CN107224589B - MR imaging polymer micelle for delivering antibody for tumor immunotherapy and preparation method thereof - Google Patents
MR imaging polymer micelle for delivering antibody for tumor immunotherapy and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1806—Suspensions, emulsions, colloids, dispersions
- A61K49/1809—Micelles, e.g. phospholipidic or polymeric micelles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
- A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- 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
- C07K16/2803—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 immunoglobulin superfamily
- C07K16/2818—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 immunoglobulin superfamily against CD28 or CD152
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- Animal Behavior & Ethology (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract
The invention discloses an MR imaging polymer micelle for delivering an antibody for tumor immunotherapy and a preparation method thereof. The polymer micelle is of a nuclear shell structure, superparamagnetic ferroferric oxide nano particles are loaded on the core, a shell layer is formed on the surface of the micelle by polypeptide coupling antibodies sensitive to matrix metalloproteinase, and monomethyl ether polyethylene glycol (mPEG) coupled by acid-sensitive bonds20kAs a shielding layer for the antibody. When the polymer micelle reaches a tumor microenvironment, namely pH is less than or equal to 6.5, the shielding layer falls off to expose the epitope of the antibody, and the coupled antibody is released under the action of matrix metalloproteinase to realize the controllable release of the antibody drug. The polymer micelle can also realize the integration of Magnetic Resonance (MR) imaging and tumor immunotherapy.
Description
Technical Field
The invention belongs to the field of polymer chemistry and biomedical engineering, and particularly relates to an MR imaging polymer micelle for delivering an antibody for tumor immunotherapy and a preparation method thereof.
Background
Malignant tumors seriously harm human health, and the common methods for treating cancer at present comprise surgery, radiotherapy, chemotherapy and immunotherapy. Tumor immunotherapy is now another effective therapy for tumor control following surgical treatment, radiotherapy and chemotherapy, and is widely used in clinical practice. Among them, immune checkpoint inhibitor antibody therapy is gaining wide attention because it shows excellent anti-tumor activity in various tumors and significantly prolongs clinical survival. However, immune checkpoint inhibitor antibody therapy is prone to immune-related adverse effects such as myocarditis, hepatitis, pneumonia, dermatitis, etc., which are thought to be caused by abnormal activation of autoreactive T cells. Therefore, there is a need for improved methods of treatment. It is therefore desirable to prepare a specific functional antibody delivery vehicle for immune checkpoint inhibitors: the antibody is shielded in a physiological environment so as to prevent the antibody from being combined with immune cells, and after the antibody reaches a tumor microenvironment, the antibody is released again so as to restore the combination function of the antibody and the immune cells. The tumor immunity is activated, and the abnormal activation rate of autoreactive T cells is reduced, so that the toxic and side effects of the tumor immunotherapy are reduced.
Magnetic Resonance (MR) imaging has the advantages of high spatial resolution, good imaging of soft and hard tissues, non-invasive observation, etc., and plays an important role in medical diagnosis, especially in molecular imaging research. With the development and combination of nanotechnology and MR molecular imaging, MR nanoprobes constructed with superparamagnetic iron oxide (SPIO) nanocrystals have gained great attention. SPIO has been used in clinical MR imaging because of its low toxicity, good biocompatibility, and magnetic resonance signal sensitivity. Therefore, the multifunctional magnetic resonance molecular probe with both antibody delivery and magnetic performance is developed, and the antibody transmission efficiency can be effectively evaluated in real time.
Disclosure of Invention
The present invention aims to overcome the above defects of the prior art and provide a multifunctional carrier with both antibody delivery and magnetic properties, namely an MR imaging polymer micelle for delivering antibodies for tumor immunotherapy.
The invention also aims to provide a preparation method of the MR imaging polymer micelle for delivering the antibody for tumor immunotherapy.
The purpose of the invention is realized by the following technical scheme:
an MR imaging polymer micelle for delivering an antibody for tumor immunotherapy, the polymer micelle having a core-shell structure: the core is loaded with superparamagnetic ferroferric oxide nano particles, the surfaces of micelles form shell layers by polypeptide coupling antibodies sensitive to matrix metalloproteinase, and monomethyl ether polyethylene glycol (mPEG) coupled by acid-sensitive bonds20kAs a shielding layer for the antibody; the monomethyl ether polyethylene glycol mPEG20kThe molecular weight is 20 KDa.
The shielding layer on the surface of the MR imaging polymer micelle is sensitive to pH, can shield immune cells in blood from being combined with the antibody under the condition that the pH of an in vivo physiological environment is 7.4, and is removed in a tumor microenvironment with the pH of 6.5, so that the antibody is released under the action of Matrix Metalloproteinases (MMPs) highly expressed in the tumor microenvironment to be combined with the immune cells to play a role in immunotherapy; on the other hand, superparamagnetic ferroferric oxide nanoparticles (SPIONs) loaded by the polymer micelle can realize non-invasive MR imaging, so that diagnosis and treatment integration is realized.
The antibody may be an immunotherapeutic antibody, such as a PD-1 antibody, or other antibodies commonly used in the art.
Preferably, the average particle size of the polymer micelle is 140nm to 190 nm.
Preferably, the superparamagnetic ferroferric oxide nanoparticle is oil-soluble.
When the polymer micelle is in a tumor microenvironment, namely pH is less than or equal to 6.5, the shielding layer falls off, and the antibody is released under the action of matrix metalloproteinase.
The mass of the antibody accounts for 1.0-2.0% of the mass of the polymer micelle, and the mass of the superparamagnetic ferroferric oxide nano particle accounts for 15-25% of the mass of the polymer micelle.
The preparation method of the MR imaging polymer micelle for delivering the antibody to be used for tumor immunotherapy comprises the following steps:
s1, taking L-phenylalanine as a raw material, and adding triphosgene to prepare L-phenylalanine-cyclic carbonic anhydride, which is expressed as L-Phe-NCA;
s2, carrying out polymerization reaction by taking L-Phe-NCA as a raw material to obtain alpha-azido-polyethylene glycol-poly (phenylalanine), wherein the expression is N3-PEG-PLPhe；
S3, preparing superparamagnetic ferroferric oxide nanoparticles (SPIONs) by using ferric acetylacetonate and 1, 2-hexadecanediol as raw materials;
s4, using N3PEG-PLPhe and SPIONs are used as raw materials, and the micelle loaded with the SPIONs is obtained by ultrasonic self-assembly and is expressed as N3-PEG-PLPhe@SPIONs；
S5, coupling an antibody on the surface of the micelle prepared by S4 by taking a PD-1 monoclonal antibody mAb and a matrix metalloproteinase substrate polypeptide Peptide as raw materials through a series of reactions to obtain mAb-Peptide-PEG-PLPhe @ SPIONs;
s6, using mPEG20k-OH is used as a raw material, and an acid-sensitive shielding layer expressed as mPEG is constructed on the surface of the micelle prepared by S5 through a series of reactions20k-(mAb-Peptide)-PEG-PLPhe@SPIONs。
The use of the polymeric micelles for MR imaging of tumor immunotherapy for the delivery of antibodies in tumor immunotherapy and MR imaging.
Compared with the prior art, the invention has the following beneficial effects:
the MR imaging polymer micelle for delivering the antibody and used for tumor immunotherapy can stably circulate in vivo and is enriched at a tumor part, so that the carried antibody drug is prevented from being combined with immune cells in a physiological environment; meanwhile, the carrier contains SPIONs kernel, so that the MR imaging radiography function can be realized; the polymer micelle also has pH and MMPs sensitivity, when reaching a tumor part, the polymer micelle can remove the shielding layer, break MMPs sensitive polypeptide, release antibody drugs, recover the combination function of antibodies and immune cells, and realize the responsive release of the antibody drugs in a tumor microenvironment, thereby increasing the tissue specificity of tumor immunity and reducing the abnormal activation rate of autoreactive T cells.
Drawings
FIG. 1 is N3-PEG-NH2L-Phe-NCA and N3Nuclear magnetic spectrum of PEG-PLPhe.
FIG. 2 shows acid-sensitive mPEG20k-CDM and mPEG20k-nuclear magnetic spectrum of pHe-alkyne.
FIG. 3 is a graph showing a distribution of particle sizes of polymer micelles under the conditions of pH7.4, pH6.5 and pH6.5& MMP-2.
FIG. 4 is a TEM image of polymer micelles (A) loaded with SPIONs without staining with stain and (B) loaded with SPIONs stained with 3% uranium acetate for 1 min.
FIG. 5 is a graph showing the binding efficiency of the polymer micelle to T lymphocytes under the conditions of pH7.4, pH6.5 and pH6.5& MMP-2.
FIG. 6 is an image of MR imaging after the polymer micelle marks tumor cells.
Detailed Description
The invention will be further explained with reference to the drawings and specific examples in the following description, which are given for illustrative and detailed purposes and are not to be construed as limiting the scope of the invention, but rather as embodying the invention in equivalent or equivalent manners within the scope of the appended claims.
In the following examples and comparative examples, all the raw materials used were commercially available products.
Example 1
A method for preparing MR imaging polymer micelle for delivering antibody for tumor immunotherapy comprises the following steps:
s1. Synthesis of L-Phe-NCA (L-phenylalanine-cyclic C-carboxyanhydride) Polymer: the specific synthesis steps are as follows: weighing 10g L-phenylalanine into a 500mL three-necked flask, adding newly steamed 100mL ethyl acetate, heating and refluxing at 90 ℃, then dripping 50mL ethyl acetate dissolved with 10g triphosgene, stirring, and heating and refluxing for about 5h until the solution is yellow clear solution. After returning to room temperature, the mixture was frozen at-40 ℃ for 0.5 h. The ethyl acetate layer was precooled with saturated NaHCO at 4 ℃ respectively3The solution (100 mL. times.2) and a saturated NaCl solution (50 mL. times.2) were washed and separated. The ethyl acetate layer was again MgSO4Drying, filtering, rotary evaporating for concentrating, precipitating in newly evaporated petroleum ether, vacuum filtering, recrystallizing the obtained solid with ethyl acetate/petroleum ether, vacuum filtering, and vacuum drying to obtain white powder L-Phe-NCA6.0g with yield of 52%.
S2.N3-synthesis of PEG-PLPhe polymer: weighing 1.0g N3-PEG-NH2(alpha-azido-omega-aminopolyethylene glycol, Mw 1.8k) in a 100mL Schlenk reaction eggplant flask, vacuum drying at 70 ℃ for 1h, introducing N2Then returned to room temperature and 30mL of freshly distilled CH added2Cl2And (4) dissolving. Dissolving 3.2g L-Phe-NCA in 3mL anhydrous DMF, adding into the above Hilack reaction eggplant bottle, sealing, reacting at 35 deg.C for 48 hr, precipitating in diethyl ether, and centrifuging to obtain white product N3-PEG-PLPhe。1Phe units by H NMR were 25 and the polymer number average molecular weight was 5525.
S3, superparamagnetism Fe3O4And (3) synthesis of nanoparticles: uniformly mixing ferric acetylacetonate (2mmol), oleylamine (6mmol), oleic acid (6mmol), 1, 2-hexadecanediol (10mmol) and benzyl ether (18mL), rapidly heating to 200 ℃ under the protection of argon, keeping the temperature for 2h, continuously heating to 300 ℃, and refluxing for 1 h. Naturally cooling to room temperature, precipitating in ethanol (200mL), centrifuging, dispersing in 35mL n-hexane, filtering, and drying to obtain Fe3O4Nanoparticles.
S4, preparing the micelle loaded with the SPIONs: mixing 3mg SPIONs and 30mg N3PEG-PLPhe was dissolved in 8mL THF, added dropwise to 30mL deionized water under sonication, dialyzed against water to remove THF (dialysis bag MWCO14k), filtered through a 220nm aqueous frit, centrifuged at 50k rpm at ultra high speed to remove micelles without encapsulated SPIONs, and redissolved in 5mM PBS (pH 7.4) to about 2.5 mg/mL. The preparation method adopts the conventional technology in the technical field.
S5, synthesizing mAb-Peptide-PEG-PLPhe @ SPIONs:
first, the PD-1 antibody (GoInVivo (TM) anti-mouse CD279, Biolegend) was thiolated, following the brief procedure: 1mL of a 1mg/mL antibody solution (pH 7.4) was added with 10. mu.L of 100mM PBS and 30mM EDTA (pH 7.4), and 0.7mg of 2-iminosulfane hydrochloride (2-IT, Mw 137.6, 5mM) was added and reacted at 20 ℃ for 45 min. Using HiTrapTMColumn for rapid desalination and purification (
Pure System), eluent composition of 10mM PBS +3mM EDTA (pH 7.4), detection wavelength: 280nm, samples were collected as 1 mL/tube. The protein-containing solutions were combined, concentrated by ultrafiltration (MWCO:10k), and quantified to 2.5 mL. The antibody concentration is measured by ultraviolet spectrophotometry or ELISA, and the recovery rate is about 80%. The amount of thiol groups was determined using DTNB, which introduced an average of 3.2 thiol groups per antibody molecule.
Secondly, the thiol group on the PD-1 antibody is added with the maleimide group of the Mal-peptide-alkyne to introduce the alkynyl (alkyne), and the brief operation steps are as follows: 2.5mg of Mal-peptide-alkyne (Mw 1013.5, 1mM) was weighed out and dissolved in 30. mu.L of DMSO, and added to the above-mentioned thiolated antibody solutionIn (1), the mixture was stirred at 4 ℃ overnight. Using HiTrapTMColumn desalination and purification (
Pure System), eluent composition 10mM PBS, pH7.4, detection wavelength: 280nm, samples were collected as 1 mL/tube. The protein-containing solutions were combined, concentrated by ultrafiltration (MWCO:10k), and quantified to 2 mL. The antibody concentration was measured by UV spectrophotometry or ELISA and the recovery rate was about 81%. Finally, the alkynyl on the PD-1 antibody and the azido on the surface of the micelle are subjected to click reaction, and the specific operation steps are as follows: 2mL of N prepared as described above3Micellar solutions of PEG-PLPhe @ SPIONs (azido of about 0.72. mu. mol) were deoxygenated by freeze-thawing after addition of 10. mu.L of Cu (II) BSC (1mg/mL), and repeated 2 times. The alkynylated antibody solution prepared above (about 0.64mg of antibody, about 0.0085. mu. mol of alkynyl group) and 2mg of vitamin C were added thereto, and reacted at room temperature overnight.
S6.mPEG20kSynthesis of- (mAb-Peptide) -PEG-PLPhe @ SPIONs:
mPEG20kthe reaction route of pHe-alkyne is as follows:
first, 2-propionic acid-3-methylmaleic anhydride (CDM) was activated with oxalyl chloride to acid chloride. Briefly described as follows: 0.37g CDM (Mw184.1,2.0mmol) was weighed out and dissolved in freshly distilled 5mLCH2Cl2Adding 50 μ L of DMF, N as catalyst2Under the protection of gas, 1.27mL of oxalyl chloride (20mmol) was added dropwise to the mixture in an ice-water bath. After the dropwise addition, the reaction was carried out at room temperature for 3 hours, and the solvent and the excess oxalyl chloride were removed by rotary evaporation in vacuo to obtain a pale yellow viscous liquid.
Second step, synthesis of mPEG20k-CDM. 1.0g mPEG20k-OH (0.05mmol) in 4mL of anhydrous CH2Cl2Then, the mixture was added to the above viscous liquid, 12mg of DMAP (Mw 122.17,0.1mmol) as a catalyst was added thereto, and a solution of 0.35mL of triethylamine (Mw101.2,2.5mmol) in 12mL of toluene was added dropwise in an ice-water bath, whereby the solution was brownish-gray and reacted for 24 hours. After the reaction is finished, the reaction solution is filtered,concentrating the filtrate, precipitating in cold diethyl ether, suction filtering, and filtering the filter cake with 50mL CH2Cl2And (4) dissolving. CH (CH)2Cl2The layers were washed with 15mL of 0.5M HCl, 15mL of saturated NaCl solution, separated, and then MgSO4Drying, filtering, concentrating and precipitating in cold ether to obtain yellowish product.1The conversion was about 80% by H NMR.
Step three, synthesizing mPEG20k-pHe-alkyne. 0.6g mPEG20k-CDM (0.03mmol) dissolved in 4mL CH2Cl2And 0.2mL DMF, adding 33 μ g propynylamine (Mw55.1, 0.6mmol), reacting at room temperature for 12h, precipitating in cold ether, centrifuging to obtain product, and adding CH2Cl2Dissolving, precipitating in ether to obtain mPEG20kThe structural formula of the pHe-alkyne is shown in the reaction scheme.
Fourth, to the overnight reacted mAb-Peptide-PEG-PLPhe @ SPIONs solution described in S5 was added 29.0mg mPEG20kpHe-alkyne (1.44. mu. mol) and 2mg vitamin C, and the reaction was continued for 8 h. After completion of the reaction, 1mL of 10mM EDTA was added, and the mixture was centrifuged at an ultra high speed of 50k rpm, and the obtained solid was washed once with PBS (pH 7.4), redissolved in 2mL of physiological saline, and stored at 4 ℃ for further use.
The solution was adjusted to pH6.5 to obtain PEG20kAfter detachment from the surface, the antibody concentration was measured by ELISA method, about 100ug/mL, and the reaction efficiency of the antibody was about 31%. The antibody accounted for 1.25% of the total mass. mPEG20kThe density of the shielding layer can be regulated and controlled by adjusting the reaction time.
Structural analysis of polymer and morphological characterization of polymer micelle: dissolving about 9mg of the product obtained in each step in a proper amount of deuterated solvent, and using1H-NMR 400MHz nuclear magnetic resonance spectrometer for testing, and detecting the polymer structure before and after reaction1H chemical shift, as shown in fig. 1 and 2. Different polymeric micelle samples were prepared, including: n is a radical of3-PEG-PLPhe、N3-PEG-PLPhe@SPIONs，mPEG20k-(mAb-Peptide)-PEG-PLPhe，mPEG20k- (mAb-Peptide) -PEG-PLPhe @ SPIONs, particle size of the polymer micelles was measured by Zeta potential and particle size analyzer (DLS), as shown in FIG. 3, with incident laser wavelength λ of 532nm and incident angle θ of 175 °At a temperature of 25 ℃; particle size values were averaged over three measurements.
The morphology of the polymer micelle is observed by a Transmission Electron Microscope (TEM) as shown in FIG. 4, wherein FIG. 4(A) shows the micelle loaded with SPIONs without dyeing, and FIG. 4(B) shows the micelle loaded with SPIONs with dyeing. It can be seen from FIG. 4(B) that the particle size of the entire polymer micelle carrier is about 170 nm. The brief operation is as follows: mu.L of the sample (0.2mg/mL) was dropped onto a pure carbon film copper net, dried at room temperature, and stained with 3% uranium acetate for 1 min. Observed with a transmission electron microscope at 120kV (the procedure was examined using techniques conventional in the art).
Dual-responsiveness detection of pH and MMPs of MR imaging polymer micelles for tumor immunotherapy: to prove that the polymer micelle prepared in example 1 has dual responsiveness to pH and MMPs, the polymer micelle is treated for 4h and 6h in the environment of MMP-2 with pH6.5 and 10nM, respectively, and then co-incubated with T lymphocytes, and the binding efficiency of the antibody and the T lymphocytes before and after the treatment is detected by using a flow cytometer, and the result is shown in FIG. 5. The result shows that the binding efficiency of the free PD-1 antibody and the T lymphocyte can reach 82.85% (FreePD 1), and the binding efficiency of the antibody and the T lymphocyte is reduced to 32.00% (Shield) after the shielding of the polymer micelle carrier, which indicates that the carrier can well shield the antibody and prevent the binding of the PD-1 antibody and the T lymphocyte under the physiological environment of pH7.4. When the polymer micelle is treated in a weakly acidic environment with pH6.5, the binding efficiency of the antibody and the T lymphocyte can be restored to 65.65 percent (pH6.5), and when MMP-2 is further used for enzyme digestion treatment, the binding efficiency of the antibody and the T lymphocyte can be restored to 75.49 percent (pH6.5 and MMP-2), which indicates that the carrier has good pH and MMPs enzyme responsiveness.
In vitro MRI imaging effect: to further demonstrate that the polymer micelle prepared in example 1 has MR imaging capability at 5X 105B16-F10 per well was inoculated in a 96-well plate, the polymer micelles prepared in example 1 were added after 24 hours of incubation so that the final iron concentration of each well was 5. mu.g/mL, 10. mu.g/mL, 15. mu.g/mL, 20. mu.g/mL, 25. mu.g/mL, 30. mu.g/mL, 35. mu.g/mL, 40. mu.g/mL, respectively, and the MR imaging ability of each well after 30min, 60min, 90min, 120min, 150min, 180min, 210min, 240min was further examined. From the figure6, it can be seen that the signals of the concentration gradient markers B16-F10 on T1WI, T/2WI, FFE and T2-mapping are gradually reduced, the degree of reduction of the signals has a certain dependence on the concentration of the polymer micelles, and the MR signal of the cells is gradually reduced along with the increase of the content of the polymer micelles. The polymer micelle has strong magnetic sensitivity and can be used for MR imaging, and the result of combining the incubation time and the iron concentration shows that the best MR imaging effect can be achieved when the iron concentration is 30 mug/mL and the incubation time is 210 min.
Example 2
A method for preparing MR imaging polymer micelle for delivering antibody for tumor immunotherapy comprises the following steps:
steps S1 to S3, and S5 to S6 are the same as those in example 1
S4, preparing the micelle loaded with the SPIONs: mixing 8mg SPION and 30mg N3PEG-PLPhe was dissolved in 10mL THF, added dropwise to 30mL deionized water under sonication, dialyzed against water to remove THF (dialysis bag MWCO14k), filtered through a 450nm aqueous frit, centrifuged at 50k rpm at ultra high speed to remove micelles without encapsulating SPIONs, and redissolved in 5mM PBS (pH 7.4) to about 2.5 mg/mL. The weight of superparamagnetic ferroferric oxide nano particles (SPIONs) accounts for 25 percent of the weight of the polymer micelle. The preparation method adopts the conventional technology in the technical field.
Example 3
A method for preparing MR imaging polymer micelle for delivering antibody for tumor immunotherapy comprises the following steps:
steps S1 to S3, and S5 to S6 are the same as those in example 1
S4, preparing the micelle loaded with the SPIONs: 4mg of SPION, 32mg of N3PEG-PLPhe was dissolved in 5mL THF, added dropwise to 25mL deionized water under sonication, dialyzed against water to remove THF (dialysis bag MWCO14k), filtered through a 450nm aqueous frit, centrifuged at 60k rpm at ultra high speed to remove micelles without encapsulated SPIONs, and redissolved in 5mM PBS (pH 7.4) to about 2.5 mg/mL. The weight of superparamagnetic ferroferric oxide nano particles (SPIONs) accounts for 15 percent of the weight of the polymer micelle. The preparation method adopts the conventional technology in the technical field.
Example 4
A method for preparing MR imaging polymer micelle for delivering antibody for tumor immunotherapy comprises the following steps:
steps S1 to S4, and S6 are the same as those in example 1
Synthesis of S4mAb-Peptide-PEG-PLPhe @ SPIONs:
first, a PD-1 antibody (GoInVivo) was introducedTManti-mouse CD279, Biolegend) thiolation, the brief procedure was as follows: 1mL of a 1mg/mL antibody solution (pH 7.4) was added with 10. mu.L of 100mM PBS and 30mM EDTA (pH 7.4), and 0.7mg of 2-iminosulfane hydrochloride (2-IT, Mw 137.6, 5mM) was added and reacted at 20 ℃ for 45 min. Using HiTrapTMColumn for rapid desalination and purification (
Pure System), eluent composition of 10mM PBS +3mM EDTA (pH 7.4), detection wavelength: 280nm, samples were collected as 1 mL/tube. The protein-containing solutions were combined, concentrated by ultrafiltration (MWCO:10k), and quantified to 2.5 mL. The antibody concentration is measured by ultraviolet spectrophotometry or ELISA, and the recovery rate is about 80%. The amount of thiol groups was determined using DTNB, which introduced an average of 3.2 thiol groups per antibody molecule.
Secondly, the thiol group on the PD-1 antibody is added with the maleimide group of the Mal-peptide-alkyne to introduce the alkynyl (alkyne), and the brief operation steps are as follows: 2.5mg of Mal-peptide-alkyne (Mw 1013.5, 1mM) was weighed out and dissolved in 30. mu.L of DMSO, and added to the above thiolated antibody solution, and stirred overnight at 4 ℃. Using HiTrapTMColumn desalination and purification (
Pure System), eluent composition 10mM PBS, pH7.4, detection wavelength: 280nm, samples were collected as 1 mL/tube. The protein-containing solutions were combined, concentrated by ultrafiltration (MWCO:10k), and quantified to 2 mL. The antibody concentration was measured by UV spectrophotometry or ELISA and the recovery rate was about 81%.
Finally, the alkynyl on the PD-1 antibody and the azido on the surface of the micelle are subjected to click reaction, and the specific operation steps are as follows: taking 0.5mL of N prepared above3Micellar solutions of-PEG-PLPhe @ SPIONs(azido of about 0.18. mu. mol), 5. mu.L of Cu (II) BSC (1mg/mL) was added, and the oxygen was removed by freeze-thawing and repeated 2 times. The alkynylated antibody solution prepared above (about 0.64mg of antibody, about 0.0085. mu. mol of alkynyl group) and 2mg of vitamin C were added thereto, and reacted at room temperature overnight. The resulting support antibody loading was greater than in example 1. And after passing through the same step S6 as in example 1, the antibody was obtained in an amount of 2.0% by mass based on the carrier.
Claims (7)
1. An MR imaging polymer micelle for delivering an antibody for tumor immunotherapy, wherein the polymer micelle has a core-shell structure: the core is loaded with superparamagnetic ferroferric oxide nano particles, the surfaces of micelles form shell layers by polypeptide coupling antibodies sensitive to matrix metalloproteinase, and monomethyl ether polyethylene glycol (mPEG) coupled by acid-sensitive bonds20kAs a shielding layer for the antibody; the monomethyl ether polyethylene glycol mPEG20kThe molecular weight is 20 KDa;
when the polymer micelle is in a tumor microenvironment, namely the pH value is less than or equal to 6.5, the shielding layer falls off, and the antibody is released under the action of matrix metalloproteinase;
the preparation method of the MR imaging polymer micelle for delivering the antibody to be used for tumor immunotherapy comprises the following steps:
s1, taking L-phenylalanine as a raw material, and adding triphosgene to prepare L-phenylalanine-cyclic carbonic anhydride, which is expressed as L-Phe-NCA;
s2, carrying out polymerization reaction by taking L-Phe-NCA as a raw material to obtain alpha-azido-polyethylene glycol-polyphenylalanine, wherein the alpha-azido-polyethylene glycol-polyphenylalanine is expressed as N3-PEG-PLPhe;
s3, preparing superparamagnetic ferroferric oxide nanoparticles (SPIONs) by using ferric acetylacetonate and 1, 2-hexadecanediol as raw materials;
s4, taking N3-PEG-PLPhe and SPIONs as raw materials, and obtaining micelles loaded with the SPIONs through ultrasonic self-assembly, wherein the expression is N3-PEG-PLPhe @ SPIONs;
s5, coupling an antibody on the surface of the micelle prepared by S4 by taking a PD-1 monoclonal antibody mAb and a matrix metalloproteinase substrate polypeptide Peptide as raw materials through a series of reactions to obtain mAb-Peptide-PEG-PLPhe @ SPIONs;
s6, taking mPEG20k-OH as a raw material, and constructing an acid-sensitive shielding layer on the surface of the micelle prepared in S5 through a series of reactions, wherein the acid-sensitive shielding layer is expressed as mPEG20k- (mAb-Peptide) -PEG-PLPhe @ SPIONs.
2. The MR imaging polymeric micelle for antibody delivery for tumor immunotherapy according to claim 1, wherein the polymeric micelle size is 140nm to 190 nm.
3. The MR imaging polymer micelle for antibody delivery for tumor immunotherapy according to claim 1, wherein the superparamagnetic ferroferric oxide nanoparticles are oil soluble.
4. The MR imaging polymer micelle used for tumor immunotherapy by conveying the antibody according to claim 1, wherein the mass of the antibody accounts for 1.0-2.0% of the mass of the polymer micelle, and the mass of the superparamagnetic ferroferric oxide nanoparticle accounts for 15-25% of the mass of the polymer micelle.
5. A method for preparing the polymeric micelle for MR imaging for the immunotherapy of tumors by delivering the antibody according to any one of claims 1 to 4, comprising the steps of:
s1, taking L-phenylalanine as a raw material, and adding triphosgene to prepare L-phenylalanine-cyclic carbonic anhydride, which is expressed as L-Phe-NCA;
s2, carrying out polymerization reaction by taking L-Phe-NCA as a raw material to obtain alpha-azido-polyethylene glycol-poly (phenylalanine), wherein the expression is N3-PEG-PLPhe；
S3, preparing superparamagnetic ferroferric oxide nanoparticles (SPIONs) by using ferric acetylacetonate and 1, 2-hexadecanediol as raw materials;
s4, using N3PEG-PLPhe and SPIONs are used as raw materials, and the micelle loaded with the SPIONs is obtained by ultrasonic self-assembly and is expressed as N3-PEG-PLPhe@SPIONs；
S5, coupling an antibody on the surface of the micelle prepared by S4 by taking a PD-1 monoclonal antibody mAb and a matrix metalloproteinase substrate polypeptide Peptide as raw materials through a series of reactions to obtain mAb-Peptide-PEG-PLPhe @ SPIONs;
s6, using mPEG20k-OH is used as a raw material, and an acid-sensitive shielding layer expressed as mPEG is constructed on the surface of the micelle prepared by S5 through a series of reactions20k-(mAb-Peptide)-PEG-PLPhe@SPIONs。
6. Use of the MR imaging polymer micelle of any one of claims 1 to 4 for the delivery of antibodies for the immunotherapy of tumors in the manufacture of a medicament for the immunotherapy of tumors.
7. Use of the polymeric micelles for MR imaging for tumor immunotherapy by delivering an antibody according to any one of claims 1 to 4 for the preparation of MR imaging micelles.
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