CN111420054B - Preparation method and application of magnetic gold nanoparticles - Google Patents
Preparation method and application of magnetic gold nanoparticles Download PDFInfo
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Abstract
The invention relates to the technical field of biological materials, in particular to a preparation method and application of magnetic gold nanoparticles. The invention mediates the composition of the gold nanoparticles and the magnetic nanoparticles by using microorganisms as carriers, and the gold nanoparticles form a colloidal gold layer with the surface appearance of the microorganisms on the surfaces of the magnetic nanoparticles by means of the microorganisms. The method has simple process steps, easy operation, low toxicity or nontoxicity of the used reagent, low preparation cost and more suitability for large-scale production; meanwhile, through microbial mediation, the magnetic nano-microspheres can adsorb more gold nanoparticles, so that more active molecules can be coupled, the binding capacity is stronger, and the titer of the magnetic gold nanoparticles coupled with the active molecules is effectively improved.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of magnetic gold nanoparticles mediated by microorganisms serving as adsorption carriers based on a microorganism template method, the magnetic gold nanoparticles obtained by the method and application of the magnetic gold nanoparticles.
Background
In recent years, magnetic nanomaterials are increasingly used in the medical field, including organ imaging and cell tracking, drug and gene delivery, tumor hyperthermia, cell separation, and the like. The surface of the magnetic nano material can be coupled with various antibodies, cells and drugs after being modified by various chemical groups to play a role. Fe 3 O 4 Magnetic particles are common magnetic cores, and due to the characteristics of high chemical activity, easy oxidation, easy agglomeration in neutral solution, and lack of functional groups, other materials are generally required to be coated to improve the stability and function of the magnetic particles. The gold (Au) nanoparticles are Fe nanoparticles with the characteristics of no toxicity, stable chemical properties, good biocompatibility, weak interaction and chemical bond coupling with biomacromolecules, easy firm combination with organic molecules containing sulfydryl and the like 3 O 4 The magnetic particles are preferably coated. The magnetic gold nanoparticles can be combined with specific antigens or antibodies through the gold nanoparticles, and have wide application in the fields of immunological detection, cell separation, targeted drug delivery and the like. However, gold nanoparticles were coated directly on Fe 3 O 4 Magnetic particles are very difficult and usually first coated with a material such as silica 3 O 4 Forming a surface-modified nuclear shell layer, and combining amino (-NH) 3 ) Then the nano-gold is coated by amino. The method has the problems of high reagent toxicity, multiple steps, high cost and the like, and also has the problems of limited specific surface area, weak adsorption capacity and the like, so that the development of a simple and convenient preparation method of the magnetic gold nanoparticles, which can realize higher gold nanoparticle composite efficiency, is urgently needed.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a high-efficiency preparation method of magnetic gold nanoparticles based on a microbial template method, the magnetic gold nanoparticles prepared by the method, application of the magnetic gold nanoparticles and a magnetic product prepared by taking the magnetic gold nanoparticles as a carrier.
In order to achieve the purpose, the invention creatively utilizes the excellent performance of the microorganism (small volume, unique surface structure and morphological characteristics, biological activity, monodispersity and light transmittance, the cell membrane structure of the microorganism can adsorb magnetic particles and metal nanoparticles such as gold nanoparticles through electrostatic attraction, van der waals force, receptor ligand interaction force and the like, the metal nanoparticles can enter the inside of an organism through free diffusion, membrane structure damage, endocytosis or pinocytosis and the like), takes the microorganism cells as a carrier and a template, mediates the composition of the gold nanoparticles and the magnetic nanoparticles, and develops a novel method for preparing the magnetic gold nanoparticles. The method has the advantages of simple steps, low toxicity and low cost. Compared with the traditional core-shell structure of the magnetic nano-microsphere, the specific surface area of the microbial cells as the carrier is greatly improved, more gold nano-particles can be adsorbed and formed into a three-dimensional structure to be compounded on the surface of the magnetic nano-microsphere, the gold nano-particle compounding rate of the magnetic gold nano-particles prepared by the method is higher, the magnetic carrier coupling active component has higher titer and stronger binding capacity.
Specifically, the technical scheme of the invention is as follows:
the invention provides a preparation method of magnetic gold nanoparticles, which mediates the composition of the gold nanoparticles and magnetic nanoparticles by taking microorganisms as carriers.
Including but not limited to bacteria, fungi, and viruses. Such bacteria include, but are not limited to, Escherichia coli, Staphylococcus aureus, and the like. The size of the microorganism may be 1nm to 1 mm.
Specifically, the preparation method comprises the following steps: adsorbing and absorbing gold ions or gold nanoparticles by using microorganisms, then interacting with the magnetic nano-microspheres, and adding a reducing agent to catalyze the gold ions or gold nanoparticles to form a colloidal gold layer on the surfaces of the microorganisms.
Preferably, the particle size of the magnetic nano-microsphere is 10-5000 nm, the surface of the magnetic nano-microsphere is positively charged and has a coupling group, and the magnetic nano-microsphere can be used as a magnetic core to form a compound.
More preferably, the magnetic nanospheres can be any magnetic nanospheres which have a particle size of 10-200 nm, have a coupling group and can be used as a magnetic core to form a compound, such as: its responsiveness to magnetic fields may be ferromagnetic, paramagnetic or superparamagnetic; it can be homogeneous magnetic nano-microsphere or a mixture of multiple magnetic nano-microspheres; it can be biodegradable or non-biodegradable magnetic nano-microspheres; it may be a magnetic nanosphere that can be monitored by optical microscopy, electron microscopy or Magnetic Resonance Imaging (MRI) or Magnetic Particle Imaging (MPI).
As an embodiment of the invention, the magnetic nanospheres are Fe 3 O 4 Magnetic nanospheres, including but not limited to Fe 3 O 4 @SiO 2 Nanospheres, Fe 3 O 4 @SiO 2 -Epoxy nanospheres, Fe 3 O 4 @SiO 2 -NH 3 AuNPs nanospheres and the like. The magnetic nano-microsphere can be a commercial product or can be prepared by adopting the conventional technical means in the field.
The preparation method of the magnetic gold nanoparticles specifically comprises the following steps:
(1) culturing microorganisms in a culture medium containing gold ions or gold nanoparticles, wherein the concentration of the gold ions or the gold nanoparticles is 0.5-15 mM;
(2) mixing the microorganism obtained in the step (1) with the magnetic nano-microspheres at 25-38 ℃ for incubation for 1-5 h, and adding a fixing agent for fixation after incubation is finished; in the incubation system, the ratio of the microorganisms to the magnetic nano microspheres is (10) 2 ～10 5 )cfu：100mg；
(3) Adding a reducing agent into the fixed reaction product obtained in the step (2) to react for 1-10 h at 30-37 ℃, and collecting precipitate after the reaction is finished;
in the step (1), the gold ions enter the microbial cells through the actions of free diffusion, membrane penetration, endocytosis or endocytosis and the like. Preferably, the initial concentration of the microorganism is 10 2 ～10 5 cfu/mL，The concentration of the gold ions or the gold nanoparticles is 0.5-5 mM. The culture is carried out under the temperature condition that the microorganism is suitable for growth.
In the step (2), the magnetic nano-microspheres are combined with the microorganisms with negative charges through acting forces such as electrostatic attraction, van der waals force, receptor ligand interaction force and the like.
Preferably, the ratio of the microorganisms to the magnetic nanospheres is (10) 2 ～10 5 )cfu：50mg。
Preferably, the fixing agent is one or more selected from glutaraldehyde, formaldehyde, acetaldehyde, paraformaldehyde, and the like. More preferably, the addition amount of the fixing agent is 1-10% of the volume percentage of the fixing agent in the reaction system.
In the step (3), gold ions are subjected to reduction reaction to generate gold nanoparticles, and a colloidal gold layer with a three-dimensional structure is formed on the surface of the microorganism according to the morphological characteristics of the microorganism.
The reducing agent may be any typical reducing agent having a reducing action, including but not limited to: active metal simple substance (such as Na, Al, Zn, Fe, etc.), active metal hydride (such as lithium aluminum hydride LiAlH) 4 Etc.), a non-metal element having a reducing action (e.g., H) 2 C, Si, etc.), non-metal elements with reducing action (e.g. C, S, etc.), alkali metal simple substances (e.g. Li, Na, K, etc.), oxides at low valence (e.g. CO, SO, etc.) 2 Etc.), non-metal hydrides (e.g. H) 2 S、NH 3 、HCl、CH 4 Etc.), salts at low valences (e.g., Na) 2 SO 3 、FeSO 4 Etc.), ascorbic acid, stannous chloride (SnCl) 2 ) Oxalic acid (H) 2 C 2 O 4 ) Potassium borohydride (KBH) 4 ) Sodium borohydride (NaBH) 4 ) Ethanol (C) 2 H 5 OH), and the like.
Preferably, the reducing agent is selected from ascorbic acid, potassium borohydride (KBH) 4 ) Sodium borohydride (NaBH) 4 ) One or more of (a). More preferably, the reducing agent is added in an amount such that the concentration thereof in the reaction system is 1 to 10 mM.
The preparation method further comprises the following steps after the precipitate is collected: washing the precipitate to remove immunogenic material.
Preferably, the removing the immunogenic substance comprises: inactivating bacteria, removing protein, nucleic acid and pyrogen.
As a preferable embodiment of the present invention, the method for preparing magnetic gold nanoparticles comprises the following steps:
(1) synthesis of Fe with positive surface charge (positive zeta potential) 3 O 4 Magnetic nanospheres (magnetic cores);
(2) the initial concentration is 10 2 ～10 5 culturing cfu/mL escherichia coli or staphylococcus aureus in a culture medium containing 0.5-2 mM chloroauric acid for 12-16 h under the condition of a temperature suitable for growth;
(3) mixing and incubating the microorganism obtained by culturing in the step (2) and the magnetic core obtained in the step (1) at the temperature of 30-37 ℃ for 1-4 h; in the incubation system, the microorganism is mixed with the Fe 3 O 4 The proportion of the magnetic nano microspheres is (10) 2 ～10 5 ) cfu: 50 mg; after the incubation is finished, adding glutaraldehyde to a final concentration of 2.5-5%, and fixing for 1-2 h;
(4) adding 2.5-5 mM ascorbic acid after the fixation reaction is finished, shaking uniformly, placing on a water bath shaking table with the temperature of 37 ℃ and the rotating speed of 100-150 r/min for reaction for 4-8 hours, and collecting precipitate after the reaction is finished;
(5) washing the precipitate collected in the step (4) by adopting a PBS buffer solution and deionized water respectively;
(6) adding 0.2-0.5% formaldehyde solution into the washed precipitate, preserving heat at 37 ℃, sterilizing for 48 hours, and then treating at 160-180 ℃ for 1.5-3 hours or at 240-250 ℃ for 25-35 minutes to remove pyrogens; and washing and drying the gold nanoparticles by using deionized water to obtain the magnetic gold nanoparticles.
On the other hand, the invention also provides the magnetic gold nanoparticles prepared by the preparation method.
The invention provides a magnetic gold nanoparticle, which comprises a magnetic nanoparticle core and gold nanoparticles distributed on the magnetic nanoparticle core according to the surface topography of microorganisms.
In another aspect, the present invention provides any one of the following applications of the preparation method or the magnetic gold nanoparticles:
(1) the application of the magnetic carrier in magnetic drug or magnetic detection reagent;
(2) the use in the manufacture of a medicament for targeted therapy, an agent for organ imaging or in vivo tracking;
(3) use in cell separation.
The invention provides a magnetic targeted drug, which comprises magnetic gold nanoparticles and an active ingredient coupled with the magnetic gold nanoparticles; the active ingredient is a substance or cell capable of coupling with gold nanoparticles.
Preferably, the active ingredient is a protein or a nucleic acid.
The bispecific antibody (BsAb) is a novel therapeutic antibody, has stronger permeability to tumors compared with the traditional therapeutic antibody, can recruit effector T cells around target cells through CD3, and has the advantages of better killing effect on tumor cells, low toxicity, low safety risk and the like. However, BsAb also exists (1) a shorter half-life, resulting in relatively weak function; (2) the effect concentration in solid tumors and large tumors is not achieved, so that the effect on the tumors is poor; (3) off-target effects on tumors and the resulting risk of cytokine storm. The magnetic gold nanoparticles provided by the invention are used as carriers to couple the bispecific antibody, so that the stability, the function and the targeting property of the bispecific antibody can be obviously improved.
The invention provides Fe 3 O 4 The bispecific antibody coupled with Au composite nano particles is Fe 3 O 4 the/Au composite nano particles are coupled with the bispecific antibody; said Fe 3 O 4 the/Au composite nano-particles are prepared by the preparation method of the magnetic gold nano-particles.
The bispecific antibody can interact with the Fe by any mode of action 3 O 4 Au complex nanoparticle coupling, for example: by Au-S bond with said Fe 3 O 4 and/Au composite nano-particle coupling.
Fe as mentioned above 3 O 4 The structure of the/Au composite nanoparticle coupled bispecific antibody is as follows: fe 3 O 4 The magnetic core, the gold nanoparticle structure formed according to the surface topography of the microorganism and the bispecific antibody combined with the gold nanoparticle. The bispecific antibodies include antibodies that target recognition of tumor antigens and antibodies that recruit, activate, immune cells.
The antibody targeted to recognize the tumor antigen can be any antibody that can recognize the tumor antigen, such as HER2, CD20, CD19, and the like.
The present invention provides Fe 3 O 4 The preparation method of the/Au composite nanoparticle coupled bispecific antibody comprises the following steps:
(1) adding two monoclonal antibodies (mass ratio of 1:1) and the Fe into PBS 3 O 4 The Au composite nano particles (the dosage of which is 250-300 times of the total mass of the monoclonal antibody) are mixed and react for 1-2 hours at room temperature on a 360-degree shaking table;
(2) centrifuging, collecting the precipitate, washing with deionized water, and drying.
The invention also provides said Fe 3 O 4 The application of the bispecific antibody coupled with Au composite nano particles in the preparation of tumor targeted therapeutic drugs.
Fe as described above 3 O 4 The application and the action mechanism of the bispecific antibody coupled with Au composite nano particles in tumor immunotherapy are as follows:
(1) recognizing and promoting the killing of tumor cells: bridging immune cells and tumor cells through two antigen recognition domains of the bispecific antibody to form a T cell-magnetic gold nanoparticle coupled bispecific antibody-tumor cell complex, inducing formation of cytotoxic immune synapse, promoting activation of T cells, mediating production of cytokines (IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-10, etc.), cytotoxic substances (perforin, granzyme), promoting proliferation of T cells and lysing target cells; further, Fe 3 O 4 The bispecific antibody coupled with Au composite nanoparticles retains the Fc segment of the antibody, and can exert effectFc segment mediated ADCC and CDC effects which are not possessed by the common bispecific antibody (the common bispecific antibody depends on single-chain coupling, does not contain an Fc segment and does not have ADCC and CDC functions); simultaneously, the specific magnetic-thermal injury effect of the magnetic nano particles can be exerted;
(2) enrichment: the targeting migration, enrichment and killing of tumor cells of immune cells at local tumor are realized through the two aspects of action of magnetic induction and tumor antigen-antibody combination;
(3) and (3) reducing transfer: can capture and enrich free tumor cells and reduce the metastasis of the tumor cells;
(4) antibody immobilization: can provide an immobilization carrier for the reaction of the CD 3/tumor antibody and a target antigen, and is more favorable for recognizing the tumor antigen, killing tumor cells and recruiting and activating effector T cells;
(5) storage of the antibody: can store the antibody, prevent the antibody from degrading outside the target site, and part of Fe is generated after the tumor cells are killed 3 O 4 The bispecific antibody coupled with the Au composite nano-particles can be released again to continuously generate secondary damage (bystander effect) to peripheral tumor cells;
(6) by Fe 3 O 4 The bispecific antibody mediated tumor killing T cells coupled with the Au composite nano particles has a tumor memory function, and can continuously kill tumors in the whole body to obtain subsequent effects;
(7)Fe 3 O 4 the/Au composite nanoparticle coupled bispecific antibody and the combined tumor cells and immune cells thereof can be detected by MRI or MPI (magnetic particle imaging system), thereby realizing in vivo tracing of the drug and the tumor site.
The invention has the beneficial effects that:
the invention provides a novel method for preparing magnetic gold nanoparticles by using microbial cells as carriers and templates to mediate the composition of the gold nanoparticles and the magnetic nanoparticles, and the method and the magnetic gold nanoparticles prepared by the method have the following advantages:
(1) the method has simple process steps, easy operation, low toxicity or nontoxicity of the used reagent and low preparation cost, and is more suitable for large-scale production;
(2) through the mediation of a microbial cell carrier, the magnetic nano-microspheres can adsorb more gold nanoparticles, so that more active molecules can be coupled, the binding capacity is stronger, and the titer of the magnetic gold nanoparticles coupled with the active molecules is effectively improved;
(3) the magnetic gold nanoparticles prepared by the method can play a role in storing and protecting small molecule active molecules (such as antibodies and the like), reduce the degradation of the magnetic gold nanoparticles in vivo and prolong the half-life period of the magnetic gold nanoparticles;
(4) the magnetic gold nanoparticles prepared by the method provide a solid phase surface for the reaction of active molecules such as antigen-antibody and the like, and play a role of an immunologic adjuvant; meanwhile, the local active molecule concentration is effectively improved, and the effect of the active molecules is improved;
(5) the magnetic gold nanoparticle coupling bispecific antibody prepared by the method can realize targeted migration under the guidance of a magnetic field, and the occurrence of off-target effect is reduced; meanwhile, tumor cells and immune cells can be locally enriched, tumor metastasis is reduced, and killing reaction is enhanced; and produces bystander effect and subsequent effect, and increases the effect of killing tumor; in addition, the magnetic gold nanoparticles can also play the functions of magnetic-thermal killing, in-vivo imaging tracing and the like.
Drawings
Fig. 1 is a schematic flow chart of a method for preparing ferromagnetic gold nanoparticles based on an escherichia coli template in embodiment 1 of the present invention.
FIG. 2 is a common optical microscope photograph of ferromagnetic gold nanoparticles based on E.coli template and ferromagnetic gold nanoparticles based on Staphylococcus aureus template in examples 1 and 2 of the present invention; wherein A is ferromagnetic gold nanoparticles based on an escherichia coli template, and B is ferromagnetic gold nanoparticles based on a staphylococcus aureus template.
Fig. 3 is a scanning electron microscope image of ferromagnetic gold nanoparticles based on staphylococcus aureus template in example 2 of the present invention.
FIG. 4 is the result of the detection of the adsorption rate of the ferromagnetic gold nanoparticles and Fe3O4@ SiO2@ Au respectively adsorbing the CD3 antibody in example 5 of the present invention; wherein A is Fe3O4@ SiO2@ Au adsorption experiment; and B is ferromagnetic gold nanoparticle adsorption experiment based on the escherichia coli template.
FIG. 5 is a graph showing the results of the in vitro mediation of the binding between LY8 tumor cells and peripheral blood mononuclear cells by ferromagnetic gold nanoparticles coupled with CD3 and CD20 bispecific antibody based on the E.coli template method in example 6 of the present invention; wherein, A is an antibody group; and B is a group without antibodies.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of ferromagnetic gold nanoparticles based on E.coli templates
This example provides a method for preparing ferromagnetic gold nanoparticles based on an escherichia coli template, which includes the following steps (a schematic flow diagram of the preparation method is shown in fig. 1):
(1) magnetic Fe 3 O 4 Synthesis of nanospheres
Weighing 9.44g FeCl 3 ·6H 2 O and 6.9022g FeCl 2· 4H 2 O, adding 160ml of deionized water for dissolution;
secondly, mechanically stirring at 500rmp, heating to 80 ℃ while stirring, slowly dripping 20ml of concentrated ammonia water by using a dropper to obtain an alkaline environment, and continuously heating and stirring for 6 hours;
standing and precipitating, separating magnetic mineral nano particles by using a magnet, standing until black particles are completely absorbed to the bottom of the cup, and discarding the liquid; washing with deionized water and anhydrous ethanol for 4 times;
drying in a vacuum drying oven to obtain magnetic Fe 3 O 4 And (4) nano microspheres.
(2) Magnetic Fe 3 O 4 @SiO 2 Synthesis of nanospheres
Taking 1g of Fe 3 O 4 Adding 100ml of deionized water and 400ml of absolute ethyl alcohol, and carrying out ultrasonic dispersion for 15min (the amplitude is 50%);
slowly dropping 5mL tetraethyl silicate (TUS) into the central vortex at the speed of about 1 drop/s under the mechanical stirring of 470 rpm; slowly dropping 5mL of strong ammonia water into the central vortex for about 1 drop/2 s; sealing, and keeping 470rpm for mechanical stirring for 5-6 h;
standing and precipitating, separating magnetic mineral nano particles by using a magnet, standing until black particles are completely absorbed to the bottom of the cup, and discarding the liquid; washing with deionized water and anhydrous ethanol for 4 times;
drying in a vacuum drying oven to obtain magnetic Fe 3 O 4 @SiO 2 And (4) nano microspheres.
(3) Magnetic Fe 3 O 4 Synthesis of @ nano microsphere
Weighing 400mg of Fe 3 O 4 @SiO 2 Adding 100ml of toluene into a round-bottom flask, and distilling at 80 ℃ under reduced pressure to extract 50ml of solution;
adding 50ml of toluene into the round-bottom flask, adding 1ml of 3-aminopropyl and triethoxysilane (APTES), adding 50mg of 4-Dimethylaminopyridine (DMAP), and sealing and reacting for 24 hours at 110 ℃ on a rotary evaporator;
taking down the round-bottom flask, standing for precipitation, separating magnetic ore nanoparticles by using a magnet, standing until black particles are completely absorbed to the bottom of the cup, and discarding the liquid; washing with anhydrous ethanol for 6 times;
drying in vacuum drying oven to obtain magnetic Fe 3 O 4 @ nano-microsphere.
(4) Streaking a refrigerated escherichia coli strain on an LB agar plate, culturing for 14 hours in a constant-temperature incubator at 37 ℃, then selecting a single colony to be inoculated in an LB liquid culture medium containing 1mM chloroauric acid, carrying out amplification culture at the constant temperature of 37 ℃ for 16 hours, centrifuging to obtain a bacterial precipitate, and washing for 2 times by PBS for later use;
(5) mixing the microorganism obtained in the step (4) and the Fe obtained in the step (3) 3 O 4 @ nano microsphere with 10 4 cfu: mixing at a ratio of 50mg, and incubating at 37 ℃ for 2 hours; after the incubation is finished, adding glutaraldehyde to a final concentration of 2.5%, and fixing for 1h at room temperature;
(6) adding 2.5mM ascorbic acid after the fixation reaction is finished, shaking uniformly, placing on a water bath shaking table with the temperature of 37 ℃ and the rotating speed of 150r/min for reaction for 6 hours, and collecting precipitate after the reaction is finished;
(7) washing the precipitate collected in the step (6) for 3 times by adopting PBS buffer solution with the volume 10 times that of the bacterial culture system, and then washing the precipitate for 3 times by adopting deionized water with the volume 10 times that of the bacterial culture system;
(8) blowing off the washed precipitate, adding 0.5% formaldehyde solution, sealing, mixing, and sterilizing at 37 deg.C for 48 hr; inoculating the inactivated product to nutrient agar plate and anaerobic broth liver culture medium, culturing at 37 deg.C for 48 hr, and performing aseptic inspection; then the mixture is treated at the high temperature of 180 ℃ for 2 hours to remove pyrogens; and washing with deionized water and drying to obtain the ferromagnetic gold nanoparticles based on the escherichia coli template.
The structural morphology of the prepared ferromagnetic gold nanoparticles based on the escherichia coli template was observed by using a common optical microscope, and the result is shown in a of fig. 2.
Example 2 preparation of ferromagnetic gold nanoparticles based on a staphylococcus aureus template
This example provides a method for preparing ferromagnetic gold nanoparticles based on a staphylococcus aureus template, which differs from example 1 only in that escherichia coli in step (4) is replaced with staphylococcus aureus.
The structural morphology of the prepared ferromagnetic gold nanoparticles based on the staphylococcus aureus template is observed by a common optical microscope and a scanning electron microscope respectively, wherein the observation result of the common optical microscope is shown as B in figure 2, and the observation result of the electron microscope is shown as figure 3.
Example 3Fe 3 O 4 Coupling of the @ E.coli-Au Complex anti-CD 3 and CD20 BsAbProduction of Heteroantibodies
This example provides a Fe3O4@ -E.coli-Au complex conjugated anti-CD 3 and CD20 bispecific antibody prepared as follows:
(1) 0.1mg of anti-CD 3 antibody, 0.1mg of anti-CD 20 antibody and 50mg of ferromagnetic gold nanoparticles based on the E.coli template prepared in example 1 were added to 1ml of PBS and mixed, and reacted for 1 hour at room temperature on a 360 ℃ shaker;
(2) centrifuging at 10000rpm for 5min, collecting precipitate, washing with deionized water, oven drying and storing.
Example 4Fe 3 O 4 Preparation of anti-CD 3 and CD20 bispecific antibody coupled with @ Staphylococcus aureus-Au complex
This example provides a Fe3O4@ -Staphylococcus aureus-Au complex conjugated anti-CD 3 and CD20 bispecific antibody prepared as follows:
(1) 0.1mg of anti-CD 3 antibody, 0.1mg of anti-CD 20 antibody and 50mg of the ferromagnetic gold nanoparticles based on the staphylococcus aureus template prepared in example 2 are added into 1ml of PBS for mixing, and the mixture is reacted for 1 hour at room temperature on a 360-degree shaking table;
(2) centrifuging at 10000rpm for 5min, collecting precipitate, washing with deionized water, oven drying and storing.
Example 5 detection of the ability of ferromagnetic gold nanoparticles based on E.coli template to adsorb CD3 antibody
Taking ferromagnetic gold nanoparticles synthesized by the method in example 1 based on an escherichia coli template, wherein the ferromagnetic gold nanoparticles contain 50mg of Fe3O4@ nano microspheres; taking Fe3O4@ SiO2@ Au with equal amount of substances as a reference;
respectively incubating ferromagnetic gold nanoparticles based on an escherichia coli template and Fe3O4@ SiO2@ Au in 1mL of PBS reaction system containing 0.2mg/mL of CD3 antibody for 1h under the conditions of 360-degree shaking table and 37 ℃;
centrifuging at 10000rpm for 5min after incubation is finished, and taking supernatant;
the absorbance of the system before and after the incubation reaction, a1 (PBS containing 0.2mg/mL CD 3) and a2 (supernatant after adsorption), were measured with a spectrophotometer, and the adsorption rate was calculated as (a1-a2)/a1 × 100%.
The results are shown in fig. 4, and show that the adsorption rate of CD3 antibody of ferromagnetic gold nanoparticles based on escherichia coli template is 80%; the adsorption rate of the CD3 antibody of Fe3O4@ SiO2@ Au is 50.2%, and the adsorption of the ferromagnetic gold nanoparticles based on the escherichia coli template is obviously better.
Example 6Fe 3 O 4 Activity assay of the anti-CD 3 and CD20 bispecific antibody coupled with the @ E.coli-Au Complex
This example utilizes in vitro binding assays to determine that the Fe3O4@ -E.coli-Au complex coupled to anti-CD 3 and CD20 bispecific antibody prepared in example 3 mediates LY-8 tumor cells (CD 20) + ) The activity of binding to peripheral blood mononuclear cells (containing T cells) is specifically as follows:
(1) isolation of peripheral blood mononuclear cells (PBMC, containing CD 3) + T cells 45% -70%);
(2) obtaining LY-8 cells in logarithmic growth phase (CD 20) + )；
(3) Preparing IMDM complete culture medium containing 10 μ L/ml glutamine, 500U/ml IL-2 and 10% fetal calf serum, resuspending LY-8 cells and PBMC with the above culture medium, inoculating into 24-well plate, adding 1 × 10 per well 5 LY-8 cells and 2X 10 6 PBMC; setting an antibody-containing group and an antibody-free group, wherein each group is provided with 3 repeated holes, and the antibody-containing group is added with a coupling bispecific antibody with a final concentration of 0.2 mg/ml; to add 1X 10 5 LY-8 cells, 2X 10 6 Wells of PBMC and media as controls; 37 ℃ and 5% CO 2 And culturing for 12h under the saturated humidity condition, and observing the result by using a common optical microscope.
The results are shown in FIG. 5, and show that the antibody group can mediate the aggregation of LY-8 tumor cells and peripheral blood mononuclear cells to form a cell mass (A of FIG. 5); the cell distribution was more dispersed in the antibody-free group (B in FIG. 5).
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (13)
1. A preparation method of magnetic gold nanoparticles is characterized in that microorganisms are used as carriers to mediate the composition of the gold nanoparticles and magnetic nanoparticles; the method comprises the following steps:
(1) culturing microorganisms in a culture medium containing gold ions or gold nanoparticles, wherein the concentration of the gold ions or the gold nanoparticles is 0.5-15 mM;
(2) mixing the microorganisms obtained in the step (1) with the magnetic nano-microspheres at 25-38 ℃ for incubation for 1-5 h, and adding a fixing agent for fixation after incubation is finished; in the incubation system, the ratio of the microorganisms to the magnetic nano microspheres is (10) 2 ～10 5 )cfu：100mg；
(3) And (3) adding a reducing agent into the fixed reaction product obtained in the step (2), reacting for 1-10 h at 30-37 ℃, and collecting the precipitate after the reaction is finished.
2. The method of manufacturing according to claim 1, comprising: adsorbing and absorbing gold ions or gold nanoparticles by using microorganisms, then interacting with the magnetic nano-microspheres, and adding a reducing agent to catalyze the gold ions or gold nanoparticles to form a colloidal gold layer on the surfaces of the microorganisms.
3. The preparation method according to claim 1 or 2, wherein the magnetic nanospheres have a particle size of 10-5000 nm, have a positive charge on the surface, have a coupling group, and can form a complex as a magnetic core.
4. The method according to claim 3, wherein the magnetic nanospheres are Fe 3 O 4 Magnetic nano-microspheres.
5. The preparation method according to claim 1, wherein the fixing agent is one or more selected from glutaraldehyde, formaldehyde, acetaldehyde, paraformaldehyde, and/or the reducing agent is one or more selected from ascorbic acid, potassium borohydride, and sodium borohydride.
6. The method according to claim 5, wherein the fixing agent is added in an amount of 1 to 10% by volume in the reaction system, and/or the reducing agent is added in an amount of 1 to 10mM in the reaction system.
7. The method according to any one of claims 1, 2, and 4 to 6, further comprising, after collecting the precipitate: washing the precipitate to remove immunogenic material.
8. The magnetic gold nanoparticle is characterized by comprising a magnetic nanoparticle core and gold nanoparticles distributed on the magnetic nanoparticle core according to the surface topography of microorganisms;
the magnetic gold nanoparticles are prepared by the preparation method of any one of claims 1 to 7.
9. The use of the magnetic gold nanoparticles of claim 8 in any one of the following (1) to (3):
(1) the application in preparing magnetic medicines or magnetic detection reagents;
(2) the use in the manufacture of a medicament for targeted therapy, an agent for organ imaging or in vivo tracking;
(3) use in cell separation.
10. A magnetic targeted drug, characterized in that it comprises the magnetic gold nanoparticle of claim 8 and an active ingredient coupled to the magnetic gold nanoparticle; the active ingredient is a substance capable of coupling with gold nanoparticles.
11. The magnetically targeted drug of claim 10, wherein the active ingredient is a protein or a nucleic acid.
12. A bispecific antibody coupled with magnetic gold nanoparticles is characterized in that the bispecific antibody is obtained by coupling the magnetic gold nanoparticles with the bispecific antibody; the magnetic gold nanoparticles are Fe 3 O 4 The magnetic gold nanoparticles are prepared by the preparation method of any one of claims 1 to 7.
13. Use of the magnetic gold nanoparticle-conjugated bispecific antibody of claim 12 for the preparation of a tumor-targeted therapeutic drug.
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