Acoustic-dynamic targeting drug-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention provides sonodynamic targeted drug-loaded nanoparticles as well as a preparation method and application thereof, and belongs to the field of targeted drug delivery preparations. The sonodynamic targeting drug-loading nanoparticle provided by the invention comprises a porous carrier, a drug loaded in the porous carrier and a targeting ligand chemically modified on the outer surface of the porous carrier, the porous carrier is a hydrogen bond organic framework; a construction unit of the hydrogen bond organic framework is a metalloporphyrin complex. After the sonodynamic targeting drug-loading nano-particle provided by the invention is injected into a 4T1 tumor-bearing BALB / c mouse body for 6 hours, fluorescence of the sonodynamic targeting drug-loading nano-particle is observed on a tumor, and a signal is continuously enhanced; after 14 days of administration, the tumor growth is obviously inhibited, and good targeting and anti-tumor effects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of targeted drug delivery preparations, and in particular to sonodynamic targeted drug-loaded nanoparticles and a preparation method and application thereof. Background Art

[0002] Nanoscale and structured drug preparations play an important role in cancer treatment, and various nanoformulations have been designed as drug delivery platforms for cancer chemotherapy. Hydrogen-bonded organic frameworks (HOFs) are an emerging and attractive class of highly crystalline porous materials with excellent biocompatibility, rich chemical functions, and adjustable porosity. Their metal-free properties and reversible bonding methods, which are different from other porous materials, give them unique advantages in the fields of biotechnology and biomedicine. However, how to safely and effectively deliver drugs to tumor sites has become an urgent problem to be solved.

[0003] In related studies, the selectivity for cancer cells can be improved by combining light, sound, magnetism and active targeting. However, the tissue penetration of near-infrared light in photodynamic therapy (PDT) is poor (about 1 to 5 cm), and it is only suitable for the treatment of superficial tumors. Radiodynamic therapy (RDT) has strong penetration ability, but ionizing radiation causes greater damage to normal tissues. In contrast, ultrasound, as a mechanical wave with a frequency beyond human hearing (>20kHz), can penetrate deep soft tissues up to 10 cm due to its unique advantage of minimal scattering in tissues, and accurately reach the tumor site to avoid damaging normal tissues. Sonodynamic therapy (SDT) is a non-invasive treatment method using ultrasound. It has received increasing attention in recent years. Its mechanism is to produce toxic reactive oxygen species (ROS, 1 O2,·OH) is used for cell apoptosis and necrosis. Therefore, compared with PDT and RDT, SDT has greater clinical translation value, but at present, the targeting of sonosensitizers in SDT in tumor cells is still not strong enough, making it difficult to achieve precise treatment. At the same time, there are also shortcomings such as side effects caused by premature drug leakage and poor anti-tumor effect. Summary of the invention

[0004] The purpose of the present invention is to provide a sonodynamic targeted drug-loaded nanoparticle and its preparation method and application. The sonodynamic targeted drug-loaded nanoparticle provided by the present invention has good targeting, can be specifically released in tumor tissue, and has good anti-tumor effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a sonodynamic targeted drug-loaded nanoparticle, comprising a porous carrier, a drug loaded in the porous carrier and a targeting ligand chemically modified on the outer surface of the porous carrier; the porous carrier is a hydrogen-bonded organic framework; the building block of the hydrogen-bonded organic framework is a metal porphyrin complex.

[0007] Preferably, the drug is an anti-tumor drug; the anti-tumor drug is doxorubicin and metformin.

[0008] Preferably, the loading amount of doxorubicin in the porous carrier is 10.9-23.3 wt %, and the loading amount of metformin in the porous carrier is 8.7-26.4 wt %.

[0009] Preferably, the amount of the targeting ligand modified on the outer surface of the porous carrier is 0.0548 to 1.154 nmol·mg -1 ;

[0010] Preferably, the targeting ligand is LHRH polypeptide, cRGD polypeptide or R8H3 polypeptide.

[0011] The present invention also provides a method for preparing the sonodynamic targeted drug-loaded nanoparticles described in the above technical solution, comprising the following steps:

[0012] (1) mixing a metal porphyrin complex, a drug, a non-protonic polar solvent, water and ethanol for self-assembly to obtain drug-loaded nanoparticles;

[0013] (2) mixing the drug-loaded nanoparticles obtained in step (1) with an activator for activation to obtain activated drug-loaded nanoparticles;

[0014] (3) The activated drug-loaded nanoparticles obtained in step (2) are mixed with a targeting ligand for chemical modification to obtain sonodynamic targeted drug-loaded nanoparticles.

[0015] Preferably, the activator in step (2) is a carboxyl activator.

[0016] Preferably, the carboxyl activating agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysulfosuccinimide.

[0017] Preferably, the mass ratio of the drug-loaded nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysulfosuccinimide in the carboxyl activator is 1:(1-5):(5-25).

[0018] The present invention also provides the use of the sonodynamic targeted drug-loaded nanoparticles described in the above technical solution or the sonodynamic targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0019] The present invention provides a sonodynamic targeted drug-loaded nanoparticle, comprising a porous carrier, a drug loaded in the porous carrier, and a targeting ligand chemically modified on the outer surface of the porous carrier; the porous carrier is a hydrogen-bonded organic framework; the building unit of the hydrogen-bonded organic framework is a metal porphyrin complex. The porous carrier in the sonodynamic targeted drug-loaded nanoparticle provided by the present invention uses the metal porphyrin complex as a building unit and has good acoustic sensitivity. The porous carrier has ultrasonic responsiveness, which can enhance the effect of sonodynamic therapy and can also respond to GSH (glutathione) in the tumor microenvironment to avoid side effects caused by premature drug leakage; by modifying the targeting ligand on the outer surface of the porous carrier, the targeting of the sonodynamic targeted drug-loaded nanoparticle is improved, and local ultrasound is performed at the tumor site to achieve precise treatment and further improve the anti-tumor effect. The results of the embodiment show that 6 hours after the sonodynamic targeted drug-loaded nanoparticles provided by the present invention were injected into 4T1 tumor-bearing BALB / c mice, the fluorescence of the sonodynamic targeted drug-loaded nanoparticles was observed on the tumor, and the signal continued to increase; 14 days after administration, the tumor growth was significantly inhibited, with good targeting and anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 TEM image of LHRH@DM@HOF-1 in Example 1;

[0021] Figure 2 It is the particle size range curve of LHRH@DM@HOF-1, HOF-1 and LHRH@HOF-1;

[0022] Figure 3 Zeta potential bar graphs of DM@HOF-1, LHRH@DM@HOF-1, HOF-1, and LHRH@HOF-1;

[0023] Figure 4 is the BET curve diagram of HOF-1;

[0024] Figure 5 The particle size distribution curve of LHRH@DM@HOF-1 under different simulated physiological environments;

[0025] Figure 6 FT-IR spectra of FeTCPPCl in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2;

[0026] Figure 7 XRD patterns of FeTCPPCl and LHRH@DM@HOF-1 in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2;

[0027] Figure 8 Thermogravimetric curves of LHRH@DM@HOF-1 in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2;

[0028] Fig. 9 This is the XPS graph of LHRH@DM@HOF-1 in Example 1;

[0029] Fig.10 It is the band gap curve diagram of FeTCPPCl and HOF-1 in comparative example 1;

[0030] Fig.11 is the energy band diagram of HOF-1 in Comparative Example 1;

[0031] Fig.12 The degradation diagram of MB by HOF-1; (a) is the curve of the absorbance of MB changing with wavelength at different time intervals, and (b) is the curve of the absorbance of MB changing with time under different conditions;

[0032] Fig.13 The degradation diagram of DPBF by HOF-1; (a) is the curve of absorbance variation of DPBF with wavelength at different time intervals, and (b) is the curve of absorbance variation of DPBF with time under different conditions;

[0033] Fig.14 : ESR spectra of FeTCPPCl and HOF-1; (a) is the ESR spectra of FeTCPPCl and HOF-1 in TEMP, (b) is the ESR spectra of FeTCPPCl and HOF-1 in DMPO;

[0034] Fig.15 The diagram is the mechanism of HOF-1 producing ROS under ultrasound;

[0035] Fig.16 is the phosphorescence intensity diagram of FeTCPPCl and HOF-1;

[0036] Fig.17 Confocal laser images of FITC@HOF-1, FITC@DM@HOF-1 and FITC-LHRH@DM@HOF-1; (a) is a confocal laser image, and (b) is a flow cytometry analysis diagram;

[0037] Fig.18 For 4T1 cells and RAW264.7 cells, the concentration of LHRH@HOF-1 ranged from 0 to 100 μg mL -1 Cell viability bar graph;

[0038] Fig.19Confocal laser images of PBS, DOX, and LHRH@HOF-1+DOX;

[0039] Fig. 20 Schematic diagram of the in vivo pharmacodynamics experiment process;

[0040] Fig.21 Fluorescence images of 4T1 tumor-bearing mice at different times under different treatment regimens;

[0041] Fig. 22 The tumor growth curves and weights of 4T1 tumor-bearing mice under different treatment regimens; (a) is the tumor growth curve, and (b) is the tumor weight;

[0042] Fig.23 These are photos of tumors after different treatment regimens on day 14;

[0043] Fig.24 H&E staining, CRT staining, HMGB1 staining, PD-L1 staining and TUNEL staining of tumor sections under different treatment regimens. DETAILED DESCRIPTION

[0044] The invention provides a sonodynamic targeted drug-loaded nanoparticle, comprising a porous carrier, a drug loaded in the porous carrier and a targeting ligand modified on the outer surface of the porous carrier.

[0045] In the present invention, the sonodynamic targeted drug-loaded nanoparticles include a porous carrier; the porous carrier is a hydrogen-bonded organic framework; the building block of the hydrogen-bonded organic framework is a metal porphyrin complex. In a specific embodiment of the present invention, the metal porphyrin complex is FeTCPPCl. The present invention uses FeTCPPCl as a building block, wherein the transition metal Fe element has high biocompatibility; and the porphyrin molecules can self-assemble into a stable HOF structure through hydrogen bonds between carboxyl groups and π-π interactions.

[0046] As an embodiment of the present invention, the pore size of the porous carrier may be 3.25-7.43 nm. The present invention limits the pore size of the porous carrier to the above range so as to better load the drug and achieve ultrasonic response.

[0047] In the present invention, the diameter of the porous carrier is preferably 80-100 nm. In the present invention, limiting the diameter of the porous carrier to the above range can facilitate the subsequent modification of the targeting ligand with an appropriate size of the sonodynamic targeting drug-loaded nanoparticles.

[0048] In the present invention, the sonodynamic targeted drug-loaded nanoparticles include drugs loaded in the porous carrier; the drugs are preferably anti-tumor drugs; the anti-tumor drugs are preferably doxorubicin and metformin; the loading amount of doxorubicin in the porous carrier is preferably 10.9-23.3wt%, more preferably 20-23wt%, and further preferably 22.1wt%; the loading amount of metformin in the porous carrier is preferably 8.7-26.4wt%, more preferably 20-25wt%, and further preferably 24.4wt%. The doxorubicin of the present invention can enhance the immunogenic cell death induced by sonodynamics, and metformin can cause PD-L1 to fall off the tumor cell membrane, thereby CD8 + T cells can continuously identify and eliminate tumor cells. The present invention limits the loading amount of doxorubicin and metformin in the porous carrier to the above range, which can improve the anti-tumor effect of the sonodynamic targeted drug-loaded nanoparticles.

[0049] In the present invention, the sonodynamic targeted drug-loaded nanoparticles include a targeting ligand chemically modified on the outer surface of the porous carrier; the modification amount of the targeting ligand on the outer surface of the porous carrier is preferably 0.0548 to 1.154 nmol·mg -1 , more preferably 0.8 to 1.10 nmol·mg -1 , more preferably 1.012nmol·mg -1 . The present invention modifies the targeting ligand on the outer surface of the porous carrier to improve the targeting of the sonodynamic targeted drug-loaded nanoparticles, achieve precision treatment, and further improve the anti-tumor effect. The present invention limits the modification amount of the targeting ligand on the outer surface of the porous carrier to the above range, which can further improve the targeting of the sonodynamic targeted drug-loaded nanoparticles and achieve precision treatment.

[0050] In the present invention, the targeting ligand is preferably LHRH polypeptide, cRGD polypeptide or R8H3 polypeptide. The present invention limits the types of targeting ligands to the above range to ensure that the sonodynamic targeted drug-loaded nanoparticles have high targeting properties.

[0051] In the present invention, the diameter of the sonodynamic targeted drug-loaded nanoparticles is preferably 120 to 180 nm, more preferably 150 to 180 nm. The present invention limits the diameter of the sonodynamic targeted drug-loaded nanoparticles to the above range so that the enhanced permeability retention effect (EPR effect) can be used to retain the nanoparticles in the tumor site, which can make the carrier have passive targeting.

[0052] The porous carrier in the sonodynamic targeted drug-loaded nanoparticles provided by the present invention uses metal porphyrin complexes as building units and has good acoustic sensitivity. The porous carrier has ultrasonic responsiveness, which can enhance the effect of sonodynamic therapy and can also respond to GSH in the tumor microenvironment to avoid side effects caused by premature drug leakage. By modifying the targeting ligand on the outer surface of the porous carrier, the targeting of the sonodynamic targeted drug-loaded nanoparticles is improved, and local ultrasound is performed at the tumor site to achieve precise treatment, further improving the anti-tumor effect.

[0053] The present invention also provides a method for preparing the sonodynamic targeted drug-loaded nanoparticles described in the above technical solution, comprising the following steps:

[0054] (1) mixing a metal porphyrin complex, a drug, a non-protonic polar solvent, water and ethanol for self-assembly to obtain drug-loaded nanoparticles;

[0055] (2) mixing the drug-loaded nanoparticles obtained in step (1) with an activator for activation to obtain activated drug-loaded nanoparticles;

[0056] (3) The activated drug-loaded nanoparticles obtained in step (2) are mixed with a targeting ligand for chemical modification to obtain sonodynamic targeted drug-loaded nanoparticles.

[0057] The invention mixes metal porphyrin complex, medicine, non-protonic polar solvent, water and ethanol for self-assembly to obtain medicine-carrying nanoparticles.

[0058] In the present invention, the mass ratio of the metal porphyrin complex to the drug is preferably (5-10): 2, more preferably (7-9): 2, and more preferably 8: 2. The present invention limits the mass ratio of the metal porphyrin complex to the drug to the above range to further improve the anti-tumor effect of the sonodynamic targeted drug-loaded nanoparticles.

[0059] In a specific embodiment of the present invention, the drugs are doxorubicin and metformin; the mass ratio of doxorubicin to metformin is 1:1.

[0060] In the present invention, the ratio of the mass of the metal porphyrin complex to the volume of the aprotic polar solvent is preferably (8-12) mg:1 mL, more preferably 10 mg:1 mL. In a specific embodiment of the present invention, the aprotic polar solvent is N,N-dimethylformamide (DMF). The present invention limits the ratio of the mass of the metal porphyrin complex to the volume of the aprotic polar solvent to the above range to ensure that the metal porphyrin complex is completely dissolved in the aprotic polar solvent.

[0061] In the present invention, the volume ratio of the aprotic polar solvent, ethanol and water is preferably (0.5-1.5): (4.5-5.5): (0.5-1.5), and more preferably 1: 5: 1. The present invention limits the volume ratio of the aprotic polar solvent, ethanol and water to the above range to ensure that the metal porphyrin complex can form good crystals.

[0062] In the present invention, the metal porphyrin complex, the drug and the aprotic polar solvent are preferably first mixed, then second mixed with water, and finally third mixed with ethanol.

[0063] The present invention has no special restrictions on the time and operation steps for the first mixing of the metal porphyrin complex, the drug and the aprotic polar solvent, as long as the metal porphyrin complex and the drug can be completely dissolved in the aprotic polar solvent.

[0064] As an embodiment of the present invention, the second mixing is performed under stirring conditions; the first mixing time can be 2 to 5 minutes, or 3 minutes. The present invention can make HOF-1 crystals grow slowly and evenly through the second mixing.

[0065] The present invention has no particular limitation on the operation and time of the third mixing, as long as the raw materials can be mixed uniformly.

[0066] As an embodiment of the present invention, the third mixing is followed by standing; the standing is preferably carried out in a light-proof condition; the standing time can be 20 to 30 minutes, or 25 minutes. The present invention can reduce the influence of light on crystal growth by standing in a light-proof condition.

[0067] After the standing is completed, the present invention preferably centrifuges, purifies and dries the product obtained by the standing in sequence.

[0068] In a specific embodiment of the present invention, the centrifugal speed is 12000 rpm; the centrifugal time is 5 min.

[0069] In the present invention, the purification solution used in the purification is preferably acetone; the number of purification is preferably 2 to 4 times, more preferably 3 times. The present invention can remove impurities in the drug-loaded nanoparticles through purification.

[0070] As an embodiment of the present invention, the drying temperature may be 40° C. and the drying time may be 2 hours. The present invention removes the residual purified liquid by drying.

[0071] After obtaining the drug-loaded nanoparticles, the present invention mixes the drug-loaded nanoparticles with an activator for activation to obtain activated drug-loaded nanoparticles.

[0072] In the present invention, the activator is preferably a carboxyl activator; the carboxyl activator is preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysulfosuccinimide.

[0073] In the present invention, the mass ratio of the drug-loaded nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and sulfo-N-hydroxysulfosuccinimide in the carboxyl activator is preferably 1:(1-5):(5-25), and more preferably 1:5:5. The present invention limits the mass ratio of the drug-loaded nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and sulfo-N-hydroxysulfosuccinimide in the carboxyl activator to the above range, which can fully activate the drug-loaded nanoparticles and is beneficial to the subsequent modification of the targeting ligand.

[0074] In the present invention, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is preferably mixed with the drug-loaded nanoparticles in the form of a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution; in a specific embodiment of the present invention, the concentration of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution is 10 mg / mL; the solvent of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution is a 4-morpholineethanesulfonic acid (MES) aqueous solution; the concentration of the 4-morpholineethanesulfonic acid (MES) aqueous solution is 1.0×10 -3 M.

[0075] In the present invention, the sulfo-N-hydroxysulfosuccinimide is preferably mixed with the drug-loaded nanoparticles in the form of a sulfo-N-hydroxysulfosuccinimide solution; in a specific embodiment of the present invention, the concentration of sulfo-N-hydroxysulfosuccinimide in the sulfo-N-hydroxysulfosuccinimide solution is 10 mg / mL; the solvent of the sulfo-N-hydroxysulfosuccinimide solution is water.

[0076] In a specific embodiment of the present invention, the drug-loaded nanoparticles and the activator are mixed by mixing the drug-loaded nanoparticles with a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution for a fourth time and then mixing the drug-loaded nanoparticles with a sulfo-N-hydroxysulfosuccinimide solution for a fifth time.

[0077] As an implementation mode of the present invention, the fourth mixing is performed under stirring conditions; the stirring speed is 80 r / min; and the fourth mixing time is 5 min.

[0078] As an embodiment of the present invention, the fifth mixing is performed under shaking conditions; the shaking frequency is 300 r / min; and the fifth mixing time is 15 min.

[0079] In the present invention, the activation temperature is preferably room temperature.

[0080] After obtaining the activated drug-loaded nanoparticles, the present invention mixes the activated drug-loaded nanoparticles with targeting ligands for chemical modification to obtain sonodynamic targeting drug-loaded nanoparticles.

[0081] In the present invention, the molar ratio of the activated drug-loaded nanoparticles to the targeting ligand is preferably 1:(5-20), more preferably 1:(10-15); the targeting ligand is preferably LHRH polypeptide. The present invention limits the molar ratio of the activated drug-loaded nanoparticles to the targeting ligand to the above range to further improve the targeting of the sonodynamic targeted drug-loaded nanoparticles.

[0082] In the present invention, the targeting ligand is preferably mixed with the activated drug-loaded nanoparticles in the form of a targeting ligand solution; the solvent of the targeting ligand solution is preferably a 0.01 M phosphate buffer solution with a pH of 7.4; the concentration of the targeting ligand in the targeting ligand solution is preferably 6×10 -5 M.

[0083] In the present invention, the chemical modification is preferably carried out under shaking conditions; the shaking frequency is preferably 300 r / min; the chemical modification temperature is preferably room temperature; and the chemical modification time is preferably 12 hours. The present invention limits the chemical modification temperature and time to the above ranges to ensure that the targeting ligand fully modifies the activated drug-loaded nanoparticles.

[0084] After the chemical modification is completed, the present invention preferably washes and dries the chemically modified product in sequence. As an embodiment of the present invention, the washing solution used for the washing can be a PBS buffer solution; the washing times can be 3 to 4 times. The present invention can remove unbound targeting ligands by washing.

[0085] The present invention has no particular limitation on the drying temperature and time, as long as the product obtained after washing can be completely dried.

[0086] The present invention also provides the use of the sonodynamic targeted drug-loaded nanoparticles described in the above technical solution or the sonodynamic targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0087] In the present invention, the anticancer drug is preferably an anti-triple-negative breast cancer chemotherapy drug, an anti-pancreatic cancer chemotherapy drug or an anti-non-small cell lung cancer chemotherapy drug.

[0088] In the present invention, the dosage form of the anticancer drug prepared by the sonodynamic targeted drug-loaded nanoparticles is preferably an injection or a tablet.

[0089] As an embodiment of the present invention, the specification of the anticancer drug can be 2.49 mg·mL -1 The dosage of the anticancer drug can be 16.6 mg kg -1 .

[0090] In an embodiment of the present invention, the method of using the anticancer drug is intravenous injection, and ultrasound is performed 24 hours after the injection; the solvent used for the intravenous injection is phosphate buffer; the pH value of the phosphate buffer is 7.4; the power of the ultrasound is 0.58W·cm -2 ; The ultrasound time is 2 minutes; The ultrasound duty cycle is 50%.

[0091] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. 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.

[0092] Example 1

[0093] A sonodynamic targeted drug-loaded nanoparticle, comprising a porous carrier, a drug loaded in the porous carrier, and a targeting ligand chemically modified on the outer surface of the porous carrier; the porous carrier is a hydrogen-bonded organic framework; the building unit of the hydrogen-bonded organic framework is FeTCPPCl; the drugs are doxorubicin and metformin; the targeting ligand is an LHRH polypeptide; the pore size of the porous carrier is 3.25-7.43 nm; the loading amount of doxorubicin in the porous carrier is 22.1 wt%; the loading amount of metformin in the porous carrier is 24.4 wt%; the modification amount of the LHRH polypeptide on the outer surface of the porous carrier is 1.012 nmol·mg -1 ;

[0094] The preparation method of the sonodynamic targeted drug-loaded nanoparticles is as follows:

[0095] (1) FeTCPPCl, doxorubicin, metformin and N,N-dimethylformamide were mixed for the first time, water was added and mixed for the second time for 3 minutes under stirring, and then ethanol was added for the third time and allowed to stand for 25 minutes under light-proof conditions, and then centrifuged at 12000 rpm for 5 minutes, the centrifuged product was purified 3 times with acetone, and dried at 40°C for 2 hours to obtain drug-loaded nanoparticles (DM@HOF-1); the mass ratio of FeTCPPCl, doxorubicin and metformin was 5:1:1; the mass ratio of FeTCPPCl to the volume of N,N-dimethylformamide was 10 mg:1 mL; the volume ratio of N,N-dimethylformamide, ethanol and water was 1:5:1;

[0096] (2) The drug-loaded nanoparticles obtained in the step (1) are mixed with a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution at room temperature at a rotation speed of 80 r / min for 5 min for a fourth mixing, and then mixed with a sulfo-N-hydroxysulfosuccinimide solution at a shaking frequency of 300 r / min for 15 min for a fifth mixing to obtain activated drug-loaded nanoparticles; the concentration of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution is 10 mg / mL; the solvent of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution is a 4-morpholineethanesulfonic acid (MES) aqueous solution; the concentration of the 4-morpholineethanesulfonic acid (MES) aqueous solution is 1.0×10 -3 M; the concentration of sulfo-N-hydroxysulfosuccinimide in the sulfo-N-hydroxysulfosuccinimide solution is 10 mg / mL; the solvent of the sulfo-N-hydroxysulfosuccinimide solution is water; the mass ratio of the drug-loaded nanoparticles to the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysulfosuccinimide in the carboxyl activator is 1:5:5;

[0097] (3) The activated drug-loaded nanoparticles obtained in step (2) are mixed with the targeting ligand solution and chemically modified for 12 hours at room temperature and under shaking conditions. The chemically modified product is washed three times with PBS buffer and dried at 50° C. for 6 hours to obtain sonodynamic targeted drug-loaded nanoparticles (LHRH@DM@HOF-1); the molar ratio of the activated drug-loaded nanoparticles to the targeting ligand is 1:10; the targeting ligand solution is an LHRH polypeptide solution; the solvent of the targeting ligand solution is 0.01 M phosphate buffer (pH 7.4); the concentration of the targeting ligand in the targeting ligand solution is 6×10 -5 M;

[0098] FeTCPPCl, doxorubicin (DOX) and metformin (MET) were purchased from Shanghai MacLean Biochemical Co., Ltd. (Shanghai, China), N,N-dimethylformamide was purchased from Aladdin Industrial Co., Ltd. (Shanghai, China), and LHRH polypeptide ((D-Lys6)-LHRH) was purchased from Shanghai Peptide Co., Ltd. (Shanghai, China).

[0099] Comparative Example 1

[0100] A porous carrier; the porous carrier is a hydrogen-bonded organic framework; the building unit of the hydrogen-bonded organic framework is FeTCPPCl; the pore size of the porous carrier is 3.25 to 7.43 nm;

[0101] After FeTCPPCl and N,N-dimethylformamide are mixed for the first time, water is added and the mixture is mixed for the second time for 3 minutes under stirring. Then, ethanol is added for the third mixing and the mixture is allowed to stand for 25 minutes under light-proof conditions. The mixture is then centrifuged at 12000 rpm for 5 minutes. The centrifuged product is purified three times with acetone and dried at 40°C for 2 hours to obtain a porous carrier (HOF-1). The ratio of the mass of the FeTCPPCl to the volume of the N,N-dimethylformamide is 10 mg:1 mL. The volume ratio of the N,N-dimethylformamide, ethanol and water is 1:5:1.

[0102] Comparative Example 2

[0103] A sonodynamic targeting nanoparticle, comprising a porous carrier and a targeting ligand chemically modified on the outer surface of the porous carrier; the porous carrier is a hydrogen bond organic framework; the building unit of the hydrogen bond organic framework is FeTCPPCl; the targeting ligand is an LHRH polypeptide; the pore size of the porous carrier is 3.25 to 7.43 nm; the modification amount of the LHRH polypeptide on the outer surface of the porous carrier is 1.012 nmol·mg -1 ;

[0104] The preparation method of the sonodynamic targeting nanoparticles is:

[0105] (1) FeTCPPCl and N,N-dimethylformamide were mixed for the first time, water was added, and the mixture was mixed for the second time for 3 minutes under stirring. Ethanol was then added for the third time, and the mixture was allowed to stand for 25 minutes under light-proof conditions. The mixture was then centrifuged at 12000 rpm for 5 minutes, and the centrifuged product was purified three times with acetone, and dried at 40°C for 2 hours to obtain a porous carrier (HOF-1); the ratio of the mass of FeTCPPCl to the volume of N,N-dimethylformamide was 10 mg:1 mL; the volume ratio of N,N-dimethylformamide, ethanol and water was 1:5:1;

[0106] (2) The porous carrier obtained in the step (1) is mixed with a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution at room temperature at a rotation speed of 80 r / min for 5 min, and then mixed with a sulfo-N-hydroxysulfosuccinimide solution at a shaking frequency of 300 r / min for 15 min to obtain an activated porous carrier; the concentration of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution is 10 mg / mL; the solvent of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution is a 4-morpholineethanesulfonic acid (MES) aqueous solution; the concentration of the 4-morpholineethanesulfonic acid (MES) aqueous solution is 1.0×10 -3 M; the concentration of sulfo-N-hydroxysulfosuccinimide in the sulfo-N-hydroxysulfosuccinimide solution is 10 mg / mL; the solvent of the sulfo-N-hydroxysulfosuccinimide solution is water; the mass ratio of the porous carrier to the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysulfosuccinimide in the carboxyl activator is 1:5:5;

[0107] (3) The activated porous carrier obtained in step (2) was mixed with the targeting ligand solution and chemically modified at room temperature and under shaking conditions for 12 hours. The chemically modified product was washed three times with PBS buffer and dried at 50° C. for 6 hours to obtain sonodynamic targeting nanoparticles (LHRH@HOF-1); the targeting ligand solution was an LHRH polypeptide solution; the solvent of the targeting ligand solution was 0.01 M phosphate buffer; the concentration of the targeting ligand in the targeting ligand solution was 6×10 -5 M.

[0108] Application Example 1

[0109] The use of the sonodynamic targeted drug-loaded nanoparticles (LHRH@DM@HOF-1) obtained in Example 1 in the preparation of an anti-breast cancer drug, wherein the dosage form of the anti-breast cancer drug is an injection, and the specification of the anti-breast cancer drug is 2.49 mg·mL -1 The dosage of the anti-breast cancer drug is 16.6 mg kg -1 ;

[0110] The method of using the anti-breast cancer drug is: intravenous injection, and ultrasound is performed 24 hours after the injection (LHRH@DM@HOF-1+US); the solvent used for the intravenous injection is phosphate buffer; the pH value of the phosphate buffer is 7.4; the power of the ultrasound is 0.58W·cm -2 ; The ultrasound time is 2 minutes; The ultrasound duty cycle is 50%.

[0111] Comparative application example 1

[0112] Application of the porous carrier (HOF-1) obtained in Comparative Example 1 in the preparation of an anti-breast cancer drug, wherein the dosage form of the anti-breast cancer drug is an injection, and the specification of HOF-1 in the anti-breast cancer drug is 2.49 mg·mL -1 The dosage of HOF-1 in the anti-breast cancer drug is 16.6 mg·kg -1 ;

[0113] The method of using the anti-breast cancer drug is: intravenous injection, and ultrasound (HOF-1+US) is performed 24 hours after the injection; the solvent used for the intravenous injection is phosphate buffer; the pH value of the phosphate buffer is 7.4; the power of the ultrasound is 0.58W·cm -2 ; The ultrasound time is 2 minutes; The ultrasound duty cycle is 50%.

[0114] Comparative Application Example 2

[0115] The use of the sonodynamic targeting nanoparticles (LHRH@HOF-1) obtained in Comparative Example 2 in the preparation of an anti-breast cancer drug, wherein the dosage form of the anti-breast cancer drug is an injection, and the specification of LHRH@HOF-1 in the anti-breast cancer drug is 2.49 mg·mL -1 The dosage of LHRH@HOF-1 in the anti-breast cancer drug is 16.6 mg·kg -1 ;

[0116] The method for using the anti-breast cancer drug is: intravenous injection; the solvent used for the intravenous injection is phosphate buffer; the pH value of the phosphate buffer is 7.4.

[0117] Comparative Application Example 3

[0118] Application of doxorubicin and metformin (DOX+MET) in the preparation of anti-breast cancer drugs, wherein the dosage of doxorubicin and metformin (DOX+MET) is 4.98 mg·mL -1 ;

[0119] The method for using the anti-breast cancer drug is: intravenous injection; the solvent used for the intravenous injection is phosphate buffer; the pH value of the phosphate buffer is 7.4.

[0120] Test Case

[0121] (I) Characterization of the sonodynamic targeted drug-loaded nanoparticles of Example 1 (LHRH@DM@HOF-1), the porous carrier of Comparative Example 1 (HOF-1), and the sonodynamic targeted nanoparticles of Comparative Example 2 (LHRH@HOF-1).

[0122] Hitachi Regulus 8100 was used to collect images of LHRH@DM@HOF-1 in Example 1, with an operating voltage of 20 kV and an operating current of 15 μA. The TEM images obtained are as follows: Figure 1 As shown. Figure 1 It can be seen that LHRH@DM@HOF-1 presents a uniform spherical morphology with a particle size of 165±2.13nm.

[0123] The particle size ranges of LHRH@DM@HOF-1 in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2 were measured using DLS (90Plus Zeta, Brookhaven); the particle size range curves of LHRH@DM@HOF-1, HOF-1, and LHRH@HOF-1 are shown in FIG. Figure 2 As shown. Figure 2 It can be seen that the particle size of HOF-1 increased after modification with LHRH peptide.

[0124] The Zeta potential of DM@HOF-1 and LHRH@DM@HOF-1 in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2 was measured by DLS (90Plus Zeta, Brookhaven). The Zeta potential data of DM@HOF-1, LHRH@DM@HOF-1, HOF-1, and LHRH@HOF-1 are shown in Table 1; the Zeta potential bar graphs of DM@HOF-1, LHRH@DM@HOF-1, HOF-1, and LHRH@HOF-1 are shown in Table 1. Figure 3 As shown. Figure 3 It can be seen that the surface charge of HOF-1 is -20.256 mV, the charge of LHRH@HOF-1 is 18.365 mV, and the charge of DM@HOF-1 is 11.254 mV, indicating that the drug (DOX+MET) is positively charged. After loading the positively charged drug (DOX+MET), the charge of LHRH@DM@HOF-1 increases to -11.254 mV, which indicates that the drug has been successfully loaded into HOF-1.

[0125] Table 1 Zeta potential data of D-M@HOF-1, LHRH@DM@HOF-1, HOF-1 and LHRH@HOF-1

[0126]

[0127]

[0128] The carbon dioxide isotherm of HOF-1 in Comparative Example 1 was measured using an automatic surface area and pore size distribution analyzer (ASAP 2460, Micromeritics), and the BET curve of HOF-1 was obtained as follows: Figure 4 As shown. Figure 4 It can be seen that HOF-1 has permanent porosity, making it an ideal candidate for drug carriers.

[0129] The stability of LHRH@DM@HOF-1 in Example 1 was tested by DLS (90Plus Zeta, Brookhaven). The particle size of LHRH@DM@HOF-1 was tested in a simulated physiological environment (10% FBS + pH7.4 PBS at 37°C) and a simulated physiological environment (pH7.4 PBS at 37°C) for 7 days. The particle size distribution curves of LHRH@DM@HOF-1 in different simulated physiological environments are shown in FIG. Figure 5 As shown, from Figure 5 It can be seen that LHRH@DM@HOF-1 can exist stably in a normal physiological environment, and its preparation process is suitable for pilot production.

[0130] The FeTCPPCl in Example 1, the HOF-1 in Comparative Example 1, and the LHRH@HOF-1 in Comparative Example 2 were subjected to FT-IR spectrum analysis using SHIMADZU IRTRACER-100, and the FT-IR spectra of the FeTCPPCl in Example 1, the HOF-1 in Comparative Example 1, and the LHRH@HOF-1 in Comparative Example 2 were obtained as shown in FIG. Figure 6 As shown. Figure 6 It can be seen that after FeTCPPCl self-assembles into HOF-1, it is located at 1720cm -1 The C=O stretching vibration of the free carboxylic acid at 1668 cm -1 , and at 3429cm -1 A broad and scattered peak appears at 3129 cm -1 、1649cm -1 、1620cm -1 The peaks at are the NH stretching vibration, C=O stretching vibration and NH bending vibration of the amide bond, respectively, indicating that the -NH2 on LHRH and the -COOH on HOF-1 undergo an amide reaction to complete the surface functionalization (chemical modification).

[0131] The FeTCPPCl and LHRH@DM@HOF-1 in Example 1, the HOF-1 in Comparative Example 1, and the LHRH@HOF-1 in Comparative Example 2 were subjected to XRD detection using Malvern Panalytical Aeris, and the XRD spectra of the FeTCPPCl and LHRH@DM@HOF-1 in Example 1, the HOF-1 in Comparative Example 1, and the LHRH@HOF-1 in Comparative Example 2 were obtained as shown in FIG. Figure 7 As shown. Figure 7 It can be seen that LHRH@DM@HOF-1 and LHRH@HOF-1 still maintain the crystal structure.

[0132] Thermogravimetric analysis was performed on LHRH@DM@HOF-1 in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2 using DSC (METTLER TOLEDO, DSC3). The thermogravimetric curves of LHRH@DM@HOF-1 in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2 were as follows: Figure 8 As shown. Figure 8 It can be seen that LHRH@DM@HOF-1 will decompose at around 120°C, which is caused by the decomposition of the loaded drug.

[0133] Thermo Scientific K-Alpha was used to perform XPS analysis on LHRH@DM@HOF-1 in Example 1, and the XPS graph of LHRH@DM@HOF-1 in Example 1 was obtained as shown in FIG. Fig. 9 As shown. Fig. 9 It can be seen that various elements in LHRH@DM@HOF-1 exist, indicating that it was successfully prepared.

[0134] (ii) Characterization of the sonodynamic properties of the porous carrier (HOF-1) of comparative example 1.

[0135] The energy band gaps of FeTCPPCl and HOF-1 in Comparative Example 1 were measured: 5 mg of HOF-1 and FeTCPPCl were dissolved in DMF, and UV-vis DRS was used to test the UV-visible diffuse reflectance spectra of the samples. The Tauc plot method was used to calculate Eg, and VB-XPS was used to measure the valence band position (Ev) of HOF, and the band gap curves of FeTCPPCl and HOF-1 were obtained as shown in the figure below: Fig.10 As shown; the energy band diagram of HOF-1 is shown Fig.11 As shown. Fig.10 and Fig.11It can be seen that the calculated band gap is about 2.57 eV between the valence band (2.08 V) and the conduction band (−0.49 V), indicating that HOF-1 can achieve electronic excitation under US irradiation to generate ROS.

[0136] Methylene blue (MB) and 1,3-diphenylisobenzofuran (DPBF) were used as probes to detect ·OH and 1 Production of O2.

[0137] MB DMSO solution (0.1 mg / mL) and HOF-1 (50 μg / mL) PBS solution were mixed and hydrogen peroxide was added. They were placed under US at different time intervals (0, 5, 10, 15, 20, 25 min) to detect ·OH. The degradation of MB under different conditions (MB+US, MB+HOF-1, MB+HOF-1+US and MB+HOF-1+US+H2O2) was observed. The degradation of MB by HOF-1 was obtained as shown in Figure 2. Fig.12 As shown, (a) is the curve of the absorbance of MB changing with wavelength at different time intervals, and (b) is the curve of the absorbance of MB changing with time under different conditions. Fig.12 It can be seen that when the MB solution containing HOF-1 was exposed to US, the absorbance peak at 668 nm in the UV–visible spectrum decreased over time, indicating that the ·OH produced by HOF-1 induced by US oxidized MB into a colorless solution. In addition, the addition of H2O2 resulted in the generation of a large amount of ·OH, which greatly reduced the peak intensity.

[0138] A DMSO solution of DPBF (100 μg / mL) was mixed with a PBS solution of HOF-1 (50 μg / mL), and the mixture was sonicated (1 W / cm2) at different time intervals (0, 5, 10, 15, 20, 25 min). 2 , 1.0MHz, 50% duty cycle), using the characteristic peak detection of DPBF at 420nm 1 The appearance of O2 was observed, and the degradation of DPBF under different conditions (DPBF+US, DPBF+HOF-1 and DPBF+HOF-1+U) was observed. The degradation of DPBF by HOF-1 was obtained as follows: Fig.13 As shown, (a) is the curve of the absorbance of DPBF changing with wavelength at different time intervals, and (b) is the curve of the absorbance of DPBF changing with time under different conditions. Fig.13 It can be seen that the solution containing DPBF and HOF-1 was exposed to US to detect 1 As the exposure time of US increased, the absorption peak of DPBF at 410 nm in the UV-visible spectrum gradually decreased, indicating that the US-induced HOF-1 produced 1O2 oxidized DPBF to colorless 1,2-dibenzoylbenzene over time, whereas negligible DPBF degradation was observed in the control groups including the US group and the HOF-1-only group.

[0139] Electron spin resonance spectroscopy (ESR) of free radicals: 2 mg of FeTCPPCl and HOF-1 were dissolved in 1 mL of DMF, H2O2 (0.1 mM, 1000 μL) and capture agents DMPO (0.02 M, 100 μL) and TEMP (0.1 M, 50 μL) were added, and the mixture was exposed to ultrasound (1 W / cm 2 , 1.0 MHz, 50% duty cycle), incubated once every 3 minutes, for a total of three incubations, the ESR spectra of FeTCPPCl and HOF-1 were obtained as shown in Fig.14 As shown, (a) is the ESR spectrum of FeTCPPCl and HOF-1 in TEMP, and (b) is the ESR spectrum of FeTCPPCl and HOF-1 in DMPO. Fig.14 It can be seen that ESR detects OH and 1 The generation of O2 was measured by using 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) as scavengers to measure ·OH and 1 O2, after FeTCPPCl was prepared into HOF-1, the ROS generation ability was enhanced.

[0140] The mechanism of HOF-1 producing ROS under ultrasound Fig.15 As shown, from Fig.15 It can be seen that at US (1.0 MHz, 0.58 W cm -2 , 2 min, 50% duty cycle), HOF-1 changes from the singlet ground state S0 to the excited singlet state, and then rapidly decays to the lowest energy level S1 of the first excited singlet state through non-radiative relaxation. After the molecule changes to the first excited triplet state T1 through the intersystem crossing process (ISC), HOF-1 relaxes to S0 mainly through phosphorescence and energy transfer. This process is called 1 O2 quenching process of O2 generation.

[0141] HOF-1 phosphorescence emission detection: FeTCPPCl and HOF-1 were dispersed in DMF at a concentration of 20 mM, excited by a 415 nm xenon lamp at 78 K, and the phosphorescence emission was tested in the range of 800-1200 nm. The phosphorescence intensity of FeTCPPCl and HOF-1 was obtained as shown in the figure. Fig.16 As shown. Fig.16It can be seen that the phosphorescence intensity of HOF-1 is 6.3% lower than that of FeTCPPCl. The decrease in phosphorescence intensity is conducive to transferring more energy to the O2 quenching process, thereby enhancing 1 Production of O2.

[0142] (III) In vitro pharmacodynamic experiments of DM@HOF-1, LHRH@DM@HOF-1 in Example 1 and HOF-1 in Comparative Example 1.

[0143] 4T1 cells were plated at 1 × 10 4 The cells were seeded at a density of 100 μg / cm2 in a 24-well plate and allowed to adhere. After FITC was conjugated to HOF-1, DM@HOF-1 and LHRH@DM@HOF-1, the obtained FITC@HOF-1, FITC@DM@HOF-1 and FITC-LHRH@DM@HOF-1 were added to the cell culture medium, incubated with the cells for 24 h, washed three times with ice-cold PBS, fixed with 4% paraformaldehyde for 30 min, stained with DAPI for 3 min, washed, and mounted with glycerol. The cells were imaged using a confocal laser scanning microscope (CLSM, 2-PLUS, Nikon), and the confocal laser images of FITC@HOF-1, FITC@DM@HOF-1 and FITC-LHRH@DM@HOF-1 were obtained as shown in the figure. Fig.17 As shown, (a) is a confocal laser image, and (b) is a flow cytometry analysis diagram. Fig.17 It can be seen that the FITC-LHRH@DM@HOF-1 group exhibited strong fluorescence in 4T1 mouse breast cancer cells compared with FITC@HOF-1 and FITC@DM@HOF-1, proving that FITC-LHRH@DM@HOF-1 has active targeting and is successfully enriched in 4T1 cells.

[0144] 4T1 cells or RAW264.7 cells were seeded in 96-well plates at a density of 1×10 4 The cells were incubated with 100 μL of culture medium for 24 h, and then treated with different concentrations of LHRH@HOF-1. Each group was repeated 3 times. After incubation for 48 h, an MTT-based cytotoxicity assay was performed to obtain the cytotoxicity of 4T1 cells and RAW264.7 cells in the range of 0 to 100 μg mL LHRH@HOF-1 concentrations. -1 The cell survival data are shown in Table 2. The survival rates of 4T1 cells and RAW264.7 cells were observed in the presence of LHRH@HOF-1 at concentrations ranging from 0 to 100 μg mL -1 The cell survival rate bar graph is as follows Fig.18 As shown, from Fig.18 It can be seen that LHRH@HOF-1 has a high concentration of 0 to 100 μg mL -1It showed no cytotoxicity over a wide concentration range.

[0145] Table 24 T1 cells and RAW264.7 cells in the LHRH@HOF-1 concentration range of 20 to 100 μg mL -1 Cell viability data

[0146] <![CDATA[Concentration (μg·mL -1 )]]> 4T1 cell survival rate (%) RAW264.7 cell survival rate (%) 0 101.58 100.78 20 99.97 100.15 40 100.97 100.84 60 98.64 102.67 80 93.64 95.78 100 92.16 82.16

[0147] 4T1 cancer cells were treated with PBS, DOX (5 μM), LHRH@HOF-1 (20 μM) + DOX (5 μM) + US. The cells were exposed to US environment (0.58 W / cm 2 , 1.0 MHz, 50% duty cycle, 5 minutes). Anti-rabbit CRT antibody (1:1000) and anti-rabbit HMGB1 antibody (1:1000) were used for immunofluorescence staining of cell surface CRT and HMGB1 at 4°C overnight, and Cy3-labeled goat anti-rabbit IgG secondary antibody (1:1000) was used at room temperature for 2 hours. After DAPI staining, CLSM observation was performed to obtain confocal laser images of PBS, DOX, and LHRH@HOF-1+DOX as shown in the figure. Fig.19 As shown. DOX is a representative anthracycline chemotherapy drug that can enhance sonodynamic therapy-induced immunogenic cell death (ICD). The release of extracellular HMGB1 and the exposure of calreticulin (CRT) are key signals for ICD. Fig.19 It can be seen that the LHRH@HOF-1+DOX+US groups all had strong red fluorescence, confirming the exposure of CRT on the 4T1 cell membrane and the release of HMGB1.

[0148] (IV) In vivo pharmacodynamics experiments of LHRH@DM@HOF-1 in Example 1, HOF-1 in Comparative Example 1, and LHRH@HOF-1 in Comparative Example 2.

[0149] In order to evaluate the anti-tumor effect of LHRH@DM@HOF-1-mediated sonodynamic combined with chemotherapeutic drugs (DOX and MET) in tumor-bearing mice, 4T1 cells were cultured at 1×10 4 The density of 50 μg / ml was injected into the left mammary fat pad of BALB / c mice (female BALB / c mice, provided by the Experimental Animal Center of Yanbian University (Yanbian, China)) to establish an orthotopic breast cancer mouse model. After 14 days, the tumor-bearing mice were obtained and randomly divided into 5 groups. The experiments were carried out with different treatment schemes (treatment schemes: blank control (Ⅰ, PBS); comparative application example 2 (Ⅱ, LHRH@HOF-1); comparative application example 1 (Ⅲ, HOF-1+US); comparative application example 3 (Ⅳ, DOX+MET); application example 1 (Ⅴ, LHRH@DM@HOF-1+US)). The schematic diagram of the experimental process is shown in the figure. Fig. 20 As shown, from Fig. 20 It can be seen that after the orthotopic breast cancer mouse model was established, the preparation was injected into the tail vein, US was applied 24 hours later, the treatment was repeated 8 times, and the mice were killed one week later.

[0150] In order to verify the ability of LHRH@DM@HOF-1 to target tumors in vivo, in vivo imaging studies were performed using 4T1 tumor-bearing BALB / c mice. In vivo fluorescence imaging was performed at different time points after intravenous injection using different treatment regimens. Fig.21 As shown, from Fig.21 It can be seen that 6 hours after injection, the fluorescence of LHRH@DM@HOF-1 was observed on the tumor, and the signal at the tumor site continued to increase (Group V). Tumor growth curves and weights of 4T1 tumor-bearing mice under different treatment regimens (data are expressed as mean ± SD, n = 5) are shown in Figure 5. Fig. 22 As shown, (a) is the tumor growth curve, (b) is the tumor weight; the photos of the tumor after different treatment regimens on day 14 are shown Fig.23 As shown. Figures 21 to 23 It can be seen that after 14 days of administration, the tumor growth of Group V was significantly inhibited, while the anticancer effects of Groups I, II and III were not obvious; unlike Group IV, the tumor growth of Group V was significantly inhibited, and the significant therapeutic effect was attributed to the enrichment of LHRH@DM@HOF-1 in the tumor site, which promoted the activation of the drug and the accumulation of ROS generated by the sonodynamic effect.

[0151] H&E staining, CRT staining, HMGB1 staining, PD-L1 staining and TUNEL staining of tumor sections under different treatment regimens (scale bar = 50 μm) Fig.24 As shown. Fig.24 It can be seen that the H&E results showed that the tumor tissue necrosis in the LHRH@DM@HOF-1+US group was aggravated and cell fragments were obvious, while no obvious apoptosis was observed in the dissected tumors in the PBS group; TUNEL detection of tumor tissue further showed that LHRH@DM@HOF-1+US could enhance ICD by reversing immunosuppression; using HMGB1 and CRT as detection indicators to examine the activation of ICD, immunohistochemistry and immunofluorescence results showed that HMGB1 release was stronger and CRT expression was enhanced in the LHRH@DM@HOF-1+US treatment group; the PD-L1 level in the DOX+MET treatment group was lower than that in the HOF-1+US group, indicating that MET inhibited the expression of PD-L1 and achieved the reversal of immunosuppression. These results confirm that LHRH@DM@HOF-1 actively targets the tumor site, the sonodynamic effect of HOF-1 enhances chemotherapy-induced ICD, and the activated MET reverses immunosuppression, thereby allowing ICD to continue to work.

[0152] Six hours after the sonodynamic targeted drug-loaded nanoparticles provided by the present invention were injected into 4T1 tumor-bearing BALB / c mice, fluorescence of the sonodynamic targeted drug-loaded nanoparticles was observed on the tumor, and the signal continued to increase; 14 days after administration, tumor growth was significantly inhibited, and the nanoparticles had good targeting and anti-tumor effects.

[0153] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sonodynamic targeted drug-loaded nanoparticle, comprising a porous carrier, a drug loaded in the porous carrier, and a targeting ligand chemically modified on the outer surface of the porous carrier; the porous carrier is a hydrogen-bonded organic framework; the building block of the hydrogen-bonded organic framework is a metal porphyrin complex.

2. The sonodynamic targeted drug-loaded nanoparticles according to claim 1, characterized in that: The drugs are anti-tumor drugs; the anti-tumor drugs are doxorubicin and metformin.

3. The sonodynamic targeted drug-loaded nanoparticles according to claim 2, characterized in that: The loading amount of doxorubicin in the porous carrier is 10.9-23.3 wt %; the loading amount of metformin in the porous carrier is 8.7-26.4 wt %.

4. The sonodynamic targeted drug-loaded nanoparticles according to claim 1, characterized in that: The modification amount of the targeting ligand on the outer surface of the porous carrier is 0.0548 to 1.154 nmol·mg -1 .

5. The sonodynamic targeted drug-loaded nanoparticles according to claim 1 or 4, characterized in that: The targeting ligand is LHRH polypeptide, cRGD polypeptide or R8H3 polypeptide.

6. The method for preparing the sonodynamic targeted drug-loaded nanoparticles according to any one of claims 1 to 5, comprising the following steps: (1) mixing a metal porphyrin complex, a drug, a non-protonic polar solvent, water and ethanol for self-assembly to obtain drug-loaded nanoparticles; (2) mixing the drug-loaded nanoparticles obtained in step (1) with an activator for activation to obtain activated drug-loaded nanoparticles; (3) The activated drug-loaded nanoparticles obtained in step (2) are mixed with a targeting ligand for chemical modification to obtain sonodynamic targeted drug-loaded nanoparticles.

7. The preparation method according to claim 6, characterized in that: The activator in step (2) is a carboxyl activator.

8. The preparation method according to claim 7, characterized in that: The carboxyl activating agents are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysulfosuccinimide.

9. The preparation method according to claim 8, characterized in that: The mass ratio of the drug-loaded nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysulfosuccinimide in the carboxyl activator is 1:(1-5):(5-25).

10. Use of the sonodynamic targeted drug-loaded nanoparticles according to any one of claims 1 to 5 or the sonodynamic targeted drug-loaded nanoparticles prepared by the preparation method according to any one of claims 6 to 9 in the preparation of anticancer drugs.

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