PH-sensitive tumor targeting nanoparticles as well as preparation method and application thereof

Abstract

The embodiment of the invention provides a pH-sensitive tumor targeting nanoparticle as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving baicalin in an organic solvent to obtain an organic phase; mixing the organic phase with an albumin solution to obtain baicalin albumin nanoparticles; dissolving folic acid in a buffer solution, adding a shrinking agent and a carboxyl activating agent, and reacting to obtain a folic acid activating solution; mixing the baicalin albumin nanoparticles with a folic acid activating solution, and reacting to obtain baicalin folic acid coupled albumin nanoparticles; the preparation method comprises the following steps: dissolving mPEG-NH2 and DCA in a buffer solution, and reacting to obtain mPEGA-DCA; adding a shrinking agent and a carboxyl activating agent into the mPEGA-DCA solution to obtain a pH sensitive polymer; and mixing the pH-sensitive polymer and the baicalin folic acid coupled albumin nanoparticles to prepare the pH-sensitive tumor targeting nanoparticles. According to the application, the toxic and side effects of the baicalin can be reduced from multiple aspects, and the tumor targeting capability of the baicalin is improved, so that the anti-tumor capability of the baicalin is enhanced.

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of pharmaceutical preparations, and specifically to a pH-sensitive tumor-targeting nanoparticle and a preparation method and use thereof. Background Art

[0002] Breast cancer is a malignant tumor that occurs in breast epithelial tissue and is one of the most common malignant tumors in women. Breast cancer is a phenomenon in which breast epithelial cells proliferate uncontrollably under the influence of multiple carcinogenic factors. Currently, among many treatment options, Chinese medicine monomers show good treatment prospects.

[0003] Among them, baicalin (BA) is a flavonoid compound extracted from the root of Scutellaria baicalensis, a plant of the Lamiaceae family. It is a glucuronic acid conjugate of baicalin and has antibacterial, antitumor, antipyretic, sedative and antispasmodic effects. The antitumor mechanism of baicalin includes inducing cell apoptosis and differentiation, inhibiting protein synthesis, inhibiting tumor cell metastasis, interfering with intercellular communication and reversing tumor resistance. However, baicalin has poor water solubility and lipid solubility, thus limiting its bioavailability in the body. In clinical practice, it usually requires a sufficiently large dose to exert a better efficacy, but increased doses are prone to adverse reactions.

[0004] Therefore, how to modify baicalin accordingly so that it can selectively accumulate in the tumor site to increase the efficacy of baicalin is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] The purpose of the present application is to provide a pH-sensitive tumor-targeting nanoparticle and a preparation method and use thereof.

[0006] To achieve the above objectives, the present application proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing pH-sensitive tumor-targeting nanoparticles, the preparation method comprising:

[0008] dissolving baicalin in an organic solvent to obtain an organic phase; mixing the organic phase with an albumin solution to obtain baicalin albumin nanoparticles;

[0009] Dissolving folic acid in a buffer solution, adding a shrinking agent and a carboxyl activator, and reacting to obtain a folic acid activation solution; mixing baicalin albumin nanoparticles with the folic acid activation solution, and reacting to obtain baicalin folic acid-coupled albumin nanoparticles;

[0010] Dissolving methoxy polyethylene glycol-amino and 3,4,5,6-tetrahydrophthalic anhydride in a buffer solution to react and obtain mPEGA-DCA; preparing an mPEGA-DCA solution, adding a shrinking agent and a carboxyl activator to the mPEGA-DCA solution to obtain a pH-sensitive polymer;

[0011] After mixing the pH-sensitive polymer and the baicalin folic acid-conjugated albumin nanoparticles, pH-sensitive tumor-targeting nanoparticles are prepared.

[0012] As an embodiment, the organic solvent is methanol and / or ethyl acetate.

[0013] As an embodiment, the buffer solution is Tris-HCL buffer solution.

[0014] As an embodiment, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0015] As an embodiment, the carboxyl activator is N-hydroxysuccinimide.

[0016] As an embodiment, the albumin solution is at least one of human serum albumin solution, bovine serum albumin solution, and recombinant human serum albumin solution.

[0017] As an embodiment, the mass ratio of the pH-sensitive polymer to the baicalin folic acid-conjugated albumin nanoparticles is 2-3:1.

[0018] In a second aspect, an embodiment of the present application provides a pH-sensitive tumor-targeting nanoparticle, which is prepared by the preparation method described in the first aspect.

[0019] In a third aspect, an embodiment of the present application provides the use of the pH-sensitive tumor-targeting nanoparticle described in the second aspect in the preparation of a drug for preventing, alleviating or treating breast cancer.

[0020] As an embodiment, the drug is a drug for improving the drug resistance of TAM in the treatment of breast cancer.

[0021] As an embodiment, the drug is an injectable solution or a drug for oral administration.

[0022] The embodiments of the present application have at least the following beneficial effects:

[0023] In the embodiments of the present application, baicalin is first encapsulated in an albumin solution to prepare baicalin albumin nanoparticles, then the FA molecule of folic acid is coupled with the baicalin albumin nanoparticles to obtain baicalin folic acid conjugated albumin nanoparticles, and then a pH-sensitive polymer is combined with the baicalin folic acid conjugated albumin nanoparticles to obtain pH-sensitive tumor-targeted nanoparticles. Among them, mPEGA in the pH-sensitive polymer can not only form a hydration film on the surface of the nanoparticles by virtue of its hydrophilic property, significantly improving the stability and water solubility of the drug, avoiding recognition by the reticuloendothelial system in vivo, and prolonging the half-life of the drug, but also react with FA on the surface of the baicalin folic acid conjugated albumin nanoparticles to generate an amide bond under alkaline conditions, and this bond is easily broken under acidic conditions, causing the dissolution of the nanoparticle skeleton.

[0024] Therefore, in the pH-sensitive tumor-targeted nanoparticles prepared in the embodiments of the present application, the long-chain mPEG exists on the surface of the albumin nanoparticles and has the function of charge inversion; the ligand FA molecule can be hidden in the pH-responsive outer shell to avoid binding to normal cells without folate receptors or with folate receptors. Among them, based on the EPR effect, the pH-sensitive tumor-targeted nanoparticles are first passively accumulated in the acidic tumor microenvironment and respond to its weak acidic pH, the acid-sensitive amide bond connecting mPEG-NH2 and the albumin nanocarrier is hydrolyzed, the long-chain mPEG falls off and degrades the acid-sensitive outer shell, and then it is actively recognized by breast cancer cells overexpressing folate receptors, enters the cells to increase the specific accumulation amount of the drug and improve the bioavailability, thereby reducing the off-target effect at non-specific sites. Thus, it is indicated that the pH-sensitive tumor-targeted nanoparticles have good application prospects in the preparation of drugs for treating breast cancer.

[0025] Additional aspects and advantages of the present application will be given in part in the following description, which will become apparent from the following description, or can be understood through the practice of the present application. Description of the Drawings

[0026] Figure 1 It is a schematic diagram of the synthesis route of the pH-sensitive tumor-targeted nanoparticles in Example 1;

[0027] Figure 2 It is a schematic diagram of the electron microscopy test results of the nanoparticles in Example 2;

[0028] Figure 3 It is a schematic diagram of the particle size and potential test results of the nanoparticles in Example 2;

[0029] Figure 4 It is a schematic diagram of the infrared spectrum test results of the nanoparticles in Example 2;

[0030] Figure 5 It is a schematic diagram of the thermogravimetric analysis test results of the nanoparticles in Example 2;

[0031] Figure 6Schematic diagram of the in vitro release test results of the nanoparticles in Example 2;

[0032] Figure 7 Schematic diagram of the cytotoxicity test results of the nanoparticles in Example 2;

[0033] Figure 8 Schematic diagram of the in vivo efficacy test results of the nanoparticles in Example 2. Detailed implementation manners

[0034] Next, in combination with the embodiments of the present application and the drawings, the technical solutions in the embodiments will be clearly and completely described. Obviously, the embodiments to be described below are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present application.

[0035] It should be understood that when used in this specification and the appended claims, the terms "comprising" and "including" 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.

[0036] 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.

[0037] First, some terms and materials involved in this embodiment will be explained below to facilitate the understanding of those skilled in the art.

[0038] FA: Folic acid.

[0039] BA: Baicalin, and the structural formula of BA is

[0040] NHS: N-Hydroxysuccinimide.

[0041] BSA: Bovine serum albumin.

[0042] mPEG-NH2: Or mPEGA, methoxypolyethylene glycol-amine.

[0043] DCA: 3,4,5,6-Tetrahydrophthalic anhydride.

[0044] EDC·HCl: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0045] BA@BSANPs: Baicalin albumin nanoparticles.

[0046] BA@FABSANPs: Baicalin folic acid conjugated albumin nanoparticles.

[0047] mPEGA-DCA-NHS: pH-sensitive polymer.

[0048] BA@mPEGAFABSANPs: Or Baicalin pH-sensitive folic acid conjugated albumin nanoparticles, namely the target product pH-sensitive tumor-targeted nanoparticles.

[0049] Unless otherwise specified, the water used in this example is ultrapure water.

[0050] The following will detail the pH-sensitive tumor-targeted nanoparticles of this example, their preparation method and uses.

[0051] First, the preparation method of the pH-sensitive tumor-targeted nanoparticles in the first aspect of this example will be described.

[0052] Preparation method

[0053] Those skilled in the art know that Baicalin (BA) has antibacterial, antitumor, antipyretic, sedative and antispasmodic effects, etc. Its antitumor mechanism of action includes inducing apoptosis and differentiation, inhibiting protein synthesis, inhibiting tumor cell metastasis, interfering with cell communication and reversing tumor drug resistance, etc. However, Baicalin has poor water solubility and lipid solubility, so its in vivo bioavailability is limited. Clinically, a sufficiently large dosage is usually required to exert better efficacy. However, increasing the dosage is likely to cause adverse reactions.

[0054] In recent years, albumin has the potential to avoid adverse immune reactions and reduce drug toxicity due to its biodegradability, non-toxicity and non-immunogenicity, and can appropriately target the delivery of a variety of drugs and endogenous molecules, and has great potential in controlling drug delivery. However, the targeting ability of albumin as a carrier is limited. Therefore, it is also necessary to modify or improve the surface of albumin to enhance its active targeting as a carrier.

[0055] In view of this, this example provides a preparation method of pH-sensitive tumor-targeted nanoparticles, and this preparation method includes:

[0056] (1) Dissolve Baicalin in an organic solvent to obtain an organic phase; mix the organic phase with an albumin solution to obtain Baicalin albumin nanoparticles.

[0057] In step (1), after dissolving Baicalin with an organic solvent, mix the organic phase with the albumin solution, thereby encapsulating Baicalin in the albumin solution to obtain Baicalin albumin nanoparticles.

[0058] Subsequently, in the subsequent steps, baicalin albumin nanoparticles will be subjected to various modifications.

[0059] (2) Dissolve folic acid in a buffer solution, add a condensing agent and a carboxyl activating agent, and react to obtain a folic acid activation solution; mix the baicalin albumin nanoparticles with the folic acid activation solution and react to obtain folic acid-conjugated baicalin albumin nanoparticles.

[0060] In this step, a folic acid activation solution is prepared based on folic acid, and the baicalin albumin nanoparticles are mixed with the folic acid activation solution; among them, the FA molecule of folic acid is conjugated with the baicalin albumin nanoparticles to obtain folic acid-conjugated baicalin albumin nanoparticles.

[0061] Specifically, since folate receptors are highly expressed on the membranes of most tumor cells and are hardly expressed in most other normal tissues, tumor cells can be selectively targeted; in step (2), by conjugating FA molecules on the surface of albumin, the targeting property of the nanoparticles is improved.

[0062] (3) Dissolve methoxypolyethylene glycol-amine and 3,4,5,6-tetrahydrophthalic anhydride in a buffer solution and react to obtain mPEGA-DCA; prepare an mPEGA-DCA solution, add a condensing agent and a carboxyl activating agent to the mPEGA-DCA solution to obtain a pH-sensitive polymer.

[0063] The purpose of step (3) is to prepare a pH-sensitive polymer and provide reactants for the subsequent coupling reaction (step 4).

[0064] Specifically, the mPEG-NH2 selected in step (3) has amphiphilicity and good biocompatibility. Using mPEG-NH2 to modify the surface of the nanopreparation can increase the relative molecular mass and reduce renal elimination; at the same time, by virtue of its hydrophilic property, a hydration film can be formed on the surface of the nanopreparation, significantly improving the stability and water solubility of the drug, avoiding recognition by the reticuloendothelial system in the body, and prolonging the half-life of the drug.

[0065] (4) After mixing the pH-sensitive polymer and the folic acid-conjugated baicalin albumin nanoparticles, pH-sensitive tumor-targeting nanoparticles are prepared.

[0066] In step (4), by mixing the pH-sensitive polymer and the folic acid-conjugated baicalin albumin nanoparticles, mPEGA can not only form a hydration film on the surface of the nanoparticles by virtue of its hydrophilic property, significantly improving the stability and water solubility of the drug, avoiding recognition by the reticuloendothelial system in the body, and prolonging the half-life of the drug, but also react with the FA on the surface of the folic acid-conjugated baicalin albumin nanoparticles under alkaline conditions to form an amide bond, which is easily broken under acidic conditions, causing the nanoparticle skeleton to dissolve.

[0067] Specifically, the amide bond is designed as a linker for baicalin as a conjugate. It has not only been proven to have acid-sensitive properties but also can remain relatively stable at pH 7.4, which allows the conjugate to remain stable before reaching the target and rapidly release the drug after reaching the lesion site.

[0068] Therefore, this embodiment essentially optimizes the delivery of baicalin and develops a multifunctional composite baicalin-targeted drug delivery nanoparticle integrating passive targeting, active targeting, and stimulus-responsive mechanisms, which can reduce the toxic and side effects of baicalin from multiple aspects, increase the tumor targeting ability, and thus enhance the anti-tumor ability of baicalin.

[0069] In summary, in the pH-sensitive tumor-targeted nanoparticles prepared in this embodiment, the long-chain mPEG exists on the surface of albumin nanoparticles and has the function of charge inversion; the ligand FA molecules can be hidden in the pH-responsive outer shell to avoid binding to normal cells without folate receptors or with folate receptors. Among them, based on the EPR effect, the pH-sensitive tumor-targeted nanoparticles are first passively accumulated in the acidic tumor microenvironment and respond to its weak acidic pH. The acid-sensitive amide bond connecting mPEG-NH2 and the albumin nanocarrier undergoes hydrolysis, and the long-chain mPEG falls off and degrades the acid-sensitive outer shell. Then, it is actively recognized by breast cancer cells overexpressing folate receptors, enters the cells to increase the specific drug accumulation amount and improve the bioavailability, thereby reducing the off-target effect at non-specific sites. Thus, it is indicated that the pH-sensitive tumor-targeted nanoparticles have good application prospects in the preparation of drugs for treating breast cancer.

[0070] The following will further explain the relevant steps of the above preparation method.

[0071] In step (1), the organic solvent is methanol and / or ethyl acetate.

[0072] It can be understood that the organic solvent can be selected from one of methanol or ethyl acetate, or from any ratio mixture of methanol and ethyl acetate.

[0073] Preferably, the organic solvent is selected from any ratio mixture of methanol and ethyl acetate; more preferably, the organic solvent is selected from an equal volume ratio mixture of methanol and ethyl acetate.

[0074] In step (1), the albumin solution is at least one of human serum albumin solution, bovine serum albumin solution, and recombinant human serum albumin solution.

[0075] Among them, the preparation method of the albumin solution can be prepared by existing known conventional methods. For example:

[0076] Dissolve albumin in ultrapure water to obtain an albumin solution.

[0077] As an implementation manner, the albumin is at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

[0078] Preferably, the albumin is bovine serum albumin (BAS). In this embodiment, BAS is used as an exemplary albumin for verification and illustration.

[0079] Generally, in step (1), the mixing reaction process of the organic phase and the albumin solution is carried out under ultrasonic conditions; after the reaction, usually an evaporator is used to spin (45°C) to remove the excess organic solvent in the reactants, and ultra-pure water dialysis purification is adopted to obtain baicalin albumin nanoparticles with higher purity.

[0080] In steps (2) and (3), the buffer solution is Tris-HCL buffer solution, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the carboxyl activating agent is N-hydroxysuccinimide.

[0081] Exemplarily, the Tris-HCL buffer solution can be a Tris-HCL buffer solution with a pH of 8.5 - 9.6 and a concentration of 1M.

[0082] In step (3), the mPEGA-DCA solution is an aqueous solution, and the mPEGA-DCA solution can be prepared by existing known preparation methods. For example:

[0083] After preparing mPEGA-DCA, dissolve mPEGA-DCA in ultra-pure water to prepare the mPEGA-DCA solution.

[0084] Exemplarily, the concentration of the mPEGA-DCA solution is 70 - 90 mg / mL. For example, the concentration can be selected as 70 mg / mL, 72 mg / mL, 74 mg / mL, 75 mg / mL, 78 mg / mL, 80 mg / mL, 81 mg / mL, 83 mg / mL, 84 mg / mL, 85 mg / mL, 86 mg / mL, 87 mg / mL, 88 mg / mL, 89 mg / mL, 90 mg / mL.

[0085] In steps (2) and (3), after preparing the corresponding target product, dialysis is usually carried out.

[0086] For example, in step (2), mix the baicalin albumin nanoparticles with the folic acid activation solution, stir with a magnetic stirrer for 8 h, and then dialyze with 10 mmol / L NaHCO3 solution for 12 - 24 h and ultra-pure water for 12 - 24 h to obtain the baicalin folic acid conjugated albumin nanoparticles.

[0087] In step (4), after mixing the pH-sensitive polymer and the folic acid-conjugated albumin nanoparticles of baicalin, the pH-sensitive tumor-targeting nanoparticles are prepared as follows:

[0088] After mixing the pH-sensitive polymer and the folic acid-conjugated albumin nanoparticles of baicalin, the mixture is stirred by a magnetic stirrer, and then dialyzed with 10 mmol / L NaHCO3 solution and ultrapure water to obtain the pH-sensitive tumor-targeting nanoparticles.

[0089] Among them, the stirring time by the magnetic stirrer is 2 - 10 h, such as 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h.

[0090] The dialysis time with NaHCO3 is 12 - 36 h, and the dialysis time with ultrapure water is 12 - 36 h; such as 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 35 h, 36 h.

[0091] As a preferred embodiment, the mass ratio of the pH-sensitive polymer to the folic acid-conjugated albumin nanoparticles of baicalin is 2 - 3:1. For example, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1.

[0092] Generally, in the above steps (1)-(4), stirring is carried out during the reaction process to accelerate the reaction or make the reaction more complete.

[0093] In this example, unless otherwise specified, the relevant reaction processes of this example can be carried out at room temperature.

[0094] Next, the synthesis process of the pH-sensitive tumor-targeting nanoparticles will be further clarified in combination with the specific preparation method.

[0095] Exemplarily, the preparation method of the pH-sensitive tumor-targeting nanoparticles includes:

[0096] (1) Preparation of baicalin albumin nanoparticles (BA@BSANPs).

[0097] Precisely weigh 200.0 mg of BSA and dissolve it in 10 mL of ultrapure water to prepare the aqueous phase; precisely weigh 10.0 mg of baicalin (BA) and dissolve it in 2 mL of methanol and 2 mL of ethyl acetate organic solvents to prepare the organic phase;

[0098] The organic phase was injected into the aqueous phase with a 2.5 mL syringe, and ultrasonicated for 8 min under ice bath conditions. After the ultrasonication, the primary emulsion was quickly placed in a rotary evaporator to remove the organic solvent at 45 °C, and dialyzed with ultrapure water for 12 - 24 h to obtain baicalin albumin nanoparticles (BA@BSANPs).

[0099] (2) Preparation of baicalin folic acid conjugated albumin nanoparticles (BA@FABSANPs).

[0100] 25 mg of folic acid was dissolved in 25 mL of 1 M Tris-HCL (pH 9.6) solution, and 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 12 mg of N-hydroxysuccinimide (NHS) were added. The mixture was stirred at room temperature for 12 h to prepare the folic acid activation solution, which was stored temporarily at 4 °C.

[0101] The BA@BSANPs prepared in step (1) were taken, and the folic acid activation solution was added. The molar ratio of folic acid to albumin was 10:1; the mixture was stirred with a magnetic stirrer for 8 h, dialyzed with 10 mmol / L NaHCO3 solution for 24 h, and then dialyzed with ultrapure water for 12 - 24 h to obtain baicalin folic acid conjugated albumin nanoparticles (BA@FA BSANPs).

[0102] (3) Preparation of pH-sensitive polymer (mPEGA-DCA-NHS).

[0103] 1.0 g of mPEG-NH2 (mPEGA) and 82.0 mg of 3,4,5,6-tetrahydrophthalic anhydride (DCA) were dissolved in 10 mL of 1 M Tris-HCL (pH 8.8) and stirred with a magnetic stirrer for 12 hours. Then the mixture was dialyzed with 3KDa for 12 h to obtain the mPEGA-DCA polymer, and the dialyzed solution was freeze-dried.

[0104] Then EDC·HCl (150 mg) and NHS (100 mg) were dissolved in 8 mL of 80 mg / mL mPEGA-DCA solution and stirred with a magnetic stirrer at room temperature for 10 hours. The mixture was dialyzed with 3KDa for 12 h to prepare the pH-sensitive polymer with activated -COOH (i.e., mPEGA-DCA-NHS). The freeze-dried mPEGA-DCA polymer was dissolved in water to make an 80 mg / mL mPEGA-DCA solution.

[0105] (4) Preparation of baicalin pH-sensitive folic acid conjugated albumin nanoparticles (BA@mPEGA FA BSANPs).

[0106] Take the BA@FABSANPs prepared in step (2), add the activated pH-sensitive polymer. The mass ratio of the pH-sensitive polymer to BA@FABSANPs is 2:1. Stir with a magnetic stirrer for 8 h, dialyze with 10 mmol / L NaHCO3 solution for 24 h and then with ultrapure water for 24 h to obtain baicalin pH-sensitive folic acid-conjugated albumin nanoparticles (BA@mPEGA FA BSANPs).

[0107] Secondly, the pH-sensitive tumor-targeting nanoparticles of the second aspect of this embodiment will be described.

[0108] pH-sensitive tumor-targeting nanoparticles

[0109] Based on the description of the first aspect, the pH-sensitive tumor-targeting nanoparticles (or baicalin pH-sensitive folic acid-conjugated albumin nanoparticles) prepared in this embodiment are obtained by mixing the pH-sensitive polymer and baicalin folic acid-conjugated albumin nanoparticles. mPEGA can not only form a hydrated film on the surface of the nanoparticles by virtue of its hydrophilic property, significantly improving the stability and water solubility of the drug, avoiding recognition by the reticuloendothelial system in vivo and prolonging the half-life of the drug, but also react with FA on the surface of baicalin folic acid-conjugated albumin nanoparticles to generate amide bonds under alkaline conditions. These bonds are easily broken under acidic conditions, causing the dissolution of the nanoparticle skeleton. Thus, the pH-sensitive tumor-targeting nanoparticles remain stable before reaching the target and rapidly release the drug after reaching the lesion.

[0110] In summary, in the pH-sensitive tumor-targeting nanoparticles prepared in this embodiment, long-chain mPEG exists on the surface of the albumin nanoparticles and has the function of charge inversion; the ligand FA molecules can be hidden in the pH-responsive outer shell to avoid binding to normal cells without folate receptors or with folate receptors. Among them, based on the EPR effect, the pH-sensitive tumor-targeting nanoparticles are first passively accumulated in the acidic tumor microenvironment and respond to its weak acidic pH. The acid-sensitive amide bond connecting mPEG-NH2 and the albumin nanocarrier undergoes hydrolysis, and the long-chain mPEG falls off and degrades the acid-sensitive outer shell, and then is actively recognized by breast cancer cells overexpressing folate receptors, enters the cells to increase the specific accumulation amount of the drug and improve the bioavailability, thereby reducing the off-target effect at non-specific sites.

[0111] In summary, the pH-sensitive tumor-targeting nanoparticles prepared in this embodiment have many advantages such as high targeting, controlled drug release, improving the solubility and absorption rate of poorly soluble drugs, and improving (overcoming) the drug resistance of TAM in the treatment of breast cancer. Therefore, they can enhance the efficacy of the drug and reduce the toxic and side effects, showing broad application prospects in the field of breast cancer treatment.

[0112] Next, the use of the pH-sensitive tumor-targeting nanoparticles of the third aspect of this embodiment will be described.

[0113] Use of pH-Sensitive Tumor-Targeting Nanoparticles

[0114] As described in the second aspect, the pH-sensitive tumor-targeting nanoparticles prepared in this example have multiple advantages, which can enhance the efficacy of drugs and reduce toxic and side effects.

[0115] Based on this, the pH-sensitive tumor-targeting nanoparticles provided in this example can be used to prepare drugs for preventing, alleviating or treating breast cancer.

[0116] It should be particularly noted that the pH-sensitive tumor-targeting nanoparticles of this example can improve (overcome) the drug resistance of TAM in the treatment of breast cancer.

[0117] As is known to those skilled in the art, after breast cancer patients receive tamoxifen (Tamoxifen, abbreviated as TAM) treatment, tumor cells gradually lose sensitivity to tamoxifen, resulting in a decrease or disappearance of the therapeutic effect of the drug, that is, the drug resistance of TAM in the treatment of breast cancer.

[0118] Through relevant experiments in this example, it is found that the pH-sensitive tumor-targeting nanoparticles of this example can improve (overcome) the drug resistance of TAM in the treatment of breast cancer, indicating that it can be made into a drug with corresponding effects.

[0119] Exemplarily, the above-mentioned drug is an injectable solution or a drug for oral administration.

[0120] Generally, when the above-mentioned pH-sensitive tumor-targeting nanoparticles are made into corresponding drugs, the drugs also include pharmaceutically acceptable carriers and / or adjuvants.

[0121] For the drug described in this example, the drug uses the pH-sensitive tumor-targeting nanoparticles as the active ingredient, and does not exclude changes in the formulation system and administration methods, medicinal salts after simple chemical modification and adjustment of the above-mentioned pH-sensitive tumor-targeting nanoparticles, and the combination of multiple compounds.

[0122] For example, in this example, one or more compounds in the pH-sensitive tumor-targeting 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.

[0123] For example, when the dosage form of the drug in this embodiment is a drug for oral administration, it contains a safe and effective amount of pH-sensitive tumor-targeting nanoparticles, as well as pharmaceutically acceptable carriers and / or adjuvants. The drug for oral administration can be made into common dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, suppositories, etc. In this embodiment, no specific limitations are imposed on the carriers and / or adjuvants, and the carriers and / or adjuvants can be adaptively adjusted according to the specific drug dosage form.

[0124] Generally, the "effective amount" of a compound (pH-sensitive tumor-targeting nanoparticles) refers to the amount sufficient to cause a target biological response. As understood by those of ordinary skill in the art, the effective amount of the compound in this embodiment can be changed according to the following factors: for example, components such as the vehicle in the drug, as well as the age, health condition of the subject, and breast cancer symptoms.

[0125] Among them, the effective amount includes a therapeutically effective amount and a prophylactically effective amount.

[0126] Unless otherwise specified, the "therapeutically effective amount" of the compound used in this embodiment is the amount sufficient to provide benefits during the treatment of breast cancer, or the amount that minimizes the improvement or remission of one or more symptoms (manifestations) related to the breast cancer state. The "prophylactically effective amount" of the compound used in this embodiment is the amount sufficient to prevent the occurrence of breast cancer, or the amount sufficient to prevent one or more symptoms related to the occurrence of breast cancer state.

[0127] It can be understood that the drug of this embodiment can also be made into an injection. For example, the pH-sensitive tumor-targeting nanoparticles can be made into corresponding injections with water for injection, normal saline, and glucose water under a sterile operating environment, and the above injections can be prepared by conventional methods.

[0128] Next, specific embodiments will be combined to further elaborate on this application. It should be understood that these embodiments are only used to illustrate / explain this application and not to limit the scope of this application.

[0129] In the following embodiments, the materials, reagents, and instruments used can be obtained from commercial sources without special instructions.

[0130] Example 1

[0131] This embodiment provides a preparation method of pH-sensitive tumor-targeting nanoparticles.

[0132] Please refer to Figure 1 the synthetic route diagram, and the preparation method of the pH-sensitive tumor-targeting nanoparticles in this embodiment includes:

[0133] (a) Preparation of baicalin albumin nanoparticles (BA@BSANPs).

[0134] Accurately weigh 200.0 mg of BSA and dissolve it in 10 mL of ultrapure water to prepare the aqueous phase; accurately weigh 10.0 mg of baicalin (BA) and dissolve it in 2 mL of methanol and 2 mL of ethyl acetate organic solvents to prepare the organic phase;

[0135] Inject the organic phase into the aqueous phase with a 2.5 mL syringe, ultrasonicate for 8 min under ice bath conditions, and quickly place the primary emulsion in a rotary evaporator to evaporate the organic solvent at 45 °C after ultrasonication. Dialyze with ultrapure water for 12 h to obtain baicalin albumin nanoparticles (BA@BSANPs).

[0136] (b) Preparation of folic acid-conjugated baicalin albumin nanoparticles (BA@FABSANPs).

[0137] Dissolve 25.0 mg of folic acid in 25 mL of 1 M Tris-HCL (pH 9.6) solution, add 20.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 12 mg of N-hydroxysuccinimide (NHS), and stir at room temperature for 12 h to prepare the folic acid activation solution, and store it temporarily at 4 °C.

[0138] Take the BA@BSANPs prepared in step (1), add the folic acid activation solution, where the molar ratio of folic acid to albumin is 10:1; stir with a magnetic stirrer for 8 h, then dialyze with 10 mmol / L NaHCO3 solution for 24 h, and then dialyze with ultrapure water for 12 h to obtain folic acid-conjugated baicalin albumin nanoparticles (BA@FABSANPs).

[0139] (c) Preparation of pH-sensitive polymer (mPEGA-DCA-NHS).

[0140] Dissolve 1.0 g of mPEG-NH2 (mPEGA) and 82.0 mg of 3,4,5,6-tetrahydrophthalic anhydride (DCA) in 10 mL of 1 M Tris-HCL (pH 8.8), stir with a magnetic stirrer for 12 hours, then dialyze the mixture with 3 KDa for 12 h to obtain the mPEGA-DCA polymer, and freeze-dry the dialyzed solution; dissolve the freeze-dried mPEGA-DCA polymer in water to make an 80 mg / mL mPEGA-DCA solution;

[0141] Then dissolve EDC·HCl (150 mg) and NHS (100 mg) in 8 mL of 80 mg / mL mPEGA-DCA solution, and stir magnetically at room temperature for 10 hours. Dialyze the mixture with 3 KDa for 12 h to obtain the pH-sensitive polymer with activated -COOH (i.e., mPEGA-DCA-NHS).

[0142] (e) Preparation of baicalin pH-sensitive folic acid-conjugated albumin nanoparticles (BA@mPEGAFABSANPs).

[0143] Take the BA@FABSANPs prepared in step (2), add the pH-sensitive polymer in step (3). The mass ratio of the pH-sensitive polymer to BA@FABSANPs is 2:1. Stir with a magnetic stirrer for 8 h, dialyze with 10 mmol / L NaHCO3 solution for 24 h, and then dialyze with ultrapure water for 12 h to obtain baicalin pH-sensitive folic acid-conjugated albumin nanoparticles (BA@mPEGA FABSANPs).

[0144] Comparative Example 1

[0145] (a) Preparation of baicalin albumin nanoparticles (BA@BSANPs).

[0146] Precisely weigh 200.0 mg BSA and dissolve it in 10 mL of ultrapure water to prepare the aqueous phase; precisely weigh 10.0 mg of baicalin (BA) and dissolve it in 2 mL of methanol and 2 mL of ethyl acetate organic solvents to prepare the organic phase.

[0147] Inject the organic phase into the aqueous phase with a 2.5 mL syringe, ultrasonicate for 8 min under ice bath conditions. After ultrasonication, quickly place the primary emulsion in a rotary evaporator to evaporate the organic solvent at 45 °C, and dialyze with ultrapure water for 12 h to obtain baicalin albumin nanoparticles (BA@BSANPs).

[0148] (c) Preparation of pH-sensitive polymer (mPEGA-DCA-NHS).

[0149] Dissolve 1.0 g of mPEG-NH2 (mPEGA) and 82.0 mg of 3,4,5,6-tetrahydrophthalic anhydride (DCA) in 10 mL of 1 M Tris-HCL (pH 8.8), stir with a magnetic stirrer for 12 hours, then dialyze the mixture with 3 KDa for 12 h to obtain the mPEGA-DCA polymer. Dissolve the mPEGA-DCA polymer in water to make an 80 mg / mL mPEGA-DCA solution.

[0150] Then dissolve EDC·HCl (150 mg) and NHS (100 mg) in 8 mL of 80 mg / mL mPEGA-DCA solution, stir magnetically at room temperature for 10 hours. Dialyze the mixture with 3 KDa for 12 h to obtain the pH-sensitive polymer with activated -COOH (i.e., mPEGA-DCA-NHS).

[0151] (d) Preparation of baicalin pH-sensitive albumin nanoparticles (BA@mPEGABSANPs).

[0152] Take the BA@BSANPs prepared in step (1), add the pH-sensitive polymer in step (2), and the mass ratio of the pH-sensitive polymer to BA@BSANPs is 2:1. After stirring for 8 h, dialyze with 10 mmol / mL NaHCO3 solution for 24 h, and then dialyze with ultrapure water for 12 h to obtain BA@mPEGABSANPs.

[0153] Example 2

[0154] Next, the BA@BSANPs, BA@FABSANPs, and BA@mPEGAFABSA NPs prepared in Example 1, and the BA@mPEGABSANPs prepared in Comparative Example 1 will be tested or verified.

[0155] In the following test methods, BA@BSANPs is baicalin albumin nanoparticles, BA@FABSANPs is baicalin folic acid conjugated albumin nanoparticles: BA@mPEGA BSA NPs is baicalin acid-sensitive albumin nanoparticles, and BA@mPEGAFABSANPs is baicalin acid-sensitive folic acid conjugated albumin nanoparticles.

[0156] Specifically, it includes:

[0157] 2.1 Characterization of nanoparticles

[0158] 2.1.1 Use a laser particle size analyzer (ZS90, Malvern, UK) to measure the particle size, PDI, and zeta potential of BA@BSANPs, BA@FA BSA NPs, BA@mPEGA BSA NPs, and BA@mPEGA FA BSANPs (hereinafter referred to as samples). The morphology of the nanoparticles was evaluated by staining the samples with 2% phosphotungstic acid (at pH 7.4 and 5.5 respectively) and observing them under a transmission electron microscope (TECNAI 10, Thermo Fisher, USA).

[0159] Please continue to refer to Figure 2 , Figure 2 showing the transmission electron microscope photos of each sample (at pH 7.4 and 5.5 respectively, with a scale bar of 100 nm), Figure 2 The transmission electron microscope images of

[0160] Specifically, the outermost layer of BA@mPEGABSANPs and BA@mPEGAFABSANPs nanoparticles can be clearly seen to be coated with mPEGA. Among them, they maintain a stable state under neutral conditions of pH 7.4, and the nanoparticles rupture significantly under weak acidic conditions, but BA@BSANPs and BA@FABSANPs are relatively stable under both pH conditions, suggesting that BA@mPEGABSANPs and BA@mPEGAFABSANPs have acid-sensitive properties and may be easily released quickly in the acidic microenvironment of tumors.

[0161] Please continue reading Figure 3 , Figure 3 A shows the schematic diagram of the particle size test results of each sample. Figure 3 B shows the schematic diagram of the Zeta potential test results of each sample. Figure 3 A and Figure 3 As shown in B, the particle sizes of BA@BSANPs, BA@FABSANPs, BA@mPEGABSANPs and BA@mPEGAFABSANPs prepared in this example are 89.63±3.95, 104.90±0.76, 95.57±2.52 and 112.70±3.09 nm, respectively; the PDIs are 0.13±0.02, 0.14±0.03, 0.15±0.03 and 0.09±0.01, respectively; the potentials of BA@BSANPs, BA@FABSANPs, BA@mPEGABSANPs and BA@mPEGAFABSANPs are -13.60±0.91, -13.00±1.35, -13.30±0.53 and -15.70±1.42 mV, respectively.

[0162] In summary, the four nanoparticles prepared in this example all have a particle size between 80 and 120 nm, a PDI of <0.2, and a potential between -13 and -16 mV. This size is easy to be enriched in the tumor area through the enhanced permeability retention effect (EPR).

[0163] 2.1.2 BA in BA solution and nanoparticles (hereinafter referred to as sample) was determined on a C18 column (250×4.6 mm, 5 μm; Agilent, USA) at 25°C using HPLC; the injection volume was 10 μL, the mobile phase was methanol:0.2% phosphoric acid (47:53), the flow rate was 1.0 mL / min, and the detection wavelength was 280 nm.

[0164] The above-mentioned BA solution refers to the BA standard solution, and the BA in the nanoparticles refers to the BA in the four nanoparticles: BA@BSANPs, BA@FA BSA NPs, BA@mPEGA BSANPs, and BA@mPEGAFA BSANPs.

[0165] Based on the above chromatographic conditions and detection methods, the contents of the encapsulated and free substances in each sample were separated and quantitatively determined, and the encapsulation efficiency (EE) and drug loading (DL) of BA were calculated using the following formulas:

[0166] EE (%) = (M c - M d ) / M c × 100;

[0167] DL (%) = (M c - M d ) × dilution factor / M e × 100.

[0168] Wherein, M c represents the total mass of BA, M d represents the mass of free BA, and M e represents the total mass of BA + the total mass of the carrier.

[0169] The specific test results are as follows:

[0170] The encapsulation efficiencies of BA@BSANPs, BA@FABSANPs, BA@mPEGABSANPs, and BA@mPEGAFABSANPs prepared in this example were 95.06 ± 0.11, 90.72 ± 0.22, 88.96 ± 0.14, and 87.86 ± 0.43%, respectively, and the drug loadings were 3.82 ± 0.04, 2.25 ± 0.01, 0.95 ± 0.00, and 0.56 ± 0.02%, respectively.

[0171] 2.1.3 Fourier transform infrared spectrometer (Nicolet iS50, USA, Thermo Fisher) was used to analyze BA, blank nanoparticles (mPEGAFA BSANPs), BA@BSANPs, BA@FABSANPs, BA@mPEGA BSANPs, BA@mPEGAFA BSANPs, free FA, free mPEGA, and the physical mixture of BA and BA@mPEGA FABSANPs (hereinafter referred to as samples).

[0172] Among them, the morphology of the above samples was powder, and the weight of each sample was about 2 mg.

[0173] It can be understood that this test was to further verify whether BA@BSANPs, BA@FABSANPs, BA@mPEGABSANPs, and BA@mPEGAFABSANPs successfully encapsulated BA.

[0174] Please refer to Figure 4 , Figure 4Schematic diagram showing the infrared spectroscopy test results of various samples, Figure 4 where a is BA, b is BA@BSANPs, c is BA@FABSANPs, d is BA@mPEGABSANPs, e is mPEGAFABSA NPs, f is FA, g is mPEGA, h is BA@mPEGAFABSANP, and i is the Fourier infrared spectrum of the physical mixture of BA and BA@mPEGAFA BSANPs.

[0175] It can be seen from Figure 4 that the characteristic absorption peaks of BA are, respectively, (OH) at 3390 cm -1 (C=O) at 1657 cm -1 (C=O) at 764 cm -1 and the benzene ring at 682 cm -1 (C=O) at 1725 cm -1 (C-O-C) glycosidic bond at 1066 cm -1 (C-O-C) glycosidic bond at 912 cm -1 (C-O-C) glycosidic bond at 1066 cm -1 The prepared BA@BSANPs, BA@FABSANPs, BA@mPEGABSANPs, and BA@mPEGA FABSANPs all do not have the BA characteristic absorption peaks at 764 cm -1 (C-O-C) glycosidic bond at 682 cm -1 (C-O-C) glycosidic bond at 1725 cm -1 (C-O-C) glycosidic bond at 1066 cm -1 (C-O-C) glycosidic bond at 912 cm -1 (C-O-C) glycosidic bond at 1066 cm -1 (C-O-C) glycosidic bond ( Figure 4 b, c, d, e, h in).

[0176] Specifically, the BA characteristic absorption peak can also be seen in BA + BA@mPEGAFABSANPs ( Figure 4 i in), indicating that BA has been successfully encapsulated by the four types of nanoparticles. It can be seen from Figure 4 f in that the infrared characteristic peaks of FA are mainly at 1696 cm -1 (C=O) at 1606 cm -1 and 1485 cm -1 respectively corresponding to the carboxyl group in FA, the amino group on the pteridine ring, and the vibration absorption peaks of C=C or C=N. For BA@FABSANPs, mPEGAFABSANPs, and BA@mPEGAFABSANPs ( Figure 4 c, e, h in), they are at 1654 cm -1A new characteristic peak appeared at [specific position], which belongs to the amide bond, indicating that FA was successfully loaded on the surfaces of BA@FABSANPs, mPEGAFABSANPs, and BA@mPEGAFABSANPs.

[0177] From Figure 4 at g in [reference] it can be seen that the peak at 1112 cm -1 is the characteristic absorption peak of the ether in mPEGA; the broad absorption peaks at 3430 cm -1 are respectively the O-H stretching vibration of carboxylic acid and the N-H stretching vibration of secondary amide. In the spectra of BA@mPEGABSANPs, mPEGAFABSANPs, BA@mPEGAFABSANPs, BA + BA@mPEGAFABSANPs ( Figure 4 at d, e, h, i in [reference]) an absorption peak appeared at 1654 cm -1 , indicating the presence of the amide bond; at the same time, the characteristic absorption peak of the ether at 1112 cm -1 indicates that mPEGA was successfully linked to BA@mPEGABSANPs, mPEGAFABSANPs, BA@mPEGAFABSANPs, and BA + BA@mPEGAFABSANPs.

[0178] 2.1.4 Use a thermogravimetric analyzer (TGA / DSC 3+, Switzerland, Mettler Toledo) to measure the thermal stability of BA, BA@BSANPs, BA@FABSA NPs, BA@mPEGA BSANPs, and BA@mPEGA FA BSANPs. The weight of each sample is 4 - 9 mg; the temperature range is 25 - 600 °C, and the thermogravimetric (TG) data is recorded at a nitrogen flow rate of 10 °C / min.

[0179] To verify the thermal stability of the prepared nanoparticles, in this example, the TG data of BA, BA@BSANPs, BA@FABSA NPs, BA@mPEGABSANPs, and BA@mPEGAFABSANPs were analyzed synchronously.

[0180] Please refer to Figure 5 , Figure 5 which shows the schematic diagram of the thermogravimetric analysis test results of each sample; from Figure 5 it can be seen that the degradation of BA is divided into 3 stages. The initial degradation temperature is when the weight of BA starts to decrease at 51 °C, and the weight loss rate of 3.37% is due to water loss; it starts to degrade at 179 °C, and the weight loss rate is 22.73%; after 264 °C, the weight has been decreasing. The total weight loss rate is 57.96%.

[0181] Among them, between 40 and 110 °C, the weight loss rates of BA, BA@BSANPs, BA@FABSANPs, BA@mPEGABSA NPs, and BA@mPEGAFABSANPs were 1.73, 3.60, 4.23%, 2.76, and 1.29% respectively. The reason is that BSA and FA are sensitive to temperature and have poor stability at high temperatures. The mPEGA modified on the outermost layer of BA@mPEGAFABSANPs can improve the stability of the nanoparticles. Therefore, the BA@mPEGAFA BSANPs prepared in this example have good thermal stability.

[0182] 2.1.5 The dialysis method described in the literature (Bai X, Lin Y, Gong L, Duan J, Sun X, Wang C, Liu Z, Jiang J, Zhou X, Zhou M, Zhang Z, Liu Z, Jing P, Zhong Z. Nanoparticles that target the mitochondria of tumor cells to restore oxygen supply for photodynamic therapy: Design and preclinical validation against breast cancer. J Control Release. 2023 Oct;362:356 - 370.) was used to measure the release of BA in nanoparticles in vitro. BA@BSANPs, BA@FA BSA NPs, BA@mPEGA BSA NPs, and BA@mPEGAFABSANPs were placed in dialysis bags, and the dialysis bags were immersed in phosphate buffer containing 0.3% Tween - 80 (pH 7.4, 6.5, or 5.5) and stirred at 37 °C and 100 rpm. 1 mL of samples were taken at 1, 2, 4, 8, 12, 24, 36, and 48 h respectively, and an equal volume of PBS was added to the dialysis bags. After passing through a 0.22 - um filter membrane (Guangzhou Jet Bio - filtration Co., Ltd., Guangzhou), the cumulative release amount of BA was detected by the chromatographic method described in 2.1.2 above.

[0183] Please refer to Figure 6 , Figure 6 which shows the schematic diagram of the in vitro release test results of BA in BA@BSANPs, BA@FABSANPs, BA@mPEGA BSANPs, and BA@mPEGAFABSANPs.

[0184] From Figure 6As shown by the test results of D, the cumulative release amount of BA@mPEGAFABSANPs was 73.19% at pH 5.5 for 24 h, 39.05% at pH 6.5, but there was no obvious drug release of BA at pH 7.4, only 10.66%, showing obvious pH-dependent drug release. This may be attributed to the modification of the outermost mPEGA. mPEGA reacts with FA on the surface of BA@FABSANPs to form an amide bond under alkaline conditions, and this bond is easily broken under acidic conditions, causing the dissolution of the nanoparticle skeleton.

[0185] The release of BA@mPEGABSANPs ( Figure 6 C) was similar to that of BA@mPEGAFABSANPs. The release of BA@BSA NPs and BA@FABSANPs was relatively stable at pH 5.5, 6.5, and 7.4 ( Figure 6 A and 6B).

[0186] It can be understood that efficient and controllable drug release behavior is particularly important for enhancing the anti-cancer activity of anti-cancer drug delivery. The in vitro release behavior of BA@mPEGAFABSANPs prepared in this example indicates that it maintains a stable state in the blood circulation. When it reaches the tumor part, due to the acidic microenvironment of the tumor microenvironment, the nanoparticles can rapidly release at this time, thereby improving the pharmacological activity of BA.

[0187] Therefore, the BA@mPEGAFABSANPs prepared in this example is actually a baicalin-targeted drug delivery nanoparticle that remains stable before reaching the target and rapidly releases drugs after reaching the lesion, and it can be used as an excellent delivery system targeting the acidic microenvironment of tumors.

[0188] 2.2 Cytotoxicity

[0189] The in vitro cytotoxicity test of this example used the CCK-8 method. Normal breast cancer cells MCF-7 and tamoxifen-resistant breast cancer cells LCC9 were inoculated in 96-well plates (5.0×10 3 cells / well), cultured for 24 h to allow them to adhere, and then MCF-7 cells were administered with different concentrations of TAM and BA (0.78 - 100 μg / mL), and blank medium and blank cells were set as controls. Different concentrations of TAM, BA, TAM+BA, TAM+BA@BSANPs, TAM+BA@FA BSA NPs, TAM+BA@mPEGABSANPs, TAM+BA@mPEGAFABSANPs (0.78 - 100 μg / mL) were administered to LCC9 cells, and blank medium and blank cells were set as controls.

[0190] Among them, the concentration of TAM in the TAM co - administration group was 1 μg / mL. 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 put back into 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).

[0191] The calculation formula for cytotoxicity is as follows:

[0192] Cell viability (%) = (OD a - OD b ) / (OD c - OD b ) × 100;

[0193] Resistance index (RI) = IC50 of TAM against drug - resistant cells / IC50 of TAM against sensitive cells;

[0194] Among them, OD a is the OD value of the sample to be tested (containing cells, CCK - 8 and drug - containing DMEM medium), OD c is the OD value of the control group (containing DMEM medium and CCK - 8 solution), and OD b is the OD value of the blank well (containing cells, DMEM medium and CCK - 8 solution).

[0195] Please refer to Figure 7 , Figure 7 which shows the test results of cytotoxicity; Figure 7 In it, A: are the cell viability graphs of MCF - 7+TAM, LCC9+TAM and LCC9+BA+TAM respectively. B: are the cell viability graphs of MCF - 7+BA and LCC9+BA respectively. C: are the cell viability graphs of LCC9 cells administered with mPEGAFABSANPs, BA@BSANPs, BA@FABSANPs, BA@mPEGABSANPs, BA@mPEGAFABSANPs under the conditions of pH 7.4 and 6.5. The data are expressed as mean ± SD (n = 3).

[0196] In this example, a CCK - 8 kit was used to detect the cytotoxicity of TAM against MCF - 7 and LCC9 cells respectively, and the IC 50 of TAM against MCF - 7 and LCC9 cells were calculated to be 1.855 and 24.84 μg / mL respectively( Figure 7 A), the resistance index was 13.39, and the IC 50 of BA against MCF - 7 and LCC9 cells was similar, being 210.8 and 229.6 μg / mL respectively( Figure 7B).

[0197] To prove whether BA can enhance the anti-tumor ability of TAM in LCC9 cells, in this example, BA and TAM were used to treat tumor cells together, showing that the combination of BA and TAM increased the sensitivity of TAM, and the IC 50 of TAM decreased from 24.84 to 2.588 μg / mL( Figure 7 A).

[0198] To improve the bioavailability of BA, BA was prepared into BA@BSANPs, BA@FABSANPs, BA@mPGEABSANPs, and BA@mPGEAFABSANPs respectively, and the IC 50 of LCC9 cells was detected in combination with TAM. As shown in Figure 7 C, when the pH was 7.4, the cell IC 50 values were 2.333, 1.978, 186.3, and 154.9 μg / mL respectively. When the pH was 6.5 (weakly acidic environment), the cell IC50 values were 2.412, 2.053, 1.585, and 1.128 μg / mL respectively. The IC 50 of BA@mPGEA BSANPs and BA@mPGEAFABSANPs under the weakly acidic condition of pH 6.5 was lower than that under the condition of pH 7.4, indicating that the prepared BA@mPGEABSANPs and BA@mPGEAFABSANPs have acid sensitivity and are conducive to drug release in the tumor acidic microenvironment.

[0199] In addition, this example further evaluated the cytotoxicity of the mPGEAFABSANPs carrier( Figure 7 C). There was no obvious cytotoxicity to LCC9 cells under the conditions of pH 7.4 and pH 6.5, indicating that the mPGEAFABSANPs carrier is safe. Therefore, the combined administration of BA@mPGEAFABSANPs and TAM has good efficacy and safety against the growth of LCC9 cells.

[0200] 2.3 In vivo efficacy

[0201] A tamoxifen breast cancer drug resistance model was constructed, and 1×10 7LCC9 cells. All nude mice were randomly divided into 11 groups. TAM was administered by intraperitoneal injection, and the nanoparticles were administered by intravenous injection. The groups were NS group (blank control group, injected with normal saline), mPEGA FABSANPs group, BA group, TAM group, TAM+BA group, TAM+BA@BSANPs group, TAM+BA@FA BSANPs group, TAM+BA@mPEGA BSANPs group, low-dose TAM+BA@mPEGA FA BSANPs group, medium-dose TAM+BA@mPEGA FA BSANPs group, and high-dose TAM+BA@mPEGA FA BSANPs group.

[0202] Among them, the dosing doses of the above groups according to the crude drug amount were as follows: mPEGA FABSANPs group: 6 mg / kg, BA group: 6 mg / kg, TAM group: 20 mg / kg, TAM+BA group: 6 mg / kg, BA@BSANPs group: 6 mg / kg, BA@FA BSANPs group: 6 mg / kg, BA@mPEGA BSANPs group: 6 mg / kg, low-dose BA@mPEGAFA BSA NPs group: 3 mg / kg, medium-dose TAM+BA@mPEGA FABSA NPs group: 6 mg / kg, high-dose TAM+BA@mPEGA FA BSA NPs group: 9 mg / kg, and the TAM dose for combined TAM administration was 20 mg / kg.

[0203] Injections were given once every 3 days for a total of 7 times. Before each injection, the tumor size was measured with a vernier caliper, and the body weight of the nude mice was recorded. Specifically, the following formula (1) was used to calculate the tumor volume. On the 22nd day of treatment, the nude mice were sacrificed, the tumor tissues were removed, weighed, and photographed, and the tumor inhibition rate was calculated using formula (2).

[0204] Tumor volume = ab 2 / 2 (1)

[0205] Tumor inhibition rate (%) = (V NS -V 治疗 ) / V NS × 100 (2)

[0206] In the above formulas: a is the long diameter of the tumor, and b is the short diameter of the tumor. VNS is the volume of the nude mice in the group injected with normal saline, and Vtreatment is the volume of the nude mice in other groups except the NS group.

[0207] This example evaluated the effects of TAM+BA@BSANPs, TAM+BA@FABSANPs, TAM+BA@mPEGA BSANPs, and TAM+BA@mPEGAFABSANPs in nude mice with xenograft tumors. Please refer to Figure 8 ,Figure 8 The test results of in vivo pharmacodynamics are shown. Figure 8 Among them, A is the change in tumor volume during the experiment. B is the change in tumor weight. C is the tumor weight after the nude mice were euthanized. D is the tumor inhibition rate. E is the tumor tissue diagram after the nude mice were euthanized. F is the process of LCC9 cell inoculation and drug administration.

[0208] The above data are expressed as mean ± SD (n = 3). Figure 8 Among them, ****P < 0.0001, ***P < 0.001, **P < 0.01, obtained by comparison with BA@mPEGAFABSANPs.

[0209] From Figure 8 the test results, the order of the tumor inhibitory effects of the nanoparticles is BA@mPEGA FA BSA NPs > TAM + BA@mPEGA BSA NPs > TAM + BA@FA BSANPs > TAM + BA@BSANPs. Among them, the inhibition rate of the medium-dose group of BA@mPEGAFABSANPs reached 82.05% (P < 0.0001, compared with the other three formulations), which confirmed the effectiveness of the FA-conjugated albumin nanoparticles modified with mPEGA on the surface and reflected the importance of targeting the tumor acidic microenvironment.

[0210] In summary, in this example, by modifying the pH-sensitive polymer mPEGA and FA on the surface of albumin nanoparticles and loading baicalin, BA@mPEGAFABSANPs were successfully prepared to overcome the drug resistance of TAM in the treatment of breast cancer. Compared with BA@BSANPs, BA@FABSANPs, and BA@mPEGABSANPs, the sensitive BA@mPEGAFA BSANPs have more excellent biological properties. Because it has better in vivo release behavior under weak acidic conditions (pH 5.5) similar to the pH of the tumor acidic microenvironment, and the cumulative release amount reaches 73.19 in 24 h. The in vivo cytotoxicity experiment showed that BA@mPEGAFABSANPs combined with TAM administration to LCC9 cells (IC 50 is 1.128 ug / mL) can significantly improve the killing ability of TAM (compared with the IC 50 of 24.84 ug / mL for TAM administered alone). And it was verified that the mPEGAFABSA NPs carrier has good safety. The in vivo experiment showed that the tumor inhibition rate of the medium-dose group of BA@mPEGAFABSANPs co-administered with TAM can reach 82.06%.

[0211] Therefore, the BA@mPEGAFABSANPs prepared in this example can be used as a baicalin-targeted drug delivery nanoparticle that remains stable before reaching the target and rapidly releases drugs after reaching the lesion, and can also be used as a promising ideal preparation for reversing TAM-induced breast cancer drug resistance.

[0212] The technical solutions provided in the embodiments of the present application have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present application. The descriptions of the above embodiments are 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, based on 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 method for preparing pH-sensitive tumor-targeting nanoparticles, characterized in that: The preparation method comprises: dissolving baicalin in an organic solvent to obtain an organic phase; mixing the organic phase with an albumin solution to obtain baicalin albumin nanoparticles; Dissolving folic acid in a buffer solution, adding a shrinking agent and a carboxyl activator, and reacting to obtain a folic acid activation solution; mixing baicalin albumin nanoparticles with the folic acid activation solution, and reacting to obtain baicalin folic acid-coupled albumin nanoparticles; Dissolving methoxy polyethylene glycol-amino and 3,4,5,6-tetrahydrophthalic anhydride in a buffer solution to react and obtain mPEGA-DCA; preparing an mPEGA-DCA solution, adding a shrinking agent and a carboxyl activator to the mPEGA-DCA solution to obtain a pH-sensitive polymer; The pH-sensitive polymer and baicalin folic acid-coupled albumin nanoparticles were mixed to prepare pH-sensitive tumor-targeting nanoparticles.

2. The preparation method according to claim 1, characterized in that: The organic solvent is methanol and / or ethyl acetate.

3. The preparation method according to claim 1, characterized in that: The buffer solution is Tris-HCL buffer.

4. The preparation method according to claim 1, characterized in that: The shrinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

5. The preparation method according to claim 1, characterized in that: The carboxyl activator is N-hydroxysuccinimide.

6. The preparation method according to claim 1, characterized in that: The albumin solution is at least one of a human serum albumin solution, a bovine serum albumin solution, and a recombinant human serum albumin solution.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the pH sensitive polymer to the baicalin folic acid coupled albumin nanoparticles is 2 to 3:

1.

8. A pH-sensitive tumor-targeting nanoparticle, characterized in that: The pH-sensitive tumor-targeting nanoparticles are prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the pH-sensitive tumor-targeting nanoparticles according to claim 8 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 used to improve TAM treatment of breast cancer resistance.

Images