PSO / PTX targeted composite nanoparticle for breast cancer and preparation method and application thereof

Abstract

The embodiment of the invention provides PSO / PTX targeted composite nanoparticles for breast cancer as well as a preparation method and application of the PSO / PTX targeted composite nanoparticles. The PSO / PTX targeted composite nanoparticle comprises an inner core and a phospholipid composition coated on the surface of the inner core, the inner core comprises PLGA (poly (lactic-co-glycolic acid)), SPIONs (at) OA as well as PTX and PSO which are loaded on the SPIONs (at) OA, and the PLGA is coated on the surface of the SPIONs (at) OA; the phospholipid composition comprises phospholipid and polyethylene glycol modified phospholipid, and the polyethylene glycol modified phospholipid is embedded in the phospholipid. According to the embodiment of the invention, PTX, PSO and SPIONs (at) OA are coated by PLGA, and then PLGA is modified by phospholipid and modified phospholipid to form an oil-in-water emulsion layer, so that the encapsulation efficiency of PTX and PSO is improved, and the drug loading capacity and the stability of PSO and PTX are further improved.

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of drug synthesis, and particularly to a PSO / PTX targeted composite nanoparticle for breast cancer, its preparation method and use. Background Art

[0002] Triple-negative breast cancer (TNBC) refers to a subtype of breast cancer in which estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are negative. TNBC is highly invasive, has a high histological grade, extensive heterogeneity, and ductal histological features, and metastatic TNBC is associated with a low overall survival rate.

[0003] Currently, the treatment methods for TNBC clinically mainly include tumor resection, chemotherapy, radiotherapy, and immunotherapy, etc. Among them, chemotherapy is widely used, but there are still unsolved problems, such as the off-target effect leading to the toxic and side effects of chemotherapeutic drugs on normal tissues, the development of acquired drug resistance in tumor cells, etc.

[0004] In the prior art, psoralen (PSO) has immunomodulatory and anti-tumor effects. Its combination with the chemotherapeutic drug paclitaxel (PTX) can inhibit the growth of TNBC and reverse breast cancer MDR. However, the water solubility, stability, and targeting of PSO and PTX are poor, which restricts the practical application of PSO and PTX. Superparamagnetic iron oxide nanoparticles (SPIONs) are crystalline substances composed of magnetic iron oxide. Since their size is smaller than the critical value, they show magnetism in a magnetic field and no magnetism when the magnetic field is removed, so they can be used to construct magnetic targeted nanocarriers.

[0005] Therefore, developing a targeted nanoformulation for TNBC based on SPIONs is of great significance for improving the treatment methods of TNBC. Summary of the Invention

[0006] The purpose of the embodiments of the present application is to provide a PSO / PTX targeted composite nanoparticle for breast cancer, its preparation method and use.

[0007] To achieve the above purpose, the embodiments of the present application propose the following technical solutions:

[0008] In the first aspect, the embodiments of the present application propose a PSO / PTX targeted composite nanoparticle for breast cancer, and the PSO / PTX targeted composite nanoparticle includes a core and a phospholipid composition coated on the surface of the core;

[0009] The core includes PLGA, SPIONs@OA, PTX and PSO loaded on SPIONs@OA, and the PLGA is coated on the surface of SPIONs@OA;

[0010] The phospholipid composition includes phospholipids and polyethylene glycol-modified phospholipids, and the polyethylene glycol-modified phospholipids are embedded inside the phospholipids.

[0011] As an embodiment, the polyethylene glycol-modified phospholipid is DSPE-PEG2000.

[0012] In a second aspect, an embodiment of the present application provides a method for preparing the PSO / PTX targeted composite nanoparticles as described in the first aspect. The preparation method includes:

[0013] Dissolve phospholipids and polyethylene glycol-modified phospholipids in a first organic solvent. After dissolution, add water and an emulsifier, and mix evenly to obtain an aqueous phase;

[0014] Dissolve PLGA in a second organic solvent. After dissolution, add PTX, PSO, and SPIONs@OA, and mix evenly to obtain an organic phase;

[0015] Add the organic phase to the aqueous phase, and obtain the PSO / PTX targeted composite nanoparticles after ultrasonic emulsification and filtration.

[0016] As an embodiment, the preparation method of the SPIONs@OA includes:

[0017] Under an inert atmosphere, take FeCl2·4H2O and FeCl3·6H2O, add ultrapure water, and mix evenly to obtain solution A;

[0018] Add ultrapure water to ammonia water, and mix evenly to obtain solution B;

[0019] Under an ice-water bath and stirring, add solution B to solution A to obtain a mixed solution; transfer the mixed solution to a water bath and stir to prepare a SPIONs suspension;

[0020] Separate the SPIONs suspension using a magnet to obtain magnetic nanoparticles and supernatant;

[0021] Add sodium oleate to the magnetic nanoparticles, stir and react under a water bath, and separate to obtain SPIONs@OA.

[0022] As an embodiment, the step of adding sodium oleate to the magnetic nanoparticles, stirring and reacting under a water bath, and separating to obtain SPIONs@OA includes:

[0023] Add sodium oleate to the magnetic nanoparticles, stir at 40 - 60 °C for 0.5 - 3 h, then separate using a magnet, wash 2 - 3 times with ultrapure water, and centrifuge to separate and obtain SPIONs@OA.

[0024] As an embodiment, the first organic solvent is anhydrous ethanol.

[0025] As an implementation manner, the second organic solvent is chloroform.

[0026] As an implementation manner, the emulsifier is Tween.

[0027] As an implementation manner, adding the organic phase into the aqueous phase, and obtaining the PSO / PTX targeted composite nanoparticles after ultrasonic emulsification and filtration, including:

[0028] Adding the organic phase into the aqueous phase by a syringe, performing ultrasonic emulsification at an ultrasonic power of 225 W for 5 min, evaporating for 5 min at 45 °C to remove the organic solvent, and obtaining the PSO / PTX targeted composite nanoparticles after filtration.

[0029] In a second aspect, the embodiments of the present application propose the use of the PSO / PTX targeted composite nanoparticles described in the first aspect in the preparation of a drug for preventing, alleviating or treating breast cancer.

[0030] As an implementation manner, the drug is a drug for inhibiting the growth of MDA-MB-231 cells.

[0031] The embodiments of the present application at least have the following beneficial effects:

[0032] In the embodiments of the present application, PTX and PSO are first loaded on SPIONs@OA, and PLGA is coated on the surface of SPIONs@OA. Then, a phospholipid composition is modified (coated) on the surface of PLGA, wherein the polyethylene glycol-modified phospholipid is embedded inside the phospholipid; in this embodiment, PTX, PSO, and SPIONs@OA are coated by PLGA, and then PLGA is modified by phospholipid and modified phospholipid to form an oil-in-water emulsion layer, so as to improve the encapsulation rate of PTX and PSO, and further improve the drug loading amount and the stability of PSO and PTX.

[0033] At the same time, the surface of the modified SPIONs is easy to be modified and functionalized, and the phospholipid and the modified phospholipid can modify the surface of SPIONs to improve the delivery efficiency of PSO and PTX; thus, the PSO / PTX targeted composite nanoparticles prepared in this embodiment first propose to co-load psoralen (PSO) and paclitaxel (PTX) in superparamagnetic polymer lipid nanoparticles, solve the problem of dual-drug synergistic delivery. The PSO / PTX targeted composite nanoparticles have both magnetic targeting, long circulation and sustained release functions, and have high clinical transformation potential, indicating that the PSO / PTX targeted composite nanoparticles have good application prospects in the preparation of drugs for treating breast cancer.

[0034] The additional aspects and advantages of the present application will be partially given in the following description, and these will become obvious from the following description, or can be understood through the practice of the present application. Description of the Drawings

[0035] Figure 1 Shows the structural schematic diagram of the PSO / PTX targeted composite nanoparticles provided in this embodiment;

[0036] Figure 2 Shows the preparation flow chart of the PSO / PTX targeted composite nanoparticles provided in this embodiment.

[0037] Figure 3 Shows the schematic diagram of the test results of particle size and potential measurement in Example 3;

[0038] Figure 4 Shows the schematic diagram of the test results of in vitro release in Example 3;

[0039] Figure 5 Shows the schematic diagram of the test results of infrared analysis in Example 3;

[0040] Figure 6 Shows the schematic diagram of the test results of differential scanning calorimetry analysis in Example 3;

[0041] Figure 7 Shows the schematic diagram of the test results of X-ray powder diffraction analysis in Example 3;

[0042] Figure 8 Shows the schematic diagram of the test results of cytotoxicity test in Example 4. Detailed implementation manners

[0043] Next, the technical solutions in the embodiments will be clearly and completely described in conjunction with the embodiments of the present application and the accompanying drawings. Obviously, the embodiments described below are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application.

[0044] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprise" indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0045] It should also be understood that the terms used in the specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms.

[0046] First, some terms and materials involved in this embodiment are explained below to facilitate the understanding of those skilled in the art.

[0047] PSO: Psoralen.

[0048] PTX: Paclitaxel.

[0049] PEG-PE: DSPE-PEG2000, also known as polyethylene glycol modified phospholipid or modified phospholipid.

[0050] SPIONs: Superparamagnetic iron oxide nanoparticles, also known as magnetic nanoparticles.

[0051] Pluronic F-127: Chemical name: polyoxyethylene polyoxypropylene ether block copolymer, trade name: Poloxamer407; also known as PF-127 in this embodiment.

[0052] SPIONs@OA: Superparamagnetic iron oxide nanoparticles coated with oleic acid (OA).

[0053] P / P-TCNs: Also known as PSO / PTX targeted composite nanoparticles, that is, targeted composite nanoparticles.

[0054] Unless otherwise specified, the water used in this embodiment is ultrapure water or deoxygenated ultrapure water.

[0055] Next, the technical solution of the embodiment of the present application will be described in detail.

[0056] First, the PSO / PTX targeted composite nanoparticles for breast cancer in the first aspect of this embodiment are described.

[0057] PSO / PTX Targeted Composite Nanoparticles

[0058] In the prior art, due to the established pharmacological activities and application significances of psoralen and paclitaxel, various nanoparticles prepared from psoralen and paclitaxel have been reported in many domestic and foreign studies, but each type of nanoparticle has its own different limitations; for example, psoralen (PSO) has immunomodulatory and antitumor effects, and its combination with the chemotherapeutic drug paclitaxel (PTX) can inhibit the growth of TNBC and reverse breast cancer MDR, but the water solubility, stability and targeting of PSO and PTX are poor, thus restricting the practical applications of PSO and PTX.

[0059] In view of this, this embodiment proposes a PSO / PTX targeted composite nanoparticle. Please refer to Figure 1 , the PSO / PTX targeted composite nanoparticle (P / P-TCNs) includes a core and a phospholipid composition coated on the surface of the core;

[0060] The core includes PLGA, SPIONs@OA, PTX and PSO loaded on SPIONs@OA, and the PLGA coats the surface of SPIONs@OA;

[0061] The phospholipid composition includes phospholipids and polyethylene glycol-modified phospholipids, and the polyethylene glycol-modified phospholipids are embedded inside the phospholipids.

[0062] Among them, the polyethylene glycol-modified phospholipid used in this example is DSPE-PEG2000.

[0063] It can be understood that in this example, PTX and PSO are first loaded on SPIONs@OA, and PLGA coats the surface of SPIONs@OA. Then, the phospholipid composition is modified (coated) on the surface of PLGA, and among them, the polyethylene glycol-modified phospholipids are embedded inside the phospholipids. In this example, PTX, PSO and SPIONs@OA are coated by PLGA, and then PLGA is modified by phospholipids and modified phospholipids to form an oil-in-water emulsion layer, so as to improve the encapsulation efficiency of PTX and PSO, and further improve the drug loading amount and the stability of PSO and PTX.

[0064] At the same time, the surface of modified SPIONs is easy to be modified and functionalized, and phospholipids and modified phospholipids can modify the surface of SPIONs to improve the delivery efficiency of PSO and PTX. Therefore, the PSO / PTX targeted composite nanoparticles prepared in this example first propose to co-load psoralen (PSO) and paclitaxel (PTX) in superparamagnetic polymer lipid nanoparticles, solve the problem of dual-drug synergistic delivery, not only can improve the drug loading amount and stability of PSO / PTX, magnetically target the tumor site, increase the accumulation of drugs (PSO / PTX) at the tumor site, and reduce the damage to normal tissues; the chemical toxicity of PSO / PTX and the magnetic induction hyperthermia synergistic treatment effect of SPIONs improve the efficacy of drugs and reduce the toxic and side effects.

[0065] In summary, the PSO / PTX targeted composite nanoparticles have both magnetic targeting, long circulation and sustained release functions, and have high clinical transformation potential, indicating that the PSO / PTX targeted composite nanoparticles have good application prospects in the preparation of drugs for treating breast cancer.

[0066] Secondly, the preparation method of the PSO / PTX targeted composite nanoparticles in the second aspect of this example will be described.

[0067] Preparation method of PSO / PTX targeted composite nanoparticles

[0068] This example provides a preparation method of PSO / PTX targeted composite nanoparticles for preparing the PSO / PTX targeted composite nanoparticles described in the first aspect. The preparation method includes the following steps:

[0069] (1) Dissolve phospholipids and polyethylene glycol-modified phospholipids in a first organic solvent. After dissolution, add water and an emulsifier, and mix evenly to obtain an aqueous phase.

[0070] In this step, dissolve phospholipids and modified phospholipids in a first organic solvent and an emulsifier. The obtained aqueous phase can be used for subsequent modification of PLGA to form an oil-in-water emulsion layer.

[0071] Preferably, the first organic solvent is absolute ethanol.

[0072] (2) Dissolve PLGA in a second organic solvent. After dissolution, add PTX, PSO, and SPIONs@OA, and mix evenly to obtain an organic phase.

[0073] In step (2), first prepare SPIONs@OA. During step (2), PTX and PSO can be loaded on SPIONs@OA, and PTX, PSO, and SPIONs@OA can be coated with PLGA to form a core.

[0074] In this step, SPIONs@OA realizes the loading of PTX and PSO, which can effectively improve the drug loading amount of PSO / PTX and is beneficial to magnetic targeting of tumor sites through SPIONs@OA. Secondly, coating PTX, PSO, and SPIONs@OA with PLGA can improve the stability of PSO / PTX.

[0075] (3) Add the organic phase to the aqueous phase, and obtain PSO / PTX targeted composite nanoparticles through ultrasonic emulsification and filtration.

[0076] In step (3), modify the PLGA coated with PTX, PSO, and SPIONs@OA obtained in step (2) with phospholipids and modified phospholipids to form an oil-in-water emulsion layer. Among them, polyethylene glycol-modified phospholipids are embedded inside the phospholipids. Through the cooperation of polyethylene glycol-modified phospholipids and phospholipids, the encapsulation efficiency of PTX and PSO can be effectively improved to increase the drug loading amount and the stability of PSO and PTX. At the same time, the surface of modified SPIONs is easy to be modified and functionalized, and phospholipids and modified phospholipids can modify the surface of SPIONs to improve the delivery efficiency of PSO and PTX.

[0077] In step (3), organic solvents are usually removed by evaporation, and then filtration is carried out to obtain the target product.

[0078] In summary, the PSO / PTX targeted composite nanoparticles prepared in this example firstly propose to co-load psoralen (PSO) and paclitaxel (PTX) in superparamagnetic polymer lipid nanoparticles, solving the problem of dual-drug synergistic delivery. The PSO / PTX targeted composite nanoparticles have both magnetic targeting, long-circulation and sustained-release functions, and have high potential for clinical translation, indicating that the PSO / PTX targeted composite nanoparticles have good application prospects in the preparation of drugs for treating breast cancer.

[0079] Next, the preparation method of this example will be further described.

[0080] As an implementation manner, the preparation method of SPIONs@OA includes:

[0081] (2.1) Under an inert atmosphere, take FeCl2·4H2O and FeCl3·6H2O, add ultrapure water, and mix evenly to obtain solution A;

[0082] (2.2) Add ultrapure water to ammonia water and mix evenly to obtain solution B;

[0083] (2.3) Under an ice-water bath and stirring, add solution B to solution A to obtain a mixed solution; transfer the mixed solution to a water bath and stir to prepare a SPIONs suspension;

[0084] (2.4) Use a magnet to separate the SPIONs suspension to obtain magnetic nanoparticles and supernatant;

[0085] (2.5) Add sodium oleate to the magnetic nanoparticles and stir and react under a water bath to obtain SPIONs@OA.

[0086] In step (2.1), the inert atmosphere is nitrogen; for example, in a three-necked flask as the reaction vessel, place FeCl2·4H2O and FeCl3·6H2O in the three-necked flask, displace the air in the system with nitrogen, add deoxygenated ultrapure water (previously deoxygenated by nitrogen), mix evenly to obtain solution A, and place it in an ice-water bath at 5°C.

[0087] In step (2.2), after obtaining solution B, it can be placed in an ice-water bath at 5°C.

[0088] In step (2.3), the main processes are reaction and crystallization; among them, after obtaining the mixed solution, transfer the mixed solution to a water bath pot, stir at 40°C for 0.5 h, and then stir at 60°C for 1 h to prepare a SPIONs suspension.

[0089] In step (2.4), the washing and separation of the SPIONs suspension are involved. Specifically, a magnet is used to separate the magnetic nanoparticles and the reaction liquid. After the nanoparticles are adsorbed thereto, the supernatant is drawn with a syringe. Subsequently, 20 - 50 mL of deoxygenated ultrapure water is added, and after stirring for 5 - 10 min, the magnetic nanoparticles and the supernatant are separated in the same manner. Wash repeatedly 2 - 3 times until the washed supernatant is neutral to obtain magnetic nanoparticles and the supernatant.

[0090] In step (2.5), the modification of the magnetic nanoparticles is achieved by adding sodium oleate.

[0091] As an implementation manner, adding sodium oleate to the magnetic nanoparticles and stirring and reacting under a water bath to separate and obtain SPIONs@OA in step (2.5) includes:

[0092] Add sodium oleate to the magnetic nanoparticles, stir at 40 - 60 °C for 0.5 - 3 h, then use a magnet to separate, and wash 2 - 3 times with ultrapure water, and separate SPIONs@OA after centrifugation.

[0093] As a preferred implementation manner, adding sodium oleate to the magnetic nanoparticles and stirring and reacting under a water bath to separate and obtain SPIONs@OA in step (2.5) includes:

[0094] Add oleic acid to the magnetic nanoparticles, stir at 50 °C for 0.5 h, then use a magnet to separate, and wash 3 times with 30 mL of ultrapure water, and separate SPIONs@OA after centrifugation.

[0095] As an implementation manner, the second organic solvent is chloroform, and / or the emulsifier is Tween.

[0096] As an implementation manner, adding the organic phase to the aqueous phase and obtaining the PSO / PTX targeted composite nanoparticles after ultrasonic emulsification and filtration includes:

[0097] Add the organic phase to the aqueous phase, perform ultrasonic emulsification at an ultrasonic power of 225 W for 5 min, evaporate for 5 min at 45 °C to remove the organic solvent, and obtain the PSO / PTX targeted composite nanoparticles after filtration.

[0098] Generally, in the above steps (1) - (4), stirring is performed during the reaction process to accelerate the reaction or make the reaction more complete.

[0099] Next, the use of the PSO / PTX targeted composite nanoparticles in the third aspect of this embodiment will be described.

[0100] Use

[0101] As described in the first aspect, the PSO / PTX targeted composite nanoparticles prepared in this example have multiple advantages. For example: (1) It can improve the drug loading and stability of PSO / PTX, magnetically target the tumor site, increase the accumulation of the drug (PSO / PTX) at the tumor site, and reduce the damage to normal tissues; (2) The chemical toxicity of PSO / PTX and the magnetic induction heat of SPIONs have a synergistic therapeutic effect, improving the efficacy of the drug and reducing the toxic and side effects.

[0102] Based on this, the PSO / PTX targeted composite nanoparticles provided in this example can be used to prepare drugs for preventing, alleviating or treating breast cancer.

[0103] It should be particularly noted that the PSO / PTX targeted composite nanoparticles of this example can be made into drugs for inhibiting the growth of MDA-MB-231 cells.

[0104] Exemplarily, the above-mentioned drug is an injectable solution or a drug for oral administration.

[0105] Generally, when the above-mentioned PSO / PTX targeted composite nanoparticles are made into corresponding drugs, the drugs also include pharmaceutically acceptable carriers and / or adjuvants.

[0106] For the drugs described in this example, the drugs take the PSO / PTX targeted composite nanoparticles as the active ingredient, and do not exclude changes in the formulation system and administration methods, medicinal salts after simple chemical modification and adjustment of the above-mentioned PSO / PTX targeted composite nanoparticles, and the combination of multiple compounds.

[0107] For example, in this example, one or more compounds in the PSO / PTX targeted composite nanoparticles of this example can be used as active ingredients and formulated in non-toxic, inert and pharmaceutically acceptable carriers and / or adjuvants; the formulated drugs can be administered through conventional routes, including but not limited to oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal or topical administration.

[0108] For example, when the dosage form of the drug in this example is a drug for oral administration, it contains a safe and effective amount of PSO / PTX targeted composite nanoparticles and pharmaceutically acceptable carriers and / or adjuvants. The drugs for oral administration can be made into common dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, suppositories, etc.; in this example, no specific limitations are placed on the carriers and / or adjuvants, and the carriers and / or adjuvants can be adaptively adjusted according to the specific drug dosage form.

[0109] Generally, an "effective amount" of a compound (PSO / PTX targeted composite nanoparticles) refers to an amount sufficient to elicit a desired biological response. As understood by those of ordinary skill in the art, the effective amount of the compound in this example can vary depending on the following factors: for example, components such as the vehicle in the drug, as well as the age, health status of the subject, and breast cancer symptoms.

[0110] Among them, the effective amount includes a therapeutically effective amount and a prophylactically effective amount.

[0111] Unless otherwise specified, the "therapeutically effective amount" of the compound used in this example is an amount sufficient to provide a benefit during the treatment of breast cancer, or the minimum amount that improves or alleviates one or more symptoms (manifestations) related to the breast cancer condition. The "prophylactically effective amount" of the compound used in this example is an amount sufficient to prevent the occurrence of breast cancer, or an amount sufficient to prevent one or more symptoms related to the occurrence of breast cancer.

[0112] It can be understood that the drug of this example can also be made into an injection. For example, PSO / PTX targeted composite nanoparticles can be made into corresponding injections with water for injection, normal saline, and glucose solution under a sterile operating environment. The above injections can be prepared by conventional methods.

[0113] The following will further illustrate the present application in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0114] In the following examples, the materials, reagents, and instruments used, unless otherwise specified, can be obtained from commercial sources.

[0115] In the following examples, unless otherwise specified, the modified phospholipid is DSPE-PEG2000, the addition rate of the liquid is controlled at 5 mL / min, and the water bath is carried out in a water bath pot.

[0116] The following will further illustrate the present application in conjunction with specific examples.

[0117] Example 1: Preparation of SPIONs@OA.

[0118] The preparation method of SPIONs@OA specifically includes the following steps:

[0119] 1.1 Preparation of solution A: Weigh 0.696 g of FeCl2·4H2O and 1.351 g of FeCl3·6H2O into a three-necked flask, displace the air in the system with nitrogen, add 50 mL of deoxygenated ultrapure water (previously deoxygenated by nitrogen), and place it in an ice bath at 5°C.

[0120] 1.2 Preparation of solution B: Add 14 mL of ammonia water to 50 mL of deoxygenated ultrapure water and place it in an ice bath at 5°C.

[0121] 1.3. Mixing: Add liquid B to liquid A under a 5°C ice-water bath and stirring (control the addition rate at 5 mL / min), and adjust the pH value of the solution to 11. Then carry out the reaction and crystallization. Transfer the obtained solution to a water bath, stir at 40°C for 0.5 h and at 60°C for 1 h to prepare the SPIONs suspension.

[0122] 1.4. Washing: Use a magnet to separate the magnetic nanoparticles and the reaction liquid. After the nanoparticles are adsorbed, use a syringe to extract the supernatant. Subsequently, add 20 - 50 mL of deoxygenated ultrapure water, stir for 10 min, and then separate the magnetic nanoparticles and the supernatant in the same way. Wash repeatedly 2 - 3 times until the washed supernatant is neutral.

[0123] 1.5. Oleic acid coating: After washing, add the sodium oleate solution (weigh 1.5 g of sodium oleate and add it to 50 mL of ultrapure water, and dissolve it by ultrasonic treatment) to the above-mentioned magnetic nanoparticles, stir at 50°C for 0.5 h, separate with a magnet, and wash 3 times with 20 - 50 mL of ultrapure water, and then centrifuge to obtain SPIONs@OA.

[0124] Example 2: Preparation of P / P-TCNs (PSO / PTX targeted composite nanoparticles).

[0125] Please refer to Figure 2 , the preparation method of P / P-TCNs (PSO / PTX targeted composite nanoparticles) provided in this example includes:

[0126] Weigh 80 mg of phospholipids and 20 mg of DSPE-PEG2000, totaling 100 mg; add them to 0.3 mL of absolute ethanol for dissolution. After complete dissolution, add 50 mL of purified water, stir and mix evenly, then add 0.2 mL of Tween, stir evenly, and keep it at 2 - 8 °C to obtain the aqueous phase.

[0127] Weigh 100 mg of PLGA and dissolve it in 1.5 mL of chloroform by ultrasonic treatment. After dissolution, add 5 mg of PTX and 5 mg of PSO, and dissolve them by ultrasonic treatment. After dissolution, add 0.1 g of SPIONs@OA (prepared in Example 1) and disperse it by ultrasonic treatment to obtain the organic phase.

[0128] Under an ultrasonic probe, slowly inject the organic phase into the aqueous phase, with an ultrasonic power of 225 W and an ultrasonic time of 5 min. After ultrasonic treatment, rotary evaporate at 45°C for 5 min to remove the organic solvent. Sterilize and filter with a 0.22 μm polyethersulfone filter membrane to obtain P / P-TCNs.

[0129] Comparative Example 1:

[0130] The difference between Comparative Example 1 and Example 2 is only that SPIONs@OA is not added to the organic phase, and the control sample 1 is prepared, denoted as P / P-PLNs.

[0131] Comparative Example 2:

[0132] Comparative Example 2 provides control sample 2, and the preparation method of this control sample 2 includes:

[0133] Preparation of PF-127 modified SPIONs: Weigh 0.5 g of SPIONs@OA nanoparticles (prepared in Example 1) into a three-necked flask, add 100 mL of an aqueous solution of PF-127 with a concentration of 1.5%, and displace the air in the system with nitrogen. Stir at room temperature in a water bath for 12 h. After stirring, separate the magnetic nanoparticles by a magnet. Wash the nanoparticles with 20 - 50 mL of deoxygenated purified water again, and wash 2 - 3 times to obtain PF-127 modified SIOPNs.

[0134] Preparation of P / P-IOPNs: Weigh 0.1 g of PF-127 modified SPIONs, add 50 mL of deoxygenated purified water, and stir and mix well to obtain the aqueous phase. Weigh 5 mg of PTX and 5 mg of PSO and dissolve them in 1.5 mL of acetonitrile, and stir / ultrasonic dissolve to obtain the organic phase; add the organic phase to the aqueous phase at 30 °C and stir for 6 h. Sterilize and filter with a 0.22 μm polyethersulfone membrane to obtain P / P-IPONs.

[0135] Example 3

[0136] Next, the P / P-TCNs prepared in Example 2 above, the P / P-PLNs and P / P-IOPNs prepared in Comparative Example 1 and Comparative Example 2 will be tested or verified.

[0137] The performance and test results are as follows:

[0138] 3.1 Particle size and potential test

[0139] Use a laser particle size analyzer (ZS90, Malvern, UK) to measure the particle size, PDI and potential of P / P-PLNs, P / P-IOPNs, and P / P-TCNs. The test results are as Figure 3 shown.

[0140] Figure 3 In the shown particle size and potential diagrams, A and B are the particle size and potential diagrams of P / P-IOPNs respectively. C and D are the particle size and potential diagrams of P / P-PLNs respectively. E and F are the particle size and potential diagrams of P / P-TCNs respectively.

[0141] From Figure 3As can be seen from B, D, and F in the figure, the prepared P / P-IOPNs, P / P-PLNs, and P / P-TCNs all have ideal particle size dimensions of 69.1±1.01, 136.1±1.24, and 188.8±2.21 nm respectively; the PDI values are 0.153±0.16, 0.207±0.15, and 0.152±0.03 respectively; the zeta potentials are -31.9±0.73, -30.8±0.94, and -40.3±0.63 respectively. This size is easily enriched in the tumor area through the enhanced permeability and retention effect (EPR).

[0142] 3.2. In vitro release

[0143] In this example, the dialysis method was used to measure the release of PSO and PTX from the nanoparticles in vitro. P / P-PLNs, P / P-IOPNs, and P / P-TCNs were placed in dialysis bags, and the dialysis bags were immersed in phosphate buffer solution (PBS, 7.4) containing 0.3% Tween-80 and stirred at 37°C and 100 rpm. 1 mL of the sample was taken out at 1, 2, 4, 8, 12, 24, 36, and 48 h, and an equal volume of PBS was added to the dialysis bag. The mixture was filtered through a 0.22 μm filter membrane (Guangzhou Jet Bio-Filtration Co., Ltd., Guangzhou), and the cumulative release amounts of PSO and PTX were detected according to the above chromatographic method. The test results are as Figure 4 shown.

[0144] Figure 4 The schematic diagram of the test results of in vitro release is shown. In this example, the in vitro release behaviors of PSO and PTX from three kinds of nanoparticles, P / P-IOPNs, P / P-PLNs, and P / P-TCNs, in a solution with pH 7.4 were investigated. As shown by Figure 4 the test results, the cumulative release amounts of PSO from P / P-IOPNs, P / P-PLNs, and P / P-TCNs at pH 7.4 for 48 h were 75.34%, 65.37%, and 45.38% respectively, and those of PTX were 75.29%, 58.99%, and 39.60% respectively. It shows that P / P-TCNs have a better sustained-release effect at pH 7.4.

[0145] 3.3. Infrared analysis

[0146] In this example, a Fourier transform infrared spectrometer (Nicolet iS50, Thermo Fisher Scientific, USA) was used to analyze PSO, PTX, PSO+PTX, IOPNs, PLNs, TCNs, P / P-PLNs, P / P-IOPNs, P / P-TCNs, and the physical mixtures of IOPNs, PLNs, TCNs with PSO+PTX. The test results are as Figure 5 shown.

[0147] Figure 5 Schematic diagram showing the analysis and test results of the infrared spectrum. Figure 5 In it, A: PSO, B: PTX, C: PSO + PTX, D: IOPNs, E: IOPNs + PSO + PTX, F: P / P-IOPNs, G: PLNs, H: PLNs + PSO + PTX, I: P / P-PLNs, J: TCNs, K: TCNs + PSO + PTX, L: P / P-TCNs.

[0148] Figure 5 (A) has a strong absorption peak at 1720 cm -1 , which is the characteristic peak of C=O of PSO. The 1720 and 1650 cm -1 in (B) are respectively the characteristic absorption peaks of ester group C=O and amide group C=O of paclitaxel. (C) is a physical mixture of PSO and PTX, so the characteristic absorption peaks of 1720 and 1650 cm -1 both appear. This peak also appears simultaneously in the physical mixtures of IOPNs, PLNs, TCNs and PSO, PTX. See Figures E, H, K. However, the characteristic absorption peaks of 1720 and 1650 cm -1 are not seen in IOPNs, P / P-IOPNs, PLNs, P / P-PLNs, TCNs and P / P-TCNs (D, F, G, I, J, L), indicating that PSO and PTX have been well encapsulated in the nanoparticles of P / P-IOPNs, P / P-PLNs and P / P-TCNs.

[0149] 3.4, DSC analysis

[0150] In this example, PSO + PTX, P / P-IOPNs, P / P-PLNs, and P / P-TCNs were respectively prepared for differential scanning calorimetry analysis: Weigh about 5 mg and place it in an aluminum pan, cover the lid and pierce holes and compact it. Using an empty aluminum pan as a reference, introduce nitrogen for protection, and the scanning temperature range is 25 - 600 °C, with a heating rate of 10 °C per minute. The test results are as Figure 6 shown.

[0151] Figure 6 Shows the differential scanning calorimetry analysis chart; Figure 6 In it, A: PSO + PTX, B: P / P-IOPNs, C: P / P-PLNs, D: P / P-TCNs.

[0152] According to Figure 6As shown, the DSC scanning spectra of the PSO and PTX drug powder mixture (A) each have an endothermic peak near the ranges of 155 °C and 250 °C. The endothermic peak near 155 °C is the endothermic peak of PSO, while the endothermic peak of doxorubicin hydrochloride is near 250 °C; P / P-IOPNs have no endothermic peaks near 155 °C and 250 °C, but have a characteristic peak near 388 °C; P / P-PLNs (C) have endothermic peaks at 147 °C and 165 °C, and the drug endothermic peaks disappear near 155 °C and 250 °C; P / P-TCNs (D) have characteristic peaks near 108 °C and 205 °C, and the drug endothermic peaks disappear near 155 °C and 250 °C. It can be seen that the endothermic peaks of the physical mixture of PSO and PTX are different from those of P / P-IOPNs, P / P-PLNs, and P / P-TCNs, indicating that PSO and PTX are encapsulated in the nanoparticles.

[0153] 3.5, X-ray powder diffraction analysis

[0154] PSO+PTX and P / P-TCNs were respectively prepared, and the X-ray diffraction patterns were measured under the following conditions: scanning speed 5° / min, scanning range 5 - 90°. The test results are as Figure 7 shown.

[0155] Figure 7 The schematic diagram of X-ray powder diffraction is shown. From Figure 7 it can be seen that PSO+PTX (A) shows many crystal form peaks and is a crystalline substance, and the relatively strong crystalline diffraction peaks are distributed in the range of 5° - 30°. P / P-TCNs (B) do not show crystal peaks. Combining with the infrared spectroscopy results in 3.3, it can be known that PSO+PTX is encapsulated in the nanoparticles in an amorphous or disordered state.

[0156] Example 4: Cytotoxicity test.

[0157] In this example, the CCK-8 method was used to perform an in vitro cytotoxicity test on the P / P-TCNs prepared in Example 2. Specifically, triple-negative breast cancer MDA-MB-231 cells9 were seeded in a 96-well plate (5.0×10 3 cells / well), cultured for 24 h to allow them to adhere, and then different concentrations of P / P-TCNs were administered. Blank medium and blank cells were set as controls. After culturing each group in an incubator for 48 h, CCK-8 solution (10 μL) was added to each sample well, and the 96-well plate was returned to the incubator for continued culture for 4 hours. The absorbance (OD) of each sample at 450 nm was recorded using a full-wavelength microplate reader (ReadMax 1200, Shanghai Flash Spectrum Biotechnology Co., Ltd., China). The cytotoxicity was calculated as follows:

[0158] Cell viability (%) = (ODa - ODb) / (ODc - ODb) × 100;

[0159] Among them, ODa is the OD value of the sample to be tested (containing cells, CCK-8, and drug-containing DMEM medium), ODc is the OD value of the control group (containing DMEM medium and CCK-8 solution), and ODb is the OD value of the blank well (containing cells, DMEM medium, and CCK-8 solution).

[0160] Please refer to Figure 8 for the administration concentration of P / P-TCNs, and the test results are as Figure 8 shown.

[0161] According to Figure 8 the experimental results, in this example, a CCK-8 kit was used to detect the cytotoxicity of P / P-TCNs on MDA-MB-231 cells. The survival rate of MDA-MB-231 cells decreased with the increase in the concentration of P / P-TCNs, that is, the higher the concentration of P / P-TCNs, the more obvious the inhibitory effect on MDA-MB-231 cells. Combining Figure 8 the test results, the IC50 of P / P-TCNs on MDA-MB-231 cells was calculated to be 13.24 μg / mL respectively. Therefore, P / P-TCNs administration has good efficacy against the growth of MDA-MB-231 cells.

[0162] In summary, in this example, PTX and PSO were first loaded on SPIONs@OA, and SPIONs@OA and PTX and PSO loaded on SPIONs@OA were used as the core, and PLGA was coated on the surface of the core. Then, a phospholipid composition was modified on the surface of the coating layer; in this example, PTX, PSO, and SPIONs@OA were coated by PLGA, and then PLGA was modified by phospholipids and modified phospholipids (forming an oil-in-water emulsion layer) to improve the encapsulation rate of PTX and PSO, thereby improving the drug loading capacity and the stability of PSO and PTX.

[0163] At the same time, the surface of modified SPIONs is easy to be modified and functionalized, and phospholipids and modified phospholipids can modify the surface of SPIONs to improve the delivery efficiency of PSO and PTX; thus, the PSO / PTX targeted composite nanoparticles prepared in this example first proposed co-loading psoralen (PSO) and paclitaxel (PTX) in superparamagnetic polymer lipid nanoparticles, solving the problem of dual-drug synergistic delivery. The PSO / PTX targeted composite nanoparticles have both magnetic targeting, long circulation, and sustained release functions, and have high clinical transformation potential, indicating that the PSO / PTX targeted composite nanoparticles have good application prospects in the preparation of drugs for treating breast cancer.

[0164] The above has introduced in detail the technical solutions provided by the embodiments of the present application. Specific examples are used herein to elaborate on the principles and implementation manners of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those of ordinary skill in the art, according to the embodiments of the present application, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present application.

Claims

1. A PSO / PTX targeted composite nanoparticle for breast cancer, characterized in that, The PSO / PTX targeted composite nanoparticles include a core and a phospholipid composition coated on the surface of the core; The core includes PLGA, SPIONs@OA, PTX and PSO loaded on SPIONs@OA, and the PLGA coats the surface of SPIONs@OA; The phospholipid composition includes phospholipids and polyethylene glycol modified phospholipids, and the polyethylene glycol modified phospholipids are embedded inside the phospholipids.

2. The PSO / PTX targeted composite nanoparticles for breast cancer according to claim 1, characterized in that, The polyethylene glycol modified phospholipid is DSPE-PEG2000.

3. A preparation method of the PSO / PTX targeted composite nanoparticles as described in claim 1 or 2, characterized in that, The preparation method includes: Dissolve phospholipids and polyethylene glycol modified phospholipids in a first organic solvent, add water and an emulsifier after dissolution, and mix evenly to obtain an aqueous phase; Dissolve PLGA in a second organic solvent, add PTX, PSO and SPIONs@OA after dissolution, and mix evenly to obtain an organic phase; Add the organic phase to the aqueous phase, and obtain the PSO / PTX targeted composite nanoparticles after ultrasonic emulsification and filtration.

4. The preparation method according to claim 3, characterized in that, The preparation method of the SPIONs@OA includes: Under an inert atmosphere, take FeCl2·4H2O and FeCl3·6H2O, add ultrapure water, and mix evenly to obtain solution A; Add ultrapure water to ammonia water and mix evenly to obtain solution B; Add solution B to solution A under an ice-water bath and stirring conditions to obtain a mixed solution; transfer the mixed solution to a water bath and stir to prepare a SPIONs suspension; Separate the SPIONs suspension by a magnet to obtain magnetic nanoparticles and supernatant; Add sodium oleate to the magnetic nanoparticles, stir and react under a water bath, and separate to obtain SPIONs@OA.

5. The preparation method according to claim 4, wherein The step of adding sodium oleate to the magnetic nanoparticles, stirring and reacting under a water bath, and separating to obtain SPIONs@OA includes: Add sodium oleate to the magnetic nanoparticles, stir at 40-60 °C for 0.5-3 h, separate by a magnet, wash 2-3 times with ultrapure water, and centrifuge to separate to obtain SPIONs@OA.

6. The preparation method according to claim 3, wherein The first organic solvent is absolute ethanol, and / or The second organic solvent is chloroform.

7. The preparation method according to claim 3, characterized in that, The emulsifier is Tween.

8. The preparation method according to claim 3, wherein The step of adding the organic phase to the aqueous phase, and obtaining the PSO / PTX targeted composite nanoparticles after ultrasonic emulsification and filtration includes: Add the organic phase to the aqueous phase by a syringe, perform ultrasonic emulsification for 5 min at an ultrasonic power of 225 W, evaporate for 5 min at 45 °C to remove the organic solvent, and obtain the PSO / PTX targeted composite nanoparticles after filtration.

9. Use of the PSO / PTX targeted composite nanoparticles according to claim 1 or 2 in the preparation of a drug for preventing, alleviating or treating breast cancer.

10. The use according to claim 9, characterized in that, The drug is a drug for inhibiting the growth of MDA-MB-231 cells.

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