CN113004418A - Specific antibody delivery platform and preparation method and application thereof - Google Patents
Specific antibody delivery platform and preparation method and application thereof Download PDFInfo
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- C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
- C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
The invention relates to a specific antibody conveying platform and a preparation method and application thereof. This specificity transfer platform, the mode through chemical bonding combines antibody and granule more stably, but specificity discernment NK cell and combine with it, can also further promote NK cell migration under the magnetic field effect to can swiftly realize the enrichment of NK cell at the tumour position high-efficiently, can obviously improve the immunotherapy effect. The compound has wide application prospect in medical detection, blood screening and targeted drug preparation.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a specific antibody delivery platform and a preparation method and application thereof.
Background
NK cells are an important component of the innate immune system. NK cells have multiple immune functions, can directly kill cells and secrete proinflammatory factors, and enhance the immune response of organisms. NK cells have direct killing ability to tumor cells, and anti-tumor immunotherapy based on NK cells is rapidly developing in the 21 st century. Although these therapies have not been clinically as successful as adoptive T cell therapies, the results of both preclinical and clinical trials have highlighted the enormous potential for NK cell-based tumor therapy. In the process of NK cell function, NK cells need to be enriched at tumor sites under the action of proinflammatory factors, then tumors are identified through a 'missing self' mode, tumor cells are directly killed through granzyme and perforin, or the NK cells play a role through releasing cytokines such as TNF and INF-gamma. Therefore, the effect of treating the tumor is achieved by enhancing the enrichment of the NK cells at the tumor site and utilizing the nonspecific killing ability of the NK cells to kill the tumor cells at the tumor site.
The currently available NK cell-based anti-tumor immunotherapies include three types: the first is that the surface receptor of NK cells is activated by cytokines or antibodies, the killing capacity of the NK cells at the tumor site is enhanced, but the cytokines and the antibodies are distributed all over the body, so that more serious side effects can be caused; secondly, the ability of the NK cells to recognize tumor cells is enhanced by modifying CAR on the surface of the NK cells, and the enrichment of the NK cells at the tumor sites is enhanced, but CAR-NK has the limitation that the CAR-NK can only be used for treating tumors with known specific antigens, but the specific antigens of most tumors are unknown at present, and the method does not fully utilize the nonspecific killing ability of the NK cells; the third is allo-adoptive NK cell transfer, which enhances the enrichment of NK cells at the tumor site by increasing the total amount of NK cells in vivo, but has the disadvantage that KIR on the surface of the returned NK cells may not be recognized by HLA of normal tissue cells, resulting in side reactions; in addition, the purity of the returned NK cells cannot be guaranteed, and in order to improve the purity of the NK cells, the sorting efficiency is inevitably reduced, so that a greater burden is caused to the original collection of the cells.
Therefore, if the enrichment of self NK cells in vivo at a tumor site can be improved, the action of NK cells at the tumor site can be promoted and the above-mentioned various side reactions can be avoided. Unfortunately, regulation of cell migration in vivo is currently immature and remains unknown.
Disclosure of Invention
Based on this, one of the purposes of the present invention is to provide a specific antibody delivery platform, which can bind to NK cells and promote NK cell migration, indicating that the platform has a wide application prospect in medical detection, blood screening and targeted drug preparation.
The specific technical scheme for realizing the purpose is as follows:
the preparation method of the specific antibody conveying platform comprises the steps of taking NK cells as targets, modifying the surfaces of ferroferric oxide nanoparticles, and chemically bonding and combining the ferroferric oxide nanoparticles and antibodies for specifically recognizing the NK cells under the action of a coupling agent.
In some embodiments, the above preparation method comprises the following steps:
(1) adjusting the ferroferric oxide nano particles with carboxylated surfaces to a proper concentration in an aqueous solution, and carrying out activation reaction on 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 1 hour to convert partial carboxyl on the surfaces of the particles into active ester;
(2) mixing the granular aqueous solution obtained in the step (1) and an antibody aqueous solution for specifically identifying NK cells according to a certain proportion, and reacting for 12 hours at 4 ℃;
(3) and separating and purifying the nanoparticles to obtain the antibody modified magnetic nanoparticles.
In some embodiments, the concentration of the ferroferric oxide nanoparticles in the step 1 is 0.5 mg/mL; the EDCI is used at a concentration of 0.05-5mmol/L, preferably 0.5-1.5 mmol/L; the NHS is used at a concentration of 0.075 to 7.5mmol/L, preferably 0.75 to 2.25 mmol/L.
In some embodiments, the mass ratio of the antibody to the ferroferric oxide nanoparticles in the step 2 is 20%; the concentration of the antibody is 0.0125-0.2mg/mL, preferably 0.1 mg/mL.
It is also an object of the present invention to provide a specific antibody delivery platform.
The specific technical scheme for realizing the purpose is as follows:
a specific antibody delivery platform is prepared by the preparation method.
In some embodiments, the specific antibody is at least one of a CD56 antibody, an anti-NK1.1 antibody, and a CD49b antibody.
In some of these embodiments, the specific antibody delivery platform is a spherical nanoparticle with a final concentration of 0.4mg/mL of particles and a final concentration of 0.08mg/mL of bound anti-NK1.1 antibody.
The invention also aims to provide an application of the specific antibody delivery platform in tumor detection and/or curative effect monitoring and/or prognosis judgment and/or personalized medication guidance.
The invention also aims to provide an anti-tumor medicament.
The specific technical scheme for realizing the purpose is as follows:
an anti-tumor pharmaceutical formulation comprising the specific antibody delivery platform described above.
In some embodiments, the pharmaceutical preparation further includes an adjuvant, preferably glucose, and further preferably a 5% glucose solution.
Compared with the prior art, the invention has the following beneficial effects:
the inventor of the invention develops a specific antibody delivery platform, which takes NK cells as targets, modifies the surfaces of ferroferric oxide nanoparticles, and chemically bonds and combines the ferroferric oxide nanoparticles and antibodies for specifically recognizing the NK cells under the action of a coupling agent. This specificity transfer platform, the mode through chemical bonding combines antibody and granule more stably, but specificity discernment NK cell and combine with it, can also further promote NK cell migration under the magnetic field effect to can swiftly realize the enrichment of NK cell at the tumour position high-efficiently, can obviously improve the immunotherapy effect. The compound has wide application prospect in medical detection, blood screening and targeted drug preparation.
Drawings
FIG. 1 is a schematic diagram showing the preparation of MNP @ NK1.1 obtained by antibody modification of MNP in example 1.
FIG. 2 is a graph showing the results of the optimization experiment in example 1, wherein FIGS. 2A and 2B are a surface potential detection graph and a particle size characterization graph, respectively, after the surface carboxyl groups of MNP are activated into active esters by using EDCI at different concentrations, and FIGS. 2C and 2D are a binding rate statistical graph and a binding condition statistical graph of corresponding MNP @ NK1.1 particles and NK cells, respectively, when the MNP is surface-modified with antibodies of different proportions.
FIG. 3 is a graph showing the results of detection of the binding pattern of the anti-NK1.1 antibody to MNP in example 1, wherein FIG. 3A is a schematic SDS-PAGE diagram, and FIG. 3B is a graph showing the results of SDS-PAGE.
Fig. 4 is a graph showing the detection results of MNPs and their bound antibodies in example 1, in which fig. 4A is a particle size diagram and fig. 4B is a scanning electron microscope diagram.
Fig. 5 is a graph of the binding efficiency of UPLC detection of anti-NK1.1 antibody to Fe3O4 magnetic nanoparticles in example 1, wherein fig. 5A is a graph of the peak shape of standard samples at different concentrations, and fig. 5B is a representation of the supernatant antibody concentrations of the control group and the experimental group.
FIG. 6 is a flow chart of the binding of particles (MNP @ NK1.1 and MNP @ IgG2a) to NK cells in example 2, wherein FIG. 6A is a flow chart of the binding of different concentrations of particles to NK cells; FIG. 6B is a statistical plot of the binding to NK cells at different incubation times for the corresponding MNP @ NK1.1 particle concentrations in 6A; FIG. 6C is a graph showing the comparison of the binding between control and experimental MNP @ IgG2a and NK cells.
FIG. 7 is a graph of the binding of particles (MNP @ NK1.1 and MNP @ IgG2a) to NK cells and T cells in PBMCs according to example 2, wherein FIG. 7A is a graph of the binding of particles to NK cells at different concentrations, FIG. 7B is a graph of the binding of particles to NK cells at different incubation times, FIG. 7C is a graph of the binding of MNP @ NK1.1 to T cells and NK cells, respectively, FIG. 7D is a statistical graph of the mean fluorescence intensity of FIG. 7B, and FIG. 7E is a statistical graph of the mean fluorescence intensity of 7C.
FIG. 8 is a diagram showing the binding of MNP @ NK1.1 particles to NK cells in laser confocal observation in example 2, wherein FIG. 8A is a diagram showing the fluorescent co-localization of NK cells and MNP @ NK1.1 particles in control and experimental groups, and FIG. 8B is a diagram showing the binding of MNP @ NK1.1 particles to NK cells in detail.
FIG. 9 is a graph showing the results of the examination of the ability of MNP @ NK1.1 particles to promote NK cell migration under the action of a magnetic field in example 3, wherein FIG. 9A is a schematic operation diagram, and FIG. 9B is a fluorescence microscope result diagram.
FIG. 10 is a schematic diagram of the experimental procedure of MNP @ NK1.1 particle mouse in example 4.
Fig. 11 is the results of the magnetic nanoparticles of example 4 for melanoma mice treated with the magnetic nanoparticles, wherein fig. 11A is a graph of the inhibition of melanoma growth by the magnetic nanoparticles; FIG. 11B is a graph showing the change in body weight of mice during the treatment, and FIG. 11C is a statistical graph showing the growth of tumors in each mouse.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The present invention relates to abbreviations and terms defined as follows:
MNP: magnetic nanoparticles and carboxyl functionalized ferroferric oxide nanoparticles.
EDCI: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
NHS: n-hydroxysuccinimide.
PEG-PLA: polyethylene glycol polylactic acid.
SEC column: a size exclusion chromatography column.
SEM: scanning electron microscopy.
DLS: dynamic light scattering.
Example 1 preparation of MNP @ NK1.1
Experimental materials:
anti-mouse NK1.1 antibody, purchased from Bio Cell, USA; MNP, available from sienna millennium biotechnology limited; EDCI, available from tokyo chemical industries co ltd (TCI); NHS, purchased from alatin, shanghai.
The preparation process is shown in figure 1, and MNP and an activating agent are combined to carry out esterification reaction, and partial surface carboxyl is activated into active ester; and reacting the MNP with the active ester with amino on an antibody capable of specifically recognizing the NK cells to obtain the magnetic nanoparticles capable of recognizing and combining the NK cells in vivo.
Performance characterization and optimization:
1.1 optimization of activator concentration:
the carboxyl on the surface of the particle can generate esterification reaction under the action of an activating agent to generate active ester, and the active ester further interacts with amino on the antibody to connect the particle and the antibody through amido bond. The ferroferric oxide nano particles are different from particles which are formed by assembling albumin, PEG-PLA and the like through the hydrophilcity and the hydrophobicity of materials, and the ferroferric oxide particles need to maintain the key stability of the ferroferric oxide nano particles in an aqueous solution through the electrostatic repulsion of surface charges. Therefore, the concentration of the activating agent for converting the carboxyl on the surface of the ferroferric oxide into the active ester needs to be in a certain range, so that the stability of the particles can be ensured, and enough active ester on the surface of the particles is ensured to interact with the antibody to generate amide chemical bonds.
As shown in fig. 2A, as the concentration of the activator EDCI was increased from 0 to 2.1mM (the concentration of NHS was always 1.5 times that of EDCI), the negative surface charge of the particles gradually decreased, representing a process of gradually converting the carboxyl groups on the surface of the particles into active esters. As shown in FIG. 2B, at lower activator concentrations, the particles remained stable in aqueous solution, increasing particle size from 100nm to 180nm as the EDCI concentration increased to 1.6mM, indicating that the particles gradually began to agglomerate and became unstable; when the EDCI concentration is increased to 2.1mM, the particle size reaches 600nm, and the electrostatic repulsive force generated by negative charges on the particle surface is insufficient to maintain the stability of the ferroferric oxide particles in the aqueous solution, so that the EDCI concentration lower than 1.6mM is selected for further optimization.
1.2 optimization of the modification ratio of the antibody on the surface of the particle:
the antibody is modified on the surface of the ferroferric oxide particle in order to realize the specific binding of the particle and a target cell, so the amount of the antibody modified on the surface of the particle is determined by the capacity of the particle to bind to the target cell after the antibody is modified. As shown in fig. 2C, in order to verify the influence of different antibody modification ratios on the ability of particles to bind to target cells, the same concentration of Cy5 fluorescent molecules is modified on the surface of the ferroferric oxide particles, and then antibodies with different ratios are modified on the surface of the ferroferric oxide nanoparticles, wherein the mass ratios of the antibodies to the particles are 5%, 10%, 15%, 20% and 25%, respectively. Detection of the amount of antibody in its supernatant by the BCA kit helped to verify that at all these ratios the antibody was fully bound to the particle surface. The specific antibody binding rate calculation formula is as follows:
binding efficiency (%) (antibody input-amount of antibody in supernatant after centrifugation)/antibody input X100%. cndot. (1-1)
The particles prepared above at different antibody modification ratios were mixed in 1 XPBS containing 0.2% bovine serum albumin at a concentration of 1. mu.g/mL and at a density of 1X 106The fluorescence intensity of Cy5 was measured by flow cytometry at 37 ℃ after 30min of incubation of NK cells/mL. Since the surface of the particles is modified with the Cy5 fluorescent molecule, the more the particles are bound by the cells, the stronger the Cy5 fluorescence of the cells. The strength of the binding capacity of the particles to the cells can be compared under different antibody modification ratios by the mean fluorescence intensity of NK cells. As shown in fig. 2D, the mean fluorescence intensity of NK cells increased with increasing antibody modification ratio at lower antibody ratios, indicating that the ability of the particles to bind to NK cells was gradually increasing at this time. When the modification ratio reaches 20%, the average fluorescence intensity of the NK cells is not increased any more, which indicates that the antibody modification ratio on the surface of the particles is continuously increased at this time, and the binding capacity of the particles and the NK cells is not improved any more. Therefore, in subsequent experiments, the mass ratio of the antibody to the ferroferric oxide nanoparticles is 20% as the optimal antibody modification ratio.
1.3 accurate detection of antibody binding efficiency:
the anti NK1.1 antibody and other impurities with different molecular weights from the antibody can be effectively separated through a gel chromatographic column of Ultra Performance Liquid Chromatography (UPLC), and the specific detection of the antibody can be realized. When only one antibody is contained in a sample, detection effects with stronger specificity and higher accuracy can be realized by using the UPLC. The experimental method is as follows:
MNP @ NK1.1 particles are prepared according to the method for preparing MNP @ NK1.1, and after preparation, the MNP @ NK1.1 particles are centrifuged, and the supernatant is taken as a sample to be detected. Under the same experimental conditions, a control group without added particles was prepared and tested with the sample. The calculation formula of the binding efficiency of the antibody is the same as that of the formula (1-1). Control group and sample group were subjected to
The SEC column of (1), using 10mM PB buffer salt of pH7.4 as a mobile phase, can detect the peak of anti-NK1.1 antibody at 3-4.5min by detecting the absorption peak at 280nm with TUV detector. As shown in fig. 3A, B, the particle concentrations of the sample group and the control group were calculated by plotting a standard curve by the peak area of the standard sample. As shown in fig. 3C, the antibody concentration in the control group was the same as the input amount, but no antibody was detected in the supernatant of the experimental group, indicating that the antibody was completely bound to the particle surface.
The optimal preparation method is obtained by combining the conclusion of the optimization experiment:
and adding ultrapure water into the carboxylated ferroferric oxide magnetic nanoparticles to dilute the carboxylated ferroferric oxide magnetic nanoparticles to 0.5mg/mL, adding an EDCI aqueous solution to a final concentration of 1mM, and adding an NHS aqueous solution to a final concentration of 1.5 mM. Activated at 37 ℃ in the dark for 1 h.
EDCI and NHS removal: the pellet was centrifuged at 9391g × 20min, the supernatant was discarded, and the pellet was resuspended with ultrapure water to a concentration of 0.5mg/mL, and washing was repeated three times.
And (3) carrying out ultrafiltration on the anti-NK1.1 antibody for 2-3 times by using a 100kDa ultrafiltration tube, and replacing the solvent of the antibody with ultrapure water. Mixing the activated particles with the antibody, adjusting the concentration of the antibody to 0.1mg/mL, fully and uniformly mixing, and reacting at 4 ℃ for 12 h. After the reaction, the osmotic pressure was adjusted as necessary using a 25% aqueous glucose solution so that the final concentration of the particles was 0.4mg/mL, the final concentration of the antibody was 0.08mg/mL, and the final concentration of glucose was 5%.
1.4 particle size change and surface topography change before and after MNP surface modification antibody:
as shown in fig. 4A, after MNP surface modification of anti-NK1.1 antibody, the mean hydrodynamic diameter of the particles increased from 100nm to 120nm as measured by Dynamic Light Scattering (DLS). As shown in fig. 4B, through SEM shooting, the original surface of the ferroferric oxide nanoparticle has an uneven surface morphology, and when the surface is modified with an anti-NK1.1 antibody, the surface becomes smooth, forming a layer of protein crown structure.
1.5 verification of the binding mode of the antibody to the particles:
there are two common methods for modifying antibodies on the surface of particles, one is electrostatic adsorption, and the other is chemical bonding. Compared with electrostatic adsorption, the chemical bonding mode is more stable for combining the antibody and the particles, and the antibody is not easy to fall off from the surfaces of the particles. The amino group of the antibody has positive charge, and the carboxyl group on the surface of the particle provides negative charge, so the antibody may be packed on the surface of the particle by means of electrostatic adsorption, and the detection of the concentration of the antibody in the supernatant of the particle cannot prove the way the antibody is bound on the surface of the particle. We verified by non-reducing SDS-PAGE experiments that the antibody is bound on the surface of the trimaran tetroxide nanoparticle by means of chemical bonding. The experimental method is as follows: diluting 10 μ g of anti-NK1.1 antibody with water to 20 μ L, adding 5 μ L of 5 Xprotein loading buffer solution to 20 μ L of MNP @ NK1.1 particle solution containing the anti-NK1.1 antibody with the same concentration, respectively, mixing, standing at room temperature for 10min, heating in a 99 ℃ metal bath for 10min, and loading after the sample is cooled; the gel was run at 120mV for 3h, and the sample bands were photographed and stained with Coomassie Brilliant blue to visualize the bands.
As shown in fig. 5A, when the antibody is bound on the surface of the particle by electrostatic adsorption, the antibody will leave the particle under the action of the electric field, and the migrated antibody band can be observed by coomassie brilliant blue staining; the antibody bound by chemical bonding can not be separated from the particles under the electrostatic action, and the antibody can not migrate. And as shown in fig. 5B, both the free and adsorbed groups had distinct bands of free antibody, while the bound group had no bands. The antibody of MNP @ NK1.1 particles is bound on the surface of the particles through chemical bonding, so that the antibody cannot migrate from a negative electrode to a positive electrode under the action of an electric field, and the binding of the antibody on the surface of the particles is stable.
Example 2 specific binding of MNP @ NK1.1 particles to NK cells
Experimental materials: the mouse B16-F10 melanoma cell line was derived from the American Standard Biotech Collection (ATCC). SPF-grade female C57BL/6 mice, 5-6 weeks old, were purchased from the Hunan Pollachida laboratory animals Co. The mice are bred in the center of experimental animals of the university of southern China, and the animal experimental flow conforms to the relevant regulations of the experimental animal management of the university of southern China.
Anti-mouse NK1.1 antibody was purchased from Bio Cell, Inc., USA.
2.1 binding of MNP @ NK1.1 particles to NK cells
To assess the ability of the magnetic nanoparticle MNP @ NK1.1 to bind to NK cells, we first explored the ability of MNP @ NK1.1 particles to interact with NK cells. First, NK cells in the spleen of a C57B/6 mouse are sorted out by an NK cell magnetic bead sorting kit according to the operating steps of the instruction, and then the NK cells (6 multiplied by 10) are sorted out5cells/mL) were co-incubated with Cy 5-labeled MNP @ IgG2a particles and Cy 5-labeled MNP @ NK1.1 particles, respectively (particle concentrations of 0, 3.125, 6.25, 12.5 and 25 μ g/mL) at 37 ℃ and MNP @ NK1.1 targeting ability was evaluated by flow cytometry: when NK cells bind to the granules, NK cells will appear Cy5 positive, and the more granules bound on the cells, the stronger the fluorescence intensity of the cells.
As shown in FIG. 6A, the mean fluorescence intensity of NK cells increased with the increase in MNP @ NK1.1 particle concentration. As shown in fig. 6B, the mean fluorescence intensity of NK cells increased with the extension of incubation time: MNP @ NK1.1 particles and NK cells are incubated for 30min, and the average fluorescence intensity of the NK cells is obviously enhanced compared with that of the NK cells incubated for 15 min; the increase in mean fluorescence intensity of NK cells was not large at 60min of incubation compared to 30min of incubation. In contrast, control MNP @ IgG2a particles bound weakly to NK cells (fig. 6C), indicating that MNP @ NK1.1 binding to cells depends on antigen-specific recognition and binding of the carried monoclonal antibody.
2.2 binding of MNP @ NK1.1 particles to NK cells in PBMCs
Considering the complex cellular components in vivo, we also incubated the particles with Peripheral Blood Mononuclear Cells (PBMCs) and detected the binding of the particles to different immune cells by flow cytometry.
The detection result is shown in FIG. 7A, under the condition of incubation for 30min, the mean fluorescence intensity of NK cells in PBMCs is gradually increased along with the increase of the concentration of MNP @ NK1.1 particles; also, the mean fluorescence intensity of NK cells was stronger in PBMCs treated with MNP @ NK1.1 particles compared to MNP @ IgG2a particles at the same particle concentration (25. mu.g/mL) and the same incubation time (0, 30, 60, 120min), indicating stronger binding of MNP @ NK1.1 particles to NK cells (FIGS. 7B and D). Meanwhile, the NK cells and T cells in PBMCs were incubated with MNP @ NK1.1 particles (25. mu.g/mL) at 37 ℃ for 0, 30, 60, 120min, respectively, and flow cytometry results showed that MNP @ NK1.1 particles bound strongly to NK cells in PBMCs and had substantially no binding to T cells in PBMCs (FIGS. 7C and E).
2.3 MNP @ NK1.1 particle binding site detection
The binding of the particles to NK cells was photographed by a laser scanning confocal microscope (CLSM), and as a result of the photographing, the binding of the particles to NK cells was photographed by a laser scanning confocal microscope (CLSM) as shown in fig. 8, and it was observed that the particles were bound to the cell surface rather than being taken up by the cells. Therefore, MNP @ NK1.1 particles are combined on the surface of NK cells, the influence of the combination on the cells is smaller compared with the effect of the combination on the cells by cellular uptake, and the killing capacity of the NK cells can be guaranteed not to be damaged.
The above results all demonstrate that the NK1.1 receptor molecule can act as a binding site for MNP @ NK 1.1.
Example 3 MNP @ NK1.1 particles promote NK cell migration under the action of a magnetic field
To verify whether MNP @ NK1.1 particles could promote migration of NK cells under the action of an applied magnetic field, we incubated NK cells with MNP @ NK1.1 particles in a petri dish. Cell density of 1X 106cells/mL, particle concentration 25. mu.g/mL, incubation conditions were 37 ℃ incubator incubation for 30 min.
The detection result is shown in fig. 9, the NK cells are gathered at the edge of the culture dish under the action of the external magnetic field, which indicates that the particles have the ability of promoting the migration of NK cells.
Example 4 animal level antitumor treatment
32C 57BL/6 mice were injected subcutaneously into the right dorsal part of the body at a density of 5X 10. mu.L6cells/mL of B16-F10 melanoma cells. 8 days after tumor implantation, dosing treatment was initiated and mice were monitored for tumor growth and weight change. The mice were randomly divided into 4 groups of 8 mice each: the first group of mice was Control group, and 625 μ L of 5% glucose aqueous solution was injected into tail vein; the second group of mice was designated M-MNGroup P @ IgG2a, 625. mu.L of MNP @ IgG2a (particle concentration of 0.4mg/mL, antibody concentration of 0.08mg/mL, dissolved in 5% glucose aqueous solution) was injected into the tail vein and treated with an applied magnetic field at the tumor site for 2h half an hour after administration; the third group of mice was designated as MNP @ NK1.1 group, 625. mu.L of MNP @ NK1.1 (particle concentration of 0.4mg/mL, antibody concentration of 0.08mg/mL, dissolved in 5% glucose aqueous solution) was injected into the tail vein without magnetic field treatment; the mice in the fourth group were scored as M-MNP @ NK1.1 group, and 625. mu.L of MNP @ NK1.1 (particle concentration of 0.4mg/mL, antibody concentration of 0.08mg/mL, dissolved in 5% glucose aqueous solution) was injected into the tail vein, and an external magnetic field was applied to the tumor site for 2 hours after half an hour of administration.
The administration was performed every two days for a total of 4 times. Throughout the treatment, tumor size was measured daily with a vernier caliper and the body weight of each group of mice was monitored. The formula for tumor volume is as follows:
volume (mm)3) 0.5 x length x width2。
The detection results are shown in fig. 11, in which fig. 11A is a graph of magnetic nanoparticles inhibiting the growth of melanoma, fig. 11C is a statistical graph of tumor growth of each mouse, and the tumors of the Control group, the M-MNP @ IgG2a group and the MNP @ NK1.1 group grow rapidly. The M-MNP @ NK1.1 experimental group has obvious effect of inhibiting tumor growth, because the magnetic particles are firstly combined with NK cells in vivo and the NK cells are enriched at the tumor part under the action of an external magnetic field. As shown in fig. 11B, the body weight of each group of mice did not change significantly throughout the treatment process, demonstrating that each component did not cause severe toxicity to mice and that the drug had good biocompatibility.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The preparation method of the specific antibody conveying platform is characterized in that NK cells are used as targets, the surfaces of ferroferric oxide nanoparticles are modified, and the ferroferric oxide nanoparticles and antibodies for specifically recognizing the NK cells are combined through chemical bonding under the action of a coupling agent.
2. The method of claim 1, comprising the steps of:
(1) adjusting the ferroferric oxide nano particles with carboxylated surfaces to a proper concentration in an aqueous solution, and carrying out activation reaction on 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 1 hour to convert partial carboxyl on the surfaces of the particles into active ester;
(2) mixing the granular aqueous solution obtained in the step (1) and an antibody aqueous solution for specifically identifying NK cells according to a certain proportion, and reacting for 12 hours at 4 ℃;
(3) and separating and purifying the nanoparticles to obtain the antibody modified magnetic nanoparticles.
3. The preparation method according to claim 2, wherein the concentration of the ferroferric oxide nanoparticles in the step 1 is 0.5 mg/mL; the EDCI is used at a concentration of 0.05-5mmol/L, preferably 0.5-1.5 mmol/L; the NHS is used in a concentration of 0.075 to 7.5mmol/L, preferably 0.75 to 2.25 mmol/L.
4. The preparation method according to claim 2, wherein the mass ratio of the antibody to the ferroferric oxide nanoparticles in the step 2 is 20%; the concentration of the antibody is 0.0125-0.2mg/mL, preferably 0.1 mg/mL.
5. A specific antibody delivery platform prepared by the method of any one of claims 1 to 4.
6. The specific antibody delivery platform of claim 5, wherein said specific antibody is at least one of a CD56 antibody, an anti-NK1.1 antibody and a CD49b antibody.
7. The specific antibody delivery platform according to claim 5, wherein said specific antibody delivery platform is spherical nanoparticles with a final concentration of 0.4mg/mL of particles and a final concentration of 0.08mg/mL of bound anti-NK1.1 antibody.
8. Use of a specific antibody delivery platform according to any one of claims 5 to 7 for tumor detection and/or efficacy monitoring and/or prognosis and/or personalized medication guidance.
9. An anti-neoplastic pharmaceutical formulation comprising a specific antibody delivery platform according to any one of claims 5 to 7.
10. The antitumor pharmaceutical preparation according to claim 9, wherein the pharmaceutical preparation further comprises an adjuvant, preferably glucose, and further preferably a 5% glucose solution by mass.
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