Targeted precise immunotherapy by applying biological protein nano-robot to carry biological agent and preparation method of biological protein nano-robot

Abstract

The invention provides targeted precise immunotherapy by applying a biological protein nano-robot to carry a biological agent and a preparation method of the biological protein nano-robot, and relates to the field of immunotherapy, the preparation method comprises the following steps: S100, preparing a serum albumin aqueous solution with the serum albumin concentration of 5-10% w / v, and a phosphate buffer solution with the pH value of 7.4 as a solvent; and S200, preparing an organic solution of a hydrophobic anti-rheumatism drug, wherein the drug is one or more of adalimumab, jucuiliumab, ezetizumab, tetronigerb, tolzumab and rituximab. When the albumin nano-robot targeting drug is used for treating rheumatism, effective targeting can be realized by utilizing the chemotactic effect of the albumin nano-robot targeting drug for immunotherapy on high-expression iNOS and ROS in an immune microenvironment, and the released anti-rheumatism drug and nitric oxide play a synergistic effect for combined treatment; wherein the anti-rheumatism medicine and nitric oxide (NO) generated in the targeting process of the nano-robot can jointly start immune circulation in vivo, and the anti-rheumatism medicine and the nitric oxide (NO) cooperate with the chemotherapy medicine to achieve the killing effect, so that the combined treatment effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy, and specifically to the use of bioprotein nanorobots to deliver biological agents for targeted precision immunotherapy and a preparation method thereof. Background Art

[0002] Albumin nanoparticle drug delivery systems have been widely used to construct anti-tumor drug delivery systems due to the excellent biocompatibility, biodegradability, and low immunogenicity of their substrates. Albumin nanoparticle drug delivery systems can also be used for precision treatment of connective tissue diseases, where abnormal immune cells produce autoantibodies and autoantibodies attack target cells. Traditional albumin nanoparticles are often loaded with hydrophobic chemotherapy drugs to extend their circulation time in the body and improve drug treatment efficacy. However, the current construction and application of albumin nanoparticle drug delivery systems still face challenges.

[0003] Currently, albumin-based drug delivery systems typically rely on crosslinkers (such as glutaraldehyde) during the synthesis process to improve the stability of nanomedicines. However, residual crosslinkers can cause matrix complications. On the other hand, while albumin-based nanomedicines loaded with hydrophobic immunotherapies can somewhat address the short circulation time of hydrophobic drugs in the body, they still face the challenge of precise targeting. They cannot precisely target the immune microenvironment, resulting in the accumulation of immunotherapies in normal tissues, causing side effects and low drug utilization. Furthermore, these nanomedicines can typically only be treated with a single drug, limiting their effectiveness.

[0004] Therefore, we have made improvements to this and proposed the use of bioprotein nanorobots to deliver biological agents for targeted precision immunotherapy and their preparation methods. Summary of the Invention

[0005] The purpose of the present invention is to address the problems raised by the current background technology.

[0006] In order to achieve the above-mentioned purpose of the invention, the present invention provides a method for preparing a targeted precise immunotherapy using bioprotein nanorobots to deliver biological agents, so as to improve the above-mentioned problems.

[0007] The specific application is as follows:

[0008] The following steps are involved:

[0009] S100, preparing a serum albumin aqueous solution, wherein the serum albumin concentration is 5-10% w / v, and the solvent is a phosphate buffer solution at pH 7.4;

[0010] S200, preparing an organic solution of a hydrophobic antirheumatic drug, wherein the drug is one or more of adalimumab, secukinumab, ixekizumab, tetasip, tocilizumab, and rituximab, the solvent is ethanol or acetone, and the drug concentration is 40-160 mg / mL;

[0011] S300, mixing the solution of step S100 with the solution of step S200 in a mass ratio of 1:5 to 1:10, adding an acid-heat denaturant to adjust the pH to 4.0-5.0, and reacting at 60-80° C. for 1-3 hours to form denatured albumin-coated drug nanoparticles;

[0012] S400, adding L-arginine solution to the product of step S300, wherein the mass ratio of L-arginine to albumin is 1:10 to 1:20, and adsorbing L-arginine on the surface of the nanoparticles by ultrasonic dispersion;

[0013] S500, centrifugal purification, and freeze-drying to obtain nanorobot targeted drugs.

[0014] As a preferred technical solution of the present application, the acid-heat denaturant in step S300 is 0.1-0.5M hydrochloric acid or citric acid buffer, the reaction temperature is 70°C ± 2°C, and the reaction time is 2 hours.

[0015] As a preferred technical solution of the present application, the power of ultrasonic dispersion in step S400 is 300 W, the time is 20 minutes, and the particle size of the nanoparticles is controlled to be 80-120 nm.

[0016] As a preferred technical solution of the present application, the freeze-drying process in step S500 includes a pre-freezing stage and a sublimation drying stage.

[0017] The biological protein nanorobot consists of a three-layer structure, which consists of a hydrophobic anti-rheumatic drug core, a denatured albumin middle layer and an L-arginine outer loading layer from the inside to the outside. Anti-rheumatic drugs and NO can act as inducers of immunogenic cell death, enhance the production of immune antigens, and thus promote the maturation of antigen-presenting cells and the activation of T cells.

[0018] As a preferred technical solution of the present application, the denatured albumin intermediate layer has a porous network structure with a pore size of 2-5 nm, and a drug encapsulation rate of ≥90%.

[0019] As a preferred technical solution of the present application, the L-arginine external loading layer accounts for 5-15% of the total mass of the nanoparticles, and the release rate is ≥80% in an environment below pH 6.5.

[0020] As a preferred technical solution of the present application, the molar ratio of the hydrophobic anti-rheumatic drug to L-arginine is 1:2 to 1:5, and the drug acts synergistically in the lesion area through the NO-mediated mitochondrial apoptosis pathway.

[0021] The application of bioprotein nanorobots in the preparation of therapeutic drugs for connective tissue diseases, which can be administered intravenously or locally, including targeted delivery to the joint cavity, salivary glands, kidneys or skin;

[0022] Targeting Mechanism:

[0023] RA: Targets abnormally proliferating fibroblast-like synoviocytes (FLS) in the synovium of joints;

[0024] Systemic lupus erythematosus (SLE): Targets areas of overactive B cells or immune complex deposits in the kidneys, skin, or blood;

[0025] SpA: Targeting inflammatory Th17 cells or bone erosion-related osteoblasts in the spine and sacroiliac joints;

[0026] pSS: Targets infiltrating lymphocytes or glandular epithelial cells in salivary or lacrimal glands.

[0027] When used for the treatment of rheumatic diseases, the immunotherapy albumin nanorobot targeted drug can be used to achieve effective targeting by utilizing the chemotactic effect of highly expressed iNOS and ROS in the immune microenvironment, and the released anti-rheumatic drugs and nitric oxide can be used to exert a synergistic effect for combined treatment, wherein the anti-rheumatic drugs can jointly activate the body's own immune cycle with the nitric oxide (NO) produced during the nanorobot targeting process, synergizing the killing effect of the chemotherapy drugs themselves, and achieving the effect of combined treatment. The specific process is as follows: anti-rheumatic drugs and NO can act as inducers of immunogenic cell death, enhance the production of immune antigens, thereby promoting the maturation of antigen-presenting cells and the activation of T cells; further, NO can normalize abnormal blood vessels in the tissue, thereby improving the transport efficiency of T cells in the lesion site, and NO can also promote the degradation of the extracellular matrix, effectively improving the infiltration of T cells and drugs in the diseased tissue. In summary, the albumin nanorobot targeted drug releases anti-rheumatic drugs and NO in response to the immune microenvironment through matrix components, and with the help of the chemotactic behavior of the nanorobot, it becomes an important therapeutic agent for regulating multi-link intervention in the immune system, thereby forming a cascade effect in the treatment process. The cascade effect of this treatment process is achieved with the help of the effective chemotaxis of nanorobots, which is also the biggest difference between this technology and existing albumin nanoparticles. It has broad application prospects in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This application provides a structural schematic diagram of the use of bioprotein nanorobots to deliver biological agents for targeted precision immunotherapy and its preparation method. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0030] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0031] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions therein may be combined with each other.

[0032] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0033] Example 1, please refer to Figure 1 , using bioprotein nanorobots to deliver biological agents for targeted precision immunotherapy and a preparation method thereof, comprising the following steps:

[0034] S100, preparing a serum albumin aqueous solution, wherein the serum albumin concentration is 5-10% w / v, and the solvent is a phosphate buffer solution at pH 7.4;

[0035] S200, the drug is one or more of adalimumab, secukinumab, ixekizumab, tetanusip, tocilizumab, and rituximab, the solvent is ethanol or acetone, and the drug concentration is 40-160 mg / mL;

[0036] S300, mixing the solution of step S100 with the solution of step S200 in a mass ratio of 1:5 to 1:10, adding an acid-heat denaturant to adjust the pH to 4.0-5.0, and reacting at 60-80° C. for 1-3 hours to form denatured albumin-coated drug nanoparticles;

[0037] S400, adding L-arginine solution to the product of step S300, wherein the mass ratio of L-arginine to albumin is 1:10 to 1:20, and adsorbing L-arginine on the surface of the nanoparticles by ultrasonic dispersion;

[0038] S500, centrifugal purification, and freeze-drying to obtain nanorobot targeted drugs.

[0039] By replacing chemical cross-linking with acid-heat denaturation, L-arginine achieves targeted functionalization through electrostatic adsorption, avoiding the toxic cross-linking agents of formaldehyde / glutaraldehyde, and achieving the dual functions of targeted drug delivery and lesion microenvironment response.

[0040] In step S300 , the acid-heat denaturant is 0.1-0.5 M hydrochloric acid or citric acid buffer, the reaction temperature is 70° C.±2° C., and the reaction time is 2 hours.

[0041] The degree of denaturation is precisely controlled to allow albumin to form a porous network structure while preventing drug leakage.

[0042] The drug loading rate is increased to 15%-25% (traditional methods ≤10%) while maintaining the stability of the nanoparticle structure.

[0043] In step S400 , the power of ultrasonic dispersion is 300 W, the time is 20 minutes, and the particle size of the nanoparticles is controlled to be 80-120 nm.

[0044] Ultrasonic parameters were optimized to ensure uniform loading of L-arginine without destroying the drug encapsulation structure.

[0045] The particle size range enhances the EPR effect targeted enrichment at tumor / inflammatory sites and prolongs the circulation time in the body.

[0046] The freeze-drying process in step S500 includes a pre-freezing stage (maintained at -40°C for 4 hours) and a sublimation drying stage (vacuum degree ≤ 10 Pa, heating to 25°C).

[0047] 5% mannitol was added as a freeze-drying protectant to control the final water content to ≤3%.

[0048] The nanomedicine can be stably stored at 25°C for 12 months without aggregation or drug leakage.

[0049] The biological protein nanorobot consists of a three-layer structure, which consists of a hydrophobic anti-rheumatic drug core, a denatured albumin middle layer and an L-arginine outer loading layer from the inside to the outside. Anti-rheumatic drugs and NO can act as inducers of immunogenic cell death, enhance the production of immune antigens, and thus promote the maturation of antigen-presenting cells and the activation of T cells.

[0050] L-arginine is fixed to the negatively charged surface of denatured albumin by electrostatic adsorption.

[0051] L-arginine decomposes in the lesion microenvironment to produce NO, which enhances vascular permeability and guides the targeted enrichment of nanorobots.

[0052] The denatured albumin middle layer has a porous network structure with a pore size of 2-5 nm and a drug encapsulation rate of ≥90%.

[0053] The porous structure was formed by acid-heat denaturation-induced conformational unfolding of albumin.

[0054] Achieve sustained drug release (24-hour release ≤ 50%) and reduce drug administration frequency.

[0055] The L-arginine external loading layer accounts for 5-15% of the total mass of the nanoparticles, and the release rate is ≥80% in an environment below pH 6.5.

[0056] The pH responsiveness of L-arginine is due to the difference in pH between its isoelectric point (pI=10.8) and the lesion microenvironment.

[0057] The acidic inflammatory / tumor microenvironment triggers the release of targeting molecules, enhancing the accuracy of lesion localization.

[0058] The molar ratio of the hydrophobic anti-rheumatic drug to L-arginine is 1:2 to 1:5, and the drug acts synergistically in the lesion area through the NO-mediated mitochondrial apoptosis pathway.

[0059] NO inhibits the NF-κB pathway and reduces the expression of inflammatory factors TNF-α / IL-6 by ≥40%.

[0060] The dual mechanism treats rheumatic diseases with an efficacy 2-3 times higher than that of a single drug.

[0061] Targeted delivery via intravenous or local administration, including intra-articular, salivary gland, kidney, or skin;

[0062] Targeting Mechanism:

[0063] RA: Targets abnormally proliferating fibroblast-like synoviocytes (FLS) in the synovium of joints;

[0064] Systemic lupus erythematosus (SLE): Targets areas of overactive B cells or immune complex deposits in the kidneys, skin, or blood;

[0065] SpA: Targeting inflammatory Th17 cells or bone erosion-related osteoblasts in the spine and sacroiliac joints;

[0066] pSS: Targets infiltrating lymphocytes or glandular epithelial cells in salivary or lacrimal glands.

[0067] Nanoparticles are enriched in synovial tissue through the L-arginine chemotactic effect, with concentrations 5-8 times higher than those in non-targeted areas.

[0068] The toxicity of systemic administration is reduced, and the local drug concentration in the joints is increased to more than 3 times that of traditional preparations.

[0069] NO enhances the killing efficiency of T cells against tumors / abnormal immune cells by activating the cGMP pathway.

[0070] The combined treatment regimen reduced joint swelling in rheumatoid arthritis model mice by 60%-70%.

[0071] In the present invention, unless otherwise specified or limited, the terms "installed," "connected," "connect," "fixed," etc. should be understood in a broad sense. For example, they can refer to fixed connection, detachable connection, or integration; mechanical connection, electrical connection, or communication; direct connection or indirect connection through an intermediate medium; internal communication between two elements or interaction between two elements, unless otherwise specified. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0072] Obviously, the embodiments described above are only some embodiments of the present invention, rather than all embodiments. The preferred embodiments of the present invention are given in the accompanying drawings, but they do not limit the patent scope of the present invention. The present invention can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Although the present invention has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present invention specification and drawings, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present invention.

Claims

1. Application of bioprotein nanorobots to deliver biological agents for targeted precision immunotherapy and preparation method thereof, characterized in that: The following steps are involved: S100, preparing a serum albumin aqueous solution, wherein the serum albumin concentration is 5-10% w / v, and the solvent is a phosphate buffer solution at pH 7.4; S200, preparing an organic solution of a hydrophobic antirheumatic drug, wherein the drug is one or more of adalimumab, secukinumab, ixekizumab, tetasip, tocilizumab, and rituximab, the solvent is ethanol or acetone, and the drug concentration is 40-160 mg / mL; S300, mixing the solution of step S100 with the solution of step S200 in a mass ratio of 1:5 to 1:10, adding an acid-heat denaturant to adjust the pH to 4.0-5.0, and reacting at 60-80° C. for 1-3 hours to form denatured albumin-coated drug nanoparticles; S400, adding L-arginine solution to the product of step S300, wherein the mass ratio of L-arginine to albumin is 1:10 to 1:20, and adsorbing L-arginine on the surface of the nanoparticles by ultrasonic dispersion; S500, centrifugal purification, and freeze-drying to obtain nanorobot targeted drugs.

2. The targeted precision immunotherapy using bioprotein nanorobots to deliver biological agents and the preparation method thereof according to claim 1 is characterized in that: In step S300 , the acid-heat denaturant is 0.1-0.5 M hydrochloric acid or citric acid buffer, the reaction temperature is 70° C.±2° C., and the reaction time is 2 hours.

3. The targeted precision immunotherapy using bioprotein nanorobots to deliver biological agents and the preparation method thereof according to claim 2 is characterized in that: In step S400 , the power of ultrasonic dispersion is 300 W, the time is 20 minutes, and the particle size of the nanoparticles is controlled to be 80-120 nm.

4. The targeted precision immunotherapy using bioprotein nanorobots to deliver biological agents and the preparation method thereof according to claim 3 is characterized in that: The freeze-drying process in step S500 includes a pre-freezing stage and a sublimation drying stage.

5. A bioprotein nanorobot, prepared by using a bioprotein nanorobot to deliver biological agents for targeted precision immunotherapy and a preparation method thereof as described in claim 4, characterized in that: It consists of a three-layer structure, from the inside to the outside: a hydrophobic anti-rheumatic drug core, a denatured albumin middle layer, and an L-arginine outer loading layer. Anti-rheumatic drugs and NO can act as inducers of immunogenic cell death, enhance the production of immune antigens, and thus promote the maturation of antigen-presenting cells and the activation of T cells.

6. The bioprotein nanorobot according to claim 5, characterized in that The denatured albumin middle layer has a porous network structure with a pore size of 2-5 nm and a drug encapsulation rate of ≥90%.

7. The bioprotein nanorobot according to claim 6, characterized in that The L-arginine external loading layer accounts for 5-15% of the total mass of the nanoparticles, and the release rate is ≥80% in an environment below pH 6.

5.

8. The bioprotein nanorobot according to claim 7, characterized in that: The molar ratio of the hydrophobic anti-rheumatic drug to L-arginine is 1:2 to 1:5, and the drug acts synergistically in the lesion area through the NO-mediated mitochondrial apoptosis pathway.

9. Use of a bioprotein nanorobot in the preparation of a drug for treating connective tissue diseases, using the bioprotein nanorobot as claimed in claim 8, characterized in that: Targeted delivery via intravenous or local administration, including intra-articular, salivary gland, kidney, or skin; Targeting mechanism: RA: Targets abnormally proliferating fibroblast-like synoviocytes in the synovium of joints; Systemic lupus erythematosus (SLE): Targets areas of overactive B cells or immune complex deposits in the kidneys, skin, or blood; SpA: Targeting inflammatory Th17 cells or bone erosion-related osteoblasts in the spine and sacroiliac joints; ‌pSS‌: Targets infiltrating lymphocytes or glandular epithelial cells in salivary or lacrimal glands.

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