron, cobalt, nickel, and oxides thereof; preferably gamma-Fe2O3、Fe3O4、CoFe2O4And Co3O4(ii) a Further preferably Fe3O4。
8. The method of claim 6, wherein the silicon source comprises sodium silicate of ordinary, high modulus, silica sol, tetraethyl orthosilicate (TEOS), methyl orthosilicate (TMOS), and bis- [3- (triethoxysilyl) propyl ] -disulfide;
the template agent is cetyl trimethyl ammonium bromide;
the fluorescein is coumarin-6, nile red, Cy5.5, IR780, Dil and other hydrophobic fluorescein; further preferred are coumarin-6 and nile red;
the linker molecule comprises carboxyl, amino, hydroxyl, thiol, maleimide and azide groups;
the GPC3 targeting factors include GC33 monoclonal antibody, YP7 monoclonal antibody, HN3 monoclonal antibody, HS20 monoclonal antibody, GPC3 polypeptide, and combinations thereof.
9. Use of the fluorescent magnetic nanocomplex of any of claims 1 to 5 in any one or more of:
1) specifically capturing circulating tumor cells of the liver cancer and/or preparing products for specifically capturing the circulating tumor cells of the liver cancer;
2) detecting and analyzing the circulating tumor cells of the liver cancer and/or preparing a product for detecting and analyzing the circulating tumor cells of the liver cancer;
3) products for early diagnosis of liver cancer and/or early diagnosis of liver cancer.
10. A method for specifically capturing circulating tumor cells of liver cancer, which comprises adding the fluorescent magnetic nanocomposite of any one of claims 1 to 5 to a sample to be tested.
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