Use of fe3o4 nanoparticles in the preparation of a drug for treating benign prostatic hyperplasia

Abstract

The application provides application of Fe3O4 nanoparticles in preparation of a drug for treating benign prostatic hyperplasia, and relates to the technical field of biological medicine.The application of Fe3O4 nanoparticles in preparation of a drug for treating benign prostatic hyperplasia.The research result of the application emphasizes that the ferroferric oxide nanoparticles are a kind of promising candidate drug for targeted treatment of BPH and related prostate diseases, and provides a new possibility for treatment intervention of urology surgery.The inventor of the application studies the effect of Fe3O4 nanoparticles on inhibition of benign prostatic hyperplasia.The result of the inventor of the application shows that the nanoparticles not only effectively reduce the proliferation rate of prostate cells, but also improve related biochemical indexes.This shows that the nanoparticles have potential application in treatment of prostatic hyperplasia.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, in particular to application of Fe3O4 nanoparticles in preparation of a drug for treating benign prostatic hyperplasia. BACKGROUND

[0002] Benign prostatic hyperplasia (BPH) is a common urological disease in middle-aged and elderly men, which is mainly manifested as dysuria. BPH is one of the most common diseases in clinical diagnosis and treatment of urology. BPH generally occurs after the age of 40, and the incidence increases with age. About 20% of men aged 50-60, 50% of men aged 61-70, and 83% of men aged 81-90 have BPH. BPH is mainly manifested as histological hyperplasia of prostate interstitial and glandular components, anatomical enlargement of the prostate, and urodynamic obstruction of the bladder outlet, which leads to elongation, compression deformation, stenosis and increased resistance of the posterior urethra, causes high pressure in the bladder and lower urinary tract symptoms (LUTS). LUTS includes storage symptoms (frequent urination, urinary urgency, urinary incontinence, nocturia), voiding symptoms (hesitancy, dysuria, intermittent urination), and post-voiding symptoms (a sense of incomplete emptying, post-void dribbling). In addition, middle-aged and elderly men with LUTS are more likely to develop erectile dysfunction (ED), and the severity of ED is related to the severity of LUTS, which threatens the health of elderly men and significantly affects their quality of life.

[0003] Despite extensive research, there has been limited progress in the pharmacological treatment options for BPH. Existing treatments mainly provide symptomatic relief without addressing the underlying cause of prostate enlargement. Current drugs include selective alpha 1 receptor antagonists such as doxazosin (approved in 1988), terazosin (1985), alfuzosin (1988), and the highly selective alpha 1 receptor antagonist tamsulosin (1992), as well as five alpha-reductase inhibitors such as finasteride (1992) and dutasteride (2001). Other treatments such as muscarinic receptor antagonists and PDE5 inhibitors such as tadalafil (2002) have also been used. However, these therapies cannot effectively prevent prostate growth. 5ARI (1992), although the only drug that reduces prostate volume, is associated with many side effects, including decreased libido, erectile dysfunction, orgasm dysfunction, increased risk of high Gleason grade prostate cancer, heart failure and cardiovascular events observed in clinical trials, and depression. SUMMARY

[0004] In order to solve the technical problems in the prior art, the application provides application of Fe3O4 nanoparticles in preparation of a drug for treating benign prostatic hyperplasia.

[0005] Application of Fe3O4 nanoparticles in preparation of a drug for treating benign prostatic hyperplasia.

[0006] Optionally, the Fe3O4 nanoparticles are hydrophilic Fe3O4 nanoparticles.

[0007] Optionally, the hydrophilic Fe3O4 nanoparticles are prepared by a chemical coprecipitation method, and a chemical reaction formula is as follows: Fe 2+ +2Fe 3+ +8OH - = Fe3O4 + 4H2O.

[0008] Optionally, a preparation method of the hydrophilic Fe3O4 nanoparticles is as follows:

[0009] (1) FeCl2.4H2O and FeCl3.6H2O are weighed and completely dissolved in an ethanol aqueous solution to form a mixed solution;

[0010] (2) under nitrogen protection, the solution obtained in the step (1) is placed in a water bath and stirred, and ammonia water is slowly added dropwise until the pH reaches 10;

[0011] (3) after reaction for 2-4 hours, the reaction is stopped, and the separated black precipitate is washed with anhydrous ethanol;

[0012] (4) the black precipitate is vacuum dried to obtain the hydrophilic Fe3O4 nanoparticles.

[0013] A drug for treating benign prostatic hyperplasia, comprising: Fe3O4 nanoparticles.

[0014] Optionally, the Fe3O4 nanoparticles are hydrophilic Fe3O4 nanoparticles.

[0015] A treatment system for benign prostatic hyperplasia, comprising: Fe3O4 nanoparticles and a magnet, wherein the magnet is used for positioning the Fe3O4 nanoparticles at a prostate tissue in vivo.

[0016] Optionally, the Fe3O4 nanoparticles are hydrophilic Fe3O4 nanoparticles.

[0017] Optionally, the hydrophilic Fe3O4 nanoparticles are prepared by a chemical coprecipitation method, and a chemical reaction formula is as follows: Fe 2+ +2Fe 3+ +8OH -= Fe3O4 + 4H2O.

[0018] Optionally, the preparation method of the hydrophilic Fe3O4 nanoparticles is as follows:

[0019] (1) FeCl2·4H2O and FeCl3·6H2O are weighed and completely dissolved in an ethanol aqueous solution to form a mixed solution;

[0020] (2) The solution obtained in step (1) is placed in a water bath under nitrogen protection, stirred, and ammonia water is slowly added dropwise until the pH reaches 10;

[0021] (3) After reacting for 2-4 hours, the reaction is stopped, and the separated black precipitate is washed with anhydrous ethanol;

[0022] (4) The black precipitate is vacuum dried to obtain the hydrophilic Fe3O4 nanoparticles.

[0023] The technical solutions provided by the embodiments of the present application have at least the following beneficial effects:

[0024] In the present study, the present inventors developed a nanoscale inhibitor Fe3O4 nanoparticle with good stability to target prostate tissue with high precision under the guidance of an external magnetic field and effectively reduce the over-proliferation of prostate epithelial cells. Biological experiments show that this nanoscale inhibitor successfully enters the prostate epithelial cells and effectively inhibits their proliferation. These findings suggest that the nanoscale inhibitor Fe3O4 nanoparticle has potential for the treatment of BPH. This nanoparticle-based strategy provides new insights into the pathophysiology of BPH and offers a promising direction for the development of targeted therapy.

[0025] The research results of the present application emphasize that the Fe3O4 nanoparticle is a promising candidate drug for targeted therapy of BPH and related prostate diseases, providing new possibilities for therapeutic intervention in urology. The present inventors studied the role of Fe3O4 nanoparticles in inhibiting benign prostatic hyperplasia. The results of the present inventors show that these nanoparticles not only effectively reduce the proliferation rate of prostate cells, but also improve related biochemical indicators. This indicates their potential application in the treatment of prostate hyperplasia. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0027] Figures 1A-1Fis a graph of the evaluation of the inhibitory effect of different metal oxides on cell proliferation provided by Example 1 of the present application; wherein, Figure 1A is a graph of the effect of different concentrations of five different metal oxides on BPH-1 cells detected by MTT method, the bar chart represents the relative inhibition of cell proliferation, and the data represents the mean ± SD of three independent experiments; Figure 1B is a graph of the cell proliferation after BPH-1 cells were treated with different concentrations of three metal ions and Fe3O4 for 24 h detected by MTT method, the bar chart illustrates the inhibitory effect on cell proliferation, and the data represents the mean ± SD of three independent experiments; Figure 1C is a graph of the distribution of cell cycle after BPH-1 cells were treated with different concentrations of Fe3O4 (0, 0.04, 0.08, 0.12, 0.16 and 0.20 mg / mL) for 24 h analyzed by flow cytometry, and the data represents three independent experiments; Figure 1D is a graph of the apoptosis after BPH-1 cells were treated with increasing concentrations of Fe3O4 (0, 0.04, 0.08, 0.12, 0.16 and 0.20 mg / mL) for 24 h analyzed by flow cytometry, and the data represents three independent experiments; Figure 1E and Figure 1F are the quantitative analysis graphs of the distribution of cell cycle Figure 1C and Figure 1D apoptosis, respectively; Figure 1E Figure 1F

[0028] Figures 2A-2G is a graph of the preparation and characterization of hydrophilic iron oxide nanoparticles provided by Example 2 of the present application; wherein, Figure 2A is a schematic diagram of the synthesis of Fe3O4 nanoparticles, FeCl2 and FeCl3 are mixed in an alkaline ethanol aqueous solution, then heated and stirred at 80℃ for 3 h to generate Fe3O4 nanoparticles; Figure 2B is a transmission electron microscopy (TEM) image showing the morphology of Fe3O4 nanoparticles; Figure 2C and Figure 2D are element distribution maps of Fe3O4 nanoparticles showing the element composition and distribution; Figure 2E is a quantitative element analysis map of Fe3O4 nanoparticles; Figure 2F is a surface charge characterization map of Fe3O4 nanoparticles, and the results are expressed as the mean ± SD of three independent experiments; Figure 2G is a statistical distribution map of the diameter of Fe3O4 nanoparticles obtained from the TEM image in Figure 2B

[0029] Figures 3A-3M is a graph of the effect of Fe3O4 in treating BPH in a BPH mouse model provided by Example 3 of the present application; wherein Figure 3A ​​​is the experimental design diagram for the establishment and treatment of BPH mouse model, after 28 days of disease induction, the mice were divided into four groups: control group (sterile 0.9% NaCl), Fe3O4 injection (5 mg / kg, intravenous injection, once every other day), only magnetic field exposure group, Fe3O4 injection combined with magnetic field exposure group; Figure 3B is a representative image of prostate tissue and surrounding organs after modeling; Figure 3C is a bioluminescence imaging map of Fe3O4 accumulation in BPH mice using AniView100 multi-modal imaging system, the data represents three independent experiments; Figure 3D is a quantitative analysis map of bioluminescence intensity from Figure 3C , the data represents three independent experiments; Figure 3E is a bioluminescence imaging map of Fe3O4 distribution in different organs after different treatments, the data represents three independent experiments; Figure 3F is a TEM visualization map of Fe3O4 nanoparticles in prostate cells of BPH mice, the yellow arrow indicates Fe3O4; Figure 3G is a representative image of prostate after 38 days of different treatments; Figures 3H-3J is a map of measuring prostate volume (H), weight (I) and prostate index (J), the data represents five independent experiments; Figures 3K-3L is a quantitative map of dihydrotestosterone (DHT, K) and testosterone (T, L) levels in prostate tissue between different treatment groups, the data represents five independent experiments; Figure 3M is a hematoxylin and eosin (H&E) staining map of prostate tissue sections from mice with different treatments. DETAILED DESCRIPTION

[0030] The technical solutions in the present application will be described below with reference to the accompanying drawings.

[0031] Nanomaterials have unique optical, electrical and structural properties, and are attracting attention in different fields such as biosensing, bioimaging, tissue engineering and drug delivery. Although iron oxide nanoparticles have been widely used in nanomedicine due to their unique physical and chemical properties, their direct use as therapeutic agents has not been explored to a great extent. Most applications focus on their role as drug carriers. The present application explores the potential of ferroferric oxide nanoparticles in addressing prostate tissue overgrowth.

[0032] Specifically, the present inventors synthesized a stable hydrophilic nanoscale inhibitor that can precisely target prostate tissue under the guidance of an external magnetic field. This inhibitor effectively inhibited the overproliferation of prostate epithelial cells. Biological tests confirmed that the nanoinhibitor effectively entered the prostate epithelial cells and inhibited their proliferation. The research results emphasize that ferroferric oxide nanoparticles are a promising candidate drug for targeted treatment of BPH and related prostate diseases, providing new possibilities for therapeutic intervention in urology.

[0033] In the following examples, male C57 BL / 6 mice and SD rats (6-8 weeks old) were purchased from Beijing HFK Biotechnology Co., Ltd., and all animals were raised under specific pathogen-free (SPF) conditions. Animal care and experimental procedures were performed in accordance with the guidelines approved by the Chinese People's Liberation Army General Hospital Experimental Animal Welfare Ethics Committee (IACUC-2023-X19-125).

[0034] In the following examples, the cell line human benign prostatic hyperplasia cell line BPH-1 was obtained from Shanghai Jimabio Biotechnology Co., Ltd.

[0035] Example 1 Evaluation of the effect of different metal oxides on the proliferation of BPH cells

[0036] 1. BPH-1 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U / mL penicillin (Invitrogen), and 100 μg / mL streptomycin (Invitrogen).

[0037] The preparation method of Fe3O4 nanoparticles is described in Example 2 below. Al2O3 nanoparticles, Fe2O3 nanoparticles, TiO2 nanoparticles, and MnO nanoparticles were purchased from Bohua Nanotechnology (Ningbo) Co., Ltd.

[0038] Cells were exposed to different concentrations (0 to 0.2 mg / mL) of five different metal oxide nanoparticles, specifically: Fe3O4 nanoparticles, Al2O3 nanoparticles, Fe2O3 nanoparticles, TiO2 nanoparticles, and MnO nanoparticles. The concentration gradients were 0, 0.04, 0.08, 0.12, 0.16, and 0.20 mg / mL, respectively. The culture conditions were: 37°C, 5% CO2 environment.

[0039] Cells were exposed to different concentrations (0 to 0.2 mg / mL) of three metal ions and Fe3O4 nanoparticles, specifically: Fe 2+ , Fe 3+ , and Ca 2+Fe3O4 nanoparticles were used. The concentration gradients were 0, 0.04, 0.08, 0.12, 0.16, and 0.20 mg / mL. The culture conditions were 37℃ and 5% CO2.

[0040] 2. Determination of cell proliferation and inhibition rate

[0041] Use the MTT assay kit (Beyotime Biotechnology Co., Ltd.). Refer to the kit instructions for detailed operating procedures. In short, add the MTT solution to the cell culture plate and incubate for 4 hours in a cell culture incubator. Then, properly mix the Formazan solution and incubate further in a cell culture incubator. Measure the absorbance at 570 nm. The number of viable cells is directly proportional to the absorbance.

[0042] 3. Flow cytometry analysis of cytotoxicity and apoptosis

[0043] Cells were seeded in 6-well plates (2 × 10⁶ cells / well). 5 Cells were cultured at 1 cell / well for 12 h. Cells were then treated under different conditions and harvested from the culture plate using trypsin (EDTA-free). After washing twice with cold PBS (pH 7.4), cells were resuspended in 1 mL of 1× binding buffer (Annexin V-FITC / PI apoptosis detection kit, Solarbio). Cells were stained for 5 min at room temperature in the dark with 5 μL Annexin V-FITC (Beyotime Biotechnology Co., Ltd.) and 5 μL propidium iodide (PI) staining solution (Beyotime Biotechnology Co., Ltd.). After incubation, 400 μL of PBS was added to each sample, and the samples were stored on ice until analysis (within 30 min). Subsequently, flow cytometry (FACS Aria) was used. TM 3. Cell analysis (BD, USA).

[0044] 4. Flow cytometry analysis of the cell cycle

[0045] Cells were seeded in 6-well plates (2 × 10⁵ cells / well) and cultured for 12 h. Cells were then treated under different conditions, harvested from the culture plates using trypsin (EDTA-free), and washed twice with cold PBS (pH 7.4). Staining working solution was prepared by mixing RNase A (Beyotime Biotechnology Co., Ltd.) and propidium iodide (PI) at a ratio of 1:9 (v / v). 300 μL of staining working solution was added to each sample, wrapped in aluminum foil, and incubated on ice for 30 min. Subsequently, the cells were analyzed using flow cytometry (FACS Aria). TM 3. Detect cells (BD, USA).

[0046] Experimental results:

[0047] To evaluate the therapeutic potential of various metal oxide nanoparticles in inhibiting BPH cell proliferation, the inventors used the commonly studied BPH-1 cell line. In vitro, the inventors exposed the cells to different concentrations (0 to 0.2 mg / mL) of different metal oxide nanoparticles and observed corresponding levels of growth inhibition, the results of which are shown in Table 1. Figure 1A .

[0048] Among the tested nanoparticles, mixed-valence oxides such as magnetite (Fe3O4) and aluminum oxide (Al2O3) exhibited inhibitory effects on BPH cell proliferation, with Fe3O4 showing significantly more potent inhibition than iron oxide (Fe2O3). Here, the cell inhibition rate was maintained at 16.02% to 19.93% when the Fe3O4 concentration was 0.04 to 0.16 mg / mL. When the Fe3O4 concentration reached 0.20 mg / mL, the inhibitory effect was significantly enhanced.

[0049] To investigate whether the inhibitory effect was due to the specific valence of iron or a combination of both, the inventors added iron ions of different valences to the culture medium at the same concentration gradient. The inventors found that both ferrous (Fe 2+ ) and ferric (Fe 3+ ) ions inhibited cell proliferation like Fe3O4, but their individual effects were not as pronounced as Fe3O4. The results are shown in Table 2. Figure 1B .

[0050] Next, the inventors sought to determine the underlying mechanism of action by analyzing whether the inhibition of BPH-1 cell proliferation was due to a decrease in proliferation rate caused by prolonged mitotic cycles or promotion of proliferation. Flow cytometry analysis showed that as the concentration of Fe3O4 nanoparticles increased, the cell cycle distribution changed: the inhibition rate was 15.3% when the concentration was 0 mg / mL. When the concentration was 0.04 mg / mL, the inhibition rate increased to 17.3%. At 0.08 mg / mL, the inhibition rate rose to 23%. When the concentration was 0.12 mg / mL, the inhibition rate was 21.6%, and when the concentration was 0.16 mg / mL, the inhibition rate was 21%. Finally, at a concentration of 0.2 mg / mL, the S phase (the stage of DNA replication) reached 29.1%. The results are shown in Table 3. Figure 1C and 1E This indicates that Fe3O4 nanoparticles primarily inhibit cell proliferation by interfering with the DNA synthesis phase of mitosis, thereby slowing down the progression of the cell cycle.

[0051] Further apoptosis analysis by flow cytometry showed that there was no significant change in the rate of apoptosis across the concentration gradient. The results are shown in Table 4. Figure 1D and 1FThe results of the inventors show that Fe3O4 nanoparticles inhibit the proliferation of BPH-1 cells by interfering with DNA replication.

[0052] Example 2. Synthesis of highly hydrophilic Fe3O4 nanoparticles

[0053] Highly hydrophilic Fe3O4 nanoparticles were synthesized using chemical co-precipitation method with Fe 2+ +2Fe 3+ +8OH - = Fe3O4 + 4H2O as raw materials. The specific operation steps are as follows:

[0054] A certain amount of FeCl2-4H2O and FeCl3-6H2O (Fe 2+ : Fe 3+ molar ratio of 1 : 1.75) was weighed without PEG6000 or oleic acid modification. They were completely dissolved in 200 mL of ethanol-water solution (volume ratio of ethanol to water is 1 : 1) to form a mixed solution. Under nitrogen protection, the solution was placed in a 80°C water bath and mechanically stirred, and ammonia was slowly added until the pH reached 10. After 3 hours of reaction, the reaction was stopped. The black precipitate separated by magnetic separation was repeatedly washed with anhydrous ethanol several times. Then vacuum dried at 80°C for 24h to obtain hydrophilic Fe3O4 nanoparticles for further characterization.

[0055] Characterization method: Zetasizer Nano ZS (Malvern, UK) was used to measure the hydrodynamic diameter and zeta potential of the particles. Transmission electron microscopy (TEM) images were obtained using a high-resolution field emission transmission electron microscope. The chemical structure of the nanoparticles was further confirmed by elemental mapping and energy dispersive X-ray spectroscopy (EDS).

[0056] Experimental results:

[0057] Synthesis of hydrophilic iron oxide nanoparticles: In order to improve the stability of Fe3O4 nanoparticles in body fluids, thereby improving their circulation and targeted accumulation at the treatment site, the inventors synthesized hydrophilic Fe3O4 nanoparticles. Using ethanol-water solution as the reaction medium, the inventors used a co-precipitation method, which provides several advantages over traditional surface modification techniques. This method not only simplifies the synthesis process by reducing the number of reaction steps, but also improves reaction efficiency while maintaining the magnetism of the final product. Figure 2A The entire synthesis process of Fe3O4 nanoparticles is outlined, which is carried out under nitrogen protection to prevent air from affecting the heated iron ions.

[0058] Figure 2BTEM images were presented, which showed the uniform morphology of Fe3O4 with an average diameter of about 10 ± 5 nm. The chemical structure of the nanoparticles was further confirmed by elemental mapping and energy dispersive X-ray spectroscopy (EDS), the results of which are shown in Figure 2C and 2D These analyses confirmed the uniform distribution of Fe and O elements, which closely matched the relative elemental content of Fe3O4, the results of which are shown in Figure 2E In addition, the zeta potential of the nanoparticles, which is related to their surface charge, was 30 ± 0.5 mV, the results of which are shown in Figure 2F The particle size obtained in the above Figure 2B was counted, the results of which are shown in Figure 2G .

[0059] Example 3

[0060] 1. BPH mouse model

[0061] A testosterone-induced BPH model was used. Male C57 BL / 6 mice and male SD rats were subcutaneously injected with a testosterone propionate solution in soybean oil (7.5 mg / kg) or a control solution (soybean oil) every day for 28 consecutive days. After 28 days, the mice were sacrificed to evaluate prostate weight, volume, and other related parameters.

[0062] 2. Biodistribution and in vivo accumulation of Fe3O4

[0063] (1) Preparation of FITC-labeled Fe3O4 nanoparticles

[0064] 1) Preparation of FITC solution

[0065] An appropriate amount of FITC was weighed and dissolved in absolute ethanol to prepare a FITC solution with a concentration of 1-5 mg / mL. Since FITC is sensitive to light, the preparation process should be carried out in the dark.

[0066] 2) FITC-labeled Fe3O4 nanoparticles

[0067] The prepared Fe3O4 nanoparticle solution was transferred to a conical flask, and FITC solution was added dropwise under magnetic stirring, with the mass ratio of FITC to Fe3O4 controlled at 1:10-1:50. The pH of the solution was adjusted to 7-8 with dilute hydrochloric acid or sodium hydroxide solution to promote the electrostatic interaction and covalent binding between FITC and Fe3O4. The reaction was stirred at room temperature in the dark for 4-6 hours to allow the FITC to fully label the surface of the Fe3O4 nanoparticles.

[0068] 3) Separation and purification of the product

[0069] After the reaction was completed, the product solution was centrifuged (8000-10000 rpm for 15-20 minutes) to remove unreacted FITC in the supernatant. The precipitate was washed with deionized water or PBS buffer multiple times, and each time after washing, centrifugation was performed. The washed product was transferred to a dialysis bag and dialyzed with deionized water or PBS buffer for 24-48 hours, and the dialysate was replaced every 4-6 hours during the dialysis to further remove residual small molecular impurities.

[0070] (2) In order to track the biodistribution of Fe3O4 in vivo, FITC-labeled Fe3O4 nanoparticles were injected intravenously into mice, and a magnet was positioned on the lower abdomen of the mice. At the predetermined time point, the mice were imaged using the AniView100 multi-mode animal in vivo imaging system (Bolinteng Company, AniView100 multi-mode animal in vivo imaging system).

[0071] At the end of the experiment, the mice were sacrificed, and the main organs were removed for ex vivo fluorescence imaging. In order to detect the in vivo accumulation of Fe3O4 in the prostate, the mice were euthanized at the end of the experiment, and the prostate tissue was collected for TEM analysis.

[0072] 3. Animal experiment

[0073] For the BPH mouse model, the prostate was taken for evaluation on the 28th day after modeling to determine the success of modeling. On the 29th day, the mice were randomly divided into control group (sterile normal saline), simple Fe3O4 group, simple magnetic field group, Fe3O4 + magnetic field group, Al3O4 + magnetic field group, 5 in each group. Fe3O4 / Al3O4 (5.0 mg / kg) was injected intravenously, and FITC-labeled Fe3O4 and Al3O4 were injected. On the 39th day, the mice were sacrificed, and liver, heart, spleen, lung, kidney, and prostate tissues were taken for morphological evaluation by hematoxylin-eosin (H&E) staining.

[0074] 4. Hematoxylin and eosin (H&E) staining

[0075] The liver, heart, spleen, lung, kidney, and prostate tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and cut into 5 μm thick sections. The tissue sections were deparaffinized with xylene, rehydrated by ethanol gradient, and stained with hematoxylin for 5 min and eosin for 1 min. Subsequently, the sections were dehydrated by ethanol gradient, cleared with xylene, and mounted with neutral resin. The therapeutic effect and side effects of Fe3O4 nanoparticles were evaluated using an optical microscope (DMi 8, Leica, Germany).

[0076] 5. Measurement of testosterone (T)

[0077] Testosterone levels were measured using a commercially available ELISA kit (Testosterone Enzyme Immunoassay Kit) (Brickell Biotech, Inc). Blood samples (0.5 mL) were collected from the orbital vein of each group (5 mice), diluted with 0.5 mL of a 3.5% sodium citrate solution, and shaken thoroughly. Within 30 minutes of sample collection, samples were centrifuged at 1000 x g for 15 minutes and the supernatant was collected for testing. To each well, 50 μL of standard working solution and test sample were added. Subsequently, 50 μL of biotinylated testosterone antibody working solution was added to each well, the plate was sealed and incubated at 37°C for 45 min, after which the solution was discarded and the plate was tapped dry. Each well was washed with 350 μL of wash solution, soaked for 1-2 min, and tapped dry. This washing step was repeated four times. Then, 100 μL of horseradish peroxidase (HRP)-labeled streptavidin working solution was added to each well, the plate was sealed, and incubated in a 37°C incubator for 30 min. Then, 300 μL of wash solution was added to each well, and the plate was washed at 30-second intervals. After each wash, the plate was tapped dry, and this washing process was repeated four times. Finally, 90 μL of color reagent was added to each well (protected from light), the plate was sealed, and incubated at 37°C for approximately 15 min in the dark. Subsequently, 50 μL of stop solution was added to each well, and the optical density (OD) was immediately measured at a wavelength of 450 nm using a microplate reader.

[0078] 6. Measurement of Dihydrotestosterone (DHT)

[0079] DHT levels were measured using an ELISA kit (Dihydrotestosterone ELISA Kit) (Elabscience, Wuhan, China). Blood samples (0.5 mL) were collected from the orbital vein of each group (5 mice), diluted with 0.5 mL of 3.5% sodium citrate solution, and then shaken thoroughly. Within 30 minutes of sample collection, the samples were centrifuged at 1000 x g for 15 minutes, and the supernatant was collected for detection. For the content determination procedure, 50 μL of standard solution at different dilutions was added to the standard wells, 50 μL of standard solution and sample diluent was added to the blank wells, and 50 μL of test sample was added to the remaining wells. Immediately, 50 μL of prepared biotinylated antibody working solution was added to each well. Cover the plate and incubate at 37°C for 45 min. After the incubation period, discard the liquid in each well and pat the plate dry on a clean absorbent paper. Then wash each well with 350 μL of wash solution, soak for 1 min, and aspirate or flick off the liquid in the plate, then pat dry. This washing step was repeated three times. Subsequently, 100 μL of horseradish peroxidase (HRP) conjugate working solution was added to each well. Cover the plate and incubate at 37°C for 30 min. After incubation, discard the liquid in each well and wash the plate 5 times. Then, 90 μL of substrate solution (TMB) was added to each well, cover the lid, and incubate at 37°C for about 15 min in the dark. Finally, 50 μL of stop solution was added to each well to terminate the reaction. The optical density (OD) of each well was immediately measured at a wavelength of 450 nm using a microplate reader.

[0080] Results of the experiment:

[0081] Fe3O4 improves the overall outcome of BPH rodent models

[0082] To verify the in vivo therapeutic effect of Fe3O4, the inventors established a BPH rodent model by administering testosterone propionate to C57 BL / 6 mice for 28 consecutive days. Fe3O4 nanoparticles were injected into the tail vein with and without magnetic guidance, see Figures 3A-3B . Without magnetic guidance, the nanoparticles entered the bloodstream and partially accumulated in the bladder region of the lower abdomen. However, when magnetic guidance was applied, a larger amount of nanoparticles remained in the bladder region for a longer period of time, see Figures 3C-3E .

[0083] To directly observe the nanoparticles in the prostate tissue, the inventors performed ultrathin sectioning and captured images using a biological transmission electron microscope. Nanoparticles were found in the cytoplasm of prostate tissue cells, see Figure 3F .

[0084] To visually evaluate the therapeutic effect, the inventors compared the prostates of all mouse groups, see Figure 3Gand quantified the prostate tissue data, see Figures 3H-3J and Figures 3C-3E It can be seen from Figure 3G that the therapeutic effect of Fe3O4 under the guidance of a magnetic field is better than that of the other control groups. It can be seen from Figures 3H-3J and Figures 3C-3E that Figures 3H-3J respectively are the evaluation of the volume, weight and prostate index of the mouse prostate, and it is found that the therapeutic effect of Fe3O4 under the guidance of a magnetic field is better than that of the other control groups. Figures 3C-3E respectively are the live fluorescence imaging after injection of FITC-Fe3O4, the data visualization analysis of fluorescence imaging and the FITC-Fe3O4 aggregation in different organs of the mouse, and it is found that the FITC-Fe3O4 can remain in the lower abdomen of the mouse for a long time under the guidance of a magnetic field, and during the circulation in the body, more FITC-Fe3O4 can enter the prostate site, reducing the side effects on other organs. The results show that the injection of Fe3O4 can inhibit the progression of BPH.

[0085] In addition, the inventors evaluated the changes in the hormone levels of the whole body of the mouse, and found that the DHT and testosterone levels are reduced after treatment compared with the control group, see Figures 3K-3L . At present, the mainstream view is that androgens promote the occurrence of prostate hyperplasia. The reduction of DHT and testosterone levels after treatment indicates that the occurrence of benign prostatic hyperplasia is reduced.

[0086] In addition, the thickness of the prostate epithelium of the treatment group is significantly reduced compared with the control group, see Figure 3M . It can be seen from the thickness of the acinus in Figure 3M that the thickness of the prostate epithelium of the treatment group is significantly reduced. It is indicated that the progression of prostate hyperplasia of the treatment group is low and slow compared with the non-treatment group.

[0087] In the present application, the inventors studied the role of Fe3O4 nanoparticles in inhibiting benign prostatic hyperplasia. The results of the inventors show that these nanoparticles not only effectively reduce the proliferation rate of prostate cells, but also improve the relevant biochemical indicators, which indicates their potential application in the treatment of prostate hyperplasia.

[0088] The above is merely a specific implementation of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of Fe3O4 nanoparticles as an active ingredient in the preparation of a drug for treating benign prostatic hyperplasia.

2. Use according to claim 1, characterized in that, The Fe3O4 nanoparticles are hydrophilic Fe3O4 nanoparticles.

3. Use according to claim 2, characterized in that, The hydrophilic Fe3O4 nanoparticles are prepared by a chemical co-precipitation method, and a chemical reaction formula thereof is: Fe 2+ +2Fe 3+ +8OH - =Fe3O4+4H2O.

4. Use according to claim 2 or 3, characterized in that, The hydrophilic Fe3O4 nanoparticles are prepared as follows: (1) FeCl2·4H2O and FeCl3·6H2O are weighed and completely dissolved in an aqueous ethanol solution to form a mixed solution; (2) The solution obtained in step (1) is placed in a water bath under nitrogen protection, stirred, and ammonia water is slowly added dropwise until the pH reaches 10; (3) After 2-4 hours of reaction, the reaction is stopped, and the separated black precipitate is washed with anhydrous ethanol; (4) The black precipitate is vacuum dried to obtain the hydrophilic Fe3O4 nanoparticles.

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