Application of pH-responsive micelle in medicine for treating prostatic cancer

Abstract

The invention discloses an application of a pH-responsive micelle in a medicine for treating prostatic cancer, and relates to the technical field of nano-medicines. The doxorubicin-loaded pH response type micelle is used for drug delivery; the pH response type micelle is prepared by the following steps: S1, carrying out esterification reaction on propranolol and DSPE-PEG-COOH, so as to obtain a DpP micelle; s2, dissolving the DpP micelles and doxorubicin DOX in a proper amount of methanol according to a preset feeding ratio; the pH-responsive micelle prepared by the invention has a pH-responsive drug release characteristic, can quickly release drugs in a tumor acidic microenvironment, and improves the drug concentration at a tumor part, so that the treatment effect is improved; the pH response type micelle successfully inhibits the expression of SOX2 in prostate cancer cells by carrying propranolol, the abnormal expression of SOX2 is closely related to invasion, metastasis and drug resistance of prostate cancer, and inhibition of the expression of SOX2 is beneficial for improving the treatment effect.

Description

Technical Field

[0001] The present invention relates to the field of nano - medicine technology, and particularly to an application of pH - responsive micelles in drugs for treating prostate cancer. Background Art

[0002] Prostate cancer is a common malignant tumor in middle - aged and elderly men, and its incidence is on the rise globally. The development of nanotechnology has brought new opportunities for cancer treatment. Drug delivery systems based on nanomaterials can improve the stability, targeting, and bioavailability of drugs, thereby enhancing the drug efficacy and reducing side effects. 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - polyethylene glycol (DSPE - PEG) has attracted much attention due to its biocompatibility and easy chemical modification. DSPE - PEG can form a nano - drug delivery system by encapsulating traditional chemotherapeutic drugs, improving the drug efficacy and reducing side effects.

[0003] Currently, the main treatment methods for prostate cancer include surgical resection, radiotherapy, and chemotherapy. However, these treatment methods have many problems. For example, surgical resection may lead to urinary incontinence and sexual dysfunction, while radiotherapy and chemotherapy have large side effects and are prone to cause tumor drug resistance, resulting in poor treatment effects. Therefore, it is urgent to develop new treatment strategies with high efficiency and low toxicity. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies in the prior art and propose an application of pH - responsive micelles in drugs for treating prostate cancer.

[0005] To achieve the above - mentioned purpose, the present invention adopts the following technical scheme:

[0006] An application of pH - responsive micelles in drugs for treating prostate cancer, using pH - responsive micelles loaded with doxorubicin for drug delivery;

[0007] The pH - responsive micelles are prepared through the following steps:

[0008] S1: Esterify propranolol with DSPE - PEG - COOH to obtain DpP micelles;

[0009] S2: Dissolve DpP micelles and doxorubicin (DOX) in an appropriate amount of methanol according to a predetermined feeding ratio, and remove methanol by rotary evaporation under reduced pressure to form a uniform film;

[0010] S3: Add PBS buffer solution, stir and hydrate at room temperature to obtain a DpPDNPs solution, that is, obtain the product.

[0011] Preferably, the specific steps of the esterification reaction are as follows:

[0012] S11: Dissolve propranolol naphthionate in dichloromethane, add DCC and DMAP, and stir in a water bath.

[0013] S12: Add DSPE-PEG-COOH to the reaction system of S11, and continue the water bath reaction for 48 hours.

[0014] S13: After the reaction is completed, remove dichloromethane by rotary evaporation to obtain DpP micelles.

[0015] Preferably: In S11, dissolve 2.59 mg of propranolol naphthionate in 5 mL of dichloromethane, add 206.33 μg of DCC and 37.87 μg of DMAP, and stir in a water bath at 35 °C for 1.5 hours.

[0016] In S12, add 27.80 mg of DSPE-PEG-COOH to the above reaction system, and continue the water bath reaction for 48 hours.

[0017] Preferably: The characterization of the pH-responsive micelles includes:

[0018] S21: Observe its size and morphology using a transmission electron microscope.

[0019] S22: Use a Malvern Zeta potential analyzer to measure the particle size and Zeta potential of DpPDNPs at different pH values.

[0020] S23: Use an ultraviolet-visible spectrophotometer to measure the content of doxorubicin, and calculate the drug loading and encapsulation efficiency.

[0021] S24: Determine the drug release behavior of DpPDNPs at different pH values by dialysis.

[0022] Preferably: Apply the pH-responsive micelles to the study of in vitro anti-tumor efficacy, specifically including:

[0023] S31: Cytotoxicity experiment: Inoculate PC3 cells, after culturing for 24 hours, replace the culture medium with different concentrations of DpPDNPs, and continue culturing for 24 hours to detect cell viability.

[0024] S32: Cell uptake experiment: Inoculate PC3 cells in a confocal culture dish, after culturing for 24 hours, add the labeled DpPDNPs solution, incubate under different pH conditions respectively, and observe the uptake of micelles by cells through a fluorescence microscope.

[0025] S33: Cell migration and invasion experiment: Conduct Transwell invasion experiment and scratch experiment, observe and count the number of invasive cells and cell migration rate.

[0026] S34: Colony formation assay: Digest, dilute, and resuspend PC3 cells, seed them in a culture dish and culture for one week, fix them, perform crystal violet staining, and count the number of colony formations.

[0027] Preferably: In S31, seed 5×10 3 cells per well in a 96-well plate, culture for 24 hours, then replace the medium with different concentrations of DpPDNPs and continue to culture for 24 hours, and detect cell viability using a CCK-8 reagent.

[0028] Preferably: In S32, incubate under pH 6.5 and 7.4 conditions, and then observe the uptake of micelles by cells through a fluorescence microscope.

[0029] Preferably: Apply the pH-responsive micelles to in vivo anti-tumor therapy experiments, specifically including:

[0030] S41: Collect PC3 cells in the logarithmic growth phase, resuspend them in PBS, adjust the cell concentration, and seed them subcutaneously in male mice;

[0031] S42: When the tumor volume reaches 100 - 150 mm 3 ³, randomly divide the mice into groups and inject the corresponding drugs through the tail vein;

[0032] S43: Administer the drug once every 3 days for a total of 4 times;

[0033] S44: During the drug administration period, measure the major and minor axes of the mice's tumors every 2 days, calculate the tumor volume, and plot the tumor growth curve;

[0034] S45: After the treatment is completed, sacrifice the mice, remove the tumor tissues and major organs, and perform hematoxylin-eosin staining and immunohistochemical staining.

[0035] Preferably: In S41, adjust the cell concentration to 5×10 5 / mL and seed them subcutaneously in the right back of male C57 mice; In S45, the major organs include the heart, liver, spleen, lungs, and kidneys.

[0036] Preferably: The immunohistochemical staining is used to detect the expression levels of SOX2, Ki67, TKS5, Cortactin, and F-actin.

[0037] The beneficial effects of the present invention are:

[0038] 1. The pH-responsive micelles prepared by the present invention have the property of pH-responsive drug release, can rapidly release drugs in the acidic microenvironment of tumors, increase the drug concentration at the tumor site, and thus improve the therapeutic effect; the pH-responsive micelles carry propanolol and successfully inhibit the expression of SOX2 in prostate cancer cells. The abnormal expression of SOX2 is closely related to the invasion, metastasis and drug resistance of prostate cancer. Inhibiting its expression helps to improve the therapeutic effect.

[0039] 2. SOX2 of the present invention is closely related to the formation of invasive pseudopodia. The pH-responsive micelles inhibit the formation of invasive pseudopodia by inhibiting the expression of SOX2, thereby reducing the invasion and migration ability of prostate cancer cells.

[0040] 3. The pH-responsive micelles of the present invention have no obvious toxicity to major organs such as the heart, liver, spleen, lungs and kidneys, and have good biosafety.

[0041] 4. Both in vitro and in vivo experiments of the present invention show that the pH-responsive micelles significantly inhibit the proliferation, migration and invasion of prostate cancer cells, and have a significant inhibitory effect on tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a schematic diagram of the IC50 of DpPDNPs in the cytotoxicity experiment of the present invention;

[0043] Figure 2 It is a schematic diagram of the CCK8 results of different treatments in the cytotoxicity experiment of the present invention;

[0044] Figure 3 It is a schematic diagram of the effect of DpPDNPs on the invasion of PC3 cells in the in vitro detection of the present invention;

[0045] Figure 4 It is a schematic diagram of the effect of DpPDNPs on the migration of PC3 cells in the in vitro detection of the present invention;

[0046] Figure 5 It is a schematic diagram of the effect of DpPDNPs on the proliferation of PC3 cells in the in vitro detection of the present invention;

[0047] Figure 6 It is a schematic diagram of the effect of DpPDNPs on the formation of invasive pseudopodia of PC3 cells of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0048] The technical solutions of the present invention will be further described in detail below in conjunction with the specific embodiments.

[0049] Example 1:

[0050] Preparation of pH-responsive micelles, including:

[0051] ① Material preparation:

[0052] Drugs and reagents

[0053] All chemical reagents are of analytical purity. The materials used are as follows:

[0054] Chemical reagents:

[0055] Propranolol (MW = 259.34, catalog number HY - B1238; MedChemExpress);

[0056] DSPE - PEG - COOH (MW = 2780.38, CAS1403744 - 37 - 5; MACKLIN);

[0057] Doxorubicin (DOX, MW = 579.98, CAS25316 - 40 - 9; MACKLIN);

[0058] Dicyclohexylcarbodiimide (DCC, MW = 206.33, catalog number HY - Y1092; MedChemExpress);

[0059] 4 - Dimethylaminopyridine (DMAP, MW = 122.17, CAS1122 - 58 - 3; MACKLIN); Dichloromethane (DCM);

[0060] Phosphate - buffered saline (PBS, pH 7.4).

[0061] Cell culture and detection kits:

[0062] RPMI - 1640 medium containing 10% fetal bovine serum (FBS);

[0063] CCK - 8 cell viability detection kit (C0038; Beyotime);

[0064] DAPI nuclear staining solution (P0131 - 5ml; Beyotime);

[0065] Dialysis membrane (molecular weight cut - off 1000Da).

[0066] Primary antibodies:

[0067] Anti - TKS5 antibody (18976 - 1 - AP; Proteintech);

[0068] Phalloidin - AF555 (16002; ZENBIO);

[0069] Anti - Ki67 antibody (28074 - 1 - AP; Proteintech);

[0070] Anti-GAPDH antibody (R24404; ZENBIO);

[0071] Anti-SOX2 antibody (11064-1-AP; Proteintech).

[0072] Cell line: human prostate cancer cell line PC3 cells.

[0073] ② Preparation of DpPDNPs:

[0074] Esterification reaction of propanolol and DSPE-PEG-COOH

[0075] Accurately weigh 2.59 mg of propanolol (10 mmol), dissolve it in 5 mL of dichloromethane, add 206.33 μg of dicyclohexylcarbodiimide (DCC, 1.3 mmol) and 37.87 μg of 4-dimethylaminopyridine (DMAP, 0.31 mmol), and stir in a 35 °C water bath for 1.5 hours.

[0076] Subsequently, add 27.80 mg of DSPE-PEG-COOH (10 mmol) to the above reaction system, and continue the water bath reaction for 48 hours. After the reaction is completed, remove dichloromethane by rotary evaporation to obtain the product DpP micelles (DpPNPs), and store the DpP micelles in the dark at 4 °C.

[0077] Prepare DpPDNPs micelles by the thin film hydration method, that is, pH-responsive micelles. Dissolve DpP micelles and doxorubicin (DOX) in an appropriate amount of methanol according to the predetermined feeding ratio, remove methanol by rotary evaporation under reduced pressure to form a uniform thin film. Subsequently, add 5 mg / mL PBS buffer solution, stir and hydrate at room temperature for 2 hours to fully dissolve the thin film and self-assemble to form micelles, obtaining DpPDNPs solution, and store it at 4 °C. In addition, synthesize a solution of DOX-loaded micelles (DpDNPs) without propanolol by the same method for subsequent comparison.

[0078] ③ Characterization of DpPDNPs:

[0079] Morphological observation: Image DpPDNPs using a FEI Tecnai F20 analytical transmission electron microscope (TEM) to observe their size and morphology. Drop the micelle solution onto a copper grid and observe after natural drying.

[0080] Particle size and Zeta potential measurement: Use a Malvern Zeta potential analyzer to measure the particle size and Zeta potential of DpPDNPs in PBS buffer solutions at different pH values. Dilute the micelle solution to an appropriate concentration and then measure, repeat each sample 3 times, and take the average value.

[0081] Drug loading and encapsulation efficiency determination:

[0082] The content of doxorubicin (DOX) was determined using an ultraviolet-visible spectrophotometer. First, a standard curve of different concentrations of DOX was plotted. A certain amount of DpPDNPs micelle solution was taken, and an appropriate amount of methanol was added to demulsify it to completely release DOX. The absorbance at 480 nm was measured, and the drug loading and encapsulation efficiency of DOX were calculated according to the standard curve. The calculation formulas are as follows:

[0083] Drug loading (%) = (mass of DOX in micelles / total mass of micelles) × 100%;

[0084] Encapsulation efficiency (%) = (mass of DOX in micelles / total mass of added DOX) × 100%.

[0085] Determination of drug release behavior:

[0086] The dialysis method was used to determine the drug release behavior of DpPDNPs at different pH values. The DpPDNPs solution was filled into a dialysis bag, and after tying both ends tightly, it was placed in 150 mL of PBS buffer release medium with different pH values (pH = 6.5, 7.4) and oscillated in a constant temperature oscillator at 37 °C at a speed of 100 rpm.

[0087] At predetermined time points (0, 1, 2, 4, 8, 12, 24, 48, 72 h), 1 mL of the release medium was taken out and an equal volume of fresh release medium was added. The concentration of DOX in the release medium was measured by an ultraviolet-visible spectrophotometer, and the cumulative release rate was calculated according to the standard curve. The calculation formula is:

[0088] Cumulative release rate (%) = (cumulative released mass of DOX / initial mass of DOX in micelles) × 100%.

[0089] Example 2:

[0090] Based on the experimental study of the role of pH-responsive micelles, this example specifically includes, on the basis of Example 1:

[0091] ① In vitro anti-tumor efficacy study:

[0092] Human prostate cancer cell line PC3 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37 °C, 5% CO2 incubator.

[0093] CCK-8 method was used to detect cell toxicity: PC3 cells were seeded at 5 × 10 per well 3Cells were seeded in 96-well plates and cultured for 24 hours. Then, the medium was replaced with medium containing different concentrations of DpPDNPs (0, 1, 2, 4, 8, 16 μg / mL), and the cells were cultured for another 24 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for an additional 0.5 hour. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated. The formula for calculating cell survival rate was: Cell survival rate (%) = (Absorbance of experimental group / Absorbance of control group) × 100%. The experiment was repeated three times, and the average value was taken.

[0094] Cell uptake experiment: PC3 cells were seeded in confocal dishes and cultured for 24 hours. Then, the labeled DpPDNPs solution with a final concentration of 5 μg / mL was added, and the pH of the medium was adjusted to 6.5 and 7.4 using different buffers, respectively. After incubation for 0.5, 1, 2, 4, and 6 hours, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI for 5 minutes, and finally, the cell uptake of the micelles was observed using a fluorescence microscope.

[0095] Cell migration and invasion experiments:

[0096] Transwell invasion experiment: A layer of Matrigel matrix (dilution ratio 1:8) was pre-coated on the upper chamber of the Transwell insert and incubated at 37 °C for 30 minutes to solidify. Referring to the migration experiment method, the number of invading cells was observed and counted, and the experiment was repeated three times. The groups were: control group (saline), DOX group, DpDNPs group, and DpPDNPs group

[0097] Wound healing assay: PC3 cells were seeded in 6-well plates and cultured until the cell confluence reached about 90%. A scratch was made on the cell monolayer using a 200 μL pipette tip. The cells were washed three times with PBS to remove the scraped cells, and the medium containing different treatment factors was added and the cells were cultured continuously. At 0 and 36 hours, the cell migration was observed and photographed under a microscope, the scratch width was measured, and the cell migration rate was calculated. The formula was: Cell migration rate (%) = (Initial scratch width - Scratch width after treatment) / Initial scratch width × 100%. The experiment was repeated three times. The groups were: control group (saline), DOX group, DpDNPs group, and DpPDNPs group.

[0098] Colony formation assay: PC3 cells were digested, diluted, and resuspended, and then seeded in 35 mm cell culture dishes and cultured for one week. 4% paraformaldehyde was added for fixation, and after crystal violet staining, the number of colony formations was photographed and counted. The groups were: control group (saline), DOX group, DpDNPs group, and DpPDNPs group.

[0099] Immunofluorescence assay: Cells were seeded in confocal dishes. After sufficient cell attachment, the cells were fixed with 4% paraformaldehyde and blocked with goat serum for 20 minutes. The primary antibody against TKS5 was added and incubated overnight at 4°C. The secondary antibody conjugated with green fluorescence was used to stain TKS5, and phalloidin conjugated with red fluorescence was used to stain F-actin. Observation was performed using a confocal microscope.

[0100] Western blot assay: Samples were subjected to 10% SDS-PAGE gel electrophoresis. The separated protein samples were electrotransferred onto PVDF membranes and blocked with skim milk powder for two hours. The primary antibody solution was added and incubated at 4°C, and the secondary antibody solution was incubated at room temperature. The expressions of SOX2 protein and GAPDH protein were detected by the ECL method.

[0101] ② In vivo anti-tumor therapy experiment

[0102] Preparation of experimental animals:

[0103] Six-week-old male C57 nude mice, weighing 18 - 22 g, were purchased from Enzville Company.

[0104] PC3 cells in the logarithmic growth phase were collected, resuspended in PBS, and the cell concentration was adjusted to 5×10 5 / mL. 100 μL of the cell suspension was inoculated subcutaneously into the right back of male C57 mice, and the tumor growth was observed regularly. When the tumor volume reached approximately 100 - 150 mm 3 ³, the mice were randomly divided into 4 groups (5 mice in each group): control group (normal saline), DOX group, DpDNPs group, and DpPDNPs group.

[0105] Corresponding drugs were injected via the tail vein according to the grouping: the DOX group was injected with free DOX (dose 5 mg / kg), the DpDNPs group was injected with DpDNPs (dose 10 mg / kg), the DpPDNPs group was injected with DpPDNPs (dose 10 mg / kg, DOX content 5 mg / kg), and the control group was injected with an equal volume of normal saline. The drug was administered once every 3 days for a total of 4 times.

[0106] During drug administration, the major axis (L) and minor axis (W) of the tumors in mice were measured with vernier calipers every 2 days. The tumor volume was calculated according to the formula V = L×W 2 ² / 2, and the tumor growth curve was plotted to observe the inhibitory effect of different treatment groups on tumor growth.

[0107] After the treatment was completed, the mice were sacrificed, and the tumor tissues and major organs were removed, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to hematoxylin-eosin (H&E) staining and immunohistochemical staining (to detect the expressions of Ki-67 and SOX2 proteins). The pathological changes and cell proliferation of the tumor tissues were observed to evaluate the effectiveness and safety of drug treatment.

[0108] The tissue sections were dewaxed in an oven at 60 °C and then successively placed in gradient alcohols (100%, 95%, 85%, 75%) for 5 minutes each. The sections were boiled in sodium citrate solution for 10 minutes and cooled to room temperature naturally. After antigen retrieval, the Ki67 protein and SOX2 protein were stained using an immunohistochemistry kit.

[0109] Statistical analysis:

[0110] The experimental data were expressed as mean ± standard deviation (mean ± SD), and data analysis was performed using SPSS 22.0 statistical software. One-way ANOVA was used for comparison among multiple groups, and LSD-t test was used for pairwise comparison between groups. A P value < 0.05 was considered statistically significant.

[0111] Results:

[0112] Preparation and characterization of DpPDNPs

[0113] DpPNPs were successfully synthesized through an esterification reaction. Subsequently, DpPDNPs containing DOX were synthesized, which had good dispersibility in water and showed color changes in different pH environments. Under transmission electron microscopy (TEM), DpPDNPs were spherical, and the images clearly showed the morphological structure of typical micelles, indicating their good self-assembly properties. The particle size distribution measured by dynamic light scattering (DLS) showed that the average particle size was 21.02 nm. The Zeta potential of DpPDNPs micelles changed significantly at different pH values: it was 24.36 mV under acidic conditions (pH 6.5), and -18.56 mV and -32.76 mV under neutral (pH 7.4) and alkaline conditions, respectively. This pH-responsive potential change helps the micelles bind to negatively charged cell membranes in the tumor microenvironment and improve the cell uptake efficiency.

[0114] The ultraviolet-visible absorption spectra showed that DOX, DpDNPs, and DpPDNPs had consistent characteristic absorption peaks at 480 nm, indicating the successful loading of DOX. By the above method, the drug loading and encapsulation efficiency of DOX in DpPDNPs were determined at different DSPE-PEG to DOX feeding ratios. When DSPE-PEG:DOX was 1:1, the drug loading was 15% and the encapsulation efficiency was 85%. The micelles synthesized at this ratio had the highest encapsulation efficiency and relatively high drug loading, so 1:1 was selected as the optimal feeding ratio for subsequent experiments.

[0115] In vitro drug release experiment

[0116] The drug release behavior of DpPDNPs was determined by a dialysis system. In a weakly acidic environment with a pH of 6.5, DpPDNPs showed a rapid drug release trend within 24 hours, and the cumulative release rate reached 59% within 72 hours; while in a neutral environment with a pH of 7.4, the drug release was relatively slow, and the cumulative release rate was 29% within 72 hours. This indicates that the DpPDNPs micelles have pH-responsive drug release characteristics, can rapidly release drugs in the acidic microenvironment of tumors, and increase the drug concentration at the tumor site. Their pH responsiveness is attributed to the hydrolysis of ester bonds under acidic conditions.

[0117] Cytotoxicity experiment

[0118] As Figure 1-2 shown, the results of the standard CCK-8 experiment showed that the cytotoxicity of DpPDNPs against PC3 cells was concentration- and time-dependent ( Figure 1 ), and its half-maximal inhibitory concentration (IC50) was approximately 4.8 μg / mL. Therefore, 5 μg / mL was selected as the in vitro treatment dose. After grouped treatment, compared with free DOX and DpDNPs, DpPDNPs had a stronger inhibitory effect on cells at the same concentration.

[0119] Cell uptake experiment

[0120] The results of fluorescence microscopy showed that the uptake of DpPDNPs by PC3 cells was time-dependent. As the incubation time prolonged, the intracellular fluorescence intensity gradually increased, indicating that the micelles could be effectively taken up by the cells. At the same time, the acidic environment promoted the uptake of DpPDNPs micelles. Compared with the neutral medium at pH 7.4, PC3 cells had a higher uptake efficiency of DpPDNPs in the acidic medium at pH 6.5. This may be attributed to the pH responsiveness and charge reversal characteristics of DpPDNPs, thus improving the cell uptake efficiency.

[0121] Cell migration and invasion experiments

[0122] The results of Transwell migration and invasion experiments showed that compared with the control group, the DOX group, and the DpDNPs group, DpPDNPs treatment significantly reduced the invasion ability of PC3 cells: the number of cells passing through the Transwell chamber membrane in the DpPDNPs treatment group was significantly reduced, and the number of invasive cells was also significantly decreased, indicating that the micelles could effectively inhibit the invasion of prostate cancer cells ( Figure 3 ). The results of the scratch experiment showed that the cell migration rate in the DpPDNPs treatment group was significantly lower than that in the control group and other treatment groups. After 48 hours of scratching, the scratch width in the DpPDNPs treatment group was significantly larger than that in other groups, indicating that the micelles could inhibit the migration of prostate cancer cells ( Figure 4 ).

[0123] Cell proliferation assay

[0124] To detect the effect of DpPDNPs on the proliferation of PC3 cells, a colony formation assay was performed. The results showed that the number of PC3 cell colonies formed in the DpPDNPs group was the least, suggesting that DpPDNPs could effectively inhibit the proliferation of PC3 cells( Figure 5 ).

[0125] Effect of DpPDNPs on SOX2 expression

[0126] One of the functions of propanolol is to inhibit SOX2 expression, but there is no experimental verification of whether it can successfully inhibit SOX2 expression in prostate cancer cells. Therefore, we co-cultured DOX, DpDNPs, and DpPDNPs with PC3 cells for two weeks. Surprisingly, the expression of SOX2 increased in the DOX group and the DpDNPs group, which may be related to the induction of tumor cell drug resistance by DOX, while the expression of SOX2 was successfully inhibited in the DpPDNPs group. Therefore, we believe that propanolol carried by DpPDNPs can successfully inhibit the expression of SOX2 in PC3 cells.

[0127] DpPDNPs inhibit invadopodia formation by inhibiting SOX2 expression

[0128] Invadopodia provide tumor cells with the ability to invade and migrate. Its formation is regulated by TKS5, and F-actin constitutes the basic skeleton of invadopodia. The co-localization of the two can determine the position of invadopodia. Analysis through the GEPIA database (http: / / gepia.cancer-pku.cn / ) showed that SOX2 was positively correlated with the expression of TKS5 and F-actin. Based on this, it is speculated that DpPDNPs may inhibit invadopodia formation by inhibiting SOX2 expression.

[0129] By immunofluorescence detection of TKS5 and F-actin, it was found that the number of cells containing invadopodia was the largest in the PC3 drug-resistant cell line. Compared with the DOX group and the DpDNPs group, the inhibitory effect of DpPDNPs on invadopodia was more significant, while the effect of DOX and DpDNPs on invadopodia may be attributed to the inhibition of cell activity by anthracyclines( Figure 6 ). The above results confirmed that DpPDNPs inhibited the formation of invadopodia by inhibiting the expression of SOX2 in prostate cancer cells.

[0130] Antitumor therapy in vivo

[0131] The experimental results of the subcutaneous tumor model of prostate cancer showed that the tumor growth in the DpPDNPs treatment group was significantly inhibited. Compared with the control group, the DOX group, and the DpDNPs group, the tumor volume and tumor weight in the DpPDNPs treatment group were significantly reduced. After administration, the tumor volume growth rate in the DpPDNPs group slowed down significantly, and with the extension of the treatment time, the inhibitory effect became more significant.

[0132] The results of immunohistochemical staining showed that the expression levels of SOX2, Ki67, TKS5, Cortactin, and F-actin in the tumor tissue were significantly reduced, indicating that DpPDNPs could inhibit tumor cell proliferation and the expression of related proteins. In addition, the results of H&E staining of the main organs such as the heart, liver, spleen, lungs, and kidneys showed that the organizational structures of each organ were intact and there were no obvious pathological changes, indicating that DpPDNPs had good biosafety in vivo.

[0133] As mentioned above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent replacements or changes should be covered within the protection scope of the present invention.

Claims

1. Application of a pH-responsive micelle in a drug for treating prostate cancer, characterized in that, Drug delivery using pH-responsive micelles loaded with doxorubicin; The pH-responsive micelles are prepared by the following steps: S1: Esterify propranolol with DSPE-PEG-COOH to obtain DpP micelles; S2: Dissolve DpP micelles and doxorubicin (DOX) in an appropriate amount of methanol according to a predetermined feeding ratio, and remove methanol by rotary evaporation under reduced pressure to form a uniform thin film; S3: Add PBS buffer solution and stir and hydrate at room temperature to obtain a DpPDNPs solution, thus obtaining the product.

2. The application of a pH-responsive micelle in a drug for treating prostate cancer according to claim 1, wherein The specific steps of the esterification reaction are as follows: S11: Dissolve propranolol in dichloromethane, add DCC and DMAP, and stir in a water bath; S12: Add DSPE-PEG-COOH to the reaction system of S11 and continue the water bath reaction for 48 hours; S13: After the reaction is completed, remove dichloromethane by rotary evaporation to obtain DpP micelles.

3. Use of a pH-responsive micelle according to claim 2 in a medicament for treating prostate cancer, characterized in that, In S11, dissolve 2.59 mg of propranolol in 5 mL of dichloromethane, add 206.33 μg of DCC and 37.87 μg of DMAP, and stir in a water bath at 35 °C for 1.5 hours; In S12, add 27.80 mg of DSPE-PEG-COOH to the above reaction system and continue the water bath reaction for 48 hours.

4. Use of a pH-responsive micelle according to claim 2 in a medicament for treating prostate cancer, characterized in that, The characterization of the pH-responsive micelles includes: S21: Observe its size and morphology using a transmission electron microscope; S22: Measure the particle size and Zeta potential of DpPDNPs at different pH values using a Malvern Zeta potential analyzer; S23: Determine the doxorubicin content using an ultraviolet-visible spectrophotometer and calculate the drug loading and encapsulation efficiency; S24: Determine the drug release behavior of DpPDNPs at different pH values by dialysis method.

5. Use of a pH-responsive micelle according to claim 2 in a medicament for treating prostate cancer, characterized in that, Apply the pH-responsive micelles to the study of in vitro anti-tumor efficacy, specifically including: S31: Cytotoxicity experiment: Inoculate PC3 cells, after culturing for 24 hours, replace the culture medium with different concentrations of DpPDNPs and continue culturing for 24 hours, and detect cell viability; S32: Cell uptake experiment: Inoculate PC3 cells in a confocal culture dish, after culturing for 24 hours, add the labeled DpPDNPs solution, incubate under different pH conditions respectively, and observe the cell uptake of micelles through a fluorescence microscope; S33: Cell migration and invasion experiment: Conduct Transwell invasion experiment and scratch experiment, observe and count the number of invasive cells and cell migration rate; S34: Colony formation experiment: Digest, dilute and resuspend PC3 cells, inoculate them in a culture dish and culture for one week, fix them and then perform crystal violet staining, and count the number of colony formations.

6. Use of a pH-responsive micelle according to claim 5 in a medicament for treating prostate cancer, characterized in that, In S31, PC3 cells were seeded at 5×10 3 cells per well in a 96-well plate. After culturing for 24 hours, the medium was replaced with a medium containing different concentrations of DpPDNPs, and the cells were cultured for another 24 hours. Cell viability was detected using a CCK-8 reagent.

7. Use of a pH-responsive micelle according to claim 5 in a drug for treating prostate cancer, characterized in that, In S32, incubate under pH 6.5 and 7.4 conditions, and then observe the cell uptake of micelles through a fluorescence microscope.

8. The application of a pH-responsive micelle in a drug for treating prostate cancer according to claim 2, wherein, Apply the pH-responsive micelles to in vivo anti-tumor therapy experiments, specifically including: S41: Collect PC3 cells in the logarithmic growth phase, resuspend them in PBS, adjust the cell concentration, and inoculate them subcutaneously in male mice; S42: When the tumor volume reaches 100 - 150 mm 3 , randomly divide the mice into groups and inject the corresponding drugs through the tail vein; S43: Administer the drug once every 3 days for a total of 4 times; S44: During the drug administration period, the major and minor axes of the tumors in the mice were measured every two days, the tumor volumes were calculated, and tumor growth curves were plotted. S45: After the treatment was completed, the mice were sacrificed, the tumor tissues and major organs were removed, and hematoxylin-eosin staining and immunohistochemical staining were performed.

9. Use of a pH-responsive micelle according to claim 8 in a medicament for treating prostate cancer, characterized in that, In S41, the cell concentration was adjusted to 5×105 / mL and inoculated subcutaneously into the right back of male C57 mice; in S45, the major organs included the heart, liver, spleen, lungs, and kidneys.

10. Use of a pH-responsive micelle according to claim 8 in a medicament for treating prostate cancer, characterized in that, The immunohistochemical staining was used to detect the expression levels of SOX2, Ki67, TKS5, Cortactin, and F-actin.

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