Polymer to be subjected to drug loading, drug-loading nano-framework and preparation method thereof

Abstract

The invention belongs to the technical field of material synthesis, particularly relates to the technical field of material synthesis, and provides a polymer to be subjected to drug loading, a drug-loading nano framework and a preparation method thereof.The preparation method comprises the steps that amino acid hydrochloride and dihydric alcohol are mixed and subjected to a dehydration reaction, and a monomer substance is obtained; the preparation method comprises the following steps: mixing a diisocyanate precursor and alpha-allyl glyceryl ether to obtain a first mixture, adding a first catalyst and an antioxidant into the first mixture, carrying out a mixing reaction to obtain a prepolymer, adding the monomer substances into the prepolymer, and carrying out a mixing reaction to obtain a polymer to be subjected to drug loading. According to the polymer to be subjected to drug loading prepared by the scheme, a coating can be formed on the surface of the iron oxide nanoparticles, so that the dispersity of the iron oxide nanoparticles is improved.

Description

Technical Field

[0001] The present application belongs to the technical field of material synthesis, and in particular relates to a drug-loaded polymer, a drug-loaded nanostructure, and a preparation method thereof. Background Art

[0002] At present, macrophage regulation provides a new anti-tumor treatment strategy, that is, eliminating tumor cells by activating the immune surveillance of the tumor microenvironment. Since macrophages have phagocytic effects, foreign nanomaterials entering the physiological system will first interact with macrophages and affect macrophage polarization, which provides the possibility of regulating macrophage-mediated tumor immune activity and activating tumor immune surveillance. Iron oxide nanoparticles have been widely studied in the field of tumor diagnosis as imaging and cell labeling agents. In addition to being used as magnetic resonance imaging contrast agents, they have great potential in the field of immune regulation of the tumor microenvironment. Iron oxide nanoparticles have regulatory activity on macrophage polarization, can induce the production of reactive oxygen species, and affect macrophages by regulating intracellular redox and iron metabolism. However, due to the poor dispersion stability of unmodified iron oxide nanoparticles in the physiological environment in vivo, they are easy to adsorb proteins and precipitate, and cannot achieve immune regulation of macrophages. Summary of the invention

[0003] The purpose of the present application is to provide a drug-loaded polymer, a drug-loaded nanostructure and a preparation method thereof, aiming to solve the problem of poor dispersion stability of iron oxide nanoparticles.

[0004] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0005] In a first aspect, the present application provides a polymer to be loaded with drugs, wherein the structure of the polymer to be loaded with drugs is:

[0006]

[0007] Among them, R1 is a diisocyanate residue, R2 is a diol residue, and R3 is an amino acid residue.

[0008] Preferably, the diisocyanate comprises at least one of hexamethylene diisocyanate, 4,4'-methylene diphenyl diisocyanate, tetramethylene diisocyanate, toluene diisocyanate and / or isophorone diisocyanate; and / or,

[0009] The diol comprises at least one of ethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol and / or 1,8-octanediol; and / or,

[0010] The amino acids include at least one of arginine, phenylalanine, valine, leucine and / or glycine.

[0011] In a second aspect, the present application provides a method for preparing a drug-loaded polymer, the preparation method comprising:

[0012] The amino acid hydrochloride and the diol are mixed and subjected to a dehydration reaction to obtain a monomer substance;

[0013] mixing a diisocyanate precursor and α-allyl glyceryl ether to obtain a first mixture;

[0014] Adding a first catalyst and an antioxidant to the first mixture, mixing and reacting to obtain a prepolymer;

[0015] The monomer material is added into the prepolymer, and mixed and reacted to obtain the polymer to be loaded with drugs.

[0016] Preferably, the amino acid hydrochloride includes L-arginine hydrochloride, and the diol includes ethylene glycol. Correspondingly, the amino acid hydrochloride and the diol are mixed and dehydrated to obtain a monomer substance, including:

[0017] Mixing L-arginine hydrochloride and ethylene glycol, adding a second catalyst and a first organic solvent, and performing a reflux reaction at a first reaction temperature for a first preset time to obtain a reactant;

[0018] The reactant is cooled to room temperature and filtered to obtain a filtered solution;

[0019] The filtered solution is placed in isopropanol and repeatedly recrystallized and dried to obtain a purified product;

[0020] The purified product is dissolved in deionized water, the pH is adjusted to a preset pH, and L-arginine ethane-1,2-diester is obtained after drying.

[0021] Preferably, the first catalyst includes 2-ethyl-stannous hexanoate, and the antioxidant includes 2,6-di-tert-butyl-4-methylphenol. Correspondingly, the first catalyst and the antioxidant are added to the first mixture, and the mixed reaction obtains the prepolymer, comprising:

[0022] adding 2-ethyl-stannous hexanoate and 2,6-di-tert-butyl-4-methylphenol to the first mixture to obtain a second mixture;

[0023] The second mixture is heated to a second preset temperature under nitrogen protection and reacted for a second preset time to obtain a prepolymer.

[0024] Preferably, the monomer substance is added to the prepolymer, and mixed and reacted to obtain the drug-loaded polymer, comprising:

[0025] Adding the L-arginine ethane-1,2-diester to the prepolymer, heating to a third preset temperature under nitrogen protection, and reacting for a third preset time to obtain a first product;

[0026] Repeatedly placing the first product in ethyl acetate and water for precipitation to obtain a second product;

[0027] The second product is subjected to a solid-liquid separation operation to obtain a solid phase drug-loaded polymer.

[0028] In a third aspect, the present application provides a drug-carrying nanostructure, wherein the drug-carrying nanostructure comprises:

[0029] A drug-loaded polymer, the drug-loaded polymer comprising the drug-loaded polymer according to claim 1 and a drug coupled to the drug-loaded polymer;

[0030] and oxide nanoparticles, wherein the drug-loaded polymer coats the oxide nanoparticles.

[0031] In a fourth aspect, the present application provides a method for preparing a drug-loaded nanostructure, the preparation method comprising:

[0032] Mixing and dissolving the modifier, the initiator and the polymer to be loaded with drugs, and reacting to precipitate the modified polymer to be loaded with drugs; wherein the polymer to be loaded with drugs is prepared by the method according to any one of claims 3 to 6;

[0033] Adding the drug to be loaded into the polymer solution of the modified polymer to be loaded with the drug, reacting and precipitating to obtain the drug-loaded polymer;

[0034] The oxide nanoparticle solution is added to the drug-loaded polymer solution of the drug-loaded polymer to react to obtain the drug-loaded nanostructure.

[0035] Preferably, the modifier includes 4-mercaptobenzoic acid, and the initiator includes azobisisobutyronitrile. Correspondingly, the modifier, the initiator and the polymer to be loaded with drugs are mixed and dissolved, and the modified polymer to be loaded with drugs is precipitated by reaction, comprising:

[0036] Mixing 4-mercaptobenzoic acid, azobisisobutyronitrile and the drug-loaded polymer, and dissolving the mixture in a second organic solvent to obtain a third mixture;

[0037] The third mixture is heated to a fourth preset temperature under nitrogen protection, and reacted for a fourth preset time to obtain a third product;

[0038] Repeatedly placing the third product in ethyl acetate for precipitation and purification to obtain a purified product;

[0039] The purified product is subjected to a solid-liquid separation operation to obtain a solid-phase modified polymer to be loaded with drugs.

[0040] Preferably, the step of adding the drug to be loaded to the polymer solution of the modified drug-loaded polymer to obtain the drug-loaded polymer by reaction and precipitation comprises:

[0041] dissolving the modified drug-loaded polymer in a third organic solvent to obtain a polymer solution;

[0042] adding the drug to be carried into the polymer solution and stirring for reaction to obtain a fourth product;

[0043] Repeatedly precipitating the fourth product in excess ethanol to obtain a precipitate;

[0044] The precipitate is subjected to a solid-liquid separation operation to obtain a solid-phase drug-loaded polymer.

[0045] Preferably, the step of adding the oxide nanoparticle solution to the drug-loaded polymer solution to obtain the drug-loaded nanostructure comprises:

[0046] dissolving the drug-carrying polymer in a fourth organic solvent to obtain a drug-carrying polymer solution;

[0047] Mixing the drug-loaded polymer solution and the oxide nanoparticle solution to obtain a mixed solution;

[0048] The mixed solution is immersed in deionized water and dialyzed for a fifth preset time to obtain the drug-loaded nanostructure.

[0049] Beneficial effects of this application

[0050] The present application provides a drug-loaded polymer, which is an amino acid-based polyureaurethane polymer. The polymer includes two main segment structures, namely an amino acid-based polyurea segment and a polyurethane segment. The urea group and the urethane group can form a stable coating on the surface of iron oxide nanoparticles to improve the dispersion stability of the iron oxide nanoparticles. In addition, the urea group and the urethane group can produce a synergistic effect with the iron oxide nanoparticles to stimulate the polarization of macrophages in the tumor site, further induce immune surveillance and tumor immune activation, thereby providing a new treatment system for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0052] Figure 1 is a flow chart of a method for preparing a drug-loaded polymer provided in an embodiment of the present application;

[0053] Figure 2 Schematic diagram of the synthesis of arginine-based polyureaurethane polymers provided in the examples of the present application;

[0054] Figure 3 The amino acid-based polyurea-urethane polymer provided in the embodiments of the present application is 1 H-NMR characterization diagram;

[0055] Figure 4 Schematic diagram of an amino acid-based polyureaurethane polymer provided in an embodiment of the present application as a drug-carrying coating;

[0056] Figure 5 It is a flow chart of a method for preparing a drug-carrying nanostructure provided in an embodiment of the present application;

[0057] Figure 6 It is a schematic diagram of the synthesis principle of the drug-carrying nanostructure provided in the embodiment of the present application;

[0058] Figure 7 The modified amino acid-based polyurea-urethane polymer provided in the embodiments of the present application is 1 H-NMR characterization diagram;

[0059] Figure 8 Schematic diagram of molecular weights of modified arginine-based polyureaurethane polymers and drug-carrying polymers provided in the examples of the present application;

[0060] Fig. 9 It is an electron microscope image of coated iron oxide nanoparticles and uncoated iron oxide nanoparticles provided in the examples of the present application;

[0061] Fig.10 It is a drug release characterization diagram and a polymer molecular weight change trend diagram provided in the examples of the present application;

[0062] Fig.11 is a macrophage polarization ratio diagram provided in the examples of the present application;

[0063] Fig.12 It is a diagram of the difference in secretion of inflammatory cytokines provided in the examples of the present application;

[0064] Fig.13 is a schematic diagram of tumor inhibition provided in an embodiment of the present application;

[0065] Fig.14 This is a magnetic variation trend diagram of the drug-loaded nanostructure provided in the embodiment of the present application. DETAILED DESCRIPTION

[0066] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments 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 creative work are within the scope of protection of the present invention.

[0067] For the convenience of description, the descriptions of "first", "second", etc. in the present invention are only set for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various implementation methods can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0068] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0069] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0070] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0071] In the prior art, macrophage regulation provides a new anti-tumor treatment strategy, which is to eliminate tumor cells by activating the immune surveillance of the tumor microenvironment. Macrophages have strong plasticity, which is manifested in polarization into different phenotypes and performing different functions under different microenvironmental stimuli. For example, macrophages will produce different levels of reactive oxygen and cytokines (superoxide, TNF-α, NO, IL-12, etc.) in the classical activation (M1) mode, participate in auxiliary Th1 polarization regulation to promote inflammation and resist pathogens, and inhibit tumor cell growth and induce tumor cell apoptosis. In the uninterventional tumor microenvironment, tumor-associated macrophages (TAMs) lose their anti-tumor properties and also hinder the immunoregulatory function of other immune cells. For example, alternatively activated (M2) macrophages produce immunosuppressive factors such as IL-10 and TGF-β, and upregulate the expression of PD-L1 in tumor macrophages to inhibit cytotoxic T cell function. TAMs can also recruit regulatory T cells into the tumor microenvironment, produce an immunosuppressive microenvironment, and induce tumor immune tolerance. At the same time, the expression of arginase I in TAMs is significantly upregulated, depleting L-arginine in the tumor microenvironment, thereby inhibiting T cell proliferation and further inducing tumor cell immune escape.

[0072] Due to the phagocytosis of macrophages, foreign nanomaterials entering the physiological system will first interact with macrophages and affect macrophage polarization, providing the possibility of regulating macrophage-mediated tumor immune activity and activating tumor immune surveillance. Iron oxide nanoparticles have regulatory activity on macrophage polarization, can induce the production of reactive oxygen species (ROS), and affect macrophages by regulating intracellular redox and iron metabolism. However, due to the poor dispersion stability of unmodified iron oxide nanoparticles in the physiological environment in vivo, they are prone to adsorb proteins and precipitate, and cannot achieve immune regulation of macrophages. The in vivo application of iron oxide nanoparticles must first solve the problems of surface modification and dispersion stability, and on this basis, introduce immune regulatory active molecules to assist tumor immunotherapy through macrophage regulation.

[0073] In this regard, the present application proposes a drug-loaded polymer, a drug-loaded nanostructure, and a preparation method thereof.

[0074] Figure 1 A flow chart of a method for preparing a drug-loaded polymer provided in an embodiment of the present application is shown.

[0075] Reference Figure 1 The present invention provides a method for preparing a drug-loaded polymer, comprising the following steps:

[0076] Step S100: mixing amino acid hydrochloride and diol, and performing a dehydration reaction to obtain a monomer substance;

[0077] Step S101: mixing a diisocyanate precursor and α-allyl glyceryl ether to obtain a first mixture;

[0078] Step S102: adding a first catalyst and an antioxidant to the first mixture, mixing and reacting to obtain a prepolymer;

[0079] Step S103: adding the above-mentioned monomer substances to the prepolymer, mixing and reacting to obtain the drug-loaded polymer.

[0080] The amino acid hydrochloride may be at least one of arginine hydrochloride, phenylalanine hydrochloride, valine hydrochloride, leucine hydrochloride and / or glycine hydrochloride.

[0081] The diol may be at least one of ethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol and / or 1,8-octanediol.

[0082] Wherein, the dehydration reaction can be carried out through a reflux device.

[0083] The diisocyanate precursor refers to a raw material or intermediate product required for synthesizing a diisocyanate residue.

[0084] The drug-loaded polymer prepared by the above steps is an amino acid-based polyureaurethane polymer, and the general structural formula is as follows:

[0085]

[0086] Among them, R1 is a diisocyanate residue, R2 is a diol residue, and R3 is an amino acid residue.

[0087] It should be noted that the diisocyanate includes: at least one of hexamethylene diisocyanate, 4,4'-methylene diphenyl diisocyanate, tetramethylene diisocyanate, toluene diisocyanate and / or isophorone diisocyanate; and / or the diol includes: at least one of ethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol and / or 1,8-octanediol; and / or the amino acid includes: at least one of arginine, phenylalanine, valine, leucine and / or glycine.

[0088] Correspondingly, R1 can be: -(CH2)6-, etc.; R2 may be: -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)6-, -(CH2)8-, etc.; R3 may be: -CH3, etc.

[0089] It should be noted that the general structural formula of the amino acid-based polyureaurethane polymer includes two main segment structures: 1) a polyurea segment based on amino acids; 2) a polyurethane segment, the side groups of the polyurethane segment are double bonds, and the molar ratio of the polyurea segment to the polyurethane segment is 0.9:0.1 to 0.5:0.5. Among them, the urea group and the urethane group of the amino acid-based polyureaurethane polymer can form a stable coating on the surface of the iron oxide nanoparticles.

[0090] Amino acid-based polyurea urethane polymers In the synthesis of a series of amino acid-based polyurea urethane polymers, arginine-based polyurea urethane polymers (Arginine-based poly(urea uranthane, APU) are taken as an example. Figure 2 The synthesis principle diagram of arginine-based polyureaurethane polymer is shown, and its general structure is as follows:

[0091]

[0092] The polymer can regulate arginine metabolism in macrophages through the nitric oxide synthase (iNOS) and arginase pathways. Arginine, as an important component of proteins and a signal regulatory factor, plays a regulatory role in various physiological activities in the body and can affect the macrophage activation mode (M1 and M2) to regulate immune responses. Among them, arginine-based polyureaurethane can also promote the production of nitric oxide (NO) in classically activated (M1) macrophages, while enhancing the secretion of TNF-α, promoting the inflammatory response of macrophages, enhancing the immune activation of the tumor microenvironment, and further activating immune surveillance in the tumor microenvironment, which has potential application value in tumor immunotherapy.

[0093] In an achievable embodiment of the present application, the amino acid hydrochloride is L-arginine hydrochloride, and the diol is ethylene glycol. Correspondingly, step S100 includes:

[0094] Mixing L-arginine hydrochloride and ethylene glycol, adding a second catalyst and a first organic solvent, and performing a reflux reaction at a first reaction temperature for a first preset time to obtain a reactant;

[0095] The reactant is cooled to room temperature and filtered to obtain a filtered solution;

[0096] The filtered solution is placed in isopropanol and repeatedly recrystallized and dried to obtain a purified product;

[0097] The purified product is dissolved in deionized water, the pH is adjusted to a preset pH, and L-arginine ethane-1,2-diester is obtained after drying.

[0098] Exemplarily, 0.13 mol of L-arginine hydrochloride, 0.06 mol of ethylene glycol and 0.135 mol of p-toluenesulfonic acid monohydrate are mixed with 250 mL of toluene; the mixture is refluxed at a first reaction temperature of 130° C. for a first preset time of 24 h until a total of 4.3 mL of water (0.24 mol) is collected by a Dean-Stark apparatus; the reactants are cooled to room temperature and filtered to obtain a filtered solution, the product is purified by repeated recrystallization in isopropanol, and dried in a vacuum oven; the dried purified product is dissolved in deionized water, the pH value is adjusted to a preset pH of 9.5 with a sodium bicarbonate solution and lyophilized to obtain L-arginine ethane-1,2-diester (Arg-2 monomer), the synthesis principle of which is as follows Figure 2 as shown in step 1 in .

[0099] In an achievable embodiment of the present application, in step S101, the diisocyanate precursor may be hexamethylene diisocyanate.

[0100] Exemplarily, step S101 includes: continuously stirring HDI (0.05 mol) in 30 mL of dimethyl sulfoxide (DMSO) and 20 mL of α-allyl glyceryl ether (0.025 mol) to obtain a first mixture.

[0101] In an embodiment that can be implemented in the present application, in step S102, the first catalyst is a substance that can increase the chemical reaction rate without changing the chemical equilibrium in a chemical reaction, and its own quality and chemical properties do not change before and after the chemical reaction; the antioxidant is a substance that prevents the adverse effects of oxygen.

[0102] Exemplarily, the first catalyst is 2-ethyl-stannous hexanoate, and the antioxidant is 2,6-di-tert-butyl-4-methylphenol. Correspondingly, step S102 includes:

[0103] Add 2-ethyl-stannous hexanoate and 2,6-di-tert-butyl-4-methylphenol to the first mixture to obtain a second mixture; heat the second mixture to a second preset temperature under nitrogen protection, and react for a second preset time to obtain a prepolymer.

[0104] Exemplarily, 2-ethyl-stannous hexanoate (accounting for 2wt% of the total mass of the reactants) and 2,6-di-tert-butyl-4-methylphenol (accounting for 1wt% of the total mass of the reactants) are added to the first mixture to obtain a second mixture, and the second mixture is heated to a second preset temperature of 60° C. under the protection of dry nitrogen, and reacted for a second preset time of 5 hours to obtain a prepolymer. The principle of the prepolymerization reaction is as follows Figure 2 as shown in step 2 in .

[0105] In an achievable embodiment of the present application, step S103 includes:

[0106] Adding the L-arginine ethane-1,2-diester to the prepolymer, heating to a third preset temperature under nitrogen protection, reacting for a third preset time to obtain a first product; repeatedly placing the first product in ethyl acetate and water for precipitation to obtain a second product;

[0107] The second product is subjected to a solid-liquid separation operation to obtain a solid phase drug-loaded polymer.

[0108] Among them, the solid-liquid separation operation can be vacuum drying, freeze drying, static drying, etc.

[0109] Exemplarily, 0.025 mol of L-arginine ethane-1,2-diester is dissolved in 80 mL of DMSO solution, and the prepolymer solution is added, and the mixture is heated to a third preset temperature of 60° C. under the protection of dry nitrogen, and the reaction is performed for a third preset time of 18 hours to obtain a first product, which is then cooled to room temperature, and the first product is repeatedly precipitated in ethyl acetate and water to remove residual catalyst, solvent and unreacted monomer to obtain a second product, which is vacuum dried to obtain an arginine-based polyureaurethane polymer. The chain extension reaction principle is as follows: Figure 2 As shown in step 3 of the above, the obtained polymer 1 H-NMR characterization (solvent is d6-DMSO) Figure 3 shown.

[0110] It should be noted that the double bond side groups in the amino acid-based polyurea-urethane polymer can be further introduced into benzoic acid groups through chemical reactions with 4-mercaptobenzoic acid (4-MBA), and the benzoic acid groups can interact with encapsulated drugs through electrostatic interactions and π-π interactions, such as macrophage TLR7 / 8 agonist R848. R848, also known as resiquimod, can induce a strong Th1 biased response in the tumor microenvironment and promote the M1 polarization of macrophages, stimulate a variety of proinflammatory cytokines with anti-tumor effects, and thus activate tumor immunity. In addition to macrophages, R848 also provides regulatory effects on dendritic cells, B lymphocytes, and T lymphocytes. However, the poor water solubility of R848 severely limits its application in the biomedical field. In this regard, the amino acid-based polyurea-urethane polymer provided in this application can effectively improve the bioavailability of R848 in vivo.

[0111] Figure 4 A schematic diagram of amino acid-based polyureaurethane polymer as a drug-carrying coating is shown, referring to Figure 4 As shown, R848 drug molecules can form a complex non-covalent complex structure with the polymer, and the polymer can form a stable polymer coating on the surface of iron oxide nanoparticles after drug loading. Amino acid-based polyureaurethane polymers as drug-loaded coatings can improve the colloidal stability of nanoparticles in a physiological environment and enhance the imaging and tumor treatment effects of nanoparticles.

[0112] Figure 5 A flow chart showing a method for preparing a drug-carrying nanostructure provided in an embodiment of the present application is shown; Figure 6 A schematic diagram of the synthesis of drug-loaded nanoarchitectures is shown.

[0113] Reference Figure 5 The present invention provides a method for preparing a drug-loaded nanostructure, comprising the following steps:

[0114] Step S200: mixing and dissolving the modifier, the initiator and the polymer to be loaded with drugs, and reacting to precipitate the modified polymer to be loaded with drugs;

[0115] Step S201: adding the drug to be loaded into the polymer solution of the modified drug-loaded polymer to obtain the drug-loaded polymer by reaction precipitation;

[0116] Step S202: adding the oxide nanoparticle solution to the drug-loaded polymer solution of the drug-loaded polymer to react to obtain the drug-loaded nanostructure.

[0117] Exemplarily, the modifier is 4-mercaptobenzoic acid, and the initiator is azobisisobutyronitrile. Correspondingly, step S200 includes:

[0118] Mixing 4-mercaptobenzoic acid, azobisisobutyronitrile and the drug-loaded polymer, and dissolving the mixture in a second organic solvent to obtain a third mixture;

[0119] The third mixture is heated to a fourth preset temperature under nitrogen protection, and reacted for a fourth preset time to obtain a third product;

[0120] Repeatedly placing the third product in ethyl acetate for precipitation and purification to obtain a purified product;

[0121] The purified product is subjected to a solid-liquid separation operation to obtain a solid-phase modified polymer to be loaded with drugs.

[0122] Exemplarily, 1mmol 4-MBA, 1.5mmol azobisisobutyronitrile (AIBN) and 0.85g arginine-based polyureaurethane polymer are dissolved in 50mL of the second organic solvent N,N-dimethylformamide (DMF) and degassed to obtain a third mixture. The third mixture is heated to a fourth preset temperature of 65°C under nitrogen protection, and reacted for a fourth preset time of 24h to obtain a third product, and the third product is repeatedly precipitated and purified in ethyl acetate to obtain a purified product, and after vacuum drying at room temperature, a solid phase modified drug-loaded polymer is obtained, i.e., 4-mercaptobenzoic acid (4-MBA) modified arginine-based polyureaurethane polymer (4-MBA-APU).

[0123] Modified arginine-based polyureaurethane polymer (4-MBA-APU)1 H-NMR characterization Figure 7 As shown, compared to Figure 3 In the unmodified arginine-based polyureaurethane polymer, the double bond peak of 4-MBA-APU at the chemical shift of 5-6 ppm was significantly weakened, indicating that the click reaction of 4-MBA consumed the double bond. In addition, the characteristic peak from 4-MBA phenyl group (7-8 ppm) appeared in the NMR spectrum of 4-MBA-APU.

[0124] It should be noted that the preparation method of the arginine-based polyureaurethane polymer is as described in the above examples.

[0125] In an achievable embodiment of the present application, step S201 includes:

[0126] The modified drug-loaded polymer is dissolved in a third organic solvent to obtain a polymer solution; the drug to be loaded is added to the polymer solution and stirred for reaction to obtain a fourth product; the fourth product is repeatedly precipitated in excess ethanol to obtain a precipitate; and the precipitate is subjected to a solid-liquid separation operation to obtain a solid-phase drug-loaded polymer.

[0127] Among them, the typical characteristics of the drug to be loaded include 1) containing aliphatic amine or aromatic amine structure; 2) pKa close to or slightly greater than 7.4; 3) containing functional groups such as aromatic rings and heterocyclic rings that can form π-π interactions. Weakly basic drugs with the above structure can form electrostatic complexes with mercaptobenzoic acid side groups in aqueous solution, and can further enhance the interaction between the polymer and the drug through π-π interactions. Specific drugs include: at least one of daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, irinotecan, topotecan, minbosten, vinopin and venkostin.

[0128] Exemplarily, 1 g of 4-MBA-APU is dissolved in 10 mL of the third organic solvent DMSO, and then 0.1 g of the drug to be loaded R848 is added to the polymer solution and stirred continuously, and the fourth product R848-APU drug-loaded polymer obtained is repeatedly precipitated in excess ethanol to remove free R848, and the precipitate is vacuum dried and stored at -20°C for later use to obtain a solid phase drug-loaded polymer. The molecular weight of 4-MBA-APU was characterized by gel permeation chromatography (GPC) with DMF as the elution phase. Figure 8 The molecular weights of the modified arginine-based polyureaurethane polymer and the drug-loaded polymer are shown, as Figure 8 As shown, the average molecular weight of 4-MBA-APU is 22350 Da (PDI=3.22), and the average molecular weight of R848-APU is 26765 Da (PDI=2.59).

[0129] In an achievable embodiment of the present application, step S202 includes:

[0130] The drug-loaded polymer is dissolved in a fourth organic solvent to obtain a drug-loaded polymer solution; the drug-loaded polymer solution and the oxide nanoparticle solution are mixed to obtain a mixed solution; the mixed solution is immersed in deionized water and dialyzed for a fifth preset time to obtain a drug-loaded nanostructure.

[0131] The oxide nanoparticles may be iron oxide nanoparticles, titanium dioxide nanoparticles, cerium oxide nanoparticles, zinc oxide nanoparticles, copper oxide nanoparticles, silver nanoparticles and silicon dioxide nanoparticles.

[0132] Exemplarily, taking iron oxide nanoparticles (IONP) as an example, the drug-carrying polymer is R848-APU, the fourth organic solvent is DMSO, a 0.5 mg / mL R848-APU DMSO solution is prepared, and the solution is mixed with an IONP solution (1 mg Fe / mL), ultrasonically treated for 5 min, and the above mixed solution is further stirred for 4 h to obtain a mixed solution, and the mixed solution is immersed in deionized water for a fifth preset time of 48 h (dialysis bag molecular weight cutoff is 100 kDa) to obtain a drug-carrying nanostructure (R848-APU-IONP). The obtained drug-carrying nanostructure solution is fixed to volume and stored at 4 ° C for use.

[0133] Fig. 9 The transmission electron microscopy morphology of coated iron oxide nanoparticles and uncoated iron oxide nanoparticles is shown. Fig. 9 , the uncoated IONP is in an aggregated state and forms a precipitate in the aqueous solution, while the R848-APU coating improves the dispersibility of IONP. The aqueous solution of R848-APU-IONP presents a uniform brown appearance, and no nanoparticle precipitation is observed after storage for 2 months, indicating that the R848-APU coating significantly improves the dispersion stability of IONP in aqueous solution. The particle size and zeta potential characteristics of R848-APU-IONP are shown in Table 1. The uncoated IONP is negatively charged (-30.5±7.2mV) due to the presence of DMSA capping. After coating with R848-APU, R848-APU-IONP exhibits a slight positive charge (5.6±2.3mV), which originates from the arginine residues in R848-APU.

[0134] Table 1

[0135] Zeta potential in saline DLS Dimensions Not coated with IONP -30.5±7.2mV 37.5±8.2nm R848-APU-IONP 5.6±2.3mV 58.5±6.1nm

[0136] It should be noted that in the amino acid-based polyureaurethane drug-loaded coating in the above embodiments of the present application, the drug molecules account for 1% to 25% of the total mass of the polymer, the particle size range of the iron oxide nanoparticles that can be used to implement the coating is 10 to 1000 nm, and the mass ratio of the amino acid-based polyureaurethane polymer to the iron element in the iron oxide nanoparticles is in the range of 0.05:1 to 0.25:1.

[0137] The drug-loaded nanostructure prepared by the above steps includes:

[0138] A drug-loaded polymer, the drug-loaded polymer comprising a polymer to be drug-loaded and a drug coupled to the polymer to be drug-loaded; and oxide nanoparticles, the drug-loaded polymer coating the oxide nanoparticles.

[0139] The oxide nanoparticles may be iron oxide nanoparticles, titanium dioxide nanoparticles, cerium oxide nanoparticles, zinc oxide nanoparticles, copper oxide nanoparticles, silver nanoparticles and silicon dioxide nanoparticles.

[0140] In other embodiments of the present application, the R848-APU polymer contains a biodegradable ester group, and the polymer degradation initiated by esterase can trigger the release of R848, and the drug release is characterized by Fig.10 As shown in A, after incubating R848-APU polymer with lipase (1 mg / mL) in physiological saline (37°C), the concentration of R848 in the supernatant was characterized by 320 nm UV absorbance at different time points. After incubation with lipase for 48 hours, the cumulative release of R848 in the supernatant exceeded 60% of the total amount of R848. At the same time, the molecular weight of R848-APU polymer detected by GPC decreased significantly ( Fig.10 B). After incubation with lipase for 48 h, the molecular weight of R848-APU decreased from 26765 Da to 7516 Da. The degradation of the polymer led to the dissociation and release of R848.

[0141] Macrophages in the tumor microenvironment are usually in an immunosuppressive state, which becomes the main obstacle to anti-tumor immunity. The M1 polarization of macrophages helps to activate anti-tumor immunity and use the patient's own immune system to eliminate tumor cells. The arginine-based polyureaurethane drug-loaded polymer-coated iron oxide nanoparticles provided in this application have macrophage regulatory activity, can significantly promote macrophage M1 polarization, and activate tumor immunity.

[0142] After the R848-APU-IONP prepared in the above example was incubated with unactivated M0 macrophages (mouse RAW264.7 and human THP-1 macrophages) for 24 h, the macrophage surface M1 marker CD80 was detected by flow cytometry, and the proportion of CD80+ macrophages was quantitatively analyzed and compared with uncoated IONP, APU polymer (without iron oxide nanoparticles) or R848-APU polymer (without iron oxide nanoparticles). Fig.11 Flow cytometric analysis results showed that R848-APU-IONP could significantly stimulate the M1 polarization of macrophages and increase the proportion of CD80+ macrophages ( Fig.11). In addition, the regulatory potential of R848-APU-IONP on macrophage M1 polarization was further demonstrated by detecting representative pro-inflammatory cytokines. Fig.12 As shown, compared with IONP, APU polymer, and R848-APU polymer, the secretion of TNF-α, interleukin 6 (IL-6), and interleukin 12 (IL-12) by mouse bonemarrow-derived macrophages incubated with R848-APU-IONP for 24 hours was significantly increased. The above representative pro-inflammatory cytokines (TNF-α, IL-6, and IL-12) are essential for tumor killing.

[0143] The antitumor effect of R848-APU-IONP was confirmed in a mouse orthotopic 4T1 mammary tumor model in balb / c mice. Tumor-bearing mice received intravenous injection of R848-APU-IONP (20 mg Fe / kg), and the size of 4T1 tumors was continuously monitored for 21 days after injection. Fig.13 As shown in the figure, compared with IONP, APU polymer, and R848-APU polymer, the R848-APU-IONP treatment group achieved a more effective tumor inhibition effect. The inhibitory effect of R848-APU-IONP on 4T1 tumors is related to the polarization of macrophage M1 ( Fig.11 ) and the production of pro-inflammatory cytokines ( Fig.12 ) are consistent.

[0144] In addition to its macrophage immunomodulatory activity and tumor-suppressing effects, R848-APU-IONP can be used as a diagnostic imaging agent, such as a contrast agent for magnetic resonance imaging (MRI). Fig.14 As shown, in T2-weighted MRI, the transverse relaxation rate (R2) of R848-APU-IONP showed a good linear relationship with the iron concentration. The relaxation constant (r2) of R848-APU-IONP in the 2T MRI field was 55.2mM -1 s -1 , which is comparable to the existing MRI contrast agent (Ferumoxytol iron oxide nanoparticles) on the market, indicating the potential of R848-APU-IONP in the field of tumor imaging and tumor diagnosis.

[0145] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A drug-loaded polymer, characterized in that: The structure of the drug-loaded polymer is: Among them, R1 is a diisocyanate residue, R2 is a diol residue, and R3 is an amino acid residue.

2. The drug-loaded polymer according to claim 1, characterized in that: The diisocyanate includes at least one of hexamethylene diisocyanate, 4,4'-methylene diphenyl diisocyanate, tetramethylene diisocyanate, toluene diisocyanate and / or isophorone diisocyanate; and / or, The diol comprises at least one of ethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol and / or 1,8-octanediol; and / or, The amino acids include at least one of arginine, phenylalanine, valine, leucine and / or glycine.

3. A method for preparing a drug-loaded polymer, characterized in that: The preparation method comprises: The amino acid hydrochloride and the diol are mixed and subjected to a dehydration reaction to obtain a monomer substance; mixing a diisocyanate precursor and α-allyl glyceryl ether to obtain a first mixture; Adding a first catalyst and an antioxidant to the first mixture, mixing and reacting to obtain a prepolymer; The monomer material is added into the prepolymer, and mixed and reacted to obtain the polymer to be loaded with drugs.

4. The method for preparing a drug-loaded polymer according to claim 3, characterized in that: The amino acid hydrochloride includes L-arginine hydrochloride, and the diol includes ethylene glycol. Correspondingly, the amino acid hydrochloride and the diol are mixed and dehydrated to obtain a monomer substance, including: Mixing L-arginine hydrochloride and ethylene glycol, adding a second catalyst and a first organic solvent, and performing a reflux reaction at a first reaction temperature for a first preset time to obtain a reactant; The reactant is cooled to room temperature and filtered to obtain a filtered solution; The filtered solution is placed in isopropanol and repeatedly recrystallized and dried to obtain a purified product; The purified product is dissolved in deionized water, the pH is adjusted to a preset pH, and L-arginine ethane-1,2-diester is obtained after drying.

5. The method for preparing a drug-loaded polymer according to claim 3, characterized in that: The first catalyst includes 2-ethyl-stannous hexanoate, and the antioxidant includes 2,6-di-tert-butyl-4-methylphenol. Correspondingly, the first catalyst and the antioxidant are added to the first mixture, and the prepolymer is obtained by mixed reaction, including: adding 2-ethyl-stannous hexanoate and 2,6-di-tert-butyl-4-methylphenol to the first mixture to obtain a second mixture; The second mixture is heated to a second preset temperature under nitrogen protection and reacted for a second preset time to obtain a prepolymer.

6. The method for preparing a drug-loaded polymer according to claim 4, characterized in that: The step of adding the monomer substance to the prepolymer and mixing and reacting to obtain the drug-loaded polymer comprises: Adding the L-arginine ethane-1,2-diester to the prepolymer, heating to a third preset temperature under nitrogen protection, and reacting for a third preset time to obtain a first product; Repeatedly placing the first product in ethyl acetate and water for precipitation to obtain a second product; The second product is subjected to a solid-liquid separation operation to obtain a solid phase drug-loaded polymer.

7. A drug-carrying nanostructure, characterized in that: The drug-carrying nanostructure comprises: A drug-loaded polymer, the drug-loaded polymer comprising the drug-loaded polymer according to claim 1 and a drug coupled to the drug-loaded polymer; and oxide nanoparticles, wherein the drug-loaded polymer coats the oxide nanoparticles.

8. A method for preparing a drug-loaded nanostructure, characterized in that: The preparation method comprises: Mixing and dissolving the modifier, the initiator and the polymer to be loaded with drugs, and reacting to precipitate the modified polymer to be loaded with drugs; wherein the polymer to be loaded with drugs is prepared by the method according to any one of claims 3 to 6; Adding the drug to be loaded into the polymer solution of the modified polymer to be loaded with the drug, reacting and precipitating to obtain the drug-loaded polymer; The oxide nanoparticle solution is added to the drug-loaded polymer solution of the drug-loaded polymer to react to obtain the drug-loaded nanostructure.

9. The method for preparing the drug-loaded nanostructure according to claim 8, characterized in that: The modifier includes 4-mercaptobenzoic acid, and the initiator includes azobisisobutyronitrile. Correspondingly, the modifier, the initiator and the polymer to be loaded with drugs are mixed and dissolved, and the modified polymer to be loaded with drugs is precipitated by reaction, comprising: Mixing 4-mercaptobenzoic acid, azobisisobutyronitrile and the drug-loaded polymer, and dissolving the mixture in a second organic solvent to obtain a third mixture; heating the third mixture to a fourth preset temperature under nitrogen protection, and reacting for a fourth preset time to obtain a third product; Repeatedly placing the third product in ethyl acetate for precipitation and purification to obtain a purified product; The purified product is subjected to a solid-liquid separation operation to obtain a solid-phase modified polymer to be loaded with drugs.

10. The method for preparing the drug-loaded nanostructure according to claim 8, characterized in that: The step of adding the drug to be loaded into the polymer solution of the modified drug-loaded polymer to react and precipitate to obtain the drug-loaded polymer comprises: dissolving the modified drug-loaded polymer in a third organic solvent to obtain a polymer solution; adding the drug to be carried into the polymer solution and stirring for reaction to obtain a fourth product; Repeatedly precipitating the fourth product in excess ethanol to obtain a precipitate; The precipitate is subjected to a solid-liquid separation operation to obtain a solid-phase drug-loaded polymer.

11. The method for preparing the drug-loaded nanostructure according to claim 8, characterized in that: The step of adding the oxide nanoparticle solution to the drug-carrying polymer solution to obtain the drug-carrying nanostructure by reaction comprises: dissolving the drug-carrying polymer in a fourth organic solvent to obtain a drug-carrying polymer solution; Mixing the drug-loaded polymer solution and the oxide nanoparticle solution to obtain a mixed solution; The mixed solution is immersed in deionized water and dialyzed for a fifth preset time to obtain the drug-loaded nanostructure.

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