Antibody modified nano-particles based on heparin carrier as well as preparation method and application of antibody modified nano-particles

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an antibody modified nanoparticle based on a heparin carrier as well as a preparation method and application of the antibody modified nanoparticle. According to the nanometer particle, a pH sensitive Atezolizumab-pH-DOX conjugate or a cathepsin B sensitive Atezolizumab-vc-DOX conjugate is used as a targeting part, and the targeting part is coupled with a PTX-loaded heparin nanometer particle through a covalent bond; the PTX-loaded heparin nano particles are amphiphilic heparin derivative nano particles loaded with PTX (Protein Transportation Xinase). According to the invention, the Atezolizumab antibody is coupled with the nanoparticles, and the anti-tumor effect of the chemotherapeutic drug is effectively improved by improving the targeting property of the chemotherapeutic drug and the killing property of an immune system. Results show that the nanoparticles can effectively kill PD-L1 high-expression cell lines, and chemotherapy and immunotherapy are effectively combined.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an antibody-modified nanoparticle based on a heparin carrier, a preparation method thereof, and an application thereof. Background Art

[0002] Paclitaxel (PTX) and doxorubicin (DOX) are commonly used chemotherapy drugs in clinical practice. Clinical studies have shown that the combined use of paclitaxel and doxorubicin in the treatment of patients with advanced breast cancer has a total effective rate of up to 68%. Compared with single drug use, the combined use of drugs has significantly improved efficacy. At the same time, since the mechanisms of action of the two drugs are completely different, the generation of drug resistance can be prevented to a certain extent. However, the use of free PTX and DOX is prone to cause serious toxicity to normal tissues. Therefore, how to achieve the efficient targeting of two different drugs to tumor tissues simultaneously and control the synchronous release of different drugs is the biggest difficulty and challenge currently faced.

[0003] Atezolizumab is a humanized IgG1 monoclonal antibody that targets programmed death ligand 1 (PD-L1). By blocking the binding of PD-L1 to PD-1 and B7-1 (CD80) on immune cells, it relieves the inhibition of tumors on T cells and restores the killing effect of the immune system on cancer cells. Currently, atezolizumab is mainly used in combination with paclitaxel (albumin-bound paclitaxel) to treat triple-negative breast cancer. However, doxorubicin has strong cardiotoxicity, and its combination with immunotherapy may increase the risk of myocarditis, thus limiting its combination with immunotherapy. The combined use of atezolizumab with paclitaxel and doxorubicin is not common in clinical practice. Summary of the Invention

[0004] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an antibody-modified nanoparticle based on heparin that can load multiple chemotherapy drugs, a preparation method thereof, and an application thereof, which can improve the targeting ability of chemotherapy drugs, reduce toxic and side effects, promote the combination of chemotherapy and immunotherapy, and achieve the maximum therapeutic effect.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is: an antibody-modified nanoparticle based on a heparin carrier; the nanoparticle uses a pH-sensitive Atezolizumab-pH-DOX conjugate or a cathepsin B-sensitive Atezolizumab-vc-DOX conjugate as a targeting part, and is coupled to a PTX-loaded heparin nanoparticle through a covalent bond; the PTX-loaded heparin nanoparticle is an amphiphilic heparin derivative nanoparticle loaded with PTX.

[0006] Preferably, the pH-sensitive Atezolizumab-pH-DOX conjugate uses Atezolizumab as the antibody, maleimidocaproic acid hydrazide as the linker, and DOX as the cytotoxin, and the antibody is linked to the cytotoxin through the linker.

[0007] Preferably, the cathepsin B-sensitive Atezolizumab-vc-DOX conjugate uses Atezolizumab as the antibody, a cathepsin B-sensitive Val-Cit derivative as the linker, and DOX as the cytotoxin, and the antibody is linked to the cytotoxin through the linker.

[0008] Preferably, the amphiphilic heparin derivative is composed of non-anticoagulant heparin and vitamin E succinate linked by an esterification reaction; it self-assembles into nanoparticles in an aqueous solution.

[0009] Preferably, in the PTX-loaded heparin nanoparticles, the drug loading of PTX is 3-5%, and the encapsulation efficiency is 70-95%.

[0010] Preferably, the mass ratio of the cytotoxin to the antibody is (1-8):1.

[0011] The present invention also provides a method for preparing the heparin carrier-based antibody-modified nanoparticles, which includes coupling the PTX-loaded heparin nanoparticles with the pH-sensitive Atezolizumab-pH-DOX conjugate or the cathepsin B-sensitive Atezolizumab-vc-DOX conjugate through an amide bond using an EDC / NHS reaction system.

[0012] The present invention also provides the application of the heparin carrier-based antibody-modified nanoparticles, and these nanoparticles are used to prepare anti-tumor drugs; the anti-tumor drugs are tumor cell activity inhibitors or immune cell activity enhancers.

[0013] The present invention also provides an anti-tumor drug, and the anti-tumor drug contains an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the heparin carrier-based antibody-modified nanoparticles described above.

[0014] The beneficial effects of the present invention are as follows: The heparin-based antibody-modified nanoparticles (GTP-Ate-pH-DOX or GTP-Ate-vc-DOX) provided by the present invention have good anti-tumor activity and immune activation ability, have high killing effect and targeting ability on MDA-MB-231 breast cancer cells with high expression of PD-L1, and the loaded PTX and DOX have good synergistic effects. The killing effect on MCF-7 cells with low expression of PD-L1 is reduced, but it can still effectively inhibit the proliferation of MCF-7. The GTP-Ate-pH-DOX or GTP-Ate-vc-DOX nanoparticles can effectively restore the tumor killing ability of T cells and macrophages and activate the body's immunity. Description of the Drawings

[0015] Figure 1 Shows the morphology of the nanoparticles prepared in the examples of the present invention; wherein, A. GTP; B. GTP-Ate-pH-DOX; C. GTP-Ate-vc-DOX; Figure 2 Shows the drug release curves of the heparin-based antibody-modified nanoparticles prepared in Example 5 of the present invention; wherein, A. Release behavior of PTX in GTP-Ate-pH-DOX; B. Release behavior of PTX in GTP-Ate-vc-DOX; C. Release behavior of DOX in GTP-Ate-pH-DOX; D. Release behavior of DOX in GTP-Ate-vc-DOX; Figure 3 Shows the inhibition rate curves of the heparin-based antibody-modified nanoparticles prepared in Example 5 of the present invention on MDA-MB-231 cells; wherein, A. Survival rate of MDA-MB-231 cells after treatment with GTP-Ate-pH-DOX; B. Survival rate of MDA-MB-231 cells after treatment with GTP-Ate-vc-DOX; Figure 4 Shows the inhibition rate curves of the heparin-based antibody-modified nanoparticles prepared in Example 5 of the present invention on MCF-7 cells; wherein, A. Survival rate of MCF-7 cells after treatment with GTP-Ate-pH-DOX; B. Survival rate of MCF-7 cells after treatment with GTP-Ate-vc-DOX; Figure 5 Shows the targeting verification of the heparin-based antibody-modified nanoparticles prepared in Example 5 of the present invention on MDA-MB-231 cells; Figure 6 Shows the verification of the activation of T cells by the heparin-based antibody-modified nanoparticles prepared in Example 5 of the present invention; Figure 7 Shows the verification of the activation of macrophages by the heparin-based antibody-modified nanoparticles prepared in Example 5 of the present invention. Detailed Implementation Modes

[0016] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be elaborated in detail below in combination with specific embodiments.

[0017] The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can all be obtained through commercial channels.

[0018] Example 1: Preparation of Amphiphilic Heparin Derivative (GSHP-TOS) The amphiphilic heparin derivative is composed of non-anticoagulant heparin (GSHP) and vitamin E succinate (TOS) connected by an esterification reaction, and can self-assemble into nanoparticles in an aqueous solution. The structure of the amphiphilic heparin derivative is as follows: .

[0019] The preparation method is as follows: Weigh TOS, EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and DMAP (4-dimethylaminopyridine) according to a mass ratio of 3:6:4:1 and dissolve them using DMF (N,N-dimethylformamide), stir and activate for 3 h. After the activation is completed, add the GSHP formamide solution while stirring and continue stirring for 12 h. Collect the reaction solution, purify it by dialysis to obtain GSHP-TOS.

[0020] Example 2: Preparation of PTX-Loaded Heparin Nanoparticles (GTP) Load PTX into the GSHP-TOS nanoparticles by the emulsification-ultrasonic method to obtain GTP nanoparticles.

[0021] The preparation method is as follows: Dissolve GSHP-TOS and PTX in a PBS / methanol mixed solution according to a mass ratio of 10:1, mix them using a magnetic stirrer for 2 h. Then, place the mixed solution under probe ultrasound (50 W, 2 s, 4 s) for ultrasonic treatment and dialysis purification, filter through a filter membrane and freeze-dry to obtain GTP nanoparticles.

[0022] Example 3: Preparation of Atezolizumab-pH-DOX The pH-sensitive Atezolizumab-pH-DOX conjugate uses Atezolizumab as the antibody, maleimidocaproic acid hydrazide as the linker, and DOX as the cytotoxin, and the antibody is connected to the cytotoxin through the linker. The structure of the pH-sensitive Atezolizumab-pH-DOX conjugate is as follows: .

[0023] The preparation method is as follows: Mix DOX and EMCH in a molar ratio of 1:3, and react for 24 h under the action of trifluoroacetic acid. Select ethyl acetate as the poor solvent and purify by recrystallization to obtain the DOX-EMCH conjugate.

[0024] Select TCEP as the reducing agent to reduce the thiol group of Atezolizumab, add 10 times the mass of DOX-EMCH and react for 12 h, add cysteine to stop the reaction, and purify using a Sephadex column to obtain the Atezolizumab-pH-DOX conjugate.

[0025] Example 4: Preparation of Atezolizumab-vc-DOX The cathepsin B-sensitive Atezolizumab-vc-DOX conjugate, with Atezolizumab as the antibody, the cathepsin B-sensitive Val-Cit derivative as the linker, and DOX as the cytotoxin, and the antibody is linked to the cytotoxin through the linker. The structure of the cathepsin-sensitive Atezolizumab-vc-DOX conjugate is as follows: .

[0026] The preparation method is as follows: Dissolve DOX and MC-Val-Cit-PABC-PNP in NMP in a molar ratio of 1:1, and react for 48 h under the action of DIEA. Select dichloromethane as the poor solvent and purify by recrystallization and thin-layer chromatography to obtain the DOX-VC conjugate.

[0027] Select TCEP as the reducing agent to reduce the thiol group of Atezolizumab, add 10 times the mass of DOX-VC and react for 12 h, add cysteine to stop the reaction, and purify using a Sephadex column to obtain the Atezolizumab-vc-DOX conjugate.

[0028] Example 5: Preparation of GTP-Ate-pH-DOX and GTP-Ate-vc-DOX The preparation method is as follows: Dissolve GTP, EDCI, and NHS in PBS in a mass ratio of 1:3:2, react in a magnetic stirrer for 45 min, adjust the solution pH to neutral, add the pH-sensitive Atezolizumab-pH-DOX conjugate or the cathepsin B-sensitive Atezolizumab-vc-DOX conjugate and continue to react for 24 h, and obtain the heparin-based antibody-modified nanoparticles GTP-Ate-pH-DOX and GTP-Ate-vc-DOX of the present invention after dialysis purification.

[0029] Example 6: Morphological Observation of GTP-Ate-pH-DOX and GTP-Ate-vc-DOX The experimental method is as follows: Dissolve the above antibody-modified nanoparticles in an aqueous solution, take an appropriate amount of the solution, drop it on a copper grid and stain it, and observe it using a transmission electron microscope.

[0030] GTP-Ate-pH-DOX and GTP-Ate-vc-DOX exist in a nano-spherical structure in an aqueous solution and have good dispersibility, as Figure 1 shown.

[0031] Example 7: Drug Release of GTP-Ate-pH-DOX and GTP-Ate-vc-DOX The experimental method is as follows: Dissolve GTP-Ate-pH-DOX or GTP-Ate-vc-DOX to an appropriate concentration and place it in phosphate buffer solutions with different pH values or acetate solutions containing cathepsin B. Collect the release solution at specific time points and detect PTX or DOX in it, and calculate the cumulative drug release amount.

[0032] GTP-Ate-pH-DOX has good acid-sensitive characteristics and can effectively release PTX and DOX in an acidic environment; GTP-Ate-vc-DOX also shows certain acid-sensitive characteristics, releasing PTX in a low pH environment. At the same time, under the stimulation of cathepsin B, GTP-Ate-vc-DOX can respond and release DOX, as Figure 2 shown.

[0033] Example 8: Cytotoxicity of GTP-Ate-pH-DOX and GTP-Ate-vc-DOX The experimental method is as follows: Collect MDA-MB-231 cells or MCF-7 cells in the logarithmic growth phase, then adjust to an appropriate concentration, and inoculate them on a 96-well plate at a number of 1×10 4 per well, and culture them overnight in a cell incubator. Add different concentrations of GTP-Ate-pH-DOX or GTP-Ate-vc-DOX to the cell culture plate and treat for 48 h, and add CCK-8 to detect cell viability.

[0034] As Figure 3 and Figure 4 shown, GTP-Ate-pH-DOX or GTP-Ate-vc-DOX can effectively inhibit the proliferation of MDA-MB-231 and MCF-7 cells. As the drug concentration increases, the cell survival rate decreases significantly.

[0035] Example 9: Targeting of GTP-Ate-pH-DOX and GTP-Ate-vc-DOX The experimental method is as follows: Collect MDA-MB-231 cells in the logarithmic growth phase, then adjust to an appropriate concentration, and inoculate 50×10 4 cells per well on a 12-well plate and culture overnight in a cell incubator. Pretreat with PD-L1 antibody for 1 h, and add GTP-Ate-pH-DOX and GTP-Ate-vc-DOX to treat the cells for 8 h. Then, stain the cell nuclei with DAPI and observe the intracellular fluorescence using a laser confocal microscope.

[0036] As Figure 5 shown, after pretreatment with PD-L1 antibody, the fluorescence intensities of GTP-Ate-pH-DOX and GTP-Ate-vc-DOX in MDA-MB-231 cells are significantly reduced. Therefore, it can be proved that GTP-Ate-pH-DOX and GTP-Ate-vc-DOX have good targeting properties and can selectively enter MDA-MB-231 cells with high PD-L1 expression under the mediation of Atezolizumab.

[0037] Example 10: T cell and macrophage activation experiments of GTP-Ate-pH-DOX and GTP-Ate-vc-DOX The experimental method is as follows: Establish co-culture systems of Jurkat T cells & MDA-MB-231 cells and RAW264.7 macrophages & 4T1 cells, add GTP-Ate-pH-DOX and GTP-Ate-vc-DOX to treat the cells for 48 h, and add CCK-8 to detect cell viability.

[0038] As Figure 6 and Figure 7 shown, GTP-Ate-pH-DOX and GTP-Ate-vc-DOX can inhibit the proliferation of tumor cells. When Jurkat T cells or macrophages are present, the survival rate of tumor cells is further reduced. Therefore, GTP-Ate-pH-DOX and GTP-Ate-vc-DOX can effectively activate Jurkat T cells or macrophages to kill tumor cells, showing certain immune activation ability.

[0039] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Heparin carrier-based antibody-modified nanoparticles, characterized in that: The nanoparticle uses a pH-sensitive Atezolizumab-pH-DOX conjugate or a cathepsin B-sensitive Atezolizumab-vc-DOX conjugate as the targeting moiety, and is conjugated to the PTX-loaded heparin nanoparticle through a covalent bond; the PTX-loaded heparin nanoparticle is an amphiphilic heparin derivative nanoparticle loaded with PTX.

2. The antibody-modified nanoparticles based on a heparin carrier according to claim 1, characterized in that: The pH-sensitive Atezolizumab-pH-DOX conjugate uses Atezolizumab as the antibody, maleimidocaproic acid hydrazide as the linker, and DOX as the cytotoxin, and the antibody is linked to the cytotoxin through the linker.

3. The heparin carrier-based antibody-modified nanoparticles according to claim 1, wherein: The cathepsin B-sensitive Atezolizumab-vc-DOX conjugate uses Atezolizumab as the antibody, a cathepsin B-sensitive Val-Cit derivative as the linker, and DOX as the cytotoxin, and the antibody is linked to the cytotoxin through the linker.

4. The antibody-modified nanoparticles based on heparin carriers according to claim 1, wherein: The amphiphilic heparin derivative is composed of non-anticoagulant heparin and vitamin E succinate linked by an esterification reaction; it self-assembles into nanoparticles in an aqueous solution.

5. The antibody-modified nanoparticles based on heparin carriers according to claim 1, characterized in that: In the PTX-loaded heparin nanoparticle, the drug loading of PTX is 3-5%, and the encapsulation efficiency is 70-95%.

6. The heparin carrier-based antibody-modified nanoparticle according to claim 2 or 3, characterized in that: The mass ratio of the cytotoxin to the antibody is (1-8):

1.

7. The preparation method of the heparin carrier-based antibody-modified nanoparticles according to any one of claims 1-5, characterized in that: The PTX-loaded heparin nanoparticle is conjugated to the pH-sensitive Atezolizumab-pH-DOX conjugate or the cathepsin B-sensitive Atezolizumab-vc-DOX conjugate through an amide bond using an EDC / NHS reaction system.

8. Use of the heparin carrier-based antibody-modified nanoparticles according to any one of claims 1 to 6, characterized in that: The nanoparticle is used for preparing an anti-tumor drug; the anti-tumor drug is a tumor cell activity inhibitor or an immune cell activity enhancer.

9. An antitumor drug, the drug comprising an active ingredient and a pharmaceutically acceptable carrier, characterized in that: The active ingredient is the heparin-based antibody-modified nanoparticle according to any one of claims 1-6.

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