Drug-loaded nanoparticles as well as preparation method and application thereof
By loading active ingredients onto magnetic nanoparticles and coating them with erythrocyte membranes, drug-loaded nanoparticles were prepared, solving the problems of high toxicity, short half-life, and poor targeting of chemotherapy drugs in the treatment of triple-negative breast cancer. This approach achieves highly efficient treatment and good biosafety for triple-negative breast cancer.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2026-04-28
AI Technical Summary
Existing chemotherapy drugs used to treat triple-negative breast cancer suffer from high toxicity, short half-life, poor targeting, and rapid metabolism of nanoparticles, resulting in poor treatment efficacy and poor biosafety.
Drug-loaded nanoparticles, designed by loading active ingredients onto magnetic nanoparticles and coating red blood cell membranes, are prepared by rotary evaporation and co-extrusion. Combined with specific active ingredients such as long peppermint, they prolong blood circulation time and improve tumor retention.
It achieves highly efficient killing and therapeutic effects on triple-negative breast cancer by inhibiting the PI3K-AKT-mTOR pathway and inducing apoptosis, and has good biocompatibility and long-term circulation capability.
Smart Images
Figure CN121926893A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a drug-loaded nanoparticle, its preparation method, and its application, specifically to a drug-loaded nanoparticle for treating triple-negative breast cancer, its preparation method, and its application. Background Technology
[0002] Breast cancer is the most common malignant tumor among women worldwide. Among them, triple-negative breast cancer faces great challenges in clinical treatment due to its heterogeneity, poor prognosis, and lack of clear molecular targets (Mo, R., Jiang, T. et al. Emerging micro-and nanotechnology based synthetic approaches for insulin delivery. Chemical society reviews, 43(10), 3595-3629). To date, chemotherapy has been the main clinical treatment for triple-negative breast cancer, but the use of chemotherapy drugs alone has inherent defects such as high toxicity and short half-life, which hinders the clinical application of chemotherapy (Dragojevic, S. et al. Polymer-based prodrugs: improving tumor targeting and the solubility of small molecule drugs in cancer therapy. Molecules, 2015. 20(12): p. 21750-21769.).
[0003] The development of nanotechnology has provided new ideas for solving the defects of chemotherapy drugs. By designing suitable nanoparticles and loading chemotherapy drugs onto them, the inherent defects such as poor drug solubility, short half-life, and poor targeting can be solved. However, during the treatment process, nanoparticles themselves, as foreign substances from outside the body, still have defects such as fast metabolism, making it difficult to achieve the ideal therapeutic effect. Therefore, it is necessary to modify them to improve their blood circulation time. The classic modification method is mainly PEG modification, but this method has the defect of causing immunogenicity after multiple injections (Xu, Q. et al. Impact of surface polyethylene glycol (PEG) density on biodegradable nanoparticle transport inmucus ex vivo and distribution in vivo. ACS nano, 2015. 9(9): p. 9217-9227.).
[0004] Therefore, there is an urgent need for a drug delivery method that can effectively prolong blood circulation time and has good biocompatibility. Summary of the Invention
[0005] This invention addresses the shortcomings of existing chemotherapy drugs in inducing triple-negative breast cancer cell death, such as insufficient tumor accumulation, poor therapeutic effect, and poor biosafety, by providing drug-loaded nanoparticles, their preparation method, and applications. These drug-loaded nanoparticles exhibit good biosafety, long circulation capacity, and high tumor retention capacity, and can exert a highly effective killing and therapeutic effect on triple-negative breast cancer by inducing apoptosis.
[0006] To achieve the above objectives, the present invention provides the following technical solutions.
[0007] The present invention provides a drug-loaded nanoparticle for treating triple-negative breast cancer, the drug-loaded nanoparticle comprising: magnetic nanoparticles and an active ingredient loaded thereon, the drug-loaded nanoparticle further comprising a red blood cell membrane coating the magnetic nanoparticles;
[0008] The active ingredient is selected from one or more of the following: piperamide, Paris saponin II, romaine, senna acetic acid, 6-gingerol, ginkgolide, solanine, cucurbitacin B, and lycorine.
[0009] In this invention, the structural formulas of the active ingredients are as follows:
[0010] Piperamide ,
[0011] Paris saponin II ,
[0012] Mao Lansu ,
[0013] Pseudolarix amabilis acetic acid ,
[0014] 6-Gingerol ,
[0015] Ginkgo biloba ,
[0016] Solanine ,
[0017] Cucurbitacin B ,
[0018] Lycorine .
[0019] In some embodiments, the active ingredient is selected from one or more of piperazine, Paris polyphylla saponin II, cucurbitacin B, and solanine; preferably, the active ingredient is piperazine.
[0020] In some embodiments, the magnetic nanoparticles are negatively charged magnetic nanoparticles, preferably iron(III) oxide nanoparticles.
[0021] In some implementations, the magnetic nanoparticles exist in cluster form.
[0022] The present invention also provides a method for preparing drug-loaded nanoparticles, the method comprising the following steps:
[0023] S1. Loading of active ingredient: The active ingredient and magnetic nanoparticles are used to prepare a drug-loaded nanoparticle precursor by rotary evaporation; the active ingredient and magnetic nanoparticles are as defined above.
[0024] S2. Red blood cell membrane coating: The drug-loaded nanoparticle precursor and red blood cell membrane are co-extruded to obtain the drug-loaded nanoparticles.
[0025] In some embodiments, in step S1, the mass ratio of the active ingredient to the magnetic nanoparticles is (0.1-1.2):(0.5-2), preferably (0.2-0.5):(0.8-1.2), and more preferably 0.3:1.
[0026] In some embodiments, step S1 includes the following steps: mixing the magnetic nanoparticles and the active ingredient in solvent A, and then rotary evaporating.
[0027] Solvent A is preferably deionized water.
[0028] The mixing time is preferably 6-24 hours, for example 12 hours.
[0029] Preferably, the active ingredient is dissolved in solvent B before being mixed; solvent B is preferably anhydrous ethanol.
[0030] The rotary evaporation temperature is preferably 36-40°C, for example 38°C.
[0031] Preferably, the rotary evaporation step further includes a washing and drying step; the washing reagent is, for example, deionized water; and the drying is, for example, freeze drying.
[0032] In some specific implementations, step S1 includes the following specific steps:
[0033] The active ingredient was dissolved in anhydrous ethanol, and the iron oxide nanoparticles were dispersed in deionized water. The two were mixed and shaken overnight, and the active ingredient was loaded into the iron oxide nanoparticles by rotary evaporation. The resulting product was washed several times with deionized water and then freeze-dried for later use.
[0034] In some embodiments, in step S2, the mass ratio of the red blood cell membrane to the drug-loaded nanoparticle precursor is (0.5-1.5):(0.5-1.5), preferably (1-1.5):(0.5-1), for example 1:0.5.
[0035] In some implementations, step S2, the co-extrusion method includes the following steps: mixing the red blood cell membrane and the drug-loaded nanoparticle precursor, sonicating, and extruding.
[0036] The ultrasound is preferably performed under ice bath conditions.
[0037] The power of the ultrasound is preferably 50-300W, more preferably 80-150W, for example 100W.
[0038] The duration of the ultrasound is preferably 10-50 seconds, more preferably 20-40 seconds, for example 30 seconds.
[0039] The number of compressions is preferably 7-16 times, more preferably 12-16 times, for example 15 times.
[0040] The extrusion is preferably performed by an extrusion device, such as a liposome extruder.
[0041] In some specific implementations, step S2 includes the following specific steps:
[0042] The red blood cell membrane and the drug-loaded nanoparticle precursor were mixed at a certain mass ratio and sonicated under ice bath conditions for a period of time. Then, the mixture was extruded using an extrusion device to obtain the drug-loaded nanoparticles.
[0043] In some embodiments, the magnetic nanoparticles are prepared by a solvothermal method, which includes the following steps:
[0044] In solvent C, ferric chloride hexahydrate, ammonium acetate, and polyglutamic acid react, followed by a secondary reaction in a reaction vessel.
[0045] The solvent C is preferably ethylene glycol.
[0046] The preferred mass ratio of ferric chloride hexahydrate, ammonium acetate, and polyglutamic acid is (1-10):(1-10):(0.1-10), more preferably (1-2):(3-4):(0.5-0.8), for example, 1.08:3.85:0.5.
[0047] The reaction temperature is preferably 120-200°C, more preferably 150-180°C, for example 160°C.
[0048] The reaction time is preferably 0.5-2 hours, more preferably 1-1.5 hours, for example 1 hour.
[0049] The reaction is preferably carried out under stirring during heating and reflux.
[0050] Preferably, the secondary reaction is carried out in a forced-air drying oven.
[0051] The temperature of the secondary reaction is preferably 160-210°C, more preferably 180-200°C, for example 200°C.
[0052] The duration of the secondary reaction is preferably 6-24 hours, more preferably 8-14 hours, for example 12 hours.
[0053] Preferably, the secondary reaction step further includes magnetic separation, washing, and dispersion steps; the washing reagent is, for example, anhydrous ethanol and / or deionized water.
[0054] In some specific embodiments, the method for preparing the magnetic nanoparticles includes the following steps:
[0055] Weigh out ferric chloride hexahydrate, acetic acid, and polyglutamic acid, dissolve them in ethylene glycol, heat under reflux at a certain temperature and stir mechanically for a period of time, then place them in a stainless steel high-pressure reactor lined with polytetrafluoroethylene. Place the reactor in a pre-set temperature drying oven and react for a period of time. After removal, allow it to cool naturally to room temperature. Separate the product with a magnet, wash with anhydrous ethanol and deionized water to remove impurities, and finally disperse the product in deionized water for later use.
[0056] In some embodiments, the red blood cell membrane is prepared by a hypotonic membrane rupture method, which includes the following steps:
[0057] Red blood cells are suspended in a hypotonic solution, centrifuged to remove hemoglobin, and the red blood cell membrane is obtained by squeezing.
[0058] The red blood cells are preferably obtained by centrifugation of whole blood.
[0059] The whole blood is preferably derived from ICR mice.
[0060] Among them, the preferred method for collecting whole blood is to collect blood from the eye socket.
[0061] The centrifugation step is preferably performed in an anticoagulant tube containing heparin.
[0062] The centrifugal speed for centrifugal separation is preferably 1000-4000 rpm, more preferably 2000-3500 rpm, for example 3000 rpm.
[0063] The centrifugation time for centrifugation is preferably 3-20 min, more preferably 5-15 min, for example 5 min.
[0064] The suspension time is preferably 20-60 min, more preferably 20-40 min, for example 30 min.
[0065] The centrifugation speed for removing hemoglobin is preferably 8000-15000 rpm, more preferably 8000-13000 rpm, for example 10000 rpm.
[0066] The centrifugation time for removing hemoglobin is preferably 5-15 min, more preferably 3-20 min, for example 5 min.
[0067] Preferably, the step of centrifuging to remove hemoglobin also includes washing and sonication steps.
[0068] The power of the ultrasound is preferably 50-300W, more preferably 80-150W, for example 100W.
[0069] The duration of the ultrasound is preferably 10-50 seconds, more preferably 20-40 seconds, for example 30 seconds.
[0070] In some specific embodiments, the method for preparing the red blood cell membrane includes the following steps:
[0071] Whole blood was collected from ICR mice via orbital blood sampling. Fresh blood was collected in heparin-containing anticoagulant tubes and centrifuged to separate red blood cells. The resulting red blood cell pellet was washed multiple times with PBS. The red blood cells were then dispersed in a hypotonic solution under ice bath conditions and suspended for a period of time. The centrifuge tube was manually shaken every 10 minutes to accelerate the rupture of red blood cells. Hemoglobin was then removed by centrifugation. The pellet was washed multiple times with hypotonic solution and PBS in sequence. The resulting red blood cell membrane was sonicated at a certain power under ice bath conditions for a period of time. Finally, red blood cell membrane fragments were extruded using a micro extruder to obtain the red blood cell membrane.
[0072] The present invention also provides a drug-loaded nanoparticle, which is prepared by the method described above for preparing drug-loaded nanoparticles.
[0073] The present invention also provides the application of the drug-loaded nanoparticles as described above in the preparation of a drug for treating triple-negative breast cancer.
[0074] In some embodiments, the dosage form of the drug is selected from one or more of the following: injection, gel, in situ gelation system, solution, suspension, emulsion, implant, transdermal agent, and microneedle; preferably, the dosage form of the drug is an injection.
[0075] In some embodiments, the route of administration of the drug includes one or more of the following: intravenous bolus injection, intratumoral injection, intravenous infusion, intraperitoneal injection, hepatic artery infusion, intramuscular injection, subcutaneous injection, implantation, transdermal absorption, and interventional route; preferably, the route of administration of the drug is intravenous bolus injection.
[0076] In some embodiments, the effective concentration of the drug is preferably not higher than 10 μg / mL;
[0077] In some implementations, the duration of the drug's effectiveness is preferably not less than 24 hours.
[0078] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.
[0079] The reagents and raw materials used in this invention are all commercially available.
[0080] The positive and progressive effects of this invention are as follows:
[0081] This invention targets specific active ingredients against triple-negative breast cancer and constructs drug-loaded nanoparticles using a specific drug loading and erythrocyte membrane coating method. These nanoparticles exhibit good biocompatibility, long circulation capacity, and high tumor retention capacity. They can induce apoptosis of cancer cells by inhibiting the PI3K-AKT-mTOR pathway and Bcl-2 expression, thereby producing a highly effective killing and therapeutic effect on triple-negative breast cancer. Attached Figure Description
[0082] Figure 1 The image shown is a transmission electron microscope image of Fe3O4-PL in Example 1.
[0083] Figure 2 The image shows a transmission electron microscope image of Fe3O4-PL@RBC in Example 1.
[0084] Figure 3To assess the effectiveness, in Example 2, the survival rate of mouse fibroblast L929 cells was determined after incubation with different concentrations of Fe3O4@RBC for 24 hours.
[0085] Figure 4 To assess the effectiveness of the study, in Example 2, the survival rate of triple-negative breast cancer cells MDA-MB-231 was determined after 24 hours of incubation with DMEM and different concentrations of PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC.
[0086] Figure 5 To assess the effectiveness, in Example 2, after incubation for 8 hours with DMEM, PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC, the reactive oxygen species levels of triple-negative breast cancer cells MDA-MB-231 were measured.
[0087] Figure 6 To assess the effectiveness, in Example 2, after incubation for 8 hours with DMEM, PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC, the mitochondrial membrane potential level of triple-negative breast cancer cells MDA-MB-231 was determined.
[0088] Figure 7 Volcano diagram of gene expression in MDA-MB-231 triple-negative breast cancer cells after treatment with the control group and Fe3O4-PL@RBC group in Example 3.
[0089] Figure 8 This is a GO enrichment analysis diagram of significantly differentially expressed genes in Example 3.
[0090] Figure 9 The image shows the KEGG enrichment analysis of significantly differentially expressed genes in Example 3.
[0091] Figure 10 For the effect, after incubating with DMEM, PL, Fe3O4, Fe3O4-PL and Fe3O4-PL@RBC for 8 hours, respectively, the expression results of Bax, Bcl-2, p-mTOR, mTOR, p-AKT and AKT proteins in triple-negative breast cancer cells MDA-MB-231 were obtained.
[0092] Figure 11 The in vivo pharmacokinetic curves of Fe3O4 and Fe3O4@RBC in Example 4 are shown.
[0093] Figure 12 The tissue distribution of Fe3O4 and Fe3O4@RBC in Example 4 is shown after 4 hours and 24 hours.
[0094] Figure 13The tumor growth curves of tumor-bearing mice after treatment with saline, PL, Fe3O4, Fe3O4-PL and Fe3O4-PL@RBC in Example 4 are shown.
[0095] Figure 14 The images and corresponding weights of tumor-bearing mice after treatment with saline, PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC in Example 4 are shown. Detailed Implementation
[0096] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.
[0097] Example 1: Preparation of drug-loaded nanoparticles
[0098] The drug-loaded nanoparticles of this embodiment were prepared according to the following steps:
[0099] 1. Preparation of magnetic nanoparticles (Fe3O4 nanoparticles)
[0100] Weigh 1.083g of ferric chloride hexahydrate (F102742-500g, Shanghai Aladdin Biochemical Technology Co., Ltd.), ammonium acetate (NH4OAc), 0.5g of polyglutamic acid (P108506-500g, Shanghai Aladdin Biochemical Technology Co., Ltd.), and 3.85g of ammonium acetate (L011599500G, Sinopharm Chemical Reagent Co., Ltd.) and dissolve them in 70mL of ethylene glycol (EG). Heat the mixture under reflux at 160℃ with mechanical stirring for 1h. Then place it in a stainless steel high-pressure reactor lined with polytetrafluoroethylene. Place the reactor in a forced-air drying oven at a pre-set temperature of 200℃ for 12h. After removal, allow it to cool naturally to room temperature. Separate the product using a magnet and wash with anhydrous ethanol and deionized water to remove impurities. Finally, disperse the product in deionized water for later use.
[0101] 2. Preparation of drug-loaded nanoparticle precursor (Fe3O4-PL)
[0102] 3 mg (S31735-100 mg, Shanghai Yuanye Biotechnology Co., Ltd.) of long pepperamide (PL) was dissolved in 2 mL of anhydrous ethanol, and 10 mg of iron oxide nanoparticles were dispersed in 2 mL of deionized water. The two were mixed and shaken overnight (12 h). Long pepperamide was loaded into iron oxide nanoparticles by rotary evaporation at 38 °C. The product was separated by a magnet and washed twice with deionized water. The precipitate obtained was the product and was freeze-dried for storage.
[0103] 3. Preparation of red blood cell membranes (RBCs)
[0104] Red blood cell membranes were prepared using a hypotonic percolation method: Whole blood was collected from ICR mice via orbital blood collection. Fresh blood was collected in an anticoagulant tube containing heparin. Red blood cells were separated by centrifugation at 3000 rpm for 5 min at 4°C. The resulting red blood cell pellet was washed multiple times with PBS. The red blood cells were then dispersed in a hypotonic solution under ice bath conditions and suspended for 30 min. The centrifuge tube was manually shaken every 10 min to accelerate the rupture of red blood cells. Subsequently, hemoglobin was removed by centrifugation at 10000 rpm for 5 min at 4°C. The red blood cells were washed multiple times with hypotonic solution and PBS in sequence. The resulting red blood cell membranes were subjected to sonication at 100W power for 30 s under ice bath conditions. Then, red blood cell membrane fragments were extruded 15 times using a micro extruder to obtain red blood cell membrane vesicles.
[0105] 4. Preparation of drug-loaded nanoparticles (Fe3O4-PL@RBC)
[0106] Red blood cell membranes and drug-loaded nanoparticle precursors were mixed in an ice bath at a mass ratio of 1 part: 0.5 parts, and then sonicated at 100W for 30 seconds under ice bath conditions. The mixture was then extruded 15 times using an extrusion device (liposome extruder) to obtain the drug-loaded nanoparticles of this embodiment.
[0107] Example 1: Structural Characterization of Drug-Loaded Nanoparticles
[0108] The drug-loaded nanoparticles (Fe3O4-PL@RBC) obtained in Example 1, as well as the intermediate products obtained during the process (Fe3O4, Fe3O4-PL, RBC, etc.), were characterized as follows: the structure and size of the nanoparticles were observed using a high-contrast transmission electron microscope (HT7800, Hitachi High Technology Corporation); the hydration size and zeta potential of the nanoparticles were determined using a nanoparticle size-zeta potential analyzer (ZS90, Malvern Panaco Corporation).
[0109] Statistical analysis: All quantitative results are presented as mean ± standard deviation. Student's t-test was used for data comparison. * P < 0.05 ** P < 0.01, *** P < 0.001.
[0110] See results Figure 1 , Figure 2 See Table 1. Figure 1 and Figure 2 These are transmission electron microscopy (TEM) images of two types of nanoparticles (Fe3O4-PL and Fe3O4-PL@RBC). Figure 1 It can be seen that the Fe3O4-PL nanoparticles prepared by this invention have regular shapes and uniform particle size distribution. Figure 2 The image shows Fe3O4-PL@RBC obtained by co-extruding red blood cell membranes onto the surface of Fe3O4-PL. As can be seen from the figure, the particle surface is coated with a membrane, indicating the successful coating of the RBC membrane.
[0111] Table 1 shows the hydration size and zeta potential of RBC, Fe3O4-PL, and Fe3O4-PL@RBC. As can be seen from the table, the hydration size of Fe3O4-PL is 242.6 nm, and the hydration size of Fe3O4-PL@RBC increases to 261.5 nm. The increased particle size is approximately the thickness of two red blood cell membranes (8-9 nm), and the zeta potential changes from -19.2 mV to -10.5 mV (close to the zeta potential of the red blood cell membrane). All of these results indicate that the red blood cell membrane is successfully encapsulated in the drug-loaded nanoparticles.
[0112] Table 1. Hydration particle size and zeta potential of RBC, Fe3O4-PL and Fe3O4-PL@RBC
[0113]
[0114] Example 2: Validation of the in vitro anticancer effect of drug-loaded nanoparticles
[0115] The following verifications were performed on the drug-loaded nanoparticles (Fe3O4-PL@RBC) obtained in Example 1, and the intermediate products obtained during the process (Fe3O4, Fe3O4-PL, RBC, etc.):
[0116] 1. In vitro cytotoxicity test
[0117] The relative viability of L929 cells and MDA-MB-231 cells was detected using the CCK-8 assay (C0038, Shanghai Beyotime Biotechnology Co., Ltd.). L929 cells were seeded in 96-well plates and cultured overnight in a cell culture incubator. After incubation with DMEM and different concentrations of Fe3O4@RBC for 24 h, CCK-8 reagent (10 μL per well) was added, and the cells were cultured for another 2 h. The absorbance at 540 nm was then measured using a microwell apparatus. MDA-MB-231 cells were seeded in 96-well plates and cultured overnight in a cell culture incubator. After incubation with DMEM, PL, and different nanoparticles at PL and nanoparticle concentrations for 24 h, CCK-8 reagent (10 μL per well) was added, and the cells were cultured for another 2 h. The absorbance at 540 nm was then measured using a microwell apparatus. The relative cell viability of the experimental groups is shown as the percentage of cell viability compared to the DMEM-treated control group.
[0118] 2. Detection of intracellular reactive oxygen species (ROS)
[0119] ROS levels in triple-negative breast cancer MDA-MB-231 cells were detected by flow cytometry. MDA-MB-231 cells were cultured overnight in 6-well plates and then cultured for 8 hours in 1 mL DMEM, PL (5 μg / mL), Fe3O4 (27 μg / mL), Fe3O4-PL, and Fe3O4-PL@RBC nanoparticles (equivalent PL or Fe3O4 concentrations). Cells were then digested with trypsin, collected by centrifugation, and washed once with PBS. Cells were dispersed in medium supplemented with DCFH-DA (2',7'-dichlorodihydrofluorescein diacetic acid, S0033S, Shanghai Beyotime Biotechnology Co., Ltd.), transferred to 1.5 mL centrifuge tubes, and cultured for another 20 min. Finally, all cells were washed twice with PBS and analyzed by flow cytometry (Gallios, Beckman Coulter).
[0120] 3. Detection of mitochondrial membrane potential (MMP)
[0121] Changes in mitochondrial membrane potential in MDA-MB-231 cells were detected using confocal microscopy. Triple-negative breast cancer MDA-MB-231 cells were cultured overnight in 6-well plates, then incubated for 8 hours with 1 mL of DMEM, PL (5 μg / mL), Fe3O4 (27 μg / mL), Fe3O4-PL, and Fe3O4-PL@RBC nanoparticles (equivalent PL concentration). After washing the cells once with PBS, 1 mL of JC-1 staining working solution (C2006, Shanghai Beyotime Biotechnology Co., Ltd.) was added, and the cells were incubated for another 20 min. Finally, all cells were washed twice with JC-1 staining buffer and observed using a laser confocal microscope.
[0122] 4. Apoptosis detection
[0123] Apoptosis rate of triple-negative breast cancer MDA-MB-231 cells was detected by flow cytometry. MDA-MB-231 cells were seeded in 6-well plates and cultured overnight in a cell culture incubator. After 24 hours of culture, the cells were incubated with 1 mL of DMEM, PL (10 μg / mL), Fe3O4 (54 μg / mL), Fe3O4-PL, and Fe3O4-PL@RBC nanoparticles (equivalent PL or Fe3O4 concentrations). Cells were then digested with trypsin, collected by centrifugation, and stained with the Annexin V-FITC apoptosis kit (S1062S, Beyotime Biotechnology Co., Ltd.) for 10 min. Finally, the cells were analyzed by flow cytometry (Gallios, Beckman Coulter).
[0124] The results are shown in Figures 3-6 .
[0125] Figure 3The survival rate of L929 cells after incubating DMEM and different concentrations of Fe3O4@RBC with L929 cells for 24 hours was shown in the figure. It can be seen from the figure that Fe3O4@RBC has almost no cytotoxicity, which proves the biosafety of the material.
[0126] Figure 4 The survival rate of MDA-MB-231 cells after incubation with different substances (DMEM, PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC) for 24 hours was shown in the figure. The figure reveals that, compared to the DMEM group, the cell survival rate of the group containing PL was significantly reduced in a concentration-dependent manner, indicating that this nanomedicine can exert a good anti-cancer effect at the cellular level. This demonstrates that Fe3O4-PL@RBC of the present invention has excellent anti-cancer effects.
[0127] Figure 5 The levels of ROS induced in MDA-MB-231 cells by different substances (DMEM, PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC) were measured. The figures show that, compared to the DMEM group, the ROS levels in all experimental groups were increased, especially in the Fe3O4-PL@RBC group. This indicates that Fe3O4-PL@RBC induces an increase in ROS levels within MDA-MB-231 cells.
[0128] Figure 6 The study investigated the changes in mitochondrial membrane potential in MDA-MB-231 cells induced by different substances (DMEM, PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC). The figures show that, compared to the DMEM group, the mitochondrial membrane potential in the PL-containing experimental groups decreased. This indicates that Fe3O4-PL@RBC effectively induces mitochondrial disruption within the cells.
[0129] To investigate the apoptosis rate of MDA-MB-231 cells induced by different substances (DMEM, PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC), it was found that the apoptosis rate was increased in all experimental groups containing PL compared to the DMEM group, especially in the Fe3O4-PL@RBC group. This indicates that Fe3O4-PL@RBC induces cell death by inducing apoptosis in MDA-MB-231 cells.
[0130] Example 3: Validation of the in vitro anticancer mechanism of drug-loaded nanoparticles
[0131] The anticancer mechanism was verified using the following steps for the Fe3O4-PL@RBC obtained in Example 1, the drug-loaded nanoparticles (Fe3O4-PL@RBC) obtained in Example 1, and the intermediate products obtained during the process (Fe3O4, Fe3O4-PL, RBC, etc.):
[0132] Transcriptome sequencing was performed on MDA-MB-231 cells treated with Fe3O4-PL@RBC. MDA-MB-231 cells were first seeded in 6-well plates and cultured for 12 hours, then treated with 1 mL of Fe3O4-PL@RBC (equivalent to a PL concentration of 5 μg / mL). Untreated cells served as a control group. After 8 hours of incubation, intracellular RNA was extracted using Trizol reagent (Life Technologies, USA) and analyzed.
[0133] Western blot analysis was performed on proteins from MDA-MB-231 cells treated with Fe3O4-PL@RBC. First, MDA-MB-231 cells were seeded in 6-well plates and cultured for 12 hours. Then, the cells were cultured for 8 hours with 1 mL of DMEM, PL (5 μg / mL), Fe3O4 (27 μg / mL), Fe3O4-PL, and Fe3O4-PL@RBC nanoparticles (equivalent PL concentration). Cells were washed, lysed, collected, and centrifuged at 12,000 rpm for 15 minutes at 4°C. Protein quantification was then performed using BCA reagent. Next, equal volumes of protein samples were separated using SDS-PAGE and transferred to a PVDF membrane, which was blocked with rapid blocking buffer for 0.5 hours. Finally, the membrane was co-incubated overnight at 4°C with antibodies against β-actin, Bax, Bcl-2, vinculin, mTOR, p-mTOR, Akt, and p-Akt. After washing three times with TBST washing solution, incubate with secondary antibody for 1 hour, then wash three more times with TBST washing solution before development.
[0134] The results are as follows Figures 7-10 As shown.
[0135] Figure 7 The differential gene volcano plot shows that gene expression changes after Fe3O4-PL@RBC treatment.
[0136] Figure 8 The GO enrichment analysis results show that Fe3O4-PL@RBC mainly affects the biological processes (including cellular processes, biological regulation, metabolic processes, and responses to stimuli), cellular components (including cells, cellular parts, organelles, and membranes), and molecular functions (including binding, catalytic activity, molecular functional regulators, and transcription activator activities) of MDA-MB-231 cells.
[0137] Figure 9 The KEGG enrichment analysis results show that Fe3O4-PL@RBC induces apoptosis in MDA-MB-231 cells and induces apoptosis by affecting the PI3K-AKT-mTOR pathway.
[0138] Figure 10 Analysis of protein expression changes in MDA-MB-231 cells after Fe3O4-PL@RBC treatment showed that Akt, p-Akt, mTOR, p-mTOR, and Bcl-2 were downregulated after Fe3O4-PL@RBC treatment, indicating that Fe3O4-PL@RBC induces apoptosis by inhibiting the PI3K-AKT-mTOR pathway and Bcl-2 expression.
[0139] Example 4: Validation of the in vivo anticancer effect of drug-loaded nanoparticles
[0140] The following verifications were performed on the drug-loaded nanoparticles (Fe3O4-PL@RBC) obtained in Example 1, and the intermediate products obtained during the process (Fe3O4, Fe3O4-PL, RBC, etc.):
[0141] 1. Animal models
[0142] 100 μL of MDA-MB-231 cells (1x10⁻¹) were subcutaneously injected into the right axilla of female BALB / c nude mice (4-5 weeks old, 15-20 g in weight, Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). 8 A tumor model was established using cells / mL. Prior to in vivo anticancer therapy, the tumor volume should reach 70 ± 20 mm. 3 .
[0143] 2. Tissue distribution and metabolic kinetics of Fe3O4-PL@RBC
[0144] When the subcutaneous tumor volume in mice reaches 70 ± 20 mm 3 Fe3O4 and Fe3O4@RBC nanoparticles (2 mg / mL) were injected intravenously into the tail vein of mice. Four and 24 hours after injection, mice were anesthetized with chloral hydrate intraperitoneally and their hearts were perfused. Tumors and major organs (heart, liver, spleen, lung, and kidney) were then collected. Each tissue was weighed and digested with an acidic mixture (Vperchloric acid:Vhydrochloric acid = 1:4), and the mixture was placed in a fume hood to allow the acid to evaporate. After the acid solution evaporated, the samples were diluted with 5% dilute nitric acid, filtered through a 0.22 μm hydrophilic membrane, and then analyzed using ICP-AES (iCAP 7400, Thermo Fisher Scientific) to determine the iron ion concentration in different tissues, thus assessing the in vivo tissue distribution of the nanoparticles in tumor-bearing mice.
[0145] Eight ICR mice (30-35g) were randomly divided into two groups (n=4). Fe3O4 and Fe3O4@RBC nanoparticles (2 mg / mL) were injected intravenously via the tail vein. At specified time intervals (0, 5 min, 15 min, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 h), 30 μL of whole blood was collected through the mouse orbital sinus. The collected whole blood was transferred to a 5 mL centrifuge tube containing 20 μL of EDTA to prevent blood clotting. The blood samples were digested with an acidic mixture, filtered through a 0.22 μm hydrophilic membrane, and the iron ion content in the blood was detected by ICP-AES (as above).
[0146] 3. In vivo antitumor therapy of Fe3O4-PL@RBC
[0147] Balb / c nude mice bearing MDA-MB-231 tumors were randomly divided into 5 groups (n=5 per group). Tumors were treated until the tumor volume reached 70 ± 20 mm. 3 Mice were injected intravenously with saline, free PL, Fe3O4, Fe3O4-PL, and Fe3O4-PL@RBC nanoparticles, at doses equivalent to PL content of 8 mg / kg. The first administration was designated day 0, and all treatments were administered every two days for a total of four doses. Changes in body weight and tumor volume were measured before each administration. On day 14, mice were sacrificed, tumors were collected, weighed, and fixed in 4% paraformaldehyde or liquid nitrogen.
[0148] Statistical analysis: All quantitative results are presented as mean ± standard deviation. Student's t-test was used for data comparison. * P < 0.05 ** P < 0.01, *** P < 0.001.
[0149] The results are as follows Figures 11-14 As shown.
[0150] Figure 11 and Figure 12 Metabolic kinetics, 4-hour and 24-hour tissue distribution of Fe3O4 and Fe3O4@RBC nanoparticles after tail vein injection in female Balb / c nude mice, respectively; from Figure 12-13As can be seen, compared with Fe3O4, Fe3O4@RBC exhibits a longer blood circulation time and a higher tumor retention rate. 24 hours after tail vein injection, Fe3O4@RBC was present in the blood circulation and tumor tissue at concentrations of 18.0 ± 2.1 ID / mL and 3.6 ± 1.8 % ID / g, respectively, while Fe3O4@RBC was present in the blood circulation and tumor tissue at concentrations of only 5.3 ± 1.7 ID / mL and 2.5 ± 1.5 % ID / g. Furthermore, compared with Fe3O4, the accumulation of Fe3O4@RBC in the liver was significantly reduced. These results indicate that the erythrocyte membrane on the surface of the nanoparticles can enhance their biocompatibility, enabling them to escape immune signals, prolong blood circulation time, and increase tumor accumulation.
[0151] Figure 13 and Figure 14 The figures show the tumor growth curve and the experimental endpoints of tumor size and weight. It can be seen from the figures that, except for the saline group, all experimental groups containing PL can inhibit tumor growth to varying degrees. Among them, Fe3O4-PL@RBC has the best anti-tumor effect, with a tumor inhibition rate of 82.9%.
Claims
1. A drug-loaded nanoparticle for treating triple-negative breast cancer, said drug-loaded nanoparticle comprising: Magnetic nanoparticles and active ingredients loaded thereon, characterized in that the drug-loaded nanoparticles further include a red blood cell membrane coating the magnetic nanoparticles; The active ingredient is selected from one or more of the following: piperamide, Paris saponin II, romaine, senna acetic acid, 6-gingerol, ginkgolide, solanine, cucurbitacin B, and lycorine.
2. The drug-loaded nanoparticles as described in claim 1, characterized in that, The active ingredient is selected from one or more of piperazine, Paris saponin II, cucurbitacin B, and solanine. Preferably, the active ingredient is piperazine.
3. The drug-loaded nanoparticles as described in claim 1, characterized in that, The magnetic nanoparticles are negatively charged magnetic nanoparticles, preferably iron oxide nanoparticles. And / or, the magnetic nanoparticles exist in cluster form.
4. A method for preparing drug-loaded nanoparticles, characterized in that, The method for preparing the drug-loaded nanoparticles includes the following steps: S1. Loading of active ingredient: The active ingredient and magnetic nanoparticles are prepared into a drug-loaded nanoparticle precursor by rotary evaporation; the active ingredient is as defined in any one of claims 1-3; the magnetic nanoparticles are as defined in claim 3; S2. Coating with red blood cell membrane: The drug-loaded nanoparticle precursor and red blood cell membrane are co-extruded to obtain the drug-loaded nanoparticles.
5. The method for preparing drug-loaded nanoparticles as described in claim 4, characterized in that, In step S1, the mass ratio of the active ingredient to the magnetic nanoparticles is (0.1-1.2):(0.5-2), preferably (0.2-0.5):(0.8-1.2), and more preferably 0.3:1; And / or, in step S1, the rotary evaporation method includes the following steps: mixing the magnetic nanoparticles and the active ingredient in solvent A, and then rotary evaporating; Solvent A is preferably deionized water; The mixing time is preferably 6-24 hours, for example 12 hours; Preferably, the active ingredient is dissolved in solvent B before mixing; preferably, solvent B is anhydrous ethanol. The rotary evaporation temperature is preferably 36-40°C, for example 38°C; Preferably, the rotary evaporation step further includes a washing and drying step; the washing reagent is, for example, deionized water; and the drying is, for example, freeze drying.
6. The method for preparing drug-loaded nanoparticles as described in claim 4, characterized in that, In step S2, the mass ratio of the red blood cell membrane to the drug-loaded nanoparticle precursor is (0.5-1.5):(0.5-1.5), preferably (1-1.5):(0.5-1), for example 1:0.5; And / or, in step S2, the co-extrusion method includes the following steps: mixing the red blood cell membrane and the drug-loaded nanoparticle precursor, sonicating, and extruding; The ultrasound is preferably performed under ice bath conditions; The power of the ultrasound is preferably 50-300W, more preferably 80-150W, for example 100W; The duration of the ultrasound is preferably 10-50 seconds, more preferably 20-40 seconds, for example 30 seconds; The number of compressions is preferably 7-16 times, more preferably 12-16 times, for example 15 times; The extrusion is preferably performed by an extrusion device, such as a liposome extruder.
7. The method for preparing drug-loaded nanoparticles as described in claim 4, characterized in that, The magnetic nanoparticles are prepared by a solvothermal method, which includes the following steps: In solvent C, ferric chloride hexahydrate, ammonium acetate and polyglutamic acid react, followed by a secondary reaction in a reaction vessel; The solvent C is preferably ethylene glycol; The preferred mass ratio of ferric chloride hexahydrate, ammonium acetate, and polyglutamic acid is (1-10):(1-10):(0.1-10), more preferably (1-2):(3-4):(0.5-0.8), for example 1.08:3.85:0.5; The reaction temperature is preferably 120-200°C, more preferably 150-180°C, for example 160°C; The reaction time is preferably 0.5-2 hours, more preferably 1-1.5 hours, for example 1 hour; The reaction is preferably carried out under stirring while being heated under reflux; Preferably, the secondary reaction is carried out in a forced-air drying oven; The temperature of the secondary reaction is preferably 160-210°C, more preferably 180-200°C, for example 200°C; The duration of the secondary reaction is preferably 6-24 hours, more preferably 8-14 hours, for example 12 hours; Preferably, the secondary reaction step further includes magnetic separation, washing, and dispersion steps; the washing reagent is, for example, anhydrous ethanol and / or deionized water.
8. The method for preparing drug-loaded nanoparticles as described in claim 4, characterized in that, The red blood cell membrane is prepared by a hypotonic membrane rupture method, which includes the following steps: Red blood cells are suspended in a hypotonic solution, centrifuged to remove hemoglobin, and the red blood cell membrane is obtained by squeezing out. The red blood cells are preferably obtained by centrifugation of whole blood; The whole blood is preferably derived from ICR mice; Among them, the preferred method for collecting whole blood is to collect blood from the eye socket; The centrifugation step is preferably performed in an anticoagulant tube containing heparin; The centrifugal speed for centrifugal separation is preferably 1000-4000 rpm, more preferably 2000-3500 rpm, for example 3000 rpm; The centrifugation time for centrifugation is preferably 3-20 min, more preferably 5-15 min, for example 5 min; The suspension time is preferably 20-60 min, more preferably 20-40 min, for example 30 min; The centrifugation speed for removing hemoglobin is preferably 8000-15000 rpm, more preferably 8000-13000 rpm, for example 10000 rpm; The centrifugation time for removing hemoglobin is preferably 5-15 min, more preferably 3-20 min, for example 5 min; Preferably, the step of centrifuging to remove hemoglobin also includes washing and sonication steps.
9. A drug-loaded nanoparticle, characterized in that, The drug-loaded nanoparticles are prepared by the method for preparing drug-loaded nanoparticles as described in any one of claims 4-8.
10. The use of the drug-loaded nanoparticles as described in any one of claims 1-3 in the preparation of a medicament for treating triple-negative breast cancer; in, The dosage form of the drug is preferably selected from one or more of the following: injection, gel, in situ gelation system, solution, suspension, emulsion, implant, transdermal agent, and microneedle; more preferably, the dosage form of the drug is an injection. The administration route of the drug preferably includes one or more of the following: intravenous bolus injection, intratumoral injection, intravenous infusion, intraperitoneal injection, hepatic artery infusion, intramuscular injection, subcutaneous injection, implantation, transdermal absorption, and interventional route; more preferably, the administration route of the drug is intravenous bolus injection. Preferably, the effective concentration of the drug is not higher than 10 μg / mL; Preferably, the duration of the drug's effectiveness is not less than 24 hours.