Novel intracellular protein delivery carrier based on Fenton / Fenton-like reaction as well as preparation method and application of novel intracellular protein delivery carrier

Abstract

The invention provides a carrier material for supporting a protein drug. The carrier material comprises an inner core, a calcium carbonate coating layer coating the inner core and a polyethylene glycol-polyglutamic acid diblock copolymer chain segment anchored on the coating layer. The intracellular protein delivery carrier provided by the invention can effectively support various metal ions and protein drugs with the Fenton / Fenton-like reaction catalysis effect, and after the intracellular protein delivery carrier is endocytosed by cells, the protein drugs, calcium ions and the metal ions with the Fenton / Fenton-like reaction catalysis effect are released; metal ions can catalyze hydrogen peroxide in the endosome / lysosome to generate hydroxyl radicals with high oxidation activity, lipid molecules are oxidized, and a lysosome membrane is destroyed; the released calcium ions can cause mitochondrial calcium overload, improve the level of hydrogen peroxide in cells, promote generation of hydroxyl radicals in endosome / lysosome and damage of membranes of the hydroxyl radicals, induce efficient rupture of the endosome / lysosome and deliver protein drugs into cytoplasm so as to play the functions of the protein drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of apoprotein nano-drugs, and relates to a carrier material for loading protein drugs, its preparation method and application, in particular to a novel intracellular protein delivery carrier based on Fenton / Fenton-like reaction, its preparation method and application. Background Art

[0002] Protein drugs have the advantages of strong specificity, high functional activity, low side effects and low genetic risk, and have been widely used in the treatment of diseases such as cancer, diabetes and autoimmune diseases. However, the commonly used protein drugs in clinics are mainly monoclonal antibodies, insulin and cytokines, etc., which are proteins that function extracellularly. Proteins that function intracellularly, such as ribonuclease A, cytochrome C and saponin, etc., have different action mechanisms from proteins that function extracellularly, and can provide more choices for the development of new protein drugs. However, proteins have characteristics such as large molecular weight, insufficient hydrophilicity and surface positive charge, resulting in poor membrane permeability and difficulty in entering cells; more importantly, protein drugs are easily trapped in endosomes after being taken up by cells and are eventually degraded by proteases in lysosomes, thus unable to reach the cytoplasm to exert their anti-cancer function.

[0003] Currently reported intracellular protein delivery carriers include polymeric micelles, polymeric vesicles, lipid nanoparticles, nanogels, nanocapsules, inorganic nanoparticles and cell-penetrating peptides, etc. However, these methods often involve complex chemical syntheses, the carriers themselves have serious cytotoxicity, or the proteins need to be modified, which affects the activity of the proteins. At the same time, the commonly used endosomal escape mechanisms of these carriers include membrane fusion (such as lipid nanoparticles, nanocapsules), proton sponge effect (such as polymeric micelles, polymeric vesicles) and membrane perforation (cell-penetrating peptides), etc. Although these endosomal escape methods can promote the endosomal escape of protein molecules to a certain extent, their efficiency is low, and most proteins will still be trapped in endosomes / lysosomes and degraded.

[0004] Therefore, how to develop a more suitable novel intracellular protein delivery carrier to solve the above problems existing in the existing intracellular protein delivery carriers, and at the same time achieve the endosomal / lysosomal escape of intracellular functional proteins is of great significance for the development of new protein drugs. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a carrier material for loading protein drugs, its preparation method and application, especially a novel intracellular protein delivery carrier based on Fenton / Fenton-like reaction. After being endocytosed by cells, the intracellular protein delivery carrier provided by the present invention will release protein drugs, calcium ions and metal ions with catalytic effect of Fenton / Fenton-like reaction, which can destroy the lysosomal membrane, promote the generation of hydroxyl radicals in endosomes / lysosomes and the destruction of their membranes, and finally deliver the protein drugs to the cytoplasm to exert the functions of the protein drugs. Moreover, the preparation method is simple, the conditions are mild, the controllability is good, and the stability is strong, which is more suitable for large-scale popularization and application.

[0006] The present invention provides a carrier material for loading protein drugs, and the carrier material includes a core, a calcium carbonate coating layer coated on the core, and a polyethylene glycol-polyglutamic acid diblock copolymer segment anchored on the coating layer;

[0007] The core of the carrier material includes the loaded protein drug and metal ions.

[0008] Preferably, the particle size of the carrier material is 10-300 nm;

[0009] The polyethylene glycol-polyglutamic acid diblock copolymer segment is uniformly anchored on the coating layer, and the copolymer segment stretches outwards to form a villous structure;

[0010] The carrier material is specifically a carrier composite loaded with protein drugs.

[0011] Preferably, the metal ions include one or more of iron ions, copper ions, manganese ions and cobalt ions;

[0012] In the carrier material, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the protein drug is (1-100):1;

[0013] The carrier material has a micelle structure or a sea urchin structure.

[0014] Preferably, in the carrier material, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to calcium carbonate is 1:(1-200);

[0015] The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the metal ions is 1:(1-100);

[0016] The carrier material has an encapsulation structure, and the protein drug is encapsulated inside the calcium carbonate coating layer.

[0017] Preferably, the protein drugs include one or more of cytokines, chemokines, monoclonal antibodies or their fragments, and therapeutic protein drugs;

[0018] The carrier material is specifically a carrier material for intracellular protein delivery.

[0019] The present invention provides a method for preparing a carrier material for loading a protein drug, comprising the following steps:

[0020] After mixing a polyethylene glycol-polyglutamic acid diblock copolymer, a protein drug, and a carbonate solution, a calcium salt solution and a metal ion source solution are added and mixed again, and then after separation and lyophilization, a carrier material for loading a protein drug is obtained.

[0021] Preferably, the carbonate includes sodium carbonate;

[0022] The concentration of the carbonate solution is 0.1-10 M;

[0023] The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the protein drug is (0.1-1):(0.01-0.1).

[0024] Preferably, the calcium salt includes calcium chloride;

[0025] The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the calcium salt is (0.1-1):(100-1000); wherein, the calcium salt is calculated as calcium ions;

[0026] The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the metal ion source is (0.1-1):(100-1000).

[0027] Preferably, the metal ion source includes one or more of ferric chloride, copper chloride, manganese chloride, and cobalt chloride;

[0028] The temperature of the re-mixing is 4-25 °C;

[0029] The time of the re-mixing is 12-24 h.

[0030] The present invention also provides the use of the carrier material described in any one of the above technical solutions or the carrier material prepared by the preparation method described in any one of the above technical solutions in the cytoplasmic delivery of protein drugs.

[0031] The present invention provides a carrier material for loading protein drugs. The carrier material includes a core, a calcium carbonate coating layer coated on the core, and a polyethylene glycol-polyglutamic acid diblock copolymer segment anchored on the coating layer; the core of the carrier material includes the loaded protein drug and metal ions. Compared with the prior art, the present invention believes that it is a feasible research direction to induce lipid peroxidation by Fenton / Fenton-like reaction, which means that in the presence of certain metal ions, hydrogen peroxide in cells can be catalytically decomposed into hydroxyl radicals with higher oxidation activity, and these hydroxyl radicals can cause the lipids in the cell membrane structure to be peroxidized, thereby destroying membrane-structured organelles (such as mitochondria, lysosomes, etc.). In addition, calcium carbonate composite nanoparticles prepared by calcium carbonate mineralization using a degradable polymer as a template have been used to separately load hydrophilic metal ions or protein drugs. Such nanoparticles have the advantages of simple preparation process, high stability, good biocompatibility and degradability. Most importantly, the process of loading proteins does not require chemical modification of the proteins and can maintain the biological activity of the proteins. Moreover, after being endocytosed by cells, the calcium carbonate composite nanoparticles can degrade in the acidic environment of endosomes / lysosomes and release the loaded drugs.

[0032] Based on this, the present invention specifically designs a carrier material for loading protein drugs with a specific structure and composition. This is a novel intracellular protein delivery carrier based on Fenton / Fenton-like reaction, which has a simple structure, good biocompatibility, does not affect protein activity, and can efficiently promote the escape of protein drugs from endosomes / lysosomes. The present invention uses a polyethylene glycol-polyglutamic acid diblock copolymer as a template to prepare novel calcium carbonate composite nanoparticles that simultaneously encapsulate protein drugs and metal ions with Fenton / Fenton-like reaction catalytic effects through calcium carbonate mineralization. After being endocytosed by cells, the calcium carbonate composite nanoparticles will decompose in the acidic environment of endosomes / lysosomes, releasing the protein drugs, calcium ions, and metal ions with Fenton / Fenton-like reaction catalytic effects. The metal ions with Fenton / Fenton-like reaction catalytic effects will catalyze hydrogen peroxide in endosomes / lysosomes to generate highly oxidative hydroxyl radicals, oxidize lipid molecules in the endosome / lysosome membrane structure, and damage the lysosome membrane. In addition, the released calcium ions can cause mitochondrial calcium overload, increase the intracellular hydrogen peroxide level, further promote the generation of hydroxyl radicals in endosomes / lysosomes and the damage of their membranes, and ultimately induce efficient endosome / lysosome rupture, delivering the protein drugs to the cytoplasm to exert the functions of the protein drugs. The intracellular protein delivery carrier provided by the present invention can effectively load a variety of metal ions with Fenton / Fenton-like reaction catalytic effects and protein drugs.

[0033] The present invention selects a biocompatible polyethylene glycol-polyglutamic acid diblock copolymer and calcium carbonate as the base materials, enabling the prepared novel intracellular protein delivery carrier to have higher clinical translation potential. The novel intracellular protein delivery carrier proposed by the present invention induces endosome / lysosome lipid peroxidation through hydroxyl radicals generated by the Fenton / Fenton-like reaction, and combines with calcium ion-induced mitochondrial calcium overload to promote the endosome / lysosome escape of protein drugs. The novel intracellular protein delivery carrier proposed by the present invention can achieve the cytoplasmic delivery of intracellular anti-cancer protein drugs with different molecular weights, different structures and different surface charges, providing new ideas for the development of novel protein drugs. Brief Description of the Drawings

[0034] Figure 1 It is a schematic diagram of the structure and a schematic diagram of the preparation process of the intracellular protein delivery carrier based on the Fenton / Fenton-like reaction provided by the present invention;

[0035] Figure 2 It is a TEM imaging diagram of the intracellular protein delivery carrier prepared in Example 3 of the present invention;

[0036] Figure 3 It is the hydrodynamic diameter of the intracellular protein delivery carrier prepared in Example 3 of the present invention;

[0037] Figure 4 It is the subcellular localization of the intracellular protein delivery carrier prepared in Example 3 of the present invention in K7 mouse osteosarcoma cells;

[0038] Figure 5 It is the corresponding cell survival rate after the intracellular protein delivery carrier prepared in Example 3 of the present invention is co-incubated with K7 mouse osteosarcoma cells;

[0039] Figure 6 It is the evaluation result of the in vivo anti-tumor effect of the intracellular protein delivery carrier prepared in Example 3 of the present invention. Detailed Embodiments

[0040] In order to further understand the present invention, the preferred implementation modes of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention rather than limiting the claims of the present invention.

[0041] There is no particular limitation on the sources of all raw materials of the present invention, and they can be purchased on the market or prepared by conventional methods well-known to those skilled in the art.

[0042] There is no particular limitation on the purity of all raw materials of the present invention. The present invention preferably uses analytically pure or conventional purity in the field of protein drug carrier materials.

[0043] All the noun expressions and abbreviations in the present invention belong to the conventional noun expressions and abbreviations in the art. Each noun expression and abbreviation is clear and definite in its relevant application field, and those skilled in the art can clearly, accurately and uniquely understand them according to the noun expressions and abbreviations.

[0044] The present invention provides a carrier material for loading protein drugs, and the carrier material includes a core, a calcium carbonate coating layer coated on the core, and a polyethylene glycol-polyglutamic acid diblock copolymer segment anchored on the coating layer;

[0045] The core of the carrier material includes the loaded protein drug and metal ions.

[0046] In the present invention, the particle size of the carrier material is preferably 10-300 nm, more preferably 50-250 nm, and even more preferably 100-200 nm.

[0047] In the present invention, the polyethylene glycol-polyglutamic acid diblock copolymer segment is preferably uniformly anchored on the coating layer, and the copolymer segment stretches outwards to form a villous structure.

[0048] In the present invention, the carrier material is specifically preferably a carrier composite material loaded with protein drugs.

[0049] In the present invention, the metal ions preferably include one or more of iron ions, copper ions, manganese ions and cobalt ions, and more preferably iron ions, copper ions, manganese ions or cobalt ions.

[0050] In the present invention, in the carrier material, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the protein drug is preferably (1-100):1, more preferably (10-80):1, and even more preferably (30-60):1.

[0051] In the present invention, the carrier material preferably has a micelle structure or a sea urchin structure.

[0052] In the present invention, in the carrier material, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to calcium carbonate is preferably 1:(1-200), more preferably 1:(10-150), even more preferably 1:(30-100), and even more preferably 1:(50-80).

[0053] In the present invention, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the metal ions is preferably 1:(1-100), more preferably 1:(10-80), and even more preferably 1:(30-50).

[0054] In the present invention, the carrier material preferably has an encapsulation structure, and the protein drug is encapsulated inside the calcium carbonate coating layer.

[0055] In the present invention, the protein drug preferably comprises one or more of cytokines, chemokines, monoclonal antibodies or fragments thereof, and therapeutic protein drugs, and more preferably is cytokines, chemokines, monoclonal antibodies or fragments thereof or therapeutic protein drugs.

[0056] In the present invention, the carrier material is specifically preferably a carrier material for intracellular protein delivery.

[0057] The present invention provides a method for preparing a carrier material loaded with a protein drug, comprising the following steps:

[0058] After mixing a polyethylene glycol-polyglutamic acid diblock copolymer, a protein drug and a carbonate solution, a calcium salt solution and a metal ion source solution are added and mixed again, and then after separation and lyophilization, a carrier material loaded with the protein drug is obtained.

[0059] In the present invention, the carbonate preferably comprises sodium carbonate.

[0060] In the present invention, the concentration of the carbonate solution is preferably 0.1-10 M, more preferably 0.1-5 M, and even more preferably 0.2-1 M.

[0061] In the present invention, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the protein drug is preferably (0.1-1):(0.01-0.1), more preferably (0.3-0.8):(0.03-0.08), and even more preferably (0.5-0.6):(0.05-0.06).

[0062] In the present invention, the calcium salt preferably comprises calcium chloride.

[0063] In the present invention, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the calcium salt is preferably (0.1-1):(100-1000), more preferably (0.3-0.8):(300-800), and even more preferably (0.5-0.6):(500-600). Wherein, the calcium salt is calculated as calcium ions.

[0064] In the present invention, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the metal ion source is preferably (0.1-1):(100-1000), more preferably (0.3-0.8):(300-800), and even more preferably (0.5-0.6):(500-600).

[0065] In the present invention, the metal ion source preferably comprises one or more of ferric chloride, copper chloride, manganese chloride and cobalt chloride, and more preferably is ferric chloride, copper chloride, manganese chloride or cobalt chloride.

[0066] In the present invention, the temperature of the re - mixing is preferably 4 - 25°C, more preferably 9 - 20°C, and even more preferably 14 - 15°C.

[0067] In the present invention, the time of the re - mixing is preferably 12 - 24 h, more preferably 14 - 22 h, and even more preferably 16 - 20 h.

[0068] The present invention provides the application of the carrier material described in any one of the above - mentioned technical solutions or the carrier material prepared by the preparation method described in any one of the above - mentioned technical solutions in the cytoplasmic delivery of protein drugs.

[0069] In order to complete and refine the overall technical solution, better ensure the composition and structure of the intracellular protein delivery carrier, and further improve the effectiveness, stability and delivery performance of the intracellular protein delivery carrier, the above - mentioned novel intracellular protein delivery carrier based on the Fenton / Fenton - like reaction, its preparation method and application specifically may include the following contents:

[0070] A novel intracellular protein delivery carrier based on the Fenton / Fenton - like reaction, the novel intracellular protein delivery carrier based on the Fenton / Fenton - like reaction comprises the following components: polyethylene glycol - polyglutamic acid diblock copolymer, calcium carbonate, metal ions, protein drugs.

[0071] A preparation method of a novel intracellular protein delivery carrier based on the Fenton / Fenton - like reaction, comprising the following steps:

[0072] Dissolve the polyethylene glycol - polyglutamic acid diblock copolymer and the protein drug in a sodium carbonate solution, then add calcium chloride and a metal ion solution that have been previously dissolved in deionized water, and obtain the product after stirring, centrifuging and freeze - drying.

[0073] Specifically, the concentration of the sodium carbonate solution is 0.2 M and the pH value is 9.0.

[0074] Specifically, the metal ion solution is a ferric chloride, copper chloride, manganese chloride or cobalt chloride solution.

[0075] Specifically, the reaction amount of the polyethylene glycol - polyglutamic acid diblock copolymer is 0.1 - 1 μmol, preferably 0.7 μmol.

[0076] Specifically, the reaction amount of the protein drug is 0.01 - 0.1 μmol, preferably 0.07 μmol.

[0077] Specifically, the reaction amount of the calcium chloride is 0.1 - 1 mmol, preferably 0.1 mmol.

[0078] Specifically, the reaction amount of the metal ion solution is 0.01 - 0.05 mmol, preferably 0.02 mmol.

[0079] Specifically, the stirring temperature is 4 to 25 °C, preferably 4 °C.

[0080] Specifically, the stirring time is 12 to 24 hours, preferably 12 h.

[0081] The novel intracellular protein delivery carrier based on the Fenton / Fenton-like reaction of the present invention is prepared by the preparation method described in any one of the above technical solutions.

[0082] See Figure 1 , Figure 1 , which is a schematic diagram of the structure and a schematic diagram of the preparation process of the intracellular protein delivery carrier based on the Fenton / Fenton-like reaction provided by the present invention.

[0083] The present invention provides the application of the carrier material based on the Fenton / Fenton-like reaction prepared by the preparation method described in any one of the above in an intracellular protein delivery carrier.

[0084] The novel intracellular protein delivery carrier based on the Fenton / Fenton-like reaction prepared by the preparation method described in any one of the above of the present invention delivers the protein drug into the cytoplasm to exert the function of the protein drug.

[0085] The above content of the present invention provides a novel intracellular protein delivery carrier based on the Fenton / Fenton-like reaction, its preparation method and application. The novel intracellular protein delivery carrier based on the Fenton / Fenton-like reaction with a specific structure and composition specially designed by the present invention has a simple structure, good biocompatibility, does not affect protein activity, and at the same time can efficiently promote the endosome / lysosome escape of protein drugs. The present invention uses a polyethylene glycol-polyglutamic acid diblock copolymer as a template to prepare novel calcium carbonate composite nanoparticles that simultaneously encapsulate protein drugs and metal ions with Fenton / Fenton-like reaction catalytic effects through calcium carbonate mineralization. After being endocytosed by cells, the calcium carbonate composite nanoparticles will decompose in the acidic environment of endosomes / lysosomes, releasing protein drugs, calcium ions and metal ions with Fenton / Fenton-like reaction catalytic effects. Metal ions with Fenton / Fenton-like reaction catalytic effects will catalyze hydrogen peroxide in endosomes / lysosomes to generate highly oxidative hydroxyl radicals, oxidize lipid molecules in the endosome / lysosome membrane structure, and damage the lysosome membrane. In addition, the released calcium ions can cause mitochondrial calcium overload, increase the intracellular hydrogen peroxide level, further promote the generation of hydroxyl radicals in endosomes / lysosomes and the damage of their membranes, and finally induce efficient endosome / lysosome rupture to deliver protein drugs into the cytoplasm to exert the function of protein drugs. The intracellular protein delivery carrier provided by the present invention can effectively load a variety of metal ions with Fenton / Fenton-like reaction catalytic effects and protein drugs.

[0086] The present invention selects a biocompatible polyethylene glycol-polyglutamic acid diblock copolymer and calcium carbonate as the base materials, so that the prepared novel intracellular protein delivery carrier has higher clinical transformation potential. The novel intracellular protein delivery carrier proposed by the present invention induces endosome / lysosome lipid peroxidation through hydroxyl radicals generated by the Fenton / Fenton-like reaction, and combines calcium ion-induced mitochondrial calcium overload to promote the endosome / lysosome escape of protein drugs. The novel intracellular protein delivery carrier proposed by the present invention can achieve the cytoplasmic delivery of intracellular anti-cancer protein drugs with different molecular weights, different structures and different surface charges, providing new ideas for the development of novel protein drugs.

[0087] To further illustrate the present invention, the following is a detailed description of a carrier material for loading protein drugs, its preparation method and application provided by the present invention in combination with examples. However, it should be understood that these examples are implemented on the premise of the technical solution of the present invention, giving detailed implementation methods and specific operation processes, only to further illustrate the features and advantages of the present invention, rather than limiting the claims of the present invention. The protection scope of the present invention is not limited to the following examples.

[0088] Example 1

[0089] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9), and then add calcium chloride (0.1 mmol) and iron chloride (0.02 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, freeze-dry to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Fe 3+ (denoted as CaC@Fe-Protein).

[0090] Example 2

[0091] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9), and then add calcium chloride (0.2 mmol) and iron chloride (0.04 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, freeze-dry to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Fe 3+ (denoted as CaC@Fe-Protein).

[0092] Example 3

[0093] Dissolve the polyethylene glycol - polyglutamic acid diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9), then add calcium chloride (0.1 mmol) and iron(III) chloride (0.02 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Fe 3+ (denoted as CaC@Fe - Protein).

[0094] Example 4

[0095] Dissolve the polyethylene glycol - polyglutamic acid diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9), then add calcium chloride (0.2 mmol) and iron(III) chloride (0.04 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Fe 3+ (denoted as CaC@Fe - Protein).

[0096] Example 5

[0097] Dissolve the polyethylene glycol - polyglutamic acid diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9), then add calcium chloride (0.1 mmol) and copper(II) chloride (0.02 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Cu 2+ (denoted as CaC@Cu - Protein).

[0098] Example 6

[0099] Dissolve the polyethylene glycol - polyglutamic acid diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9), then add calcium chloride (0.2 mmol) and copper(II) chloride (0.04 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Cu 2+ (denoted as CaC@Cu - Protein).

[0100] Example 7

[0101] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.1 mmol) and copper chloride (0.02 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that simultaneously encapsulates the protein drug and Cu 2+ (denoted as CaC@Cu-Protein).

[0102] Example 8

[0103] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.2 mmol) and copper chloride (0.04 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that simultaneously encapsulates the protein drug and Cu 2+ (denoted as CaC@Cu-Protein).

[0104] Example 9

[0105] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.1 mmol) and manganese chloride (0.02 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that simultaneously encapsulates the protein drug and Mn 2+ (denoted as CaC@Mn-Protein).

[0106] Example 10

[0107] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.2 mmol) and manganese chloride (0.04 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that simultaneously encapsulates the protein drug and Mn 2+ (denoted as CaC@Mn-Protein).

[0108] Example 11

[0109] Dissolve the poly(ethylene glycol)-poly(glutamic acid) diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.1 mmol) and manganese chloride (0.02 mmol) that have been previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Mn 2+ (denoted as CaC@Mn-Protein).

[0110] Example 12

[0111] Dissolve the poly(ethylene glycol)-poly(glutamic acid) diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.2 mmol) and manganese chloride (0.04 mmol) that have been previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Mn 2+ (denoted as CaC@Mn-Protein).

[0112] Example 13

[0113] Dissolve the poly(ethylene glycol)-poly(glutamic acid) diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.1 mmol) and cobalt chloride (0.02 mmol) that have been previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Co 2+ (denoted as CaC@Co-Protein).

[0114] Example 14

[0115] Dissolve the poly(ethylene glycol)-poly(glutamic acid) diblock copolymer (0.4 μmol) and the protein drug (0.04 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.2 mmol) and cobalt chloride (0.04 mmol) that have been previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Co 2+ (denoted as CaC@Co-Protein).

[0116] Example 15

[0117] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.1 mmol) and cobalt chloride (0.02 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Co 2+ (denoted as CaC@Co-Protein).

[0118] Example 16

[0119] Dissolve the polyethylene glycol-polyglutamic acid diblock copolymer (0.7 μmol) and the protein drug (0.07 μmol) in sodium carbonate solution (0.2 M, pH = 9). Subsequently, add calcium chloride (0.2 mmol) and cobalt chloride (0.04 mmol) previously dissolved in deionized water. After stirring (4 °C, 12 h) and centrifugation, lyophilize to obtain a novel intracellular protein delivery carrier that encapsulates both the protein drug and Co 2+ (denoted as CaC@Co-Protein).

[0120] Example 17

[0121] Use transmission electron microscopy to analyze the morphology of the novel intracellular protein delivery carrier CaC@Fe-Protein. Drop the CaC@Fe-Protein obtained in Example 3 onto the surface of a copper grid, and perform the test after natural drying.

[0122] See Figure 2 , Figure 2 which is the TEM imaging diagram of the intracellular protein delivery carrier prepared in Example 3 of the present invention.

[0123] From Figure 2 the transmission electron microscopy image results of CaC@Fe-Protein in, it can be concluded that the microscopic morphology of CaC@Fe-Protein prepared in the present invention is spherical.

[0124] Example 18

[0125] Use a dynamic light scattering instrument (DLS) to analyze the size of CaC@Fe-Protein obtained in Example 3, and measure the particle size of CaC@Fe-Protein obtained in Example 3.

[0126] See Figure 3 , Figure 3 which is the hydrodynamic diameter of the intracellular protein delivery carrier prepared in Example 3 of the present invention.

[0127] From Figure 3From the particle size statistics results of CaC@Fe-Protein, the diameter of CaC@Fe-Protein nanoparticles is about 214 nm.

[0128] Example 19

[0129] Inoculate 10,000 K7 cells in a confocal dish and culture for 24 hours. Add the CaC@Fe-Protein prepared in Example 3 and continue to incubate with K7 cells for 4 hours. Select K7 cells incubated with pure Protein and PBS for 4 hours as the control. Subsequently, add Lysotracker Green (lysosome marker) with a final concentration of 200 nM to each group and culture for 1 hour. After washing with PBS, add Hoechst 33342 containing 1 μg / mL and incubate for 10 minutes. Use a laser confocal microscope to image the protein delivery of CaC@Fe-Protein. The results are shown in Figure 4 .

[0130] See Figure 4 , Figure 4 which is the subcellular localization of the intracellular protein delivery vector prepared in Example 3 of the present invention in K7 murine osteosarcoma cells.

[0131] From Figure 4 the subcellular localization results of CaC@Fe-Protein in

[0132] Example 20

[0133] Inoculate 5,000 K7 cells in a 96-well plate and culture for 24 hours. Add 20 μL of CaC@Fe-Protein and Protein with different concentrations and continue to incubate for 72 hours. Subsequently, add 20 μL of MTT solution to each well, discard the supernatant after culturing for 4 hours, add 100 μL of DMSO, and oscillate for 1 min. Detect the absorbance of the sample at 492 nm. The results are shown in Figure 5 .

[0134] See Figure 5 , Figure 5 which is the corresponding cell viability of the intracellular protein delivery vector prepared in Example 3 of the present invention after co-incubation with K7 murine osteosarcoma cells.

[0135] From Figure 5It can be seen that the cytotoxicity of CaC@Fe-Protein. Under the condition of the same concentration, CaC@Fe-Protein can effectively inhibit the activity of tumor cells compared with pure Protein, proving that CaC@Fe-Protein successfully delivers the protein drug into the cytoplasm to exert the cytotoxic function of the protein drug.

[0136] Example 21

[0137] Subcutaneously inject 1×10 6 K7 cells into the right lower abdomen of female BALB / c mice. When the tumor grows to 100 mm 3 , on the 0th, 2nd, 4th, and 6th days respectively, intravenously inject the novel intracellular protein delivery carrier CaC@Fe-Protein prepared in Example 3, and use the intravenous injection of an equal amount of pure Protein and PBS as the control group. Regularly measure the tumor volume during the treatment period, and the obtained tumor growth curve is shown in Figure 6 .

[0138] See Figure 6 , Figure 6 , which is the evaluation result of the in vivo anti-tumor effect of the intracellular protein delivery carrier prepared in Example 3 of the present invention.

[0139] It can be seen from Figure 6 that for the tumor growth curves of the control group and the mice treated with CaC@Fe-Protein, CaC@Fe-Protein can effectively inhibit tumor growth compared with pure Protein, proving that CaC@Fe-Protein successfully delivers the protein drug into the cytoplasm to exert the cytotoxic function of the protein drug.

[0140] The above provides a detailed introduction to a novel intracellular protein delivery carrier based on the Fenton / Fenton-like reaction and its preparation method and application. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above examples is only used to help understand the method and its core idea of the present invention, including the best mode, and also enables any person skilled in the art to practice the present invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The protection scope of the present invention patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements that are not different from the literal description of the claims, or if they include equivalent structural elements that have no substantial difference from the literal description of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A carrier material for loading protein drugs, characterized in that, The carrier material includes a core, a calcium carbonate coating layer coated on the core, and a polyethylene glycol-polyglutamic acid diblock copolymer segment anchored on the coating layer; The core of the carrier material includes the protein drug and metal ions carried.

2. The carrier material according to claim 1, characterized in that, The particle size of the carrier material is 10-300 nm; The polyethylene glycol-polyglutamic acid diblock copolymer segments are uniformly anchored on the coating layer, and the copolymer segments stretch outwards to form a villous structure; The carrier material is specifically a carrier composite carrying a protein drug.

3. The carrier material according to claim 1, wherein The metal ions include one or more of iron ions, copper ions, manganese ions, and cobalt ions; In the carrier material, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the protein drug is (1-100):1; The carrier material has a micelle structure or a sea urchin structure.

4. The carrier material according to claim 1, characterized in that, In the carrier material, the molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to calcium carbonate is 1:(1-200); The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the metal ions is 1:(1-100); The carrier material has an encapsulation structure, and the protein drug is encapsulated inside the calcium carbonate coating layer.

5. The carrier material according to claim 1, characterized in that, The protein drug includes one or more of cytokines, chemokines, monoclonal antibodies or their fragments, and therapeutic protein drugs; The carrier material is specifically a carrier material for intracellular protein delivery.

6. A method for preparing a carrier material for loading a protein drug, characterized in that, It includes the following steps: After mixing the polyethylene glycol-polyglutamic acid diblock copolymer, the protein drug, and the carbonate solution, adding a calcium salt solution and a metal ion source solution and mixing again, and then separating and freeze-drying, a carrier material carrying the protein drug is obtained.

7. The preparation method according to claim 6, characterized in that, The carbonate includes sodium carbonate; The concentration of the carbonate solution is 0.1-10 M; The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the protein drug is (0.1-1):(0.01-0.1).

8. The preparation method according to claim 6, characterized in that, The calcium salt includes calcium chloride; The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the calcium salt is (0.1-1):(100-1000); where the calcium salt is calculated as calcium ions; The molar ratio of the polyethylene glycol-polyglutamic acid diblock copolymer to the metal ion source is (0.1-1):(100-1000).

9. The preparation method according to claim 6, characterized in that The metal ion source includes one or more of ferric chloride, copper chloride, manganese chloride, and cobalt chloride; The temperature of the re-mixing is 4-25 °C; The time of the re-mixing is 12-24 h.

10. Use of the carrier material according to any one of claims 1-5 or the carrier material prepared by the preparation method according to any one of claims 6-9 in the cytoplasmic delivery of protein drugs.

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