IRGD peptide modified cabazitaxel albumin nanoparticles and preparation method thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and provides an iRGD peptide modified cabazitaxel albumin nanoparticle (iRGD-HSA-CTX NPs) and a preparation method thereof, and the iRGD-HSA-CTX NPs is prepared from the following raw materials in parts by weight: 2.5 to 10 parts of cabazitaxel, 50 to 200 parts of albumin, 20 to 100 parts of phospholipid, 500 to 2000 parts of iRGD peptide, 1500 to 6000 parts of cross-linking agent and 0.005 to 0.3 part of freeze-drying protective agent. The iRGD-HSA-CTX NPs prepared by the preparation method disclosed by the invention is relatively small in particle size, uniform in particle size distribution and high in drug encapsulation efficiency, the toxic and side effects of CTX are reduced, the tumor targeting property is improved, and the anti-tumor activity is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to iRGD-modified cabazitaxel albumin nanoparticles and a preparation method thereof. Background Art

[0002] Cabazitaxel (CTX) is a second-generation taxane drug. Compared with the first-generation taxane drugs paclitaxel (PTX) and docetaxel (DTX), it is methylated at the C7 and C10 sites in the structure, resulting in a reduced affinity for drug-resistant proteins. Therefore, CTX has stronger activity against drug-resistant tumors.

[0003] CTX mainly binds to tubulin (an important regulatory protein during cell mitosis) to promote its aggregation and inhibit microtubule depolymerization, thereby blocking the proliferation and division of tumor cells. Although the currently marketed cabazitaxel injection Jevtana shows good anti-tumor effects, it also has disadvantages such as insufficient targeting and large toxic and side effects. Therefore, developing a nano-drug delivery system with good targeting and high safety is of great significance for reducing the toxicity of CTX, enhancing its efficacy, and improving the quality of life of patients.

[0004] The preparation method of albumin nanoparticles is simple, has high biosafety, and can tightly bind small molecule and protein drugs to achieve drug circulation and release in the body. Therefore, it is an effective drug delivery system and has great advantages in tumor treatment, such as being non-toxic and non-immunogenic, and being able to bind to the gp60 receptor (a glycoprotein with a molecular weight of 60 kDa) expressed on tumor vascular endothelial cells, and then interacting with the secreted protein SPARC (an acidic and cysteine-rich extracellular matrix glycoprotein) overexpressed by various tumor cells, thus promoting the active targeting of albumin nanoparticles loaded with drugs to tumor tissues and enriching in tumor cells. At the same time, through the enhanced permeability and retention effect (EPR) of tumor tissues, the concentration of drugs in local tissues is significantly increased, enhancing the therapeutic effect. However, tumors are heterogeneous, with dense stroma, disordered blood vessels, pathological lymphatic networks, and increased interstitial fluid pressure, which hinder the effective delivery of nano-drugs to tumors, especially the deep regions far from the vascular system, resulting in unsatisfactory therapeutic effects. A cyclic polypeptide iRGD composed of 9 amino acid residues (CRGDKGPDC) reported in 2010 was confirmed to be able to target tumor sites and significantly improve the tumor tissue penetration ability of chemotherapeutic drugs, providing a new option for maximizing the therapeutic effect of drugs in the deep part of tumors. iRGD contains three parts: an RGD motif, a CendR motif (C-R-G-D-K / R), and a protease recognition site, and mainly targets and enriches in tumors through the following steps: Since integrin receptors α ν β3, α ν β5 are highly expressed on the surface of various tumor cells and tumor vascular endothelial cells, the RGD motif first binds specifically to them. Subsequently, under the action of protease, the disulfide bond of the peptide chain is cleaved to activate the CendR motif. It should be noted that specific binding to α ν β3, α ν β5 is necessary for activating the CendR motif. Finally, the activated CendR motif binds to neuropilin-1 (NRP-1) highly expressed in tumor cells, triggering endocytosis or phagocytosis of cells and the trans-tissue transport pathway. As a key regulatory node of vascular permeability, NPR-1 has been proven to play an important role in tumor metastasis. The interaction between the CendR sequence and NRP-1 is the basis for the high penetrability of drugs in tumor tissues. Through the above steps, the iRGD peptide significantly improves the tumor targeting and the penetration ability within the tumor parenchyma of drugs or carriers, enhancing the anti-tumor effect.

[0005] The present invention prepares an iRGD peptide-modified albumin nano-drug delivery system. Using human serum albumin (HSA) as an anti-tumor drug carrier, iRGD peptide-modified cabazitaxel albumin nanoparticles with uniform particle size are prepared, which can effectively reduce the toxic side effects of cabazitaxel while improving its tumor targeting and enhancing the drug efficacy. Summary of the Invention

[0006] The purpose of the embodiment of the present invention is to prepare a stable iRGD peptide-modified cabazitaxel albumin nanoparticle to reduce the toxic side effects of cabazitaxel and improve the drug targeting.

[0007] The embodiment of the present invention is implemented as follows. An iRGD-HSA-CTX NPs includes the following raw materials in parts by weight:

[0008] Cabazitaxel 2.5 - 10 parts, albumin 50 - 200 parts, phospholipid 20 - 100 parts, iRGD peptide 500 - 2000 parts, cross-linking agent 1500 - 6000 parts, lyoprotectant 0.005 - 0.3 parts

[0009] As a preference of the present invention, the albumin carrier material includes one or both of human serum albumin and bovine serum albumin, and the preferred material is human serum albumin.

[0010] As a preference of the present invention, the average particle size range of the iRGD-HSA-CTX NPs is 50 - 200 nm, and more preferably 70 - 150 nm.

[0011] As a preference of the present invention, the lyoprotectant of the freeze-dried preparation of the nanoparticles is mannitol, sodium octanoate, arginine, glycine, lactose, glucose, sucrose or trehalose, and more preferably sodium octanoate.

[0012] As a preference of the present invention, the dosage of the lyoprotectant of the freeze-dried preparation of the nanoparticles is 0.05% - 3%, and more preferably 1%.

[0013] Another purpose of the embodiment of the present invention lies in a preparation method of iRGD-HSA-CTX NPs, which is characterized in that the method includes the following steps:

[0014] 1) Weigh cabazitaxel and phospholipid according to the amount and dissolve them in an organic solvent, preferably a mixed solvent of any combination of chloroform, dichloromethane, ethanol and acetone, to form an oil phase; in the oil phase, the concentration of cabazitaxel is 0.05 - 0.2 mg / mL;

[0015] 2) Disperse albumin in pure water to form an aqueous phase; in the aqueous phase, the concentration of albumin is 1 - 4 mg / mL;

[0016] 3) At a stirring speed of 300 - 1200 r / min, slowly inject the oil phase in step 1) into the water phase in step 2) using a syringe, and under ice bath conditions, perform ultrasonic treatment to form a primary emulsion. The ultrasonic power is 20% - 60%, and the ultrasonic time is 2 - 10 min;

[0017] 4) The primary emulsion prepared in step 3) is continuously stirred at room temperature for 2 - 6 h to remove the organic solvent, and a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm is obtained.

[0018] 5) Take a certain amount of the cabazitaxel albumin nanoparticle colloidal solution and centrifuge and purify it in an ultrafiltration tube. The centrifugation speed is 5000 - 20000 rpm, and the time is 5 - 20 min. Use PBS to adjust the pH of HSA - CTX NPs to between 7.0 and 7.5.

[0019] 6) Add a certain amount of cross - linker solution (sulfo - SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1 - 4 h.

[0020] 7) Centrifuge the activated nanoparticle solution in an ultrafiltration tube. The centrifugation speed is 5000 - 20000 rpm, and the time is 5 - 20 min to remove the excess cross - linker and keep the pH between 7.0 and 7.5;

[0021] 8) Add iRGD, stir overnight at room temperature, and then centrifuge and purify it in an ultrafiltration tube. The centrifugation speed is 5000 - 20000 rpm, and the time is 5 - 20 min to remove the excess iRGD, and iRGD - HSA - CTX NPs are obtained.

[0022] 9) Weigh the lyoprotectant according to the amount, and perform lyophilization on the obtained nanomaterial in step 8) and the lyoprotectant to prepare the lyophilized preparation of iRGD - modified cabazitaxel albumin nanoparticles.

[0023] An iRGD - HSA - CTX NPs material provided by an embodiment of the present invention is composed of cabazitaxel, albumin, and iRGD peptide material. The obtained nanoparticles have uniform particle size, good biocompatibility and biodegradability. HSA, as an endogenous albumin, is often used as a drug carrier to transport and load exogenous molecular drugs; the cyclic polypeptide iRGD has been proven to be able to target tumor sites and significantly improve the tumor tissue penetration ability of chemotherapeutic drugs, enabling the drugs to exert the maximum efficacy deep in the tumor. Therefore, the nanoparticles of the present invention have extremely small side effects caused by the preparation itself and good targeting properties.

[0024] The nanoparticles provided by the present invention have small particle size, uniform distribution, good stability and targeting properties, and good anti - tumor activity. Description of the Drawings

[0025] Figure 1 Particle size distribution diagram of iRGD-HSA-CTX NPs;

[0026] Figure 2 Transmission electron microscope image of iRGD-HSA-CTX NPs;

[0027] Figure 3 is SDS-PAGE electrophoresis diagram of iRGD-HSA-CTX NPs, where A is HSA-CTX NPs, B is iRGD-HSA-CTX NPs, and C is Marker;

[0028] Figure 4 SDC map of iRGD-HSA-CTX NPs and physical mixture of iRGD and HSA-CTX NPs;

[0029] Figure 5 Stability of iRGD-HSA-CTX NPs in different media;

[0030] Figure 6 Appearance morphology of iRGD-HSA-CTX NPs before and after freeze-drying, where A is the colloidal solution, B is the freeze-dried preparation, C is the freeze-dried powder, D is the reconstituted solution of the freeze-dried preparation, and E is the change in particle size and encapsulation efficiency before and after freeze-drying;

[0031] Figure 7 Cumulative drug release curve of iRGD-HSA-CTX NPs under different pH conditions;

[0032] Figure 8 In vitro hemolysis of iRGD-HSA-CTX NPs;

[0033] Figure 9 In vitro anti-tumor effect of iRGD-HSA-CTX NPs, where Part A and Part B show the cell viability of 4T1 cells after incubation with free CTX, HSA-CTX NPs, and iRGD-HSA-CTX NPs at different CTX concentrations for 24 h and 48 h; Part C and Part D show the cell viability of A549 cells after incubation with free CTX, HSA-CTX NPs, and iRGD-HSA-CTX NPs at different CTX concentrations for 24 h and 48 h;

[0034] Figure 10Uptake of iRGD-HSA-CTX NPs in tumor cells. Among them, A is the fluorescence microscopy images of cells after incubation of iRGD-HSA-DiD NPs and HSA-DiD NPs with 4T1 cells for 4 h; B is the intracellular CTX concentration after incubation of iRGD-HSA-CTX NPs and HSA-CTX NPs with 4T1 cells and A549 cells for 4 h. Detailed implementation manners

[0035] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Example 1

[0037] iRGD-HSA-CTX NPs include the following raw materials: 5 mg of cabazitaxel; 50 mg of human serum albumin; 80 mg of phospholipid; 500 mg of iRGD peptide; 1500 mg of crosslinking agent.

[0038] Preparation process: Weigh cabazitaxel and phospholipid according to the amount, dissolve them in an organic solvent, preferably a mixed solvent of any combination of chloroform, dichloromethane, ethanol, and acetone, to form an oil phase; disperse albumin in pure water to form an aqueous phase; under a stirring speed of 300-1200 r / min, slowly inject the oil phase sucked by a syringe into the aqueous phase, and perform ultrasonic treatment under ice bath conditions to form a primary emulsion, with an ultrasonic power of 20%-60% and an ultrasonic time of 2-10 min; continue to stir the prepared primary emulsion at room temperature for 2-6 h, and volatilize the organic solvent to obtain a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm. Take a certain amount of cabazitaxel albumin nanoparticle colloidal solution in an ultrafiltration tube for centrifugal purification, with a centrifugal speed of 5000-20000 rpm and a time of 5-20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. Add a certain amount of crosslinking agent solution (sulfo-SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1-4 h. Centrifuge the activated nanoparticle solution in an ultrafiltration tube, with a centrifugal speed of 5000-20000 rpm and a time of 5-20 min, to remove the excess crosslinking agent and keep the pH between 7.0 and 7.5; add iRGD, stir overnight at room temperature, and then centrifuge and purify in an ultrafiltration tube, with a centrifugal speed of 5000-20000 rpm and a time of 5-20 min, to remove the excess iRGD, thus obtaining iRGD-HSA-CTX NPs. The particle size of this nanoparticle is 90.56 nm, and the encapsulation efficiency is 90.42%.

[0039] Example 2

[0040] iRGD-HSA-CTX NPs, comprising the following raw materials: 10 mg of cabazitaxel; 50 mg of human serum albumin; 80 mg of phospholipid; 500 mg of iRGD peptide; 1500 mg of crosslinking agent.

[0041] Preparation process: Weigh cabazitaxel and phospholipid according to the amount, dissolve them in an organic solvent, preferably a mixed solvent of any combination of chloroform, dichloromethane, ethanol, and acetone, to form an oil phase; disperse albumin in pure water to form an aqueous phase; under a stirring speed of 300 - 1200 r / min, slowly inject the oil phase sucked by a syringe into the aqueous phase, and under ice bath conditions, perform ultrasonic treatment to form a primary emulsion, with an ultrasonic power of 20% - 60% and an ultrasonic time of 2 - 10 min; the obtained primary emulsion is continuously stirred at room temperature for 2 - 6 h to evaporate the organic solvent, and a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm is obtained. Take a certain amount of the cabazitaxel albumin nanoparticle colloidal solution and centrifuge and purify it in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. Add a certain amount of crosslinking agent solution (sulfo-SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1 - 4 h. The activated nanoparticle solution is centrifuged in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess crosslinking agent and keep the pH between 7.0 and 7.5; add iRGD, stir overnight at room temperature, and then centrifuge and purify it in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess iRGD, and iRGD-HSA-CTX NPs are obtained. The particle size of this nanoparticle is 83.71 nm, and the encapsulation efficiency is 92.79%.

[0042] Example 3

[0043] iRGD-HSA-CTX NPs, comprising the following raw materials: 10 mg of cabazitaxel; 50 mg of human serum albumin; 50 mg of phospholipid; 500 mg of iRGD peptide; 1500 mg of crosslinking agent.

[0044] Preparation process: Weigh cabazitaxel and phospholipids according to the amount, dissolve them in an organic solvent, preferably a mixed solvent composed of any combination of chloroform, dichloromethane, ethanol, and acetone, to form an oil phase; disperse albumin in pure water to form an aqueous phase; under a stirring speed of 300 - 1200 r / min, slowly inject the oil phase sucked by a syringe into the aqueous phase, and perform ultrasonic treatment under ice bath conditions to form a primary emulsion, with an ultrasonic power of 20% - 60% and an ultrasonic time of 2 - 10 min; continue to stir the obtained primary emulsion at room temperature for 2 - 6 h, and volatilize the organic solvent to obtain a colloidal solution of cabazitaxel albumin nanoparticles with an average particle size not exceeding 150 nm. Take a certain amount of the colloidal solution of cabazitaxel albumin nanoparticles and centrifuge and purify it in an ultrafiltration tube at a centrifugation speed of 5000 - 20000 rpm for 5 - 20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. Add a certain amount of crosslinking agent solution (sulfo-SMCC) to the colloidal solution of cabazitaxel albumin nanoparticles, and activate it at room temperature for 1 - 4 h. Centrifuge the activated nanoparticle solution in an ultrafiltration tube at a centrifugation speed of 5000 - 20000 rpm for 5 - 20 min to remove the excess crosslinking agent and keep the pH between 7.0 and 7.5; add iRGD, stir overnight at room temperature, and then centrifuge and purify it in an ultrafiltration tube at a centrifugation speed of 5000 - 20000 rpm for 5 - 20 min to remove the excess iRGD, thus obtaining iRGD-HSA-CTX NPs. The particle size of this nanoparticle is 187.46 nm, and the encapsulation efficiency is 78.39%

[0045] Example 4

[0046] iRGD-HSA-CTX NPs include the following raw materials: 10 mg of cabazitaxel; 50 mg of human serum albumin; 100 mg of phospholipids; 500 mg of iRGD peptide; 1500 mg of crosslinking agent.

[0047] Preparation process: Weigh cabazitaxel and phospholipids according to the amount, dissolve them in an organic solvent, preferably a mixed solvent composed of any combination of chloroform, dichloromethane, ethanol, and acetone, to form an oil phase; disperse albumin in pure water to form an aqueous phase; under a stirring speed of 300 - 1200 r / min, slowly inject the oil phase sucked by a syringe into the aqueous phase, and perform ultrasonic treatment under ice bath conditions to form a primary emulsion, with an ultrasonic power of 20% - 60% and an ultrasonic time of 2 - 10 min; continue to stir the obtained primary emulsion at room temperature for 2 - 6 h, and evaporate the organic solvent to obtain a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm. Take a certain amount of the cabazitaxel albumin nanoparticle colloidal solution and centrifuge and purify it in an ultrafiltration tube at a centrifugation speed of 5000 - 20000 rpm for 5 - 20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. Add a certain amount of crosslinking agent solution (sulfo-SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1 - 4 h. Centrifuge the activated nanoparticle solution in an ultrafiltration tube at a centrifugation speed of 5000 - 20000 rpm for 5 - 20 min to remove the excess crosslinking agent and keep the pH between 7.0 and 7.5; add iRGD, stir overnight at room temperature, and then centrifuge and purify it in an ultrafiltration tube at a centrifugation speed of 5000 - 20000 rpm for 5 - 20 min to remove the excess iRGD, thus obtaining iRGD-HSA-CTX NPs. The particle size of this nanoparticle is 90.45 nm, and the encapsulation efficiency is 89.91%

[0048] Example 5

[0049] iRGD-HSA-CTX NPs comprise the following raw materials: 10 mg of cabazitaxel; 100 mg of human serum albumin; 80 mg of phospholipids; 500 mg of iRGD peptide; 1500 mg of crosslinking agent.

[0050] Preparation process: Weigh cabazitaxel and phospholipids according to the amount, dissolve them in an organic solvent, preferably a mixed solvent of any combination of chloroform, dichloromethane, ethanol, and acetone, to form an oil phase; disperse albumin in pure water to form an aqueous phase; under a stirring speed of 300 - 1200 r / min, slowly inject the oil phase sucked by a syringe into the aqueous phase, and perform ultrasonic treatment under ice bath conditions to form a primary emulsion, with an ultrasonic power of 20% - 60% and an ultrasonic time of 2 - 10 min; continue to stir the obtained primary emulsion at room temperature for 2 - 6 h, and volatilize the organic solvent to obtain a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm. Take a certain amount of cabazitaxel albumin nanoparticle colloidal solution in an ultrafiltration tube for centrifugal purification, with a centrifugal speed of 5000 - 20000 rpm and a time of 5 - 20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. Add a certain amount of crosslinking agent solution (sulfo-SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1 - 4 h. Centrifuge the activated nanoparticle solution in an ultrafiltration tube, with a centrifugal speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess crosslinking agent and keep the pH between 7.0 and 7.5; add iRGD, stir overnight at room temperature, and then centrifuge and purify in an ultrafiltration tube, with a centrifugal speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess iRGD, thus obtaining iRGD-HSA-CTX NPs. The particle size of this nanoparticle is 111.31 nm, and the encapsulation efficiency is 92.02%

[0051] Example 6

[0052] iRGD-HSA-CTX NPs, comprising the following raw materials: 10 mg of cabazitaxel; 50 mg of human serum albumin; 80 mg of phospholipids; 500 mg of iRGD peptide; 1500 mg of crosslinking agent.

[0053] Preparation process: Weigh cabazitaxel and phospholipids in accordance with the dosage, dissolve them in an organic solvent, preferably a mixed solvent of any combination of chloroform, dichloromethane, ethanol, and acetone, to form an oil phase; disperse albumin in pure water to form an aqueous phase; under a stirring speed of 300 - 1200 r / min, slowly inject the oil phase sucked by a syringe into the aqueous phase, and under ice bath conditions, perform ultrasonic treatment to form a primary emulsion, with an ultrasonic power of 20% - 60% and an ultrasonic time of 2 - 10 min; the obtained primary emulsion is continuously stirred at room temperature for 2 - 6 h to remove the organic solvent, and a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm is obtained. Take a certain amount of the cabazitaxel albumin nanoparticle colloidal solution and centrifuge and purify it in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. Add a certain amount of cross-linking agent solution (sulfo-SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1 - 4 h. Centrifuge the activated nanoparticle solution in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess cross-linking agent and keep the pH between 7.0 and 7.5; add iRGD, stir overnight at room temperature, and then centrifuge and purify it in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess iRGD, and iRGD-HSA-CTX NPs are obtained. The particle size of this nanoparticle is 84.37 ± 3.44 nm, the Zeta is -4.87 ± 2.09 mV, and the encapsulation efficiency is 93.67 ± 2.56%.

[0054] Example 7

[0055] Preparation of the freeze-dried preparation of iRGD-HSA-CTX NPs, including the following raw materials: 10 mg of cabazitaxel; 50 mg of human serum albumin; 80 mg of phospholipids; 500 mg of iRGD peptide; 1500 mg of cross-linking agent; 1% sodium caprylate.

[0056] Preparation process: Weigh cabazitaxel and phospholipids according to the amount, dissolve them in an organic solvent, preferably a mixed solvent of any combination of chloroform, dichloromethane, ethanol, and acetone, to form an oil phase; disperse albumin in pure water to form an aqueous phase; under a stirring speed of 300 - 1200 r / min, slowly inject the oil phase sucked by a syringe into the aqueous phase, and under ice bath conditions, perform ultrasonic treatment to form a primary emulsion, with an ultrasonic power of 20% - 60% and an ultrasonic time of 2 - 10 min; the obtained primary emulsion is continuously stirred at room temperature for 2 - 6 h to remove the organic solvent, and a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm is obtained. Take a certain amount of the cabazitaxel albumin nanoparticle colloidal solution and centrifuge and purify it in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. Add a certain amount of cross-linking agent solution (sulfo-SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1 - 4 h. The activated nanoparticle solution is centrifuged in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess cross-linking agent and keep the pH between 7.0 and 7.5; add iRGD, stir overnight at room temperature, and then centrifuge and purify it in an ultrafiltration tube, with a centrifugation speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess iRGD, and iRGD-HSA-CTX NPs are obtained. Add 1% sodium caprylate to the prepared nanoparticle solution, vortex to dissolve it, place it in a freeze dryer with a pre-set freeze-drying program, take it out after 24 h, stopper and seal it to obtain the freeze-dried preparation of iRGD-HSA-CTX NPs. After reconstitution with ultrapure water, the particle size is measured with a laser particle size analyzer. The average particle size of the reconstituted freeze-dried preparation is about 87.98 nm, and the encapsulation efficiency is about 93.04%.

[0057] Characterization and pharmacodynamic experiments

[0058] 1. Particle size distribution of iRGD-HSA-CTX NPs

[0059] The particle size distribution of iRGD-HSA-CTX NPs is detected by a laser particle size analyzer. The measurement results of Example 6 are as Figure 1 shown. The measurement results show that the average particle size of iRGD-HSA-CTX NPs is 84.37 ± 3.44 nm, the polydispersity index is 0.237 ± 0.02, and the average Zeta potential is -4.87 ± 2.09 mV.

[0060] 2. Transmission electron microscope characterization

[0061] Figure 2Transmission electron microscope image of iRGD-HSA-CTX NPs prepared in Example 6. It can be seen from the figure that the microscopic morphology of iRGD-HSA-CTX NPs is spherical or quasi-spherical, and the size is relatively uniform.

[0062] 3. SDS-PAGE electrophoresis experiment

[0063] Figure 3 is SDS-PAGE diagram of iRGD-HSA-CTX NPs prepared in Example 6. It was observed that the position of iRGD-HSA-CTX NPs was higher than that of HSA-CTX NPs, that is, the molecular weight of iRGD-HSA-CTX NPs was larger, proving that iRGD was modified onto HSA-CTX NPs. The reason for the unclear separation may be that the molecular weight of iRGD is small and the molecular weight of HSA is large, and the difference in the total molecular weight between iRGD-HSA-CTX NPs and HSA-CTX NPs is small, so the difference is not significant.

[0064] 4. Differential scanning calorimetry (DSC)

[0065] Figure 4 DSC spectrum of iRGD-HSA-CTX NPs prepared in Example 6. The test results showed that iRGD-HSA-CTX NPs had a characteristic endothermic peak at 208.5 °C, and the physical mixture of iRGD and HSA-CTX NPs had a characteristic endothermic peak at 234.3 °C, indicating that there was a chemical bond binding between iRGD and HSA-CTX NPs, proving that iRGD was successfully modified onto HSA-CTX NPs.

[0066] 5. Stability of iRGD-HSA-CTX NPs

[0067] Figure 5 Results of the stability investigation of iRGD-HSA-CTX NPs solution prepared in Example 6. Four different media, namely ultrapure water, normal saline, PBS (pH = 7.4), and 1640 complete medium, were used to simulate physiological conditions and experimental conditions, and the particle size changes of iRGD-HSA-CTX NPs within five days were measured. The experimental results are shown in Figure 5 . The experimental results showed that within 5 days, the particle size of iRGD-HSA-CTX NPs had an increasing trend in ultrapure water, but there was no obvious change in the particle size of the nanoparticles in normal saline, PBS (pH = 7.4), and 1640 complete medium, indicating good stability.

[0068] 6. Influence of the freeze-drying process on the stability of iRGD-HSA-CTX NPs.

[0069] Observe the appearance of the freeze-dried preparation of iRGD-HSA-CTX NPs prepared in Example 7. It is a white or off-white loose block solid, without shrinkage or collapse. See Figure 6 parts B and C in Figure 6 Part A in Figure 6 is a photo of the colloidal solution of iRGD-HSA-CTX NPs before freeze-drying. Reconstitute the freeze-dried preparation of iRGD-HSA-CTX NPs prepared in Example 7 with ultrapure water and observe its redispersibility. Figure 6 Part D in

[0070] Table 1 shows the redispersibility of the freeze-dried preparation in three kinds of solvents.

[0071]

[0072] 7. In vitro release test of iRGD-HSA-CTX NPs

[0073] The freeze-dried preparation of iRGD-HSA-CTX NPs prepared in Example 6 was reconstituted with ultrapure water. 3 mL of the reconstituted solution was taken and placed in a dialysis bag (Mw = 3500), and both ends were tied tightly. Then it was placed in a centrifuge tube containing 30 mL of release medium. A CTX solution with the same concentration was prepared and carried out according to the above steps. Using an air-bath constant temperature oscillator, the centrifuge tubes containing iRGD-HSA-CTX NPs and CTX samples were placed in it and oscillated (conditions: 37 °C, 100 r / min). At 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h time points, 2 mL of the drug-containing release medium was aspirated on time and detected by HPLC. After sampling, 2 mL of the corresponding release medium was added respectively. As Figure 7 shown, under the conditions of pH 7.4 and 5.0, the drug release rate of iRGD-HSA-CTX NPs reached about 60% after 48 h, showing a certain sustained-release effect.

[0074] 8. Hemolysis experiment of iRGD-HSA-CTX NPs

[0075] Take 8 dry and sterile 1.5 mL centrifuge tubes, numbered 1-8 respectively. Set tube 1 as the positive control group, tube 2 as the negative control group, and tubes 3-8 as the experimental groups. The positive control group was added with ultrapure water, the negative control group was added with normal saline, and the experimental groups were respectively added with a certain amount of iRGD-HSA-CTX NPs prepared in Example 6. Then the prepared red blood cell suspension was added to the above centrifuge tubes, and 3 groups were prepared in parallel. After mixing, it was placed in a constant temperature air-bath shaker (37 ± 0.5 °C) for incubation for 3 h, then centrifuged at 1500 rpm for 6 min, the supernatant was collected, and its absorbance value was measured at 540 nm, and the cell hemolysis rate was calculated. The experimental results are as Figure 8 shown. In the positive control group, complete hemolysis occurred, and there was no hemolysis or agglutination in the negative control group and the experimental groups. The cell hemolysis rates of iRGD-HSA-CTX NPs at different concentrations were all less than 5%, which was consistent with the naked-eye observation, indicating that the prepared iRGD-HSA-CTX NPs had good biosafety.

[0076] 9. Investigation of the in vitro anti-tumor ability of iRGD-HSA-CTX NPs

[0077] Investigate the in vitro anti-tumor activity of iRGD-HSA-CTX NPs prepared in Example 6. Using mouse breast cancer 4T1 cells and human non-small cell lung cancer A549 cells as models, the CCK-8 reagent method was used to investigate the in vitro anti-tumor activities of CTX, HSA-CTX NPs, and iRGD-HSA-CTX NPs. As Figure 9, in the drug concentration range of 5-100 μg / mL, as the CTX concentration increases, the cell survival rate gradually decreases, and the cytotoxicity increases over time. That is, the cytotoxicity of free CTX, HSA-CTX NPs, and iRGD-HSA-CTX NPs against 4T1 and A549 cells shows both concentration dependence and time dependence. The cytotoxicity of HSA-CTX NPs and iRGD-HSA-CTX NPs is higher than that of free CTX at the same concentration. It may be that HSA interacts with gp60 and SPARC of tumor cells, resulting in more endocytosis of nanoparticles by 4T1 and A549 cells, thus leading to an increase in CTX concentration and cytotoxicity. There is a statistically significant difference in the cytotoxicity between HSA-CTX NPs and iRGD-HSA-CTX NPs. Compared with HSA-CTX NPs, iRGD-HSA-CTX NPs show higher cytotoxicity. The reason may be that both 4T1 and A549 tumor cells highly express α ν β3, α ν β5 and NRP-1. iRGD further improves the tumor penetration ability of iRGD-HSA-CTX NPs by binding to α ν β3, α ν β5 and interacting with NRP-1, resulting in a significant increase in its cytotoxicity.

[0078] 10. Investigation of the uptake ability of cells to iRGD-HSA-CTX NPs

[0079] The in vitro cell uptake of the iRGD-HSA-CTX NPs prepared in Example 6 was investigated. Using mouse breast cancer 4T1 cells and human non-small cell lung cancer A549 cells as models, after incubating with HSA-CTX NPs and iRGD-HSA-CTX NPs with a CTX concentration of 80 μg / mL for 4 h, they were washed with PBS, the cells were digested with trypsin and centrifuged. The obtained cell pellet was incubated with 2% sodium dodecyl sulfate (SDS) solution overnight and sonicated to completely rupture the cells. After centrifugation, the supernatant was taken, and the CTX content in the cells was determined by HPLC. The measurement results are as shown in Figure 10 Part B. The CTX concentration in the cells incubated with iRGD-HSA-CTX NPs was significantly higher than that in the cells incubated with HSA-CTX NPs, that is, the uptake of iRGD-HSA-CTX NPs by 4T1 and A549 tumor cells was significantly higher than that of HSA-CTX NPs. The oil-soluble fluorescent dye DiD was used to label albumin nanoparticles, and the uptake of HSA-DiD NPs and iRGD-HSA-DiD NPs by 4T1 cells was observed through a fluorescence inverted microscope, as shown in Figure 10As shown in Part A, the fluorescence intensity of DiD in 4T1 cells incubated with iRGD-HSA-DiD NPs was significantly higher than that in 4T1 cells incubated with HSA-DiD NPs. The above results indicate that more nanoparticles modified with iRGD can be taken up by 4T1 and A549 cells, resulting in an increase in intracellular drug concentration, demonstrating that iRGD modified on the surface of nanoparticles can bind to highly expressed α ν β3, α ν β5 and NRP-1, improving the tumor targeting of iRGD-HSA-CTX NPs.

[0080] The above-described embodiments merely represent several implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

[0081] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An iRGD peptide-modified cabazitaxel albumin nanoparticle, characterized in that, Comprising the following raw materials in parts by weight: Cabazitaxel 2.5 - 10 parts, albumin 50 - 200 parts, phospholipid 20 - 100 parts, iRGD peptide 500 - 2000 parts, crosslinking agent 1500 - 6000 parts, lyoprotectant 0.05 - 0.3 parts.

2. The iRGD-HSA-CTX NPs according to claim 1, wherein The albumin material is one or both of human serum albumin and bovine serum albumin.

3. The iRGD-HSA-CTX NPs according to claim 1, wherein The average particle size range of the nanoparticles is 50 - 200 nm, preferably 70 nm - 150 nm.

4. The lyophilized preparation of iRGD-HSA-CTX NPs according to claim 1, characterized in that, The lyoprotectant of the freeze-dried nanoparticle preparation is one or more of mannitol, sodium caprylate, arginine, glycine, lactose, glucose, sucrose or trehalose.

5. The lyophilized preparation of iRGD-HSA-CTX NPs according to claim 1, characterized in that, The dosage of the lyoprotectant in the freeze-dried nanoparticle preparation is 0.5% - 3%.

6. A preparation method of iRGD peptide-modified cabazitaxel albumin nanoparticles, characterized in that, Comprising: 1) Weigh cabazitaxel and phospholipid in accordance with the amount, dissolve them in an organic solvent, preferably in a mixed solvent of any combination of chloroform, dichloromethane, ethanol and acetone, to form an oil phase; in the oil phase, the concentration of cabazitaxel is 0.05 - 0.2 mg / mL; 2) Disperse albumin in pure water to form an aqueous phase; in the aqueous phase, the concentration of albumin is 1 - 4 mg / mL; 3) Under a stirring speed of 300 - 1200 r / min, use a syringe to slowly inject the oil phase in step 1) into the aqueous phase in step 2), and under ice bath conditions, perform ultrasonic treatment to form a primary emulsion, with an ultrasonic power of 20% - 60% and an ultrasonic time of 2 - 10 min; 4) The primary emulsion prepared in step 3) is continuously stirred at room temperature for 2 - 6 h, and the organic solvent is evaporated to obtain a cabazitaxel albumin nanoparticle colloidal solution with an average particle size not greater than 150 nm. 5) Take a certain amount of cabazitaxel albumin nanoparticle colloidal solution in an ultrafiltration tube for centrifugal purification, with a centrifugal speed of 5000 - 20000 rpm and a time of 5 - 20 min, and adjust the pH of HSA-CTX NPs to between 7.0 and 7.5 with PBS. 6) Add a certain amount of crosslinking agent solution (sulfo-SMCC) to the cabazitaxel albumin nanoparticle colloidal solution and activate it at room temperature for 1 - 4 h. 7) Centrifuge the activated nanoparticle solution in an ultrafiltration tube, with a centrifugal speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess crosslinking agent and keep the pH between 7.0 and 7.5; 8) Add iRGD, stir overnight at room temperature and then centrifuge and purify in an ultrafiltration tube, with a centrifugal speed of 5000 - 20000 rpm and a time of 5 - 20 min, to remove the excess iRGD, thus obtaining iRGD-HSA-CTX NPs. 9) Weigh the lyoprotectant in accordance with the amount, and perform freeze-drying on the lyoprotectant and the nanomaterial obtained in step 8) to prepare the freeze-dried preparation of iRGD-modified cabazitaxel albumin nanoparticles. 10) The freeze-drying step in step 9) is: pre-freeze at -80°C for 3 h, perform sublimation drying to remove the excess water by programmed heating at 0.9°C / h, and then heat up to 20°C for desorption drying to obtain the freeze-dried preparation.

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