Acid-activated bio-orthogonal click amphiphilic polymer and nanoparticles thereof, preparation method and application thereof

Abstract

The application belongs to the technical field of medicine and chemical industry, and particularly relates to acid-activated biological orthogonal click amphiphilic polymer and nanoparticles, a preparation method and application thereof. The nanoparticles are composed of precursor nanoparticles self-assembled by acid-responsive amphiphilic polymers grafted and modified by click reaction groups and corresponding effect nanoparticles loaded with multiple therapeutic agents and modified by click reaction groups. After the acid-responsive polymer precisely responds to the extracellular micro-acid environment of tumors and the intracellular acid environment for targeted delivery and in-situ activation and release of biological orthogonal click reaction groups in tumors, the in-situ biological orthogonal click reaction of tissues is used to improve the accumulation of therapeutic agents in tumor tissues, improve the tumor treatment effect, and reduce toxic side effects. The acid-responsive polymer labeled with the biological orthogonal click group has the structural formula shown in the following formula 1 and 2.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and chemical technology, specifically relating to acid-activated bioorthogonal click amphiphilic polymers and their nanoparticles, preparation methods, and applications. Background Technology

[0002] Malignant tumors have become one of the major diseases threatening the health of the Chinese people. During tumor treatment, the accumulation of therapeutic agents in normal tissues leads to severe toxic side effects. For example, platinum-based drugs commonly used in chemotherapy can cause severe nausea and vomiting (cisplatin), and leukopenia and thrombocytopenia (carboplatin); PD-1 / PD-L1 blocking antibodies commonly used in immunotherapy can cause interstitial pneumonia and respiratory failure. Bioorthogonal click chemistry, due to its high reaction rate, high reaction specificity, and good biocompatibility, has been developed to improve the targeting of drugs to tumors, thereby enhancing the therapeutic effect and reducing toxic side effects. However, tissue-insensitive bioorthogonal click reactions still cannot improve the accumulation of therapeutic agents in tumor tissues. Therefore, this invention designs a tumor microacid environment-activated bioorthogonal click reaction nanoplatform, which consists of precursor nanoparticles formed by the self-assembly of acid-responsive amphiphilic polymers grafted with click reactive groups and corresponding click reactive group-modified nanoparticles loaded with multiple therapeutic agents. By utilizing acid-responsive polymer targeted delivery and in-situ activation and release of bioorthogonal click reactive groups in tumors, the accumulation of therapeutic agents in tumor tissues is enhanced through in-situ bioorthogonal click reactions, thereby improving the efficacy of tumor treatment and reducing toxic side effects. Summary of the Invention

[0003] Based on the above background, the present invention provides an acid-responsive diblock polymer modified with a bioorthogonal click reactive group and a tumor tissue acid-responsive nanoparticle prepared from the diblock polymer. These nanoparticles activate the bioorthogonal click reaction through the extracellular microacid environment of the tumor, enabling the precise delivery of various therapeutic agents and achieving precision tumor treatment.

[0004] This invention provides an acid-responsive polymer modified with bioorthogonal click reactive groups, wherein the acid-responsive polymer modified with bioorthogonal click reactive groups comprises an extracellular acid-responsive amphiphilic block polymer as shown in Formula 1 and an intracellular acid-responsive amphiphilic polymer as shown in Formula 2:

[0005]

[0006] in,

[0007] R1 is N,N-ethylpropylamino or hexamethyleneamino; preferably, it is N,N-ethylpropylamino.

[0008] R2 is carboxyl-modified dibenzocyclooctylene (DBCO) or carboxyl-modified transcyclooctene (TCO); preferably, it is carboxyl-modified DBCO.

[0009] x is an integer between 10 and 145; preferably 113.

[0010] y is an integer between 50 and 100; preferably 60.

[0011] And z is an integer from 1 to 5; preferably 3.

[0012] The polymer shown in Formula 1 of this invention is a linear polymer, including but not limited to grafted polymers, and has tumor extracellular acid responsive properties.

[0013]

[0014] in,

[0015] R3 is an azide group or a tetrazine group; preferably, it is an azide group.

[0016] R4 is N,N-diethylamino, N,N-diisopropylamino, N,N-di-tert-butylamino, N,N-ethylpropylamino, pentamethyleneamino, hexamethyleneamino; preferably, it is N,N-diisopropylamino.

[0017] m is an integer between 10 and 145; preferably, it is 113.

[0018] n is an integer between 20 and 100; preferably, it is 50.

[0019] o is an integer from 1 to 10; preferably, it is 5.

[0020] The polymer shown in Formula 2 of this invention is a linear polymer, including but not limited to grafted polymers.

[0021] This invention also provides a method for preparing the extracellular acid-responsive amphiphilic block polymer shown in Formula 1, wherein the extracellular acid-responsive amphiphilic block polymer is synthesized by reversible addition-fragmentation chain transfer polymerization as shown in the following reaction route I:

[0022]

[0023] Reaction route I

[0024] Step a: Synthesis of polyethylene glycol-b-poly(R1-r-methylpropene hydroxyethyl ester) copolymer

[0025] A RAFT chain transfer agent-terminated polyethylene glycol macromolecular initiator was dissolved in N,N-dimethylformamide in a molar ratio of 1:50-100:1-10:0.1 (preferably 1:70-5:0.1) with ethyl methacrylate monomer containing R1, hydroxyethyl methacrylate monomer, and initiator azobisisobutyronitrile. The reaction was carried out at 70°C for 24 hours under anaerobic conditions. The reaction product was diluted with an appropriate amount of DMF and dialyzed with deionized water. The product was then freeze-dried in a dialysis bag with a capacity cutoff of 3500 Daltons to obtain a polyethylene glycol-b-poly(R1-r-hydroxyethyl methacrylate) copolymer.

[0026]

[0027] Reaction route a

[0028] Step b: Coupling R2 with polyethylene glycol-b-poly(R1-r-hydroxyethyl methacrylate) copolymer

[0029] The block copolymer obtained in step a was dissolved in anhydrous N,N-dimethylformamide to obtain a solution A with a mass concentration of 10% w / v. A 30% w / v solution B was prepared by mixing carboxyl-modified R2 with N,N-diisopropylethylamine, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a molar ratio of 1:1.5:1.5:1.5 in N,N-dimethylformamide. The reaction was allowed to proceed for 1.5 h. Then, solution A was added dropwise to solution B, and the reaction continued for 24 h. The mixture was then dialyzed with N,N-dimethylformamide and deionized water using a dialysis bag with a cutoff of 3500 Daltons. The mixture was freeze-dried to obtain the extracellular acid-responsive amphiphilic block polymer of formula 1, covalently modified with R2.

[0030]

[0031] Reaction route b

[0032] The definitions of R1, R2, x, y, and z are shown in Equation 1.

[0033] In one specific embodiment, the preparation of the extracellular acid-responsive amphiphilic block polymer of Formula 1 specifically includes the following steps: synthesizing a polyethylene glycol-b-poly(ethyl propylamino methacrylate-r-dibenzocyclooctylene) block copolymer via reversible addition-fragmentation chain transfer polymerization as shown in reaction route I:

[0034]

[0035] Reaction route I

[0036] Step a': Synthesis of polyethylene glycol-b-poly(ethyl propyl aminoethyl methacrylate-r-hydroxyethyl methacrylate) copolymer

[0037]

[0038] A RAFT chain transfer agent-terminated polyethylene glycol macromolecular initiator was dissolved in an appropriate amount of N,N-dimethylformamide in a molar ratio of 1:50-100:1-5:0.1 with ethyl propylaminoethyl methacrylate, hydroxyethyl methacrylate monomer, and initiator azobisisobutyronitrile. The reaction was carried out at 70°C for 24 hours under anaerobic conditions. The reaction product was diluted with an appropriate amount of DMF and dialyzed with deionized water. The product was then freeze-dried in a dialysis bag with a capacity cutoff of 3500 Daltons to obtain a polyethylene glycol-b-poly(ethyl propylaminoethyl methacrylate-r-hydroxyethyl methacrylate) copolymer.

[0039] Step b': Polyethylene glycol-b-poly(ethyl propyl aminoethyl methacrylate-r-hydroxyethyl methacrylate) copolymer coupled with DBCO

[0040] The block copolymer obtained in step a' was dissolved in anhydrous N,N-dimethylformamide to obtain a solution A with a mass concentration of 10% w / v. DBCO, N,N-diisopropylethylamine, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed in a molar ratio of 1:1.5:1.5:1.5 in N,N-dimethylformamide to prepare a 30% w / v solution B. The reaction was carried out for 1.5 h. Then, solution A was added dropwise to solution B, and the reaction was continued for 24 h. The mixture was then dialyzed with N,N-dimethylformamide and then with deionized water. The dialysis bag with a cutoff of 3500 Daltons was used. The mixture was freeze-dried to obtain the block polymer shown in Formula 1, which was covalently modified with DBCO.

[0041]

[0042] R1, x, y, and z are as shown in Equation 1.

[0043] This invention also provides a method for preparing an intracellular acid-responsive amphiphilic polymer, which specifically includes the following steps:

[0044] A polyethylene glycol macromolecular RAFT chain transfer agent with a pairing group of bioorthogonal click reaction of Formula 1 was dissolved in an appropriate amount of N,N-dimethylformamide with ethyl methacrylate monomer, hydroxyethyl methacrylate monomer, and initiator azobisisobutyronitrile containing R4 in a molar ratio of 1:20-100:1-10:0.1 (preferably 1:50-5:0.1). The reaction was carried out at 70°C for 24 h under anaerobic conditions. The reaction product was diluted with an appropriate amount of DMF and dialyzed with deionized water. The product was then freeze-dried in a dialysis bag with a cutoff of 3500 Daltons to obtain polyethylene glycol-b-poly(diisopropylaminoethyl methacrylate) copolymer.

[0045] The intracellular acid-responsive amphiphilic polymer was synthesized via reversible addition-fragmentation chain transfer polymerization as shown in reaction route II below:

[0046]

[0047] Reaction Route II

[0048] R3, R4, m, n and o are shown in Equation 2.

[0049] This invention also provides two types of nanoparticles composed of acid-responsive diblock polymers modified with bioorthogonal click reactive groups, including precursor nanoparticles (extracellular acid-responsive nanoparticles) and effector nanoparticles (intracellular acid-responsive nanoparticles), wherein:

[0050] The extracellular acid-responsive nanoparticles are prepared from the extracellular acid-responsive amphiphilic block polymer shown in Formula 1; used for delivering the R2 group in the extracellular acid-responsive amphiphilic block polymer shown in Formula 1 via bioorthogonal click reaction in the tumor extracellular microenvironment; and loaded with therapeutic agents from the nanoparticles.

[0051] The intracellular acid-responsive nanoparticles are prepared from an intracellular acid-responsive amphiphilic polymer as shown in Formula 2, other materials that can self-assemble into nanoparticles, or a loaded therapeutic agent, wherein the intracellular acid-responsive amphiphilic polymer as shown in Formula 2 accounts for 10%-20% of the total mass of the nanoparticles; the distribution of the therapeutic agent in tumor tissue is improved by undergoing a bioorthogonal reaction with the extracellular acid-responsive nanoparticles.

[0052] In one specific embodiment, the precursor nanoparticles comprise an extracellular acid-responsive amphiphilic block polymer of Formula 1, wherein the precursor nanoparticles comprise any one of a variety of tumor therapeutic agents loaded with polyethylene glycol hydrophilic outer layer, poly(ethyl propylaminoethyl methacrylate-r-dibenzocyclooctylene) hydrophobic core, and a loaded tumor therapeutic agent.

[0053] In one specific embodiment, the effect nanoparticles include any nanoparticles of the intracellular acid-responsive amphiphilic polymer shown in Formula 2, including liposomes, micelles, vesicles, etc.; wherein, the effect nanoparticles include any one of the following: a polyethylene glycol hydrophilic outer layer modified with groups that pair with the bioorthogonal click reaction of the extracellular acid-responsive amphiphilic block polymer shown in Formula 1; an inner layer of other self-assembled nanoparticle materials such as lipids, block polymers, dendritic macromolecules, etc.; and a load of various tumor therapeutic agents.

[0054] The present invention also provides a method for preparing the extracellular acid-responsive nanoparticles, the method comprising dissolving the extracellular acid-responsive amphiphilic block polymer and the therapeutic prodrug shown in Formula 1 simultaneously in an organic solvent at a mass ratio of 99-4 to obtain a solution containing the extracellular acid-responsive amphiphilic block polymer and the therapeutic prodrug, adding the mixed solution dropwise to deionized water under ultrasonic conditions at a volume ratio of 1:9 with water, and then removing the organic solvent or the loaded therapeutic prodrug by ultrafiltration or dialysis to obtain polymer nanoparticles loaded with the therapeutic prodrug.

[0055] In one specific embodiment, the method for preparing the nanoparticles includes the following steps:

[0056] The extracellular acid-responsive amphiphilic block copolymer and the therapeutic prodrug shown in Formula 1 are dissolved in an organic solvent to obtain a solution containing the amphiphilic block copolymer and the therapeutic prodrug. The solution is then dropped into deionized water under ultrasonication, wherein the volume ratio of the organic solvent is less than 10%. The organic solvent and the unloaded therapeutic prodrug are then removed by ultrafiltration or dialysis to obtain the acid-responsive nanoparticles.

[0057] The present invention also provides a method for preparing the intracellular acid-responsive nanoparticles, the method comprising dissolving the acid-responsive amphiphilic polymer shown in Formula 2 and other excipients such as lipids, amphiphilic block polymers, dendritic macromolecules, etc., which can be assembled into nanoparticles, and a therapeutic prodrug in an organic solvent, wherein the acid-responsive amphiphilic polymer shown in Formula 2 accounts for 1.0%-20% of the total mass, and the therapeutic prodrug accounts for 1.0%-20% of the total mass; and preparing nanoparticles by ultrasonic precipitation or thin film dispersion.

[0058] In one specific embodiment, the method for preparing the nanoparticles includes the following steps:

[0059] The amphiphilic polymer shown in Formula 2 and other materials that can self-assemble into nanoparticles, such as lipids, amphiphilic block polymers, and dendritic macromolecules, as well as the therapeutic prodrug, are dissolved in an organic solvent to obtain a mixed solution. This solution is then dropped into deionized water under ultrasonication, wherein the volume ratio of the organic solvent is less than 10%. The organic solvent and the unloaded therapeutic prodrug are then removed by ultrafiltration or dialysis to obtain the acid-responsive nanoparticles.

[0060] The organic solvent is preferably selected from at least one of tetrahydrofuran, methanol, N,N-dimethylformamide and N,N-dimethylacetamide.

[0061] The therapeutic agent is a small molecule prodrug selected from chemotherapeutic agents, targeted agents, photodynamic therapy agents, immunotherapy agents, and synergistic combinations of these therapeutic agents;

[0062] The chemotherapy agents are oxaliplatin, paclitaxel, camptothecin, and doxorubicin; the targeted agents are palbociclib, pomaconazole, ribociclib, and G1T28; the photodynamic therapy agent is PPa; and the immunotherapy agents are the IDO inhibitor NLG919, the PD-L1 inhibitor JQ1, the STING agonist, and the TLR agonist.

[0063] This invention also provides an application of two types of nanoparticles composed of acid-responsive diblock polymers modified with bioorthogonal click reactive groups, wherein:

[0064] The precursor nanoparticles exhibit responsiveness to the extracellular acidic environment of tumor tissue. Based on the tumor microacidic environment, the diblock polymer can specifically label the tumor extracellular microenvironment with bioorthogonal click reactive groups and release specific tumor tissue therapeutic agents. The precursor nanoparticles self-assemble into stable nanoparticles under normal physiological conditions (e.g., pH = 7.4), with a hydrodynamic particle size of approximately 70 nanometers. They exhibit good stability during blood circulation, preventing leakage of bioorthogonal click reactive groups and therapeutic prodrugs. In the extracellular microacidic environment of tumor tissue, ethylpropylaminoethyl methacrylate in the precursor nanoparticles is protonated, exposing bioorthogonal click reactive groups and therapeutic prodrugs.

[0065] The effect nanoparticles undergo a bioorthogonal click reaction with the activated precursor nanoparticles in the tumor extracellular microenvironment, promoting the accumulation of effect nanoparticles in tumor tissue and prolonging the retention time. Under normal physiological conditions (e.g., pH=7.4), the effect nanoparticles self-assemble into stable nanoparticles, exhibiting good stability during blood circulation.

[0066] This invention also provides a bioorthogonal click reaction nanoplatform activated by the tumor extracellular acidic environment for synergistic tumor therapy, enhancing tumor immunogenicity and inhibiting tumor immune escape. The nanoparticles are used for in-situ bioorthogonal click reactions in tumor tissue, improving the precise delivery of various therapeutic agents and achieving precision tumor treatment. The precursor nanoparticles can be loaded with prodrugs for enhancing tumor immunogenicity, such as photodynamic therapy agents, chemotherapy agents, and molecularly targeted therapy agents; the effector nanoparticles can be loaded with prodrugs for inhibiting tumor immune escape, such as IDO inhibitors and PD-L1 inhibitors; or any combination of therapeutic agents satisfying the purposes of this invention.

[0067] The amphiphilic acid-responsive polymer precursor nanoparticles of formula 1 are prepared by solution self-assembly and have a multilayer structure, including a polyethylene glycol outer layer and a poly(R1-r-R2) inner layer. The average hydrodynamic particle size of the precursor nanoparticles is preferably 70 nanometers.

[0068] The precursor nanoparticles possess a tumor extracellular acidic environment responsiveness, giving them multifunctionality. The polyethylene glycol hydrophilic layer prevents protein adsorption, ensuring the precursor nanoparticles remain stable in the bloodstream, reducing clearance by the endothelial reticulum system, and prolonging blood circulation time. After reaching tumor tissue via the EPR effect, the precursor nanoparticles are protonated, causing dissociation and exposing bioorthogonal click reactive groups for labeling the tumor extracellular environment. This in-situ bioorthogonal click reaction enhances the accumulation and retention of effector nanoparticles in tumor tissue; and the specific release of the therapeutic prodrug is triggered by the tumor extracellular acidic environment.

[0069] The amphiphilic acid-responsive polymer effect nanoparticles of formula 2 were prepared by solution self-assembly and specifically include nanoparticles such as liposomes, micelles, and vesicles.

[0070] The effect nanoparticles can be selected from various types, giving them multifunctionality. The effect nanoparticles have a biblock polymer outer layer capped with the bioorthogonal click reactive group described in Formula 2, as well as other self-assembling nanoparticle materials and therapeutic agent inner layers. The effect nanoparticles maintain good hydrodynamic stability in blood circulation. Upon reaching tumor tissue, they undergo a bioorthogonal click reaction with activated precursor nanoparticles, promoting retention of the effect nanoparticles in the tumor tissue and achieving precise and efficient delivery of the therapeutic agent.

[0071] This invention also provides a therapeutic agent precision delivery nanoplatform for use in synergistic tumor therapy, such as the precise delivery of chemotherapy agents and immunotherapeutic agents, primarily for synergistic immunotherapy of tumors. The therapeutic agent prodrug, for the purposes of this invention, can be selected from any therapeutic agent that can enhance tumor immunogenic death, including at least one from the group consisting of chemotherapy agents, photodynamic therapy agents, molecularly targeted therapy agents, and immunotherapeutic agents, or multiple therapeutic agent combinations can be achieved according to the needs of synergistic therapy.

[0072] The use of acid-responsive polycationic micelle nanoparticles provided by the present invention in the treatment of cancer, the cancer types being mainly breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, or prostate cancer, etc.

[0073] The beneficial effects of this invention include:

[0074] 1) This invention utilizes two types of acid-responsive polymers to precisely distinguish between the extracellular microacidic environment and the intracellular acidic environment, accurately achieving extracellular activation and release of bioorthogonal click reactive groups (such as DBCO, TCO, etc.). Then, through in-situ bioorthogonal click reaction, it enhances the accumulation of therapeutic agents modified with matching bioorthogonal click reactive groups in tumor tissues (including small molecules, antibodies, nanoparticles, etc.), improving therapeutic efficacy while reducing toxic side effects.

[0075] 2) The key technological innovation of this invention lies in the fact that the precursor nanoparticles activated by tumor extracellular microacids can specifically label the extracellular microenvironment of tumor tissue with bioorthogonal click reactive groups, while they cannot label the extracellular microenvironment of normal tissue; thereby increasing the accumulation of therapeutic agents in tumor tissue and reducing their distribution in normal tissue.

[0076] 3) The innovation and uniqueness of this invention lies in the broad-spectrum and specific physiological environment response of the precursor nanoparticles to the extracellular microacidic environment of solid tumors, which makes this invention broad-spectrum and applicable to all solid tumor treatment scenarios. Attached Figure Description

[0077] Figure 1 The polymer structure in Examples 1 and 2 of this invention is a polyethylene glycol-b-poly(ethyl propylamino ethyl methacrylate-r-dibenzocyclooctylene) diblock copolymer (mPEG). 113 -bP(EPA 40 -r-DBCO3)), the polymer structure is azide-polyethylene glycol-GG-poly(diisopropylaminoethyl methacrylate-r-hydroxyethyl methacrylate)(N3-GG-PEG). 113 -b-PDPA 50 The proton NMR spectrum of -r-HEMA5).

[0078] Figure 2 The particle size variation of the acid-activated bioorthogonal nanoplatform prepared in Example 3 of this invention in different pH buffer solutions.

[0079] Figure 3 This invention provides an example of how, in vivo, the precursor nanoparticles were investigated to respond to the tumor microacidic environment at the in vivo level in Example 4 of this invention.

[0080] Figure 4 The NP1 and N3-CyOH prepared in Example 5 of this invention; (A) in vivo distribution imaging of mice after combined injection and injection alone; and (B) semi-quantitative fluorescence analysis of tumor tissue.

[0081] Figure 5 (A) In vivo distribution and (B) semi-quantitative analysis of tumor tissue for NP1 and NP3 prepared in Example 6 of this invention, when NP3 was injected alone and when NP1 and NP3 were injected together.

[0082] Figure 6 The tumor inhibition curves of the nanoparticles PEID, N3PGDJ and PGDJ prepared in Example 7 of this invention are shown. Detailed Implementation

[0083] The present invention will be described with reference to the following specific embodiments and accompanying drawings, but the present invention is not limited to these specific embodiments.

[0084] The methoxy-terminated polyethylene glycol 5000, 4-cyano-4-[(dodecylthioalkylthiocarbonyl)thioalkyl]valeric acid, diisopropylaminoethyl methacrylate, ethylpropylaminoethyl methacrylate, and hydroxyethyl methacrylate used in the examples were purchased from Sigma-Aldrich (China) Co., Ltd. Hydroxyethyl methacrylate was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd. Unless otherwise specified, all other reagents and solvents used were purchased from Sinopharm Group (Shanghai) Chemical Reagent Co., Ltd.

[0085] 4T1 breast cancer cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. DMEM culture medium and fetal bovine serum were purchased from Gibco (product number 11965118).

[0086] Sample data were determined using the following instruments: 1H NMR spectroscopy (NMR spectroscopy). 1 H NMR was performed using a BRUKERAVANCE NEO 500 NMR spectrometer, with TMS as an internal standard, and chemical shift units were expressed in ppm. -1 ;

[0087] The hydrodynamic particle size and surface potential of the cationic micelles were determined by a MALVERNNANOSIZER particle size analyzer and the transmission electron microscopy images were obtained by a Talos L120C transmission electron microscope.

[0088] Unless otherwise specified, all equipment and testing methods used in this application are conventional in the field.

[0089] To address the severe toxic side effects caused by the accumulation of therapeutic agents in normal tissues during tumor treatment, this invention proposes a tumor-microacidic environment-activated bioorthogonal click-reaction nanoplatform. This nanoplatform consists of precursor nanoparticles formed by the self-assembly of acid-responsive amphiphilic polymers grafted with click-reactive groups, and corresponding click-reactive group-modified nanoparticles loaded with various therapeutic agents. By utilizing the targeted delivery of acid-responsive polymers and the in-situ activation and release of bioorthogonal click-reactive groups in the tumor, the accumulation of therapeutic agents in tumor tissue is enhanced through in-situ bioorthogonal click reactions, thereby improving tumor treatment efficacy and reducing toxic side effects.

[0090] The nanoplatform provided by this invention includes acid-responsive amphiphilic polymers grafted with click reactive groups and precursor nanoparticles formed by their self-assembly, and corresponding effect nanoparticles loaded with various therapeutic agents modified with click reactive groups.

[0091] Bioorthogonal click reaction (BCR) groups, including prodrug molecules, antibodies, and nanoparticles modified with BCR groups. The polymers are linear polymers modified with polyethylene glycol, but are not limited to linear polymers, including grafted polymers; BCR groups include azides, dibenzocyclooctylene, tetrazine, and transcyclooctene, etc.

[0092] Example 1: Polyethylene glycol-b-poly(ethyl propyl aminoethyl methacrylate-r-dibenzocyclooctylene) diblock copolymer (mPEG) 113 -bP(EPA 40 Preparation of -r-DBCO3)

[0093] Take a polyethylene glycol macromolecular initiator (mPEG) with a molecular weight of 5000 Da. 113 0.2 g of β-CTA, 0.517 g of ethyl propylaminoethyl methacrylate (EPA-MA), 0.048 g of hydroxyethyl methacrylate (HEMA), and 0.0004 g of azobisisobutyronitrile (AIBN) were dissolved in 1.0 mL of N,N-dimethylformamide (DMF). After deoxygenation under vacuum, the mixture was reacted at 70 °C for 24 hours. After the reaction was complete, the product was purified by dialyzing with ethanol and deionized water, with a residue cutoff of 3.5 kDa. Lyophilization yielded 0.529 g of the product, with a yield of 69.2%. Using deuterated chloroform as solvent, the polymer structure was determined by 1H NMR spectroscopy to be polyethylene glycol-b-poly(ethyl propylaminoethyl methacrylate-r-hydroxyethyl methacrylate) diblock copolymer (mPEG). 113 -bp(EPA 40 -r-HEMA7)).

[0094] Take polyethylene glycol-b-poly(ethyl propyl aminoethyl methacrylate-r-hydroxyethyl methacrylate) diblock copolymer (mPEG) 113 -bp(EPA 40 0.2 g of -r-HEMA7 was dissolved in 5 mL of DMF and set aside. 0.010 g of carboxyl group DBCO, 0.008 g of DMAP, and 0.009 g of EDCI were dissolved in 2 mL of DMF. The reaction was carried out for 1.5 hours, and then the polymerization solution was added dropwise to the above solution, and the reaction continued for 24 hours. After the reaction was complete, the product was dialyzed against ethanol and deionized water, with a rejection ratio of 3.5 kDa. Lyophilization yielded 0.168 g of product, with a yield of 81.3%. Using deuterated chloroform as solvent, the polymer structure was confirmed by 1H NMR spectroscopy to be polyethylene glycol-b-poly(ethyl propyl aminoethyl methacrylate-r-dibenzocyclooctylene) diblock copolymer (mPEG). 113 -bP(EPA 40 -r-DBCO3)), see Figure 1 .

[0095] Example 2: Azide-polyethylene glycol-GG-poly(diisopropylaminoethyl methacrylate-r-hydroxyethyl methacrylate) (N3-GG-PEG) 113 -b-PDPA 50 Preparation of -r-HEMA5)

[0096] A 5000 Da azido-terminated polyethylene glycol macromolecular initiator (N3-PEG) 113 0.15 g of PEG-GG-CTA, 0.322 g of diisopropylaminoethyl methacrylate (DPA-MA), 0.048 g of hydroxyethyl methacrylate (HEMA), and 0.0004 g of azobisisobutyronitrile (AIBN) were dissolved in 1.0 mL of N,N-dimethylformamide (DMF). After deoxygenation under vacuum, the mixture was reacted at 70 °C for 24 hours. After the reaction was complete, the product was purified by dialyzing with ethanol and deionized water, with a residue cutoff of 3.5 kDa. Lyophilization yielded 0.429 g of the product, with a yield of 85.1%. Using deuterated chloroform as solvent, the polymer structure was determined by 1H NMR spectroscopy to be azide-polyethylene glycol-GG-poly(diisopropylaminoethyl methacrylate-r-hydroxyethyl methacrylate) (N3-GG-PEG). 113 -b-PDPA 50 -r-HEMA5), see Figure 1 .

[0097] Example 3: Verification of Acid-Activated Bioorthogonal Reaction in Solution

[0098] Take the mPEG prepared in Example 1 of this invention 113 -bP(EPA 40 NP1 nanoparticles prepared from the acid-responsive polymer (-r-DBCO3) were taken from the N3-GG-PEG prepared in Example 2 of this invention. 113 -b-PDPA 50 Acid-responsive polymers and mPEG 113 -GG-bP(DPA 50 -r-JQ1) acid-responsive polymer co-assembled nanoparticles NP2; NP1 and NP2 were dispersed in PBS buffers at pH 7.4 and 6.5, respectively, and co-incubated at 37°C for 30 min. The micelle hydrodynamic radius was measured using a dynamic light scattering instrument, and the change in nanoparticle size was observed using a transmission electron microscope. The test results are as follows: Figure 2 As shown in the figure. Experimental results indicate that the microacidic environment of the tumor can trigger the cross-linking of NP1 and NP2 via a bioorthogonal click reaction, forming larger particles.

[0099] Example 4: Response of precursor nanoparticles to tumor microacid rings

[0100] The Balb / c model of triple-negative breast cancer 4T1 was used to evaluate the response of precursor nanoparticles in an acid-activated bioorthogonal nanoplatform to the tumor microacidic environment. The tumor volume was increased to 100 mm². 3 At that time, they were randomly divided into two groups, one group was injected alone with (iv) NP1 (mPEG prepared in Example 1 of this invention). 113 -bP(EPA 40 In another group, mice were pretreated with 2-deoxy-D-glucose (2-DG) for 24 hours followed by (iv) injection of NP1. 2-DG is a glycolysis inhibitor that inhibits the formation of an acidic extracellular microenvironment in tumor cells by suppressing glycolytic metabolism. Figure 3 As shown, compared with the NP1 group alone, the fluorescence of tumor tissues in mice pretreated with 2-DG was significantly inhibited, indicating that the precursor nanoparticles have tumor extracellular microacid responsiveness.

[0101] Example 5: Acid-activated bioorthogonal nanoplatform promotes the accumulation of small molecule probes (or drugs) in tumor tissue.

[0102] The Balb / c model of triple-negative breast cancer 4T1 was used to evaluate the effect of acid-activated bioorthogonal nanoplatform promoting the accumulation of small molecule drugs in tumor tissue; azide-modified hemicyanine (N3-CyOH) was selected as the model molecule. Tumor-bearing mice were injected with (1) NP1 (DBCO 1.0 mg / kg) + N3-CyOH (2.0 mg / kg); (2) N3-CyOH (2.0 mg / kg); and NP1 (mPEG prepared in Example 1 of this invention) was injected into the tail vein. 113 -bP(EPA 40 Two hours after injection of nanoparticles prepared from the acid-responsive polymer (-r-DBCO3), N3-CyOH was injected via the tail vein. Four hours later, both groups of mice were imaged using a small animal in vivo imaging system, and tumor tissue was subjected to semi-quantitative fluorescence analysis. The experimental results are as follows: Figure 4 As shown, the tumor microacidic environment activated the bioorthogonal nanoplatform, resulting in an approximately two-fold increase in the accumulation of N3-CyOH in tumor tissue.

[0103] Example 6: Acid-activated bioorthogonal nanoplatform promotes nanoparticle accumulation in tumor tissue

[0104] The Balb / c model of triple-negative breast cancer 4T1 was used to evaluate whether acid-activated bioorthogonal nanoplatforms could accumulate nanoparticles in tumor tissue; mPEG prepared in Example 1 of this invention was used. 113 -bP(EPA 40 NP1 nanoparticles prepared from the acid-responsive polymer (-r-DBCO3) were taken from the N3-PEG prepared in Example 2 of this invention. 113 -GG-bP(DPA 50-r-HEMA5) acid-responsive polymer with mPEG loaded with photosensitizer PPa 113 -GG-bP(DPA 50 NP3 nanoparticles were co-assembled from acid-responsive polymers (-r-PPa). Tumor-bearing mice were injected with (1) NP1 (DBCO 1.0 mg / kg) + NP3 (PPa 5.0 mg / kg); (2) NP3 (PPa 5.0 mg / kg); NP1 was injected via the tail vein, followed by NP3 via the tail vein 2 hours later. After 72 hours, the two groups of mice were imaged using a small animal in vivo imaging system, and the tumor tissue was semi-quantitatively analyzed using fluorescence. The experimental results are as follows: Figure 5 As shown, compared to the NP3-only injection group, the fluorescence intensity of PPa in mouse tumor tissues in the NP1 and NP3 combination group increased by approximately 3 times after 3 days, indicating that the acid-activated nanoplatform can effectively enhance the accumulation of therapeutic agents in tumor tissues.

[0105] Example 7: Acid-activated bioorthogonal nanoplatform enhances the efficacy of antitumor immunotherapy

[0106] The efficacy of acid-activated bioorthogonal nanoplatforms in enhancing antitumor immunotherapy was evaluated using a Balb / c model of triple-negative breast cancer 4T1; mPEG prepared in Example 1 of this invention was used. 113 -bP(EPA 40 Extracellular acid-responsive polymers (-r-DBCO3) were loaded with the photothermal agent ICG to prepare nanoparticles (PEID) co-loaded with DBCO and ICG; ICG mediated photothermal therapy to induce inflammatory responses and enhance tumor immunogenicity. The N3-GG-PEG prepared in Example 2 of this invention was used. 113 -b-PDPA 50 -r-HEMA5) intracellular acid-responsive polymerization and mPEG loaded with therapeutic agent JQ1 113 -GG-bP(DPA 50 -r-JQ1) acid-responsive polymers were co-assembled into azide-modified nanoparticles (N3PGDJ), in which mPEG 113 -GG-bP(DPA 50 -r-JQ1) acid-responsive polymers self-assembled into azide-free nanoparticles (PGDJ) as a control.

[0107] Mice carrying 4T1 tumors were randomly divided into 5 groups (n=6): PBS as control; PEID nanoparticle monotherapy group; N3PGDJ monotherapy group; PEID@PGDJ combination therapy group; and PEID@N3PGDJ combination therapy group. ICG and JQ1 were administered at doses of 2.0 mg / kg and 10.0 mg / kg, respectively. The nanoparticles were administered intravenously (iv) to the animals every 3 days for a total of 3 injections. In the combination therapy group, PEID nanoparticles were injected into the tail vein 2 hours later, followed by injection of PGDJ or N3PGDJ nanoparticles. Mice were anesthetized with 2% (v / v) isoflurane and then irradiated with an 808 nm laser at a consistently stable temperature of 45°C for 5 minutes. Tumor size was measured every two days using digital calipers and calculated as (width 2 × length) / 2. At the end of the experiment, the mice were euthanized.

[0108] like Figure 6 As shown, after treatment with PEID@N3PGDJ, the tumor gradually shrank after day 10 and did not recur; while treatment with PEID@PGDJ inhibited tumor growth, but the tumor recurred on day 10. The tumor inhibition experiment demonstrates that the acid-activated bioorthogonal platform, due to the in situ tumor extracellular microacid environment activating the bioorthogonal reaction, increases the accumulation of drugs in tumor tissue, thereby significantly inhibiting tumor growth.

Claims

1. A nanoplatform composed of an acid-responsive diblock polymer modified with bioorthogonal click reactive groups, characterized in that, Including extracellular acid-responsive nanoparticles and intracellular acid-responsive nanoparticles, The extracellular acid-responsive nanoparticles are prepared from the extracellular acid-responsive amphiphilic block polymer shown in Formula 1; used for delivering the R2 group in the extracellular acid-responsive amphiphilic block polymer shown in Formula 1 via bioorthogonal click reaction in the tumor extracellular microenvironment; and loaded with therapeutic agents from the nanoparticles. Formula 1 in, R1 is N,N-ethylpropylamino or hexamethyleneamino; R2 is carboxyl-modified dibenzocyclooctylene (DBCO) and carboxyl-modified transcyclooctene (TCO); x is an integer between 10 and 145; y is an integer between 50 and 100; And z is an integer between 1 and 5; The intracellular acid-responsive nanoparticles are prepared from materials including an intracellular acid-responsive amphiphilic polymer as shown in Formula 2, other materials capable of self-assembling into nanoparticles, or loaded therapeutic agents, wherein the intracellular acid-responsive amphiphilic polymer as shown in Formula 2 accounts for 10%-20% of the total mass of the nanoparticles; the distribution of the therapeutic agent in tumor tissue is improved by undergoing a bioorthogonal reaction with the extracellular acid-responsive nanoparticles. Formula 2 in, R3 is an azide group or a tetrazine group; R4 is N,N-diethylamino, N,N-diisopropylamino, N,N-di-tert-butylamino, N,N-ethylpropylamino, pentamethyleneamino, hexamethyleneamino; m is an integer between 10 and 145. n is an integer between 20 and 100; o is an integer from 1 to 10.

2. The nanoplatform composed of an acid-responsive diblock polymer modified with bioorthogonal click reactive groups as described in claim 1, characterized in that, Preparation method of the extracellular acid-responsive amphiphilic block polymer Includes the following steps: Step a: Synthesis of polyethylene glycol-b-polypolymer A RAFT chain transfer agent-terminated polyethylene glycol macromolecular initiator was dissolved in N,N-dimethylformamide in a molar ratio of 1:50-100:1-5:0.1 with ethyl methacrylate monomer, hydroxyethyl methacrylate monomer, and initiator azobisisobutyronitrile containing R1. The reaction was carried out at 70°C for 24 h under anaerobic conditions. The reaction product was diluted with an appropriate amount of DMF and dialyzed with deionized water. The product was then freeze-dried in a dialysis bag with a cutoff of 3500 Daltons to obtain polyethylene glycol-b-polypolymer. Reaction route a Step b: Coupling of R2 with polyethylene glycol-b-polypolymer The block copolymer obtained in step a was dissolved in anhydrous N,N-dimethylformamide to obtain a solution A with a mass concentration of 10% w / v. A 30% w / v solution B was prepared by mixing carboxyl-modified R2 with N,N-diisopropylethylamine, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a molar ratio of 1:1.5:1.5:1.5 in N,N-dimethylformamide solution. The reaction was allowed to proceed for 1.5 h. Then, solution A was added dropwise to solution B, and the reaction continued for 24 h. The mixture was then dialyzed with N,N-dimethylformamide and then with deionized water using a dialysis bag with a cutoff of 3500 Daltons. The resulting product was freeze-dried to obtain the block polymer shown in Formula 1, covalently modified with R2. Reaction route b Wherein, R1 is N,N-ethylpropylamino or hexamethyleneamino; R2 is carboxyl-modified dibenzocyclooctylene (DBCO) and carboxyl-modified transcyclooctene (TCO); x is an integer between 10 and 145; y is an integer between 50 and 100; And z is an integer between 1 and 5.

3. The nanoplatform composed of an acid-responsive diblock polymer modified with bioorthogonal click reactive groups as described in claim 1, characterized in that, The preparation method of the intracellular acid-responsive amphiphilic polymer includes the following steps: A RAFT chain transfer agent-terminated polyethylene glycol macromolecular initiator, ethyl methacrylate monomer containing R4, hydroxyethyl methacrylate monomer, and initiator azobisisobutyronitrile are dissolved in an appropriate amount of N,N-dimethylformamide at a molar ratio of 1:20-100:0.1; the reaction is carried out at 70°C for 24 h under anaerobic conditions; the reaction product is diluted with an appropriate amount of DMF and dialyzed with deionized water using a dialysis bag with a cutoff of 3500 Daltons; the product is then freeze-dried to obtain the intracellular acid-responsive amphiphilic polymer. The intracellular acid-responsive amphiphilic polymer was synthesized via reversible addition-fragmentation chain transfer polymerization as shown in reaction route II below: Reaction Route II R3 is an azide group or a tetrazine group; R4 is N,N-diethylamino, N,N-diisopropylamino, N,N-di-tert-butylamino, N,N-ethylpropylamino, pentamethyleneamino, hexamethyleneamino; m is an integer between 10 and 145. n is an integer between 20 and 100; o is an integer from 1 to 10.

4. The nanoplatform composed of an acid-responsive diblock polymer modified with bioorthogonal click reactive groups as described in claim 1, characterized in that, The method for preparing the extracellular acid-responsive nanoparticles includes simultaneously dissolving the acid-responsive diblock polymer and the therapeutic prodrug as shown in Formula 1 as described in claim 1 in an organic solvent at a mass ratio of 99-4 to obtain a solution containing the acid-responsive diblock polymer and the therapeutic prodrug. The mixed solution is then added dropwise to deionized water under ultrasonic conditions at a volume ratio of 1:9 with water. The organic solvent or the loaded therapeutic prodrug is then removed by ultrafiltration or dialysis to obtain polymer nanoparticles loaded with the therapeutic prodrug.

5. The nanoplatform composed of an acid-responsive diblock polymer modified with a bioorthogonal click reactive group as described in claim 1, characterized in that, The method for preparing the intracellular acid-responsive nanoparticles includes dissolving the acid-responsive diblock polymer as described in Formula 2 as in claim 1, a lipid or amphiphilic block polymer or dendritic macromolecule that can be assembled into nanoparticles, and a therapeutic prodrug in an organic solvent, wherein the acid-responsive diblock polymer as described in Formula 2 accounts for 1.0%-20% of the total mass, and the therapeutic prodrug accounts for 1.0%-20% of the total mass; and preparing the nanoparticles using an ultrasonic precipitation method or a thin film dispersion method.

6. The nanoplatform composed of an acid-responsive diblock polymer modified with a bioorthogonal click reactive group as described in claim 4 or 5, characterized in that, The organic solvent is selected from at least one of tetrahydrofuran, methanol, N,N-dimethylformamide and N,N-dimethylacetamide; And / or, the therapeutic agent is a small molecule prodrug selected from chemotherapeutic agents, targeted agents, photodynamic therapy agents, immunotherapy agents, and synergistic combinations of these therapeutic agents; The chemotherapy agents are oxaliplatin, paclitaxel, camptothecin, and doxorubicin; the targeted agents are palbociclib, pomaconazole, ribociclib, and G1T28; the photodynamic therapy agent is PPa; and the immunotherapy agents are the IDO inhibitor NLG919, the PD-L1 inhibitor JQ1, the STING agonist, and the TLR agonist.

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