Folate receptor-mediated manganese ion coordination type cabazitaxel-loaded albumin nanoparticles as well as preparation method and application of folate receptor-mediated manganese ion coordination type cabazitaxel-loaded albumin nanoparticles

Abstract

The invention discloses folate receptor mediated manganese ion coordination type cabazitaxel-loaded albumin nanoparticles as well as a preparation method and application thereof. Human serum albumin is covalently linked with folic acid through an amide condensation reaction to obtain a folic acid-albumin modifier with folic acid targeting; further combining with manganese ions through metal coordination to form a folic acid-albumin-manganese ion modifier with a targeting function; finally, efficient loading of cabazitaxel is achieved through a simple heating polymerization method, and the folate receptor mediated cabazitaxel-loaded albumin nanoparticles are formed. According to the nanoparticles provided by the invention, the particle size is about 140 nm, the stability is good, and the drug loading capacity and the encapsulation efficiency of cabazitaxel are high; the compound has a slow release effect, is good in in-vitro release behavior, and has important application value in the aspect of targeted delivery of drugs to tumors. Compared with other traditional nanoparticles, the nanoparticles show a remarkable anti-tumor effect.

Description

Technical Field

[0001] This invention relates to a folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticle, its preparation method, and its application, belonging to the field of targeted anti-tumor drug technology. Background Technology

[0002] Malignant tumors have become one of the major diseases seriously threatening human health. Traditional chemotherapy drugs, due to their non-specific distribution and lack of tumor selectivity, often cause severe systemic toxicity in clinical applications, while also causing significant damage to normal tissues and organs (such as bone marrow and heart). Furthermore, because of the low efficiency of drug accumulation in tumor tissues, increased dosages are required to achieve effective therapeutic concentrations, which further exacerbates adverse reactions and may induce multidrug resistance. Therefore, developing a drug delivery system that can efficiently target tumor tissues, improve drug bioavailability, and reduce systemic toxicity has become an urgent need in the field of cancer treatment.

[0003] Nanoparticles, with their unique size effect and tunable surface physicochemical properties, can selectively accumulate in tumor tissues through enhanced permeability and retention (EPR) effects, thereby improving drug pharmacokinetic behavior and enhancing therapeutic efficacy. Among numerous nanocarriers, albumin (such as human serum albumin HSA or bovine serum albumin BSA) is widely considered an ideal drug delivery carrier due to its excellent biocompatibility, biodegradability, non-immunogenicity, and long circulating half-life. Abraxane®, a paclitaxel-albumin conjugate nanoparticle approved by the US FDA in 2016, was the world's first marketed albumin nanomedicine. Its clinical success not only demonstrated the effectiveness of albumin carriers but also provided important practical evidence for subsequent research. Furthermore, the albumin molecule surface is rich in active functional groups such as carboxyl and amino groups, facilitating the connection of targeting ligands or functional molecules through chemical modification to construct multifunctional nanodelivery systems. In addition, albumin, as an endogenous protein, can further enhance its accumulation at the tumor site by interacting with SPARC protein (a secretory protein that is acidic and rich in cysteine) overexpressed by tumor cells.

[0004] To enhance the recognition and internalization efficiency of nanoparticles on tumor cells, active targeting strategies have been widely applied in the design of nanocarriers. Folic acid is one of the most extensively studied small-molecule targeting ligands. Its advantages include: folic acid has nanomolar affinity for the folic acid receptor (FR); the FR is significantly overexpressed on the surface of various epithelial-derived tumor cells (such as ovarian cancer, breast cancer, lung cancer, and cervical cancer), while its expression level is extremely low or absent in normal tissues; and the folic acid-receptor complex can efficiently enter cells through receptor-mediated endocytosis and is transported via the endosome-lysosome pathway.

[0005] Cabazitaxel is a novel second-generation taxane microtubule stabilizer. Its chemical structure has undergone key modifications compared to paclitaxel and docetaxel (such as methoxylation at C-7 and C-10). This modification significantly reduces its properties as a P-glycoprotein (P-gp) substrate, effectively overcoming the problem of tumor multidrug resistance (MDR) mediated by P-gp overexpression. Clinical studies have demonstrated that cabazitaxel exhibits significant survival benefits in patients with metastatic castration-resistant prostate cancer (mCRPC) who have progressed after docetaxel treatment, and it has become an important second-line treatment option in this field.

[0006] Metal ions can form more stable nanostructures through coordination with albumin, thereby effectively improving the encapsulation efficiency of hydrophobic drugs. Studies have shown that nanoparticles prepared by coordinating zinc ions with albumin exhibit significantly improved encapsulation efficiency of hydrophobic model drugs compared to uncoordinated systems. This invention innovatively selects manganese ions as the coordination center ion for albumin nanoparticles. Manganese ions possess a similar ability to zinc ions to construct stable nanonetworks through coordination with albumin, thereby efficiently encapsulating hydrophobic drugs (such as taxanes). Summary of the Invention

[0007] This invention addresses the problems of poor selectivity of existing chemotherapy drugs and incomplete drug release from traditional nanoparticles. It provides a folic acid receptor-mediated manganese ion-coordinated carbamate albumin-loaded nanoparticle, its preparation method, and its application. The nanoparticle has strong active targeting ability, high encapsulation rate, complete drug release, and can effectively reduce drug toxicity and side effects.

[0008] The technical solution to achieve the objective of this invention is to provide a method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles, comprising the following steps:

[0009] Step 1: Preparation of folic acid-albumin modified product

[0010] Folic acid was dissolved in dimethyl sulfoxide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added. The mixture was activated in the dark, and the precipitate was removed by filtration to obtain a folic acid solution. Albumin solution was dissolved in carbonate buffer at pH 9.0 and then slowly added dropwise to the folic acid solution to obtain the folic acid-albumin modified solution FA-HSA.

[0011] Step 2: Preparation of folic acid-albumin-manganese ion modified compounds

[0012] MnCl2 was added to the folic acid-albumin modified solution prepared in step 1 and reacted at room temperature for 4 h. The reaction solution was placed in a dialysis bag and dialyzed in carbonate buffer at pH 9.0 for 12 h. After freeze drying, folic acid-albumin-manganese ion modified compound FA-HSA@Mn was obtained.

[0013] Step 3: Preparation of drug-loaded nanoparticles

[0014] The folic acid-albumin-manganese ion modified compound prepared in step 2 was dissolved in an aqueous solution. The solution was placed in a 37°C constant temperature stirred water bath and stirred rapidly for 10 min. 1 mg of cabazitaxel was dissolved in 100 µL of anhydrous ethanol and then slowly added to the above solution. After incubation for another 30 min, the solution was rapidly cooled. Free drug and organic solvent were removed by ultrafiltration to obtain folic acid receptor-mediated manganese ion coordinated cabazitaxel-loaded albumin nanoparticles FA-HSA@Mn-Cbz.

[0015] The above-mentioned method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles, wherein the albumin is human serum albumin.

[0016] In the above-mentioned method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate albumin-loaded nanoparticles, in step 1, the volume ratio of dimethyl sulfoxide to pH 9.0 carbonate buffer to human serum albumin solution is 3-7: 3-7: 2-6, and the concentration range of human serum albumin is 10 mg / mL-50 mg / mL.

[0017] In the above-mentioned method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate albumin nanoparticles, in step 1, the molar ratio of folic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:1 to 2:1 to 2.

[0018] In the above-mentioned method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate albumin nanoparticles, in step 3, the volume ratio of water dissolving the folic acid-albumin-manganese ion modifier to anhydrous ethanol dissolving carbamate is 1:0.05-0.2.

[0019] In the above-mentioned method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate albumin nanoparticles, in steps 1 and 2, the mass ratio of folic acid, albumin, and MnCl2 is 1250:1000:3.

[0020] The above-mentioned method for preparing folic acid receptor-mediated manganese ion-coordinated cabazitaxel-albumin nanoparticles has a folic acid-albumin-manganese ion modifier: cabazitaxel ratio of 10-20:1 by mass.

[0021] Application of folic acid receptor-mediated manganese ion-coordinated carbamate albumin nanoparticles prepared by the above method in pharmaceutical preparation.

[0022] The drug delivery system provided by this invention consists of human serum albumin modified with folic acid and manganese ions and cabazitaxel. The human serum albumin is covalently linked to folic acid via an amide condensation reaction and further binds to manganese ions through metal coordination to form a FA-HSA@Mn composite carrier with targeting function. Finally, cabazitaxel is efficiently loaded using a simple heating polymerization method, constituting a folic acid receptor-mediated cabazitaxel-loaded albumin nanoparticle. This nanoparticle can significantly improve the efficiency of tumor-targeted drug delivery and intracellular accumulation, effectively reduce systemic toxicity, and prolong the drug's circulation time in vivo.

[0023] The principle of this invention lies in the coordination binding of folic acid-modified human serum albumin with manganese ions, and further encapsulating the drug into nanoparticles through intermolecular forces. These nanoparticles are designed to undergo the following processes in vivo: 1. Human serum albumin itself carries a negative charge, which can to some extent prevent it from being recognized and cleared by the reticuloendothelial cell phagocytic system, thereby improving circulatory stability; 2. The nanoparticles have a suitable particle size, which can accumulate at the tumor site through enhanced permeability and retention (EPR) effects, while folic acid modification can actively target the overexpressed folic acid receptors on the surface of tumor cells, enhancing cellular uptake efficiency; 3. The manganese ions introduced into the nanoparticles can enhance drug encapsulation efficiency by regulating protein conformation and promote drug release in the intracellular environment, improving therapeutic efficacy.

[0024] The folic acid receptor-mediated manganese ion-coordinated carbazoline-loaded albumin nanoparticles prepared by the above method have the following characteristics and advantages:

[0025] 1. Active targeting capability: Folic acid modified on the surface of human serum albumin can specifically bind to the folic acid receptors that are highly expressed on the surface of tumor cells, enhancing the recognition and accumulation of nanoparticles on tumor tissues and increasing the accumulation of drugs at the target site.

[0026] 2. Manganese ion-assisted drug delivery: Manganese ions introduced into nanoparticles can enhance drug encapsulation efficiency by regulating protein conformation and promote drug release in the intracellular environment, thereby improving therapeutic efficacy.

[0027] 3. Good biocompatibility and circulatory stability: Human serum albumin is an endogenous protein with high safety and low immunogenicity; its surface is negatively charged, which can reduce phagocytosis by the macrophage system, prolong the circulation time in the body, and help reduce the systemic toxicity of cabazitaxel. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the preparation process of the folic acid-albumin-manganese ion modified compound FA-HSA@Mn provided by the present invention.

[0029] Figure 2 The infrared spectra of FA, HSA, and FA-HSA prepared in Example 1 of this invention are shown.

[0030] Figure 3 The UV-Vis spectra of FA-HSA and FA-HSA@Mn prepared in Example 1 of this invention are shown.

[0031] Figure 4 The image shows the DLS particle size distribution of FA-HSA@Mn prepared in Example 1 of this invention.

[0032] Figure 5 The particle size change of the folic acid receptor-mediated manganese ion-coordinated carbamate albumin nanoparticles prepared in Example 1 of this invention over 96 hours is shown in the figure.

[0033] Figure 6 The in vitro release curves of folic acid receptor-mediated manganese ion-coordinated carbamate albumin nanoparticles and Tween-CTX prepared in this embodiment of the invention are shown.

[0034] Figure 7 Cytotoxicity of different formulations prepared for embodiments of the present invention against mouse prostate cancer RM-1 cells (n=3). Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] The reagents and chemicals used in the embodiments of this invention are as follows:

[0037] Human serum albumin, cabazitaxel, folic acid (Shanghai Sangon Biotech Co., Ltd.); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide (Adamas Reagent Co., Ltd.), manganese chloride, dimethyl sulfoxide, sodium carbonate buffer, phosphate buffer, potassium bromide.

[0038] Example 1

[0039] The specific preparation method of this embodiment includes the following steps:

[0040] 1. Synthesis of folic acid-albumin modified compounds

[0041] 20 mg of folic acid (FA) was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and sonicated for 5 min. 11 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 7 mg of N-hydroxysuccinimide (NHS) were added with stirring. The mixture was activated at room temperature in the dark for 2 h, and the precipitate was removed by filtration. 0.8 mL of human serum albumin (HSA) solution was dissolved in 1 mL of pH 9.0 carbonate buffer. This solution was added dropwise to the activated folic acid solution to obtain the folic acid-albumin modified solution (denoted as FA-HSA).

[0042] 2. Synthesis of folic acid-albumin-manganese ion modified compounds

[0043] Add 3 mL of MnCl2 (16 µg / mL) to the solution obtained in step 1, react at room temperature for 4 h, place the reaction solution in a dialysis bag and dialyze for 12 h with carbonate buffer at pH 9.0 as the external dialysis solution, and then freeze dry to obtain folic acid-albumin-manganese ion modified product (denoted as FA-HSA@Mn).

[0044] 3. Preparation of drug-loaded nanoparticles:

[0045] 15 mg of FA-HSA@Mn lyophilized nanoparticles were dissolved in 1 mL of aqueous solution. The solution was placed in a 37°C constant temperature stirring water bath and stirred rapidly for 10 min. 1 mg of cabazitaxel was dissolved in 100 µL of anhydrous ethanol and slowly added to the above solution. After incubation for 30 min, the solution was rapidly cooled. Free drug and organic solvent were removed by ultrafiltration to obtain purified folic acid receptor-mediated manganese ion-coordinated cabazitaxel-loaded albumin nanoparticles (denoted as FA-HSA@Mn-Cbz). The measured particle size was 141.7 nm ± 3.5 nm.

[0046] Example 2

[0047] The specific preparation method of this embodiment includes the following steps:

[0048] 1. Synthesis of folic acid-albumin modified compounds

[0049] 20 mg of folic acid (FA) was dissolved in 1 mL of dimethyl sulfoxide (DMSO), sonicated for 5 min, and 8.7 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 5.2 mg of N-hydroxysuccinimide (NHS) were added with stirring. The mixture was activated in the dark for 2 h, and the precipitate was removed by filtration. 0.67 mL of human serum albumin (HSA) solution was dissolved in 1 mL of pH 9.0 carbonate buffer. This solution was added dropwise to the activated folic acid to obtain the folic acid-albumin modified solution (denoted as FA-HSA).

[0050] 2. Synthesis of folic acid-albumin-manganese ion modified compounds

[0051] Add 3 mL (16 µg / mL) of MnCl2 to the solution obtained in step 1, react at room temperature for 4 h, place the reaction solution in a dialysis bag and dialyze for 12 h with carbonate buffer at pH 9.0 as the external dialysis solution, and then freeze dry to obtain folic acid-albumin-manganese ion modified product (denoted as FA-HSA@Mn).

[0052] 3. Preparation of drug-loaded nanoparticles:

[0053] 15 mg of FA-HSA@Mn lyophilized product was dissolved in 1 mL of aqueous solution. The solution was placed in a 37°C constant temperature stirred water bath and stirred rapidly for 10 min. 1 mg of cabazitaxel was dissolved in 100 µL of anhydrous ethanol and slowly added to the above solution. After incubation for 30 min, the solution was rapidly cooled. Free drug and organic solvent were removed by ultrafiltration to obtain purified folic acid receptor-mediated manganese ion-coordinated cabazitaxel-loaded albumin nanoparticles (denoted as FA-HSA@Mn-Cbz). The particle size of the nanoparticles was measured to be 150 nm ± 2.5 nm. Following the procedure in Example 7, the particle size of the FA-HSA@Mn-Cbz nanoparticles prepared in this example did not change significantly within 96 hours. Furthermore, following the procedure in Example 8, the encapsulation efficiency of cabazitaxel by the FA-HSA@Mn-Cbz prepared in this example was measured to be 85.10% ± 1.5%.

[0054] Example 3

[0055] The specific preparation method of this embodiment includes the following steps:

[0056] 1. Synthesis of folic acid-albumin modified compounds

[0057] 20 mg of folic acid (FA) was dissolved in 1 mL of dimethyl sulfoxide (DMSO), sonicated for 5 min, and then 17.4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10.4 mg of N-hydroxysuccinimide (NHS) were added with stirring. The mixture was activated in the dark for 2 h, and the precipitate was removed by filtration. 0.86 mL of human serum albumin (HSA) solution was dissolved in 1 mL of pH 9.0 carbonate buffer. This solution was added dropwise to the activated folic acid to obtain the folic acid-albumin modified solution (denoted as FA-HSA).

[0058] 2. Synthesis of folic acid-albumin-manganese ion modified compounds

[0059] Add 3 mL (16 µg / mL) of MnCl2 to the solution obtained in step 1 and react at room temperature for 4 h. Place the reaction solution in a dialysis bag and dialyze for 12 h with carbonate buffer at pH 9.0 as the external dialysis solution. Then freeze-dry to obtain folic acid-albumin-manganese ion modified product (denoted as FA-HSA@Mn).

[0060] 3. Preparation of drug-loaded nanoparticles:

[0061] 15 mg of FA-HSA@Mn lyophilized drug was dissolved in 1 mL of aqueous solution. The solution was placed in a 37°C constant temperature stirred water bath and stirred rapidly for 10 min. 1 mg of cabazitaxel was dissolved in 100 µL of anhydrous ethanol and slowly added to the above solution. After incubation for 30 min, the solution was rapidly cooled. Free drug and organic solvent were removed by ultrafiltration to obtain purified folic acid receptor-mediated manganese ion-coordinated cabazitaxel-loaded albumin nanoparticles (denoted as FA-HSA@Mn-Cbz). The particle size of the nanoparticles was measured to be 160 nm ± 2.5 nm. Following the procedure in Example 7, the particle size of the FA-HSA@Mn-Cbz nanoparticles prepared in this example did not change significantly within 96 hours. Following the procedure in Example 8, the encapsulation efficiency of cabazitaxel by the FA-HSA@Mn-Cbz prepared in this example was measured to be 87.60% ± 1.5%.

[0062] Comparative Example 1

[0063] Dissolve 15 mg of FA-HSA lyophilized product in an aqueous solution. Place the solution in a 37°C constant temperature stirring water bath and stir rapidly for 10 min. Dissolve 1 mg of cabazitaxel in 100 µL of anhydrous ethanol and slowly add it to the above solution. Continue incubation for 30 min and then cool rapidly. Remove free drug and organic solvent by ultrafiltration to obtain purified folic acid-albumin nanoparticles (denoted as FA-HSA-Cbz). The measured particle size is 165 nm ± 3.5 nm.

[0064] Comparative Example 2

[0065] Dissolve 15 mg of lyophilized HSA in an aqueous solution. Place the solution in a 37°C constant temperature stirring water bath and stir rapidly for 10 min. Dissolve 1 mg of cabazitaxel in 100 µL of anhydrous ethanol and slowly add it to the above solution. Continue incubation for 30 min and then cool rapidly. Remove free drug and organic solvent by ultrafiltration to obtain purified albumin nanoparticles (denoted as HSA-Cbz). The measured particle size is 135 nm ± 2.5 nm.

[0066] Experimental Example 1

[0067] This embodiment verifies the connection of the FA-HSA prepared in Example 1.

[0068] Take 1 mg each of FA and HSA, FA-HSA prepared in Example 1, and 100 mg of potassium bromide. Mix each sample thoroughly with the potassium bromide and grind until homogeneous. Press the mixture into transparent thin films for infrared spectroscopy analysis. The Fourier transform infrared spectroscopy results are shown below. Figure 2 The infrared spectra of FA, HSA, and FA-HSA are shown in the figure, with FA at 1697 cm⁻¹. -1 A characteristic absorption peak appeared at 1656 cm⁻¹, with HSA at 1656 cm⁻¹. -1 1545cm -1 Characteristic absorption peaks appear; FA-HSA shows a peak at 1691 cm⁻¹. -1 1527cm -1 The appearance of characteristic absorption peaks and the significant changes in the spectrum of the conjugate compared to the original spectrum indicate that FA and HSA have successfully achieved covalent coupling.

[0069] Experimental Example 2

[0070] This embodiment verifies the connection of FA-HSA@Mn prepared in Example 1.

[0071] Accurately weigh 5.0 mg each of FA-HSA and FA-HSA@Mn prepared in Example 1, and dilute to 10 mL in a volumetric flask with phosphate buffered saline (PBS) at pH 7.4 to prepare a 0.5 mg / mL test solution. Place an appropriate amount of solution in a quartz cuvette and scan using a UV-Vis spectrophotometer in the wavelength range of 200–800 nm. The UV-Vis absorption spectra are shown below. Figure 3 The figure shows the average spectra of FA-HSA and FA-HSA@Mn, respectively. As can be seen from the figure, compared with FA-HSA, the absorption peak of FA-HSA@Mn shows a red shift, increased intensity, and broadened peak shape, confirming the presence of Mn. 2+ The electronic structure of FA-HSA was altered through coordination, indicating that manganese ions have successfully achieved coordination linkage with FA-HSA.

[0072] Experimental Example 3

[0073] This embodiment investigates the particle size of FA-HSA@Mn-Cbz prepared in Example 1.

[0074] The particle size of FA-HSA@Mn-Cbz nanoparticles was determined by taking approximately 1 mL of the FA-HSA@Mn-Cbz prepared in this example, placing it in a cuvette, immersing it in the sample cell, and measuring the particle size using a Malvern particle size analyzer. The results are shown below. Figure 4 ,Depend on Figure 4 It can be seen that the particle size of the nanoparticle is 141.7nm ± 3.5nm.

[0075] Test Example 4

[0076] This experiment investigates the stability of FA-HSA@Mn-Cbz prepared in Example 1 at 25°C.

[0077] FA-HSA@Mn-Cbz nanoparticles were diluted with an appropriate amount of phosphate-buffered saline (PBS) and stored at a constant temperature of 25°C. Samples were taken at 0 h, 24 h, 48 h, 72 h, and 96 h, and their particle size was determined using dynamic light scattering (DLS) to evaluate the stability of the nanoparticles. The results are shown in [link to results]. Figure 5 As shown in the figure, the particle size of FA-HSA@Mn-Cbz did not change significantly within 96 hours, indicating that the nanoparticles have good storage stability in PBS.

[0078] Experimental Example 5

[0079] This experiment investigates the encapsulation efficiency of HSA-Cbz prepared in Comparative Example 2, FA-HSA-Cbz prepared in Comparative Example 1, and FA-HSA@Mn-Cbz prepared in Example 1.

[0080] Establishing the standard curve equation for cabazitaxel (Cbz): Accurately weigh an appropriate amount of Cbz standard and use methanol-PBS mixed solution as solvent to gradually dilute to obtain standard solutions with concentrations of 50 µg / mL, 25 µg / mL, 12.5 µg / mL, 6.25 µg / mL, 3.125 µg / mL, 1.56 µg / mL, and 0.78 µg / mL. The peak area of ​​the standard at each concentration was determined by high-performance liquid chromatography (HPLC). Linear regression was performed on the peak area against the Cbz concentration to obtain the standard curve equation: Y = 2 × 10⁻⁶. 10 x+88830 R=0.9977.

[0081] The encapsulation efficiency of the nanoparticles was calculated using formula (1).

[0082] The encapsulation efficiency of nanoparticles is calculated using the formula (1): (amount of drug loaded in nanoparticles / total amount of drug added) × 100%; where the amount of drug loaded in nanoparticles is calculated by dissolving and precipitating the nanoparticles after ultrafiltration and centrifugation, and determining the Cbz content by HPLC; the total amount of drug added is the total amount of Cbz added during the preparation process, which is confirmed by HPLC quantitative analysis.

[0083] The encapsulation efficiency of the nanoparticles prepared in Example 1 is shown in Table 1.

[0084] Table 1 Encapsulation efficiency of cabazitaxel by different nanoparticles (n=3) Nanoparticle formulation Encapsulation rate HSA-Cbz 77.07%±1.3% FA-HSA-Cbz 82.30%±2.4% FA-HSA@Mn-Cbz 94.10%±1.5%

[0085] Experimental Example 6

[0086] This experimental example investigates the in vitro release behavior of FA-HSA@Mn-Cbz prepared in Example 1.

[0087] The in vitro drug release behavior of FA-HSA@Mn-Cbz nanoparticles was investigated using the dialysis bag method: The concentration of the FA-HSA@Mn-Cbz nanoparticle suspension was adjusted to 1 mg / mL. Cabazitaxel was dissolved in a mixed solvent containing 13% ethanol (w / w) and polysorbate-80 (15:57, w / w) as a control (simulating commercially available cabazitaxel injection, denoted as Tween-Cbz) (n=3). 1 mL of each sample was placed in a dialysis bag (MWCO=3000 Da), and the dialysis bag was immersed in PBS release medium containing 0.5% polysorbate-80. The mixture was shaken at 100 r / min in a constant temperature shaker at 37℃. 0.5 mL of release medium was collected at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h, and an equal volume of fresh medium at the same temperature was added simultaneously. All samples were filtered through a 0.22 µm microporous membrane, and the content of cabazitaxel was determined by HPLC. The cumulative release percentage was calculated according to formula (2). The results are shown in [reference needed]. Figure 6 . Figure 6 The results showed that FA-HSA@Mn-Cbz nanoparticles exhibited a slow and increasing trend in the initial stage (0-12 h) due to the release of a small amount of free drug on the carrier surface, and gradually released completely in the later stage (12-48 h) as the carrier structure loosened. In contrast, the Tween-Cbz control group had a significantly faster drug release rate, indicating that the nanoparticles have obvious sustained-release characteristics.

[0088] Formula (2): Cumulative release (%) = Amount of carbatazone in the dialysis fluid taken at different time points / Total amount of drug in nanoparticles.

[0089] In formula (2), the amount of carbatazone in the dialysis fluid refers to the concentration of FA-HSA@Mn-Cbz diluted to a certain concentration and then determined by HPLC, which is then substituted into the standard curve equation 1.

[0090] Experimental Example 7

[0091] In this experiment, the MTT assay was used to evaluate the in vitro killing effect of FA-HSA@Mn-Cbz prepared in Example 1 on mouse prostate cancer cells (RM-1).

[0092] RM-1 cells in logarithmic growth phase were seeded into 96-well plates and cultured for 24 hours until adherence. The medium was then replaced with culture medium containing different concentration gradients of the test drugs. The test drugs included Tween-Cbz, FA-HSA@Mn-Cbz, FA-HSA-Cbz, HSA-Cbz, and corresponding blank vectors (FA-HSA@Mn, FA-HSA, HSA). All groups containing cabazitaxel were set at six gradients based on cabazitaxel concentrations: 128, 64, 32, 16, 8, and 4 µg / mL. After another 24 hours of culture, MTT reagent was added to each well and incubated for 4 hours. Formazan crystals were then dissolved, and absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated. Results showed that cell viability in all blank vector groups was above 85%, indicating that the vectors themselves were non-toxic. At all test concentrations, as shown in the attached... Figure 7 The order of cytotoxicity was: Tween-Cbz > FA-HSA@Mn-Cbz > FA-HSA-Cbz > HSA-Cbz (** represents p≦0.01, *** represents p≦0.001, **** represents p≦0.0001). In vitro cytotoxicity experiments showed that the direct diffusion of Tween-Cbz and the potential synergistic toxicity of ethanol (as a free drug solvent) with polysorbate 80 resulted in lower cell survival rates in the Tween-Cbz group. The FA-HSA@Mn-Cbz of this invention still exhibited significantly better final killing effects than HSA-Cbz and FA-HSA-Cbz. This demonstrates that the product of this invention achieves a potent killing effect on tumor cells due to its combination of folic acid targeting and the synergistic therapeutic effects of manganese ions.

Claims

1. A method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles, characterized in that, Includes the following steps: Step 1: Preparation of folic acid-albumin modified product Folic acid was dissolved in dimethyl sulfoxide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added. The mixture was activated in the dark, and the precipitate was removed by filtration to obtain a folic acid solution. Albumin solution was dissolved in carbonate buffer at pH 9.0 and then slowly added dropwise to the folic acid solution to obtain the folic acid-albumin modified solution FA-HSA. Step 2: Preparation of folic acid-albumin-manganese ion modified compounds MnCl2 was added to the folic acid-albumin modified solution prepared in step 1 and reacted at room temperature for 4 h. The reaction solution was placed in a dialysis bag and dialyzed in carbonate buffer at pH 9.0 for 12 h. After freeze drying, folic acid-albumin-manganese ion modified compound FA-HSA@Mn was obtained. Step 3: Preparation of drug-loaded nanoparticles The folic acid-albumin-manganese ion modified compound prepared in step 2 was dissolved in an aqueous solution. The solution was placed in a 37°C constant temperature stirred water bath and stirred rapidly. Cabazitaxel dissolved in anhydrous ethanol was slowly added to the above solution. After incubation for 30 min, the solution was rapidly cooled. Free drug and organic solvent were removed by ultrafiltration to obtain folic acid receptor-mediated manganese ion-coordinated cabazitaxel-albumin nanoparticles FA-HSA@Mn-Cbz.

2. The method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles according to claim 1, characterized in that, The albumin in question is human serum albumin.

3. The method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles according to claim 2, characterized in that, In step 1, the volume ratio of dimethyl sulfoxide to pH 9.0 carbonate buffer to human serum albumin solution is 3-7 : 3-7 : 2-6, and the concentration range of human serum albumin is 10 mg / mL-50 mg / mL.

4. The method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles according to claim 1, characterized in that, In step 1, the molar ratio of folic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:1 to 2:1 to 2.

5. The method for preparing folic acid receptor-mediated manganese ion-coordinated carbataxel-loaded albumin nanoparticles according to claim 1, characterized in that, In step 3, the volume ratio of water used to dissolve the folic acid-albumin-manganese ion modifier to anhydrous ethanol used to dissolve cabazitaxel is 1:0.05 to 0.

2.

6. The method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles according to claim 1, characterized in that... In steps 1 and 2, the mass ratio of folic acid to albumin and MnCl2 is 1250:1000:

3.

7. The method for preparing folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles according to claim 1, characterized in that, The mass ratio of folic acid-albumin-manganese ion modifier to cabazitaxel is 10–20:

1.

8. The application of folic acid receptor-mediated manganese ion-coordinated carbamate-loaded albumin nanoparticles prepared by the preparation method according to any one of claims 1-7 in the preparation of pharmaceuticals.

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