CN112426536B - Magnetic targeting Fe3O4Nano-composite and preparation method and application thereof - Google Patents
Magnetic targeting Fe3O4Nano-composite and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a magnetic targeting Fe3O4A nano-particle compound and a preparation method and application thereof belong to the technical field of biological medicine. The invention provides a simple, easy and efficient shallow solid tumor treatment mode, namely NK-Fe formed by CD56 antigen-antibody action under the action of external magnetic force, namely applying strong magnetic field on a tumor part3O4The compound can sense the action force of a magnetic field, so as to reach a tumor part and kill and eliminate the tumor, thereby having good value of practical application.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to magnetic targeting Fe3O4A nano-composite, 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.
The incidence and mortality of tumors are high, and the trend is rising in recent years, the current main tumor treatment modes include several modes, including traditional operation treatment, radiotherapy and chemotherapy, and in recent years, targeted treatment and immunotherapy appear, wherein the targeted treatment and the immunotherapy have great potential. Targeted therapies are directed primarily to molecules of the tumor, such as specific antibodies. Immunotherapy mainly comprises TCR-T, TIL, CIK, DC-CIK, CAR-NK, CAR-T and the like, and at present, the immunotherapy mode becomes a new field of tumor therapy and continuously brings surprise to human beings.
Although numerous, the current methods for treating tumors still have unsatisfactory treatment effects, including some FDA approved therapies, and although significant, the high cost of treatment is prohibitive. Particularly in the aspect of solid tumors, the tumor microenvironment is very complex, hypoxia and low Ph are realized, and a large number of immunosuppressive cells exist, so that the immunosuppressive effector cells are inhibited in different modes to play a role in protecting the growth and division of the tumors. NK cells are innate immune cells that function independently of antigen presentation, are not MHC-dependent, and are not prone to GVHD, while T cell functions are limited by these limitations, and NK cells have completed treatment of hematological tumors (Kheng New Chinese medicine receptor T-cell Therapy for solid tumors. molecular Therapy Oncology.2007; 3,16006), but have failed to achieve satisfactory results in the treatment of solid tumors. NK-92 cells are a killer tumor cells, and since FDA approval in the United states, NK-92 cells have been used clinically for many years. Although NK-92 has a certain killing ability in vitro, especially on K562 cells, the in vivo effect is limited due to the poor targeting ability.
NK-92 cells highly express CD56 molecule, CD56, also known as neural cell addition molecule-1(NCAM-1) on their surface. NK-92CD56highsubsets primarily kill tumors by the TNF family (FasL, TRIAL, mTNF), whereas CD56lowThe substes are to kill the tumor by perforin and granzyme. CD56 is an adhesion molecule that exerts regulatory functions by cellular cognate cell-cell interactions (Hinsby, a.m., Berezin, V).&Bock, E.molecular mechanisms of NCAM function, 2004.front Biosci 9, 2227-2244.), Ghina Taouk et al report that CD56 positive breast cancer cells can form an immunological synapse with CD56 molecules and CD56 molecules on the surface of NK-92 cells, improving the cytotoxic ability of NK-92 cells to breast cancer cells (Ghina Taouk et al, CD56expressor in Breast cancer cells expressing the sensitivity to natural killer-mediated cytotoxicity treating the formation of cytotoxic immunological synthesis.2019. scientific results.9: 8756). NK cell killing is mainly through perforin-granzyme B pathway and Fas pathway to exert killing effect, and some cytokines such as IFN-gamma. The NK cell can further activate the NK cell by immune synapse formed after the NK cell recognizes the target cell, release granzyme and directly act on the target cell, so that the target cell is killed.
Fe3O4The nano particles have superparamagnetism, so the nano particles can show rapid and continuous targeting action capability under the action of a magnetic field, and have great application potential in the field of drug delivery. And Fe3O4The nanoparticles have very good biocompatibility, nontoxicity and stability, which determine their stability and safety in vivo, and in addition, Fe in vivo3O4The nanoparticles can be detected by NMR imaging technique for Fe3O4The nanoparticles are tracked.
Disclosure of Invention
In view of the problems in the prior art, the present invention is directed to providing a magnetic target of Fe3O4Nanoparticle composites and methods of making and using the same. The invention provides a simple, easy and efficient shallow solid tumor treatment mode, namely NK-Fe formed by CD56 antigen-antibody action under the action of external magnetic force, namely applying strong magnetic field on a tumor part3O4The compound can sense the action force of a magnetic field, so as to reach a tumor part and kill and eliminate the tumor, thereby having good value of practical application.
In order to solve the technical problems, the technical scheme of the invention is as follows:
in a first aspect of the invention, a magnetic target of Fe is provided3O4Nanoparticle complexes of magnetically targeted Fe3O4The nano-particle compound is NK-Fe3O4Nanoparticle composites, in particular, said Fe3O4The surface of the nanoparticle is connected with an anti-CD 56antibody, and the anti-CD 56antibody is connected with NK-92 immune cells.
In a second aspect of the present invention, there is provided the above magnetically targeted Fe3O4A method of preparing a nanoparticle composite, the method comprising:
mixing Fe3O4The nanoparticles are incubated with DMSA to form DMSA-modified Fe3O4Nanoparticles, and then adding anti-CD 56antibody to the nanoparticles and incubating to obtain Fe3O4-CD56 magnetic beads; then co-incubating with NK-92 immune cells.
In a third aspect of the present invention, there is provided the above magnetically targeted Fe3O4Use of a nanoparticle complex for the preparation of a drug delivery system.
In a fourth aspect of the present invention, there is provided a drug delivery system comprising the above magnetically targeted Fe3O4A nanoparticle composite.
In a fifth aspect of the present invention, there is provided the above-mentioned magnetically targeted Fe3O4Application of nanoparticle compound and/or drug delivery system in preparation of antitumor drugsThe application in the field of medicine.
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme reports a magnetic targeting Fe for the first time3O4Nanoparticle composites and methods of making and using the same. The invention relates to NK-Fe formed by CD56 antigen-antibody under the action of external magnetic force, namely applying strong magnetic field on tumor part3O4The compound can sense the action force of a magnetic field, so that the compound reaches a tumor part, kills and eliminates the tumor, and effectively inhibits the growth of tumor cells, thereby having good practical application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings according to the provided drawings without creative efforts.
FIG. 1 is a photograph of the detection of CD56 antigen on the surface of NK-92 cells in the examples of the present invention;
FIG. 2 is a graph of material characterization in an example of the invention;
wherein: a is Fe3O4Nanoparticle electron microscopy transmission images; b is an XRD spectrum; c is Fe3O4Carrying out superparamagnetic detection on the nanoparticles; d is Fe3O4-DMSA sample detection; e is a Zeta potential test chart; f is Fe3O4Detecting the nuclear magnetic imaging property of the nano particles; g is HRTEM imaging.
FIG. 3 shows Fe in the example of the present invention3O4Nanoparticle toxicity detection and NK-92 cell binding CD56 magnetic bead map;
wherein: a is Fe at different dosages3O4Effect of nanoparticles on 293T cell and human T cell activity; b is the effect of CD56 magnetic beads with different doses on apoptosis of 293T cells and human T cells; c is the flow result of combining CD56 magnetic beads and pure magnetic beads with NK-92 cells; d is copolymerization of CD56 magnetic beads and NK-92 cellsA scorch result; e is the scanning result of the electron microscope combining CD56 magnetic beads and pure magnetic beads with NK-92 cells; f is a flow chart, and CD56 magnetic beads are combined in NK-92 cells at different time points; g is an electron microscope scanning image, and CD56 magnetic beads are combined in NK-92 cells at different time points.
FIG. 4 is a graph showing the ability of CD56 magnetic beads to proliferate and kill NK-92 cells in an example of the present invention;
wherein: a is a line graph of the influence of CD56 magnetic beads on the proliferation capacity of NK-92 cells; b is the influence of CD56 magnetic beads with different effective target ratios and different doses on K562 cell killing by NK-92 cells; c is a graph of the influence of CD56 magnetic beads on the release of cytokines in the process of killing K562 cells by NK-92 cells under the condition of different effective target ratios; d is a graph of the effect of different doses of CD56 magnetic beads on the cytokine release during the K562 cell killing process by NK-92 cells.
FIG. 5 is a diagram showing the verification of the oriented movement of the magnetic bead complex of NK-CD56 under the action of magnetic force in the example of the present invention;
wherein: a is a screenshot of rapid movement of the NK-CD56 magnetic bead under the action of a magnet; b is a transwell experiment design drawing, an upper small chamber is NK-CD56 magnetic beads or NK-pure magnetic bead compounds, a lower small chamber is K562 cells, and a magnet is adhered to the bottom of a small hole; c is the condition of NK-92 cells with green fluorescence which migrate to the lower chamber observed under a fluorescence microscope; d is a flow chart of NK-92 cells migrating to the lower chamber; e is a histogram of the killing of K562 cells by migrating NK-92 cells; f shows the result of the cytokine released during the killing process of the migrating NK-92 cells to K562 cells.
FIG. 6 is a series of graphs of magnetic targeting mediated killing of H22 cells in NK-CD56 magnetic beads in vivo in an example of the present invention;
wherein: a is a mouse experimental flow chart, BALB/c nude mice are injected with H22 cells for molding, effect compounds are respectively returned at 7 th, 11 th and 15 th days for subsequent observation and monitoring; b is a mouse tumor nuclear magnetic resonance effect graph and a tumor site SNR histogram; c shows the results of the tumor sizes of the mice in each group at 14 days and 22 days; d is an in vitro presentation graph of tumors of each group of mice at 16 days; e is a graph of the change in body weight of the mice; f is a mouse tumor growth change diagram; g is a mouse survival curve graph; results for Granzyme B content in mouse tumor homogenate and serum at day 16H; i is prussian blue staining pattern of tumor sections at 16 days, magnification: x 400, (scale bar 50 μm); j is tumor h.e. staining and hCD45 antigen IHC staining, magnification: x 400 (scale bar 50 μm).
FIG. 7 is a series of magnetic targeting mediated killing of B16 cells in NK-CD56 magnetic beads in vivo in an example of the present invention;
wherein: a is a mouse experimental flow chart, a C57BL/6N mouse is injected with B16 cells for molding subcutaneously, and an effect compound is returned on the 7 th day for subsequent observation and monitoring; b is a graph of the change of the body weight of the mice; c is a mouse tumor growth change chart; d is the mouse survival plot.
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 example embodiments according to the present application. 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 stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, although there are numerous ways to treat tumors, the treatment effect is still unsatisfactory; NK-92 cell is a tumor cell with killing property, although NK-92 has a certain killing ability in vitro, especially for K562 cell, because of poor targeting ability, the in vivo effect is limited.
In one embodiment of the present invention, a magnetic target of Fe is provided3O4Nanoparticle complexes of magnetically targeted Fe3O4The nano-particle compound is NK-Fe3O4Nanoparticle composites, in particular, said Fe3O4The surface of the nanoparticle is connected with an anti-CD 56antibody, and the anti-CD 56antibody is connected with NK-92 immune cells.
In one embodiment of the present invention, the Fe3O4The particle size of the nano particles is 10-50 nm, and the preferable particle size is 20 nm. Magnetic targeting Fe designed by the invention3O4Nanoparticle composite, under the action of external force magnetic field, Fe3O4Can guide NK-92 immune cells to a tumor part, thereby killing the tumor cells and effectively inhibiting the growth of the tumor cells.
In one embodiment of the invention, the anti-CD 56antibody is an anti-human CD56 antibody.
In one embodiment of the present invention, there is provided the above-mentioned magnetically targeted Fe3O4A method of preparing a nanoparticle composite, the method comprising:
mixing Fe3O4The nanoparticles are incubated with DMSA to form DMSA-modified Fe3O4Nanoparticles, and then adding anti-CD 56antibody to the nanoparticles and incubating to obtain Fe3O4-CD56 magnetic beads; then co-incubating with NK-92 immune cells.
In one embodiment of the present invention, the Fe3O4The nano particles are prepared and synthesized by a ferric oleate high-temperature thermal decomposition method; specifically, the preparation method comprises the following steps: adding ferric chloride and sodium oleate into an organic solvent, heating for reaction to obtain ferric oleate, adding oleic acid and octadecene, and reacting at high temperature to obtain the product. Fe prepared by the method3O4The nano particles have uniform size and good dispersibility.
In one embodiment of the invention, the mass ratio of ferric chloride to sodium oleate is 1: 1-5.
In a specific embodiment of the invention, the organic solvent is a mixed solution of ethanol, n-hexane and water, and the volume ratio of the ethanol, the n-hexane and the water is 60-90:120-160: 40-80; preferably 80:140: 60.
In one embodiment of the present invention, Fe3O4The specific method for the incubation reaction of the nanoparticles and the DMSA comprises the following steps: mixing Fe3O4The nanoparticles were added to an aqueous DMSA solution (containing Na)2CO3) Adding tetrahydrofuran, performing ultrasonic treatment and vibrating treatment to obtain the product.
In one embodiment of the present invention, the Fe3O4The mass ratio of the nanoparticles to the DMSA is 0.5-2: 1, and preferably 1: 1.
In one embodiment of the present invention, the ultrasonic treatment is performed for 0.2 to 1 hour, and the shaking treatment is performed for 0.5 to 2 hours.
In one embodiment of the invention, the Fe is incubated with anti-CD 56antibody3O4The specific method of CD56 magnetic beads is as follows: mixing Fe3O4Dispersing the-DMSA into water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform incubation reaction, and then adding an anti-CD-56 antibody to perform incubation to obtain the anti-CD-56 antibody.
In one embodiment of the present invention, Fe3O4Control of Fe after Dispersion of DMSA in Water3O4The concentration of DMSA is 0.1-5 mg/mL, preferably 1mg/mL, and the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1-3: 4-8, preferably 2: 5;
in one embodiment of the present invention, the specific method of co-incubation with NK-92 immune cells is: combining NK-92 immune cells with Fe3O4The CD56 magnetic beads are incubated at 35-40 deg.C (preferably 37 deg.C) for 10-20min (preferably 15min), or at room temperature for 20-40 min (preferably 30 min).
In one embodiment of the present invention, there is provided the above-mentioned magnetically targeted Fe3O4Use of a nanoparticle complex for the preparation of a drug delivery system.
In one embodiment of the present invention, there is provided a drug delivery system comprising the above magnetically targeted Fe3O4A nanoparticle composite.
In one embodiment of the present invention, there is provided the above-mentioned magnetically targeted Fe3O4Nano meterUse of a particle complex and/or a drug delivery system as described above in the preparation of an anti-tumour drug.
The anti-tumor medicament provided by the invention can be prepared into different conventional dosage forms (dosage form) by adding appropriate auxiliary materials and additives by utilizing the known technology in the field, such as injection, freeze-dried powder for injection, liposome suspension, tablets, gels, implants and the like, and can be administered by intravenous injection, local injection, implantation, inhalation and other routes.
It is to be noted that tumors are used in the present invention as known to those skilled in the art. Benign tumors are defined as cellular hyperproliferation that fails to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs).
In yet another embodiment of the invention, the medicament of the invention is useful for treating malignant tumors. Examples of malignant tumors that can be treated with the drug of the present invention include solid tumors and hematological tumors. Solid tumors may be, for example, tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx, tongue and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva. Malignant tumors include hereditary cancers such as squamous cell carcinoma, melanoma, retinoblastoma, and nephroblastoma, among others. Furthermore, malignant tumors include primary tumors in the organs and corresponding secondary tumors in distant organs (tumor metastases). Hematological tumors can be, for example, aggressive and indolent forms of leukemia and lymphoma, i.e., non-hodgkin's disease, chronic and acute myeloid leukemia (CML/AML), Acute Lymphocytic Leukemia (ALL), hodgkin's disease, multiple myeloma, and T-cell type lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, carcinoid syndromes, and cancers of unknown primary site and AIDS-related malignancies.
In another embodiment of the present invention, the anti-tumor drug of the present invention is suitable for treating superficial solid tumors, such as squamous cell carcinoma, melanoma, tongue cancer, etc., in combination with a secondary targeting technique.
It should be noted that malignant tumors do not necessarily require the formation of metastases in the distal organs. Some tumors exert devastating effects on the primary organ itself through their aggressive growth properties. These can lead to structural destruction of tissues and organs, ultimately resulting in failure of function and death of the organ in question.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
1. Test method
1.1 Fe3O4Synthesis of nanoparticles and CD56 magnetic beads
Fe3O4The nano-particles are synthesized by a high-temperature thermal decomposition method of iron oleate. First, iron oleate, 10.8g FeCl, was prepared3·6H2O (Chinese medicine) and 36.5g of sodium oleate (Meclin) are added into a mixed solution of 60mL of deionized water, 80mL of ethanol (Chinese medicine) and 140mL of n-hexane (Chinese medicine), the mixed solution is heated and stirred for 4 hours at the temperature of 70 ℃, and the target product iron oleate is purified by liquid separation and distillation. Adding the obtained iron oleate into 5.7g of oleic acid (Meclin) and 200g of octadecene (avastin), mixing uniformly, heating to 320 ℃, keeping the temperature for half an hour, cooling to room temperature after the reaction is finished, adding excessive ethanol to precipitate Fe3O4The nanoparticles were washed three times with ethanol.
Washed Fe3O4The nanoparticles were modified with carboxyl groups by reaction with DMSA (2, 3-dimercaptosuccinic acid, michelin). 20mg of DMSA dissolved in Na2CO3Adding 20mg Fe into deionized water solution of the traditional Chinese medicine3O4Adding 1mL of tetrahydrofuran (traditional Chinese medicine) into the nano particles, reacting for half an hour under the ultrasonic condition, and then reacting for 1 hour on a shaking table to obtain the DMSA modified Fe3O4Particles (Fe)3O4-DMSA)。
Mixing Fe3O4Dispersing DMSA into water at a concentration of 1mg/mL, adding 2mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (source leaf) and 5mM N-hydroxysuccinimide (source leaf), standing at room temperature for half an hour, adding 50. mu.L of mouse anti-human CD56antibody (BD Pharmingen) with Pe/cy7 fluorescent dye, reacting overnight at 4 ℃, and centrifuging and purifying the obtained product to obtain Fe3O4CD56 magnetic beads (CD56 magnetic beads).
The size, uniformity and lattice information of the synthesized nano magnetic beads are characterized by a Transmission Electron Microscope (TEM) and a high-resolution transmission electron microscope (HRTEM). Fe3O4-DMSA and Fe3O4The stability of the CD56 magnetic beads was characterized by the Zeta potential test. The synthetic magnetic bead crystal structure information was tested by X-ray diffraction analysis of the pattern. And analyzing the functional group information on the surface of the magnetic bead by Fourier transform infrared spectroscopy so as to judge the modification condition of the DMSA and the CD 56. The magnetic properties of the synthetic beads were analyzed by vibrating the sample magnetometer. And magnetic imaging properties of the synthetic magnetic beads were tested by magnetic resonance imaging (MRI (GE Signa HDx 3.0T MRI, USA)) using T2 imaging mode, and whether cells reached the tumor site was verified 24 hours after tail vein infusion of NK-CD56 magnetic bead complex. Binding of CD56 magnetic beads as well as pure magnetic beads and NK92 cells was shown by scanning electron microscopy (SEM (SEM S-4800, Hitachi, Japan)). Fe was tested using a confocal fluorescence microscope (Axio-Imager LSM-800, ZESSI, Germany)3O4Binding of CD56 magnetic beads to the surface of NK92 cells. The directional movement of NK-CD56 magnetic bead complexes under a permanent magnet was observed using an inverted microscope (Olympus Corporation, IX 73). Granzyme B and IFN- γ in cell supernatants, serum and tumor homogenates were detected using the human Granzyme B kit (proteintech) and the human IFN- γ kit (proteintech), with the detection procedure being carried out strictly according to the instructions.
1.2 flow cytometry, immunohistochemical staining (IHC) and Prussian blue staining
Take 1X 105Mu.l of CD56 magnetic beads or pure magnetic beads are added into NK-92 cells, incubated for 15min at 37 ℃ in the dark or incubated for 30min at normal temperature in the dark, washed for three times with PBS, and then analyzed by a flow cytometer (Beckman).
Each group of tumor tissue sections (4. mu.M) were deparaffinized stepwise in xylene and different gradient alcohols, then in 3% H2O2Heating in the solution for antigen retrieval, sealing with goat serum at room temperature for 1h, and staining primary antibody: mouse anti-human CD45 antibody dilutions were treated for 2h at 4 ℃. And then washing with PBS for three times, washing for 10min each time, treating in horseradish peroxidase-labeled anti-mouse antibody diluent (Dako, DK) for 1h, then staining DAB, washing with running water, then counterstaining with hematoxylin, washing with running water, and finally dehydrating in xylene and gradient alcohol step by step. After the blocking of the neutral resin, the resin was observed under an Olympus OP80 microscope and photographed.
Detection of Fe on tumor slice tissue using Prussian blue detection kit (Solebao)3O4The process is strictly operated according to the specification.
1.3 CD56 magnetic beads affecting NK-92 cell proliferation and killing experiment
The CD 56-bound magnetic beads and untreated NK-92 cells were added to a 96-well plate, 3000 cells per well, 10. mu.L of Cell Counting Kit-8 (KeyGen BioTECH, CN) detection solution was added to each well before detection, and the reaction was carried out at 37 ℃ in the dark for 1h, and the absorbance value at 450nm wavelength was measured in each well on a microplate reader. Assays were performed at 0, 24, 48 and 72h after cell plating.
Experiments in which magnetic beads of CD56 affected the killing ability of NK-92 cells were evaluated by measuring the amount of Lactate Dehydrogenase (LDH) released from dead cells, which were K562 cells and B16 cells, using CytoTox
The Non-Radioactive cytoxicity Assay (Promega, USA) kit is operated strictly according to the experimental instructions, and the specific steps are as follows: adding 1X 10 to 24-well plate5Target cells were added to CD 56-bound magnetic beads or unbound NK-92 cells in a final system of 1.5 mLSetting four effective target ratios of 0.25:1, 0.5:1, 1:1 and 2:1 respectively, changing the amount of CD56 magnetic beads incubated with NK-92 to 0, 0.5, 1, 2, 5 and 10 mu L respectively under the condition that the effective target ratio is 1:1, incubating effector cells and target cells for 6h, centrifuging and collecting supernatant, and detecting the amount of LDH released by each group of target cells by using the kit. The detection formula is as follows:
killing efficiency (%) ═ experimental group LDH release amount-effector cell LDH release amount-target cell LDH release amount)/(target cell total postmortem LDH release amount-target cell LDH release amount)
1.4 Fe3O4Nanoparticle non-toxicity test
Human T cells and 293T cells with Fe respectively3O4Nanoparticle co-incubation validation of Fe3O4The nanoparticles are nontoxic, and are prepared by adding 1 × 10 nanoparticles into 24-well plate5Individual cell, with 1. mu. LFe3O4Nanoparticle co-culture for 10h, followed by CytoTox
The Non-Radioactive cytotoxin Assay kit detects the death condition of cells. In addition, FITC Annexin V Apoptosis Detection Kit I (BD biosciences) was used to detect the toxicity of CD56 magnetic beads to NK-92 cells.
1.5 migration experiment of NK-CD56 magnetic beads under magnetic force (Transwell experiment)
The Tranwell model is established as follows: lower chamber is placed at 1X 105K562 cells were cultured in MEM-alpha medium (Invitrogen, Carlsbad, Calif.) containing 10% fetal bovine serum in a system of 500. mu.L, and NK-CD56 magnetic beads or NK-pure magnetic beads prestained with CFSE were placed on the upper layer and cultured in MEM-alpha medium containing 1% fetal bovine serum in a system of 100. mu.L with an effective target ratio of 0.25:1 and 2: 1. A standard circular permanent magnet with a diameter of 1.5cm was attached to the bottom of the cell and contained 5% CO at 37 deg.C2The culture box is cultured for 12 hours, and then detection is carried out. The upper chamber was removed and the NK-CD56 magnetic beads that migrated to the lower layer were observed under an Olympus OP80 microscope; collecting the cells in the lower layer, and detecting each group of NK-92 cells migrating to the lower layer on a flow cytometer; in addition, theThe level of Granzyme B, IFN- γ and LDH in the supernatant was measured.
1.6 Effect experiment of NK-CD56 magnetic beads in mice
To determine whether magnetic targeting-mediated NK-92 cells bound to CD56 magnetic beads could function in mice, a mouse model was constructed. Female SPF-grade BALB/C nude and C57BL/6N mice 6-8 weeks old were used for modeling: BALB/c nude mice right side back subcutaneous injection 1.8X 105H22 cells, and returning NK-CD56 magnetic beads and the like for three times on days 7, 11 and 15; c57BL/6N mice right side dorsal subcutaneous injection 2X 105B16 cells, NK-CD56 magnetic beads and the like are returned once on day 7. After all mice received the reinfusion, a permanent magnet with a diameter of 1cm was attached to the tumor site and the duration of magnet action was 6-12 h. After randomized cohort, 6 mice per group were given the following reinfusion: NK-92 cells after group 1 feedback 100 μ L PBS and group 2 feedback and 100 μ L CD56 magnetic beads co-incubation, totaling 1X 106NK-92 cells after co-incubation of individual cells, group 3 feedback and 100. mu.L of pure magnetic beads, amounting to 1X 106And (4) cells. Mice were weighed 3-4 times a week and mouse tumor size was calculated according to the following formula:
Nuclear magnetic imaging was performed on nude mice on day 16, then the mice were euthanized, tumors were photographed, the tumor size was observed, then the tumors were ground, and the cytokine content in the tumor homogenate and serum was examined.
2. Test results
2.1 Synthesis of magnetic beads of NK cells-CD 56 and verification of their non-toxicity to cells
In Fe3O4The nano particles show that one layer of DMSA is stably combined to form the Fe modified by the DMSA3O4Nanoparticles (hereinafter, collectively referred to as pure magnetic beads) are then stably bound to the surface of the pure magnetic beads, and mosseanti-humanCD 56antibody (BD Pharmingen) with Pe/Cy7 fluorescence is stably bound to the surface of the pure magnetic beads (hereinafter, collectively referred to as CD 56-magnetic beads). NK-92 cells express CD56 antigen (FIG. 1), and if necessary, NK-92 cells and CD56 magnetic beads (or pure magnetic beads, as control) are incubated together at 37 deg.C for 15min or at room temperature for 30min at an incubation ratio of 1 × 105Mu.l of each NK-92 cell was added. Different gradient amounts (0, 0.5. mu.l, 1. mu.l, 2. mu.l, 5. mu.l and 10. mu.l) of Fe were added3O4Nanoparticles and CD56 magnetic beads were added to well-grown 293T cells and peripheral blood T cells of healthy human origin to evaluate their toxicity to tumor cells and normal cells.
The results show that: fe3O4The diameter of the nano-particles is 20nm, the nano-particles are not agglomerated and have good dispersibility (figure 2). The size distribution of the synthesized nano magnetic beads is in the interval of 18-22nm and has good uniformity through TEM test (FIG. 2A). High Resolution Transmission Electron Microscopy (HRTEM) imaging showed that the synthesized nanocrystals had good crystallinity (fig. 2G). By comparing XRD patterns with Fe3O4Standard card comparison showed that the synthesized product was Fe3O4(FIG. 2B), the corresponding standard cards are 19-0629. Vibrating the sample magnetometer showed that the synthesized magnetic beads had superparamagnetism (fig. 2C) and their magnetic saturation intensity was 30 emu/g. Analysis by Infrared Spectroscopy, Fe3O4The DMSA sample contained a distinct C-O, C-O vibrational peak, demonstrating that the sample contained carboxyl groups that had been successfully modified on magnetic beads, i.e., DMSA had been modified on magnetic beads (fig. 2D). Fe3O4The CD56 sample contains obvious N-H vibration peak, and the CD56antibody is proved to be contained in the sample. Zeta potential test shows that Fe3O4The potential of-DMSA is-32 mV, Fe3O4The potential of-CD 56 is-41 mV, which proves that the surface potential of the nanoparticles is negative and has a larger negative value after the DMSA is modified, so that the solution has good stability and dispersibility, and the potential is further reduced after the CD56 is connected, so that the CD56 is proved to be successfully connected with Fe3O4On the surface of DMSA (FIG. 2E). Magnetic resonance imaging showed that the synthesized nanobeads had good imaging properties (fig. 2F). Fe3O4Both nanoparticles and CD56 magnetic beads were non-toxic to tumor and non-tumor cells and did not kill them (fig. 3A and 3B). Flow-through results showed that pure magnetic beads could not bind to NK-92 cells, while CD56 magnetic beads could bind to NK-92 cells (FIG. 3C); confocal results showed that CD56 magnetic beads could bindOnto NK-92 cells (FIG. 3D); scanning by electron microscopy showed that after co-incubation, pure magnetic beads could not bind to NK-92 cells, while CD56 magnetic beads could bind to NK-92 cells (FIG. 3E); after incubation of NK-92 cells and CD56 magnetic beads, detection was carried out at 2h and 12h, and flow and electron microscope scanning results show that CD56 magnetic beads are bound to the surfaces of the NK-92 cells (FIGS. 3F and 3G).
From the above experiments and the results thereof, the following conclusions can be drawn:
Fe3O4the nano-particles have good stability and superparamagnetism, do not cause damage to cells, still have no toxicity after being coated with CD56 antibodies to form CD56 magnetic beads, and can be combined on the surface of NK-92 cells for a long time, thereby laying a stable foundation for the continuous enrichment capacity of the NK-92 cells in vivo.
2.2 testing of the proliferation and killing ability of CD56 magnetic beads on effector cells:
taking a certain amount of NK-92 cells and CD56 magnetic beads for co-incubation, adding CCK-8 reagents in 0h, 24h, 48h and 72h respectively, and detecting whether the proliferation capacity of the cells is affected; NK-92 cells were incubated with a corresponding amount of CD56 magnetic beads, and the cells were added to K562 cell line, and the amount of target cells was unchanged (1X 10)5One), effective target ratio (effector cells: target cells) are 0.25:1, 0.5:1, 1:1 and 2:1, the cells are cultured for 6h together, the LDH release amount detection mode is adopted to detect whether the killing capacity of the NK-92 cells to the target cells K562 is changed after the CD56 magnetic beads are combined, meanwhile, under the condition that the effective target ratio is not changed at 1:1, the quantity of the CD56 magnetic beads (0, 0.5 mu l, 1 mu l, 2 mu l, 5 mu l and 10 mu l) is sequentially increased, whether the killing capacity of the NK-92 cells to the target cells K562 is changed after the CD56 magnetic beads are combined is detected, and the killing effect of the NK-92 cells to the mouse melanoma cell line B16 is detected in the same mode. CD56 is an adhesion molecule expressed on the surface of NK cells, and NK cells with low expression of CD56 can produce more cytokines to exert cytotoxic effects, while NK cells with high expression of CD56 exert cytotoxic effects in contrast (Cooper MA, Caligiuri MA 2004.isolation and characterization of human natural killer cell subsets curr protocol Immunol 34.Chapter 7: Unit 7.). Centrifuging at 12000g × 5min, collecting supernatant, and detecting killer effector molecule in the supernatant by ELISA methodRelease of Granzyme B and IFN- γ.
The results show that: CD56 magnetic beads did not affect the proliferation potency of NK-92 cells (FIG. 4A). Under the conditions of different effective target ratios, the killing capacity of NK-92 cells to suspended cells K562 is not influenced by CD56 magnetic beads, the experimental group has no obvious difference compared with the control group without CD56 magnetic beads (fig. 4B left), and the release amount of two cytokines in the supernatant accounts for the same problem (fig. 4C); along with the increasing of the addition amount of CD56 magnetic beads, the killing efficiency of NK-92 cells to K562 cells is not changed (right side of figure 4B), and meanwhile, the release amount of two effector molecules in supernatant is not obviously different from that of a control group (figure 4D), which indicates that the killing capacity of NK-92 cells to K562 cells is not influenced by increasing the amount of CD56 magnetic beads.
From the above experiments and the results thereof, the following conclusions can be drawn:
NK-92 cells are combined with CD56 magnetic beads, even if CD56 antigen is blocked, the killing capability of the NK-92 cells cannot be affected, and a solid foundation is laid for the research on the NK-92 cells for killing in vivo.
2.3 in vitro experiments, the directional migration ability of NK-92 cells combined with CD56 magnetic beads under the action of magnetic force is verified:
mu.l and 10. mu.l of CD56 magnetic beads were added to 5X 10, respectively5And (3) performing co-incubation in an NK-92 cell system to form an NK-CD56 magnetic bead complex system, adding a small piece of weak magnetic magnet into the NK-CD56 magnetic bead complex system, and observing the movement condition of the complex under an inverted microscope. A Transwell experiment was also designed, and as shown in FIG. 5B, the lower chamber was laid out 1X 106K562 cells, paving an NK-CD56 magnetic bead compound for pre-staining CFSE on the upper layer, incubating pure magnetic beads and NK-92 cells for pre-staining CFSE in a control group, setting 2 effective target ratios of 0.25:1 and 2:1 respectively, adhering a magnet with the diameter of 1.5cm to the bottom of each small chamber of each hole, placing the small chambers in an aseptic culture environment for 12 hours, observing the migration amount of the NK-92 cells to the lower layer and the killing condition of the K562 cells on the lower layer, collecting supernatant, and detecting the release amounts of Granzme B and IFN-gamma of the NK-92 cells in the supernatant by ELISA to measure the migration capacity of the NK-92 cells combined with CD56 magnetic beads and unbound CD56 magnetic beads under the action of magnetic force.
The results show that:
under the action of external magnetic force, the NK-CD56 magnetic bead complex can be clearly observed to move rapidly towards the magnet direction or along the magnetic induction line direction under an inverted microscope, the migration speed of the magnetic bead group with 10 mu lCD56 added is faster (video), and FIG. 5A is a screenshot of the rapid migration of the NK-CD56 magnetic bead complex. In the Transwell experiment, the lower chamber was observed by a fluorescence microscope, green cells were NK-92 cells of pre-stained CFSE that migrated from the upper chamber to the lower chamber, and the amount of NK-92 cells migrated from the magnetic bead set of NK-CD56 was significantly greater than that of the NK-pure magnetic bead set (FIG. 5C), especially the effective target ratio was 0.5: in case 1, the effect is more remarkable. The proportion of CFSE positive cells in the lower chamber was flow-detected and the results showed: when the effective target ratio is 0.25:1, the positive cell rates of the NK-CD56 magnetic bead group and the NK-pure magnetic bead group are respectively about 11% and 4%, and when the effective target ratio is 2:1, the positive cell rates of the NK-CD56 magnetic bead group and the NK-pure magnetic bead group are respectively about 45% and 35% (fig. 5D). The killing effect of NK-92 cells migrating to the lower chamber on K562 cells is detected, and the result shows that: the NK-CD56 magnetic bead group was significantly higher than the control group (fig. 5E), although the effective target ratio was 0.25:1, there was no significant difference between the two groups. When K562 cells were killed by NK-92 cells, granzyme B and IFN-. gamma.were released into the supernatant, and the magnetic bead group of NK-CD56 was significantly higher than the control group (FIG. 5F).
From the above experiments and the results thereof, the following conclusions can be drawn:
under the action of external magnetic force, NK-92 cells combined with CD56 magnetic beads can directionally migrate, which indicates that external magnetic field is opposite to Fe3O4The acting force of the nano-particles can carry NK-92 cells to move together, while the NK-92 cells and the pure magnetic beads in the control group are not combined, so that the directional migration is not generated under the action of magnetic force. The result well simulates the directional migration capability of the NK-CD56 magnetic bead complex under the action of in vivo magnetic targeting, and provides powerful guidance for the design and implementation of in vivo experiments.
2.4 magnetic targeting of Fe3O4Mode and effect of nanoparticle-coupled NK-92 cells in vivo killing of superficial solid tumors:
the experimental mice are SPF-grade BALB/C female nude mice and C57BL/6N female mice, are 6-8 weeks old and 18-22 g in weight, and are provided by Beijing Wintorlington experiment animal center, and the raising conditions are executed according to the SPF-grade animal standard.
BALB/C nude mice are inoculated with H22 cells subcutaneously on the right back for tumor-bearing modeling, C57BL/6N mice are inoculated with B16 cells subcutaneously on the right back for tumor-bearing modeling until the tumor grows to about 50mm3At time, mice were randomly divided into 3 groups, effector cells were reinfused: group 1 returned PBS, group 2 returned NK-pure magnetic bead complex, group 3 returned NK-CD56 magnetic bead complex, and magnetic patch was adhered to the tumor site after the return (FIG. 6A and FIG. 7A). And (3) observing the survival condition of the mice, monitoring the tumors and the weight change of the mice during the period, taking blood and tumors regularly, and detecting the content of the Granzyme B in serum and tumor homogenate. And (3) taking mouse tumors, slicing, and detecting the NK-92 cell infiltration condition by adopting Prussian blue staining and immunohistochemical staining. All experiments involving mice were performed exactly as required by the institutional animal care and use committee of Shandong university, and mice were euthanized when the tumor diameter reached 12 mm.
The results show that: for BALB/c nude mouse experiments, nuclear magnetic imaging results of tumor sites show that SNR (signal to noise ratio) of NK-pure magnetic bead groups and NK-CD56 magnetic bead groups are obviously lower than that of PBS (figure 6B), which indicates that Fe exists in tumor sites3O4The Prussian blue staining of the nano-particles and the tumor tissue sections shows that a large amount of Fe exists in tumor parts of NK-pure magnetic bead groups and NK-CD56 magnetic bead groups3+Ion, whereas PBS group did not (figure 6I). On day 14, significantly smaller tumors were observed in the NK-CD56 magnetic bead group than in the other two groups, which was increasingly evident on day 22 (FIG. 6C), and NMR imaging showed minimal tumors in NK-CD56 magnetic bead group mice (FIG. 6B). On day 16 3 mice were euthanized and tumors were stripped from each group and the NK-CD56 magnetic bead group was found to have minimal lumps with no apparent difference in the other groups (FIG. 6D). During the experiment, the weight changes of mice among groups are not very different (fig. 6E), compared with PBS group, the tumor of NK-CD56 bead group mice is obviously inhibited, the tumor of NK-pure bead group mice is not obviously changed (fig. 6F), the survival time of NK-CD56 bead group mice is longest and exceeds 32 days, and the survival time of other two groups of mice is about 25 days (fig. 6G). On day 16, NK-CD56 magnetic bead group bloodThe Granzyme B content was highest in the serum and tumor homogenates, and the serum content in the NK-pure magnetic bead group was significantly higher than in the PBS group (fig. 6H). The NK-92 cells highly express CD45 antigen molecules, the tumor sections are stained with anti-human CD45 antibody, and only the NK-CD56 magnetic bead group tumor tissues have CD45 positive cells (FIG. 6J).
For the C56BL/6N mouse experiment, the weight change of mice among groups is not obviously different (figure 7B), compared with the PBS group, the tumor of the NK-CD56 magnetic bead group mice is inhibited, but the difference among groups is larger, the tumor of the NK-pure magnetic bead group mice is not inhibited (figure 7C), and the survival period of the NK-CD56 magnetic bead group mice is prolonged by 4-5 days and is better than that of the other two groups (figure 7D).
From the above experiments and their results, the following conclusions were drawn:
coupled with Fe under the action of an in vitro magnetic field3O4NK-92 cells of the nanoparticles can reach the tumor site and inhibit the growth of the tumor. A magnet was also found in the tumor site of NK-pure magnetic bead group, but the tumor-suppressing effect was not exhibited because NK-92 cells were not recruited. NK-pure magnetic bead group mice contained NK-92 cells in peripheral blood, which interacted with normal cells of mice, and released a certain amount of Granzyme B (FIG. 6H), but did not sufficiently function in tumor suppression. The C56BL/6N mouse model is not as good as a BALB/C nude mouse, probably because the C57BL/6N mouse is a non-immunodeficiency mouse, immune cells in the mouse body can reject humanized NK-92 cells, and further the anti-tumor effect of the NK-92 cells in the body is interfered, and in addition, the anti-tumor capability of the NK-92 cells in the body is weaker because the anti-tumor effect is continuously achieved only by transfusion once. In summary, the results indicate that Fe3O4The nanoparticles are subjected to magnetic force, and have the capacity of carrying NK-92 cells to a superficial tumor site and inhibiting the growth of tumors. The invention constructs a new strategy of tumor immunotherapy, solves the problem that immune cells are difficult to effectively target the neck of tumor cells in the immune cell therapy process, and can utilize a magnetic targeting mode to deliver CAR-T cells or macrophages to tumor parts or inflammation parts based on the design concept of NK cells, thereby effectively improving the capacity of targeted therapy.
Tumors remain a public health problem affecting human health, a variety ofThe number of anti-tumor means is infinite, especially the immunotherapy of tumor shows a very attractive prospect, but the therapeutic effect is still unsatisfactory, and there are various disadvantages such as high cost. Fe3O4The nano-particles are stable, non-toxic and superparamagnetic materials, and are widely applied to various medical fields. Firstly, Fe with the diameter of 20nm is synthesized3O4Nanoparticles having a ratio of diameter to NK-92 cell diameter of about 1: 1000. NK-92 cell high expression CD56 antigen molecule capable of being combined with Fe3O4And the CD56 magnetic beads with the surface hatched with the CD56antibody and taking the nanoparticles as the core are stably combined, and the stable combination time is longer than 12 h. In vitro experiments show that NK-92 cells combined with CD56 magnetic beads directionally migrate under the action of magnetic force, and the migration speed is very high when observed under a microscope, which indicates that the magnetic force applied to the CD56 magnetic beads combined on the surface of the NK-92 cells is enough to provide continuous and powerful power for the NK-92 cells to target tumors. The results of in vivo experiments in mice show that the NK-CD56 magnetic beads can exert good effects in immunodeficient mice and normal mice, and are well verified in BALB/C and C57BL/6N mice, NK-92 cells combined with CD56 magnetic beads can directionally migrate to tumor sites under the action of external magnetic force to kill tumor cells, and meanwhile, lethal effector molecules Granzyme B and IFN-gamma are released, and to a certain extent, the release amount of Granzyme B and IFN-gamma represents the killing effect of NK-92 cells (Qingming Wang, et al. enhanced Cancer Immunotherapy with Smad 3-sized NK-92 cells.2018.Cancer immunology research.6 (8)). Meanwhile, the C57NL/6N mouse is not irradiated to eliminate immune cells in the mouse, so that host-versus-graft reaction can occur to a certain extent and NK-92 cells are inhibited to play a role, and the obtained effect is relatively poor. The invention is expected to play a role of throwing the brick and leading to some valuable technical changes, for example, CAR-T technology is the current anti-tumor immunotherapy technology with very hot fire, although the CAR-T technology is provided with a GPS navigation system, the CAR-T technology can have off-target effect in most cases, and if the CAR-T technology is combined with magnetic targeting technology to provide double targeting for CAR-T, the CAR-T technology should be large to some extentThe force improves the situation. Of course, for operational reasons, the magnetic targeting techniques of the present invention can only treat a few superficial tumors, such as squamous cell carcinoma, melanoma, tongue cancer, and the like. In this experiment, the CD56antibody was bound to Fe with a diameter of 20nm3O4The surface of the nano-particles is combined with NK-92 cells to form a complex, which proves that Fe is generated under the action of an external magnetic field3O4Can guide NK-92 cells to a tumor part, thereby killing the tumor cells and effectively inhibiting the growth of the tumor cells.
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 (21)
1. Magnetic targeting Fe3O4Nanoparticle composite, characterized in that the magnetic targeting of Fe3O4The nano-particle compound is NK-Fe3O4Nanoparticle composites of said Fe3O4The surface of the nanoparticle is connected with an anti-CD 56antibody, and the anti-CD 56antibody is connected with NK-92 immune cells.
2. Magnetically targeted Fe as claimed in claim 13O4Nanoparticle composite, characterized in that the Fe3O4The particle size of the nano particles is 10-50 nm;
the anti-CD 56antibody is an anti-human CD56 antibody.
3. Magnetically targeted Fe as claimed in claim 23O4Nanoparticle composite, characterized in that the Fe3O4The particle size of the nano-particles is 20 nm.
4. Magnetically targeted Fe of claim 1 or 33O4Nanoparticle compositionA process for the preparation of a compound, said process comprising:
mixing Fe3O4The nanoparticles are incubated with DMSA to form DMSA-modified Fe3O4Nanoparticles, and then adding anti-CD 56antibody to the nanoparticles and incubating to obtain Fe3O4-CD56 magnetic beads; then co-incubating with NK-92 immune cells.
5. The method of claim 4, wherein the Fe3O4The nano particles are prepared and synthesized by a ferric oleate high-temperature thermal decomposition method; the preparation method comprises the following steps: adding ferric chloride and sodium oleate into an organic solvent, heating for reaction to obtain ferric oleate, adding oleic acid and octadecene, and reacting at high temperature to obtain the product.
6. The method according to claim 5, wherein the mass ratio of ferric chloride to sodium oleate is 1: 1-5.
7. The method according to claim 5, wherein the organic solvent is a mixed solution of ethanol, n-hexane and water, and the volume ratio of ethanol, n-hexane and water is 60-90:120-160: 40-80.
8. The method according to claim 7, wherein the volume ratio of ethanol, n-hexane and water is 80:140: 60.
9. The method of claim 4, wherein Fe3O4The incubation reaction method of the nanoparticles and the DMSA comprises the following steps: mixing Fe3O4Adding Na-containing nanoparticles2CO3Adding tetrahydrofuran into the DMSA water solution, and performing ultrasonic treatment and oscillation treatment to obtain the DMSA water solution.
10. The method of claim 9, wherein the Fe is3O4The mass ratio of the nanoparticles to the DMSA is 0.5-2: 1.
11. The method of claim 10, wherein the Fe is3O4The mass ratio of nanoparticles to DMSA was 1: 1.
12. The method of claim 9, wherein the sonication is performed for 0.2 to 1 hour and the shaking is performed for 0.5 to 2 hours.
13. The method of claim 4, wherein the Fe is incubated with the anti-CD 56antibody3O4The method of CD56 magnetic beads is: mixing Fe3O4Dispersing DMSA into water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for incubation reaction, and then adding an anti-CD-56 antibody for incubation to obtain the anti-CD-56 antibody;
the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1-3: 4-8.
14. The method of claim 13, wherein Fe is3O4Control of Fe after Dispersion of DMSA in Water3O4The concentration of the-DMSA is 0.1-5 mg/mL.
15. The method of claim 14, wherein Fe is3O4Control of Fe after Dispersion of DMSA in Water3O4The concentration of DMSA was 1 mg/mL.
16. The method of claim 13, wherein the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2: 5.
17. The method of claim 4, wherein the co-incubation with NK-92 immune cells is performed by: combining NK-92 immune cells with Fe3O4-CD56 magnetic beads incubated at 35-40 deg.C10-20min, or 20-40 min at normal temperature.
18. The method of claim 17, wherein the NK-92 immune cells are incubated with Fe3O4-CD56 magnetic beads were incubated at 37 ℃ for 15min, or at room temperature for 30 min.
19. Magnetically targeted Fe of claim 1 or 33O4Use of a nanoparticle complex for the preparation of a drug delivery system.
20. A drug delivery system comprising the magnetically targeted Fe of claim 1 or 33O4A nanoparticle composite.
21. Magnetically targeted Fe of claim 1 or 33O4Use of a nanoparticle complex and/or a drug delivery system according to claim 20 for the preparation of an anti-tumor drug.
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| Surface specifically modified NK-92 cells with CD56 antibody conjugated superparamagnetic Fe3O4 nanoparticles for magnetic targeting immunotherapy of solid tumors;Zhao, SB et al.;《NANOSCALE》;20211014;第13卷(第45期);第19109-19122页 * |
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