Application of arsenene nano preparation and / or beta-elemene in preparation of products for treating brain tumors

Abstract

The invention provides application of an arsenene nano preparation and / or beta-elemene in preparation of a product for treating brain tumors, and belongs to the technical field of biology. The arsenene nano preparation and / or beta-elemene are / is applied to brain glioma and lung cancer brain metastatic tumor model mice, so that tumor cells can be effectively killed, tumor metastasis can be inhibited, in-vivo immune response can be activated, maturation of dendritic cells can be promoted, NK cells and CD8 + T cells can be effectively activated, and the secretion amount of tumor suppression cell factors can be increased. Meanwhile, the beta-elemene effectively reduces cardiotoxicity caused by accumulation of the arsenene nano preparation in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of arsenene nanoparticles and / or β-elemene in the preparation of products for treating brain tumors. Background Technology

[0002] Intracranial malignant tumors have long faced clinical challenges due to their blood-brain barrier barrier, high heterogeneity, and immunosuppressive microenvironment, resulting in low treatment efficiency and high recurrence rates. In recent years, targeted therapy and immune checkpoint inhibitors have shown potential in various solid tumors, but their effectiveness in the treatment of brain tumors has been limited. Therefore, developing novel treatment strategies that combine efficient blood-brain barrier penetration, specific tumor-killing activity, and immune microenvironment remodeling capabilities has become a key direction for overcoming the bottlenecks in brain tumor treatment.

[0003] Arsenic trioxide (ATO) has demonstrated the potential of arsenic agents as highly effective anticancer agents by targeting the PML-RARα fusion protein. However, when treating solid tumors, traditional arsenic agents face multiple challenges, including pharmacokinetic defects, narrow therapeutic windows, and lack of targeting. Furthermore, elemental arsenic (As) remains inert in normal tissues, while hydrogen peroxide (H2O2) overexpressed in the tumor microenvironment can selectively oxidize it to highly toxic trivalent arsenic (As). 3+ This "smart activation" mechanism has been validated in various solid tumor models, including lung cancer and liver cancer. Despite the promising prospects of arsenylene, its use as a single drug still carries the risk of cumulative cardiotoxicity.

[0004] β-elemene, a natural sesquiterpene compound extracted from the traditional Chinese medicine Curcuma zedoaria, is a core component of a novel class II antitumor drug. Studies have shown that β-elemene can directly kill tumor cells, effectively inhibit tumor metastasis, reverse multidrug resistance, and reshape the immune microenvironment, exerting its antitumor effects through multiple targets and pathways. However, the lipophilic nature of β-elemene results in poor water solubility, with an oral bioavailability of only 15-20%, and its monotherapy potency is insufficient for clinical tumor treatment. Nanocarriers and combination strategies are needed to enhance efficacy. Currently, there are no findings on the application of arsenene nanoparticles and / or β-elemene in the treatment of brain tumors. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of arsenene nanoparticles and / or β-elemene in the preparation of products for treating brain tumors. Arsenene nanoparticles and / or β-elemene have the effect of inhibiting glioma and brain metastases of lung cancer, and the combination of the two also has the effect of reducing toxicity and enhancing efficacy.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides the use of arsenene nanoformulations and / or β-elemene in the preparation of products for treating brain tumors.

[0007] Preferably, the brain tumor includes glioma and lung cancer brain metastases.

[0008] This invention provides the application of β-elemene in the preparation of products that reduce the toxicity caused by arsenene nanoparticles.

[0009] Preferably, the toxicity caused by the arsenene nanoparticles is the same as the cardiotoxicity caused by arsenene nanoparticles in the treatment of brain tumors.

[0010] Preferably, the dosage of the arsenene nanoparticle is 3-7 mg / kg, and the dosage of the β-elemene is 30-50 mg / kg.

[0011] Preferably, the arsenene nanoformulation comprises arsenene nanodots and a DSPE-PEG layer coating the surface of the arsenene nanodots.

[0012] This invention provides a method for preparing the arsenene nanoparticles, comprising the following steps: mixing arsenic powder, antioxidant and N-methylpyrrolidone and sonicating; centrifuging for the first time to remove large particles that have not been peeled off; centrifuging for the second time to collect the arsenic nanoparticle precipitate; co-incubating arsenic nanoparticles with DSPE-PEG2000 or DSPE-PEG2000-cy5.5 and sonicating them; and then forming arsenene nanoparticles by rotary evaporation and thin-film hydration.

[0013] Preferably, the ratio of arsenic powder, N-methylpyrrolidone and antioxidant is 0.2-1.5g:15-25mL:100-200mg; the mass ratio of arsenic nanodots to DSPE-PEG2000 or DSPE-PEG2000-cy5.5 is 1:0.5-5.

[0014] Preferably, the parameters of the hybrid ultrasound include: power 125-175W, ice bath temperature ≤20℃, working time 8-12s / pause 3-7s, and total time 25-35h.

[0015] Preferably, the rotation speed of the first centrifugation is 4500-5500 rpm, and the rotation speed of the second centrifugation is 11500-12500 rpm.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention applies arsenene nanoparticles to mice and finds that arsenene nanoparticles can effectively cross the blood-brain barrier. When combined with β-elemene, they can effectively kill tumor cells, inhibit tumor metastasis, activate the immune response in vivo, promote the maturation of dendritic cells, and effectively activate NK cells and CD8+. +T cells and the secretion of tumor suppressor cytokines increased. Simultaneously, β-elemene effectively reduced the cardiotoxicity caused by the accumulation of arsenene nanoparticles in vivo. This invention provides a novel strategy for synergistic and toxicity-reducing antitumor activity, opening up possibilities for the widespread clinical application of arsenene nanoparticles in antitumor treatment. Attached Figure Description

[0017] Figure 1 This study presents the tissue distribution of arsenene nanoparticles and their antitumor activity when combined with β-elemene molecules. A shows electron micrographs of arsenene nanoparticles; B shows the distribution of Cy5.5-labeled arsenene nanoparticles in various tissues and organs of mice at different time points after intravenous injection; C shows the statistical distribution of Cy5.5-labeled arsenene nanoparticles in the mouse brain at different time points after intravenous injection; D shows the statistical distribution of Cy5.5-labeled arsenene nanoparticles in various tissues and organs of mice at different time points after intravenous injection; E shows the synergistic index of arsenene nanoparticles combined with β-elemene in mouse glioma cells; F shows the killing effect of different concentrations of arsenene nanoparticles on normal mouse endothelial cells (Bend3), mouse glioma cells (GL-261), and CT2A); G shows the expression of apoptosis proteins in mouse glioma cells after treatment with arsenene nanoparticles and β-elemene, individually or in combination.

[0018] Figure 2 This study presents the in vivo antitumor activity of the combination of arsenene nanoparticles and β-elemene molecules. A shows the flowchart of the construction and drug treatment of the C57BL / 6 mouse orthotopic glioma model. B demonstrates, through MRI and HE staining of glioma mouse tumor tissue, that the combination of arsenene nanoparticles and β-elemene molecules can inhibit glioma growth, with the combination significantly enhancing the antitumor effect. C shows that the combination of arsenene nanoparticles and β-elemene can prolong the survival of glioma mice. D and E show the statistical results of bioluminescence assays of the inhibition of glioma growth by arsenene nanoparticles and β-elemene alone and in combination; the differences were statistically significant. F shows the effect of ELISA on the expression of immune-related cytokines in mouse serum after treatment with arsenene nanoparticles and β-elemene. ****P<0.00001, ***P<0.0001, **P<0.001, *P<0.05.

[0019] Figure 3 To detect the effects of drug administration on CD8 levels in blood, spleen, and tumor tissue in mice using flow cytometry + The effect of T cells; A and B represent CD8 levels in mouse blood. + T cell proportion statistics; C and D represent CD8+ in mouse spleen. + T cell proportion statistics; E and F represent CD8+ in mouse tumor tissue. +T cell proportion statistics; ***P<0.0001, **P<0.001, *P<0.05.

[0020] Figure 4 To detect the effects of drug administration on NK cells and dendritic cells (DCs) in the blood, spleen, and tumor tissue of mice by flow cytometry; A: Statistical analysis of the proportion of NK cells in mouse blood; B: Statistical analysis of the proportion of DC cells in mouse blood; C: Statistical analysis of the proportion of NK cells in mouse spleen; D: Statistical analysis of the proportion of DC cells in mouse spleen; E: Statistical analysis of the proportion of NK cells in mouse tumor tissue; F: Statistical analysis of the proportion of DC cells in mouse tumor tissue; ***P<0.0001, **P<0.001, *P<0.05.

[0021] Figure 5 To detect the effects of drug administration on M2 macrophages and CD4+ in blood, spleen, and tumor tissues of mice using flow cytometry + The effect of T cells; A shows the proportion of M2 macrophages in mouse blood; B shows the proportion of CD4 in mouse blood. + T cell proportion statistics; C represents the proportion of M2 macrophages in mouse spleen; D represents the proportion of CD4 cells in mouse spleen. + T cell proportion statistics; E represents the proportion of M2 macrophages in mouse tumor tissue; F represents the proportion of CD4+ macrophages in mouse tumor tissue. + T cell proportion statistics; ***P<0.0001, **P<0.001, *P<0.05.

[0022] Figure 6 The study aimed to reverse the cardiotoxicity induced by β-elemene in arsenene nanoparticles. Figure A shows the HE and Masson staining results of heart tissue sections from the control and drug-treated mouse groups with gliomas. Figures B, C, D, and E show the statistical results of serum biochemical levels (CK, CK-MB, CTnT, and LDH) in each group of mice after drug administration. Figure F shows the statistical results of heart weight in each group of mice after drug administration. ***P<0.0001, **P<0.001, *P<0.05.

[0023] Figure 7The combination of arsenene nanoparticles and β-elemene molecules effectively inhibited the progression of lung cancer brain metastases. A is a flowchart of the construction and drug treatment process for the C57BL / 6 mouse lung cancer brain metastasis model; B shows the results of HE staining of mouse tumor tissue demonstrating that the combination of arsenene nanoparticles and β-elemene molecules can inhibit the progression of lung cancer brain metastases, with the combination significantly inhibiting brain metastases; C and E are statistical results of bioluminescence assays showing the inhibition of lung cancer brain metastasis growth by arsenene nanoparticles and β-elemene alone and in combination, with statistically significant differences; D shows the statistical changes in body weight of mice in each group during drug administration; F shows the effect of ELISA detection on the expression of immune-related cytokines in mouse serum after treatment with arsenene nanoparticles and β-elemene; ****P<0.00001, ***P<0.0001, **P<0.001, *P<0.05. Detailed Implementation

[0024] This invention provides the application of arsenene nanoparticles and / or β-elemene in the preparation of products for treating brain tumors. This invention applies arsenene nanoparticles and / or β-elemene to mouse models of orthotopic glioma and mouse models of lung cancer brain metastasis, finding that arsenene nanoparticles, used alone or in combination with β-elemene molecules, can effectively inhibit the proliferation of glioma cells and the brain metastasis of lung cancer cells in mice, exerting an anti-brain tumor effect. Unless otherwise specified, the β-elemene described in this invention is obtained through commercially known channels in the art, preferably from Aladdin, CAS: 33880-83-0.

[0025] In this invention, the arsenene nanoparticles and / or β-elemene activate an anti-brain tumor immune response. The activated anti-brain tumor immune response is achieved through one or more of the following: 1) polarizing pro-tumor M2 tumor-associated macrophages to a tumor-suppressive M1 phenotype; 2) enhancing the expression of immune-stimulating cytokines; 3) increasing CD8+ expression in vivo. + T cells, NK cells and mature dendritic cells; 4) activate the immunosuppressive tumor microenvironment; 5) activate T cell immunity.

[0026] This invention provides the use of β-elemene in the preparation of products that reduce the toxicity caused by arsenene nanoparticles. The toxicity caused by the arsenene nanoparticles described in this invention includes, but is not limited to, the cardiotoxicity caused by arsenene nanoparticles in the treatment of brain tumors, preferably the cardiotoxicity caused by arsenene nanoparticles in the treatment of brain tumors.

[0027] In this invention, the dosage of the arsenene nanoparticle formulation is 3-7 mg / kg, preferably 5 mg / kg, and the dosage of β-elemene is 30-50 mg / kg, preferably 40 mg / kg. The dosages described above are for use in model mice; dosages for humans or other animals can be calculated using conventional methods. Preferably, the dosages of the arsenene nanoparticle formulation combined with β-elemene in this invention are consistent with the dosages of the arsenene nanoparticle formulation and β-elemene alone.

[0028] In this invention, the arsenene nanoparticle formulation comprises arsenic nanodots (AsNDs) and a DSPE-PEG layer coating the surface of the arsenic nanodots. The DSPE-PEG in this invention can be replaced with DSPE-PEG-cy5.5. The lateral dimension of the arsenic nanodots in this invention is less than 10 nm.

[0029] This invention provides a method for preparing the aforementioned arsenene nanoparticles, comprising the following steps: mixing arsenic powder, an antioxidant, and N-methylpyrrolidone with ultrasonication; centrifuging for the first time to remove large particles that have not been peeled off; and centrifuging for the second time to collect the arsenic nanoparticle precipitate; co-incubating the arsenic nanoparticles with DSPE-PEG2000 or DSPE-PEG2000-cy5.5 and ultrasonically breaking them up; and then forming arsenene nanoparticles by rotary evaporation and thin-film hydration. The arsenic used in this invention is high-purity arsenic, avoiding oxidized arsenic impurities. The purpose of ultrasonication in this invention is to break up large glass particles. An antioxidant is added in this invention to inhibit arsenic oxidation. The antioxidant used in this invention is preferably vitamin C.

[0030] In this invention, the ratio of arsenic powder, N-methylpyrrolidone, and antioxidant is 0.2-1.5g:15-25mL:100-200mg, preferably 0.5g:20mL:150mL; the mass ratio of arsenic nanodots to DSPE-PEG2000 or DSPE-PEG2000-cy5.5 is 1:0.5-5, preferably 1:0.8-3.5, and more preferably 1:1.

[0031] In this invention, the parameters of the hybrid ultrasound include: power 125-175W, ice bath temperature ≤20℃, working time 8-12s / pause 3-7s, total time 25-35h, preferably power 150W, ice bath temperature ≤20℃, working time 10s / pause 5s, total time 30h. The purpose of the hybrid ultrasound in this invention is to ultrasonically ablate and break down arsenic powder into arsenic nanoparticles.

[0032] In this invention, the rotation speed of the first centrifugation is 4500-5500 rpm, preferably 5000 rpm, and the rotation speed of the second centrifugation is 11500-12500 rpm, preferably 12000 rpm.

[0033] In this invention, arsenic nanodots are PEGylated using DSPE-PEG2000 or DSPE-PEG2000-cy5.5. The preferred time for co-incubating DSPE-PEG2000 or DSPE-PEG2000-cy5.5 with AsNDs followed by ultrasonic disruption is 10-30 min, more preferably 20 min. The mixture after ultrasonic treatment with water is then rotary evaporated and processed using a thin-film hydration method to form AsNDs@PEG or AsNDs@PEG-cy5.5. The purpose of using DSPE-PEG2000 or DSPE-PEG2000-cy5.5 modification in this invention is to enhance the water solubility and blood circulation time of the arsenene nanoparticles.

[0034] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0035] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] The experimental data in the following examples are expressed as mean ± standard deviation. The data were compared and analyzed by a two-tailed Student's-T test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.00001 were considered statistically significant).

[0037] Example 1: Preparation of Arsenene Nanoparticles (1) Raw material pretreatment: High-purity arsenic is selected to avoid oxidized arsenic impurities. Vitamin C, a reducing agent, is added to N-methylpyrrolidone (NMP) organic solution to inhibit arsenic oxidation.

[0038] (2) Mixed ultrasound: 0.5g of arsenic powder (267961-10G, Sigma-Aldrich) was dispersed in 20mL of N-methylpyrrolidone (NMP, 443778-500mL, Sigma-Aldrich), and 150mg of vitamin C (Sigma-Aldrich) was added for mixed ultrasound treatment. A probe ultrasound machine (150W power) was used, and ultrasound was performed for 30h under ice bath conditions of 4℃ (intermittent mode: 10s working / 5s pause).

[0039] (3) Gradient centrifugation: First centrifugation (5000 rpm) removes large particles that have not been peeled off and removes the supernatant; second centrifugation (12000 rpm) of the supernatant collects the arsenic nanodots (AsNDs) precipitate.

[0040] (4) PEGylation modification: DSPE-PEG2000 (Maclean, CAS 147867-65-0) or DSPE-PEG2000-cy5.5 (Yusi Pharmaceutical) were co-incubated with AsNDs at a mass ratio of 1:1 and ultrasonically broken for 20 min (the other ultrasonic conditions were the same as in step (2) of this embodiment). After rotary evaporation, AsNDs@PEG or AsNDs@PEG-cy5.5 were formed by thin film hydration.

[0041] Example 2 The cells used in this embodiment were GL261 cells, CT2A cells, Lewis cells, and Bend3 cells, all of which were preserved in the laboratory.

[0042] The specific steps for resuscitation, passage, seeding, and lentiviral transfection of GL261 cells, CT2A cells, and Bend3 cells are as follows: 1. Cell resuscitation and culture (1) After removing the cells from liquid nitrogen, quickly place them in a 37°C constant temperature water bath to thaw; (2) Place the fully thawed cells in a benchtop centrifuge and centrifuge at 800 rpm for 5 min; (3) Discard the old culture medium and add 1 mL of preheated complete culture medium (containing 10% FBS, 100 units / mL penicillin + streptomycin) to resuspend the cells; (4) Transfer the cells to a 10cm cell culture dish (10×10cm), the culture volume is 7mL, and gently shake well; (5) Place the cells in a cell culture incubator (37°C, containing 5% CO2), replace with fresh complete culture medium the next day, and passage when the cell density reaches 90%.

[0043] 2. Cell passage (1) Preheat the complete culture medium, 0.25% Trypsin-EDTA, and PBS buffer; (2) Take the cells from step 1 of this embodiment, discard the old culture medium, and add 2 mL of PBS along the side wall to rinse; (3) Discard the PBS, add 2 mL of 0.25% Trypsin-EDTA to the dish, and incubate at 37°C to digest the cells; (4) After the cells are completely digested, add 2 mL of complete culture medium to stop the digestion. Use a micropipette to transfer the cells to a 10 mL centrifuge tube and centrifuge at 800 rpm for 5 min in a benchtop centrifuge. (5) Discard the supernatant, resuspend the cells in the appropriate fresh complete culture medium, and then transfer them to a 10cm culture dish and gently shake to mix. (6) Place the cells in a cell culture incubator (37°C, containing 5% CO2) and passage them every other day.

[0044] 3. Cell inoculation (1) Take cells in the logarithmic growth phase, discard the old culture medium, rinse with 2 mL PBS, add 2 mL 0.25% Trypsin-EDTA and incubate at 37℃ for complete digestion; (2) Add 2 mL of complete culture medium to neutralize 0.25% Trypsin-EDTA, transfer the cells to a 10 mL centrifuge tube using a micropipette, and centrifuge at 800 rpm for 5 min in a benchtop centrifuge. (3) Discard the supernatant, resuspend the cells in an appropriate amount of complete culture medium, and count the cells using a hemocytometer under an inverted microscope; (4) Calculate the required cell suspension volume and the corresponding culture medium volume based on the number of cells to be inoculated for the experiment (6-well plate: 2 mL; 12-well plate: 1 mL; 24-well plate: 500 μL). (5) Place the cells in a cell culture incubator (37°C, containing 5% CO2) for subsequent experiments.

[0045] 4. Luciference lentivirus transfection: (1) Cell seeding: 24 hours before infection, target cells are seeded at an appropriate density (e.g., 1-2 × 10⁶ cells per well in a 24-well plate). 5 / hole), ensuring a confluence rate of 30-50% during infection.

[0046] (2) Viral infection: Remove the old culture medium, add fresh culture medium containing virus solution, and incubate at 37°C for 48 hours.

[0047] (3) Change of medium and culture: After 48 hours, change to complete culture medium and continue to culture for 72 hours to observe fluorescence expression.

[0048] (4) Screening: Use 2 μg / ml puromycin to screen successfully transfected cells. Screening continues for 1 week in preparation for subsequent experiments.

[0049] Example 3 1. The particle size of the arsenic nanodots (AsNDs) prepared in Example 1 was characterized and detected by electron microscopy. The particle size of the arsenic nanodots was 1-8 nm.

[0050] 2. Mice were randomly divided into 4 groups. The control group was injected with AsNDs@PEG 5 mg / kg via the tail vein, while the other three groups were injected with AsNDs@PEG-cy5.5 5 mg / kg via the tail vein. The distribution of the arsenene nanoparticles in various tissues and organs of mice was observed at 1 h, 2 h, and 4 h after injection.

[0051] like Figure 1 As shown in AD, arsenene nanodots with a particle size of less than 10 nm can effectively penetrate the blood-brain barrier and accumulate in mouse brain tissue after intravenous injection, reaching the enrichment peak between 1 and 2 hours.

[0052] 3. CCK8 and synergistic index detection of the two drugs The cytotoxicity of arsenene nanoparticles (AsNDs@PEG) and the synergistic index of the two drugs on GL261, CT2A and Bend3 cells were evaluated using the Cell Count Kit-8 (CCK-8).

[0053] (1) Seed cells into 96-well plates, 5 × 10⁶ cells per well. 3 Cells were then incubated for 24 h in different concentrations of arsenene nanoparticles (0, 0.1, 1, 5, 10 μg / ml) or different concentrations of β-elemene (Aladdin, E303852, CAS: 33880-83-0) (0, 10, 20, 30 μg / ml).

[0054] (2) Add serum-free fresh culture medium containing 10% CCK-8 solution to each well. Incubate the 96-well plate at 37°C for 2 hours to allow the live cells to fully react with the CCK-8 reagent.

[0055] (3) The absorbance of each well at 450 nm was measured using an ELISA reader to quantify the cytotoxic effect. The absorbance represents the number of viable cells remaining after drug exposure. The results were analyzed using GraphPad Prism 9.0 software.

[0056] like Figure 1 As shown in F, the arsenene nanoparticles have a selective killing effect on glioma cells, but no significant inhibitory effect on normal brain endothelial cells (Bend3). Figure 1 The results showed that the combination of arsenene nanoparticles and β-elemene molecules could significantly and synergistically inhibit the growth of glioma cells.

[0057] 4. Western blot detection (1) The cells were seeded into 6-well plates and, after adhesion, were incubated for 24 hours with PBS, 5 μg / ml arsenene nanoparticles, 20 μg / ml β-elemene or a combination of both.

[0058] (2) Collect and lyse cells after drug administration by mixing RIPA buffer with 1 mM PMSF.

[0059] (3) Use the BCA protein detection kit to determine the protein concentration and add the protein sample to the prepared SDS polyacrylamide gel.

[0060] (4) After electrophoresis, the sample was transferred to a PVDF membrane, sealed, incubated with primary and secondary antibodies, and detected using chemiluminescence.

[0061] Figure 1 The results showed that the combination of arsenene nanoparticles and β-elemene molecules could induce apoptosis in glioma cells.

[0062] Example 4 1. Construction of an orthotopic mouse model of glioma (1) Anesthesia and fixation: Anesthetize the mouse by intraperitoneal injection of 4% chloral hydrate (200μL / 20g). Fix the mouse incisors to the stereotaxic instrument and adjust the ear rods to make the skull horizontal (nose aligned with the midline, tail lifted without falling off).

[0063] (2) Craniotomy and localization: After shaving and disinfection, cut the scalp along the midline (about 0.5 cm) to expose the anterior fontanelle (Bregma). Wipe away the pia mater on the surface of the skull with 4% H2O2 to clearly locate the Bregma cross.

[0064] (3) Drilling location: 0.6mm anterior to the anterior fontanelle and 1.8mm to the right of the sagittal suture, drill through the skull with a dental drill (hole diameter ≤ 1mm).

[0065] (4) Cell injection: Draw 1 μL of GL261-luci cell suspension (10,000 cells / μL, stored on ice) into a microsyringe and insert the needle vertically to 4.02 mm below the dura mater and then withdraw it 0.04 mm. Inject slowly at a rate of 0.1 μL / min, stop the needle for 5 min to prevent backflow, and seal the pore with bone wax.

[0066] (5) Suturing and recovery: Use medical adhesive to bond the scalp and keep it warm at 40°C until the patient wakes up.

[0067] like Figure 2 As shown in Figure A, mice with an orthotopic glioma were randomly divided into four groups: PBS (PBS group); 5 mg / kg arsenene nanoparticles (AsNDs@PEG group); 40 mg / kg β-elemene (ELE group); and a combination of arsenene nanoparticles and β-elemene (Combo group). The mice were administered the drugs via tail vein injection. Arsenene nanoparticles were injected every 3 days after tumor implantation, and β-elemene was injected every 2 days after tumor implantation. Mice were sacrificed on day 21, and samples were collected for analysis and statistical data collection. The combination of arsenene nanoparticles and β-elemene was a combination of the individual drugs mentioned above.

[0068] 2. Nuclear magnetic resonance imaging and bioluminescence imaging (1) Anesthesia: Isoflurane inhalation anesthesia.

[0069] (2) Intraperitoneal injection of 3 mg / animal of D-fluorescein potassium salt, biological imaging was performed 10 min later, and the exposure time was 30 s.

[0070] (3) After anesthesia, the mouse was fixed in the MRI scanner and the mouse brain was photographed.

[0071] 3. HE staining (1) Mouse brain tissue sections were soaked in xylene twice, 15 min each time, to completely dissolve the paraffin. Gradient ethanol dehydration: anhydrous ethanol I → anhydrous ethanol II → 95% ethanol → 80% ethanol → 70% ethanol (5 min each), rinsed with running water for 5 min to remove ethanol residue.

[0072] (2) Cell nuclear staining: Immerse in hematoxylin stain for 5-8 min (room temperature), then rinse with running water for 5-10 min (to remove excess stain).

[0073] (3) Differentiation, blue staining, and cytoplasmic staining: 1% hydrochloric acid ethanol (prepared with 75% ethanol) for 30s → rinse with running water for 30s (to remove non-specific staining in the cytoplasm), rinse with 0.6% ammonia or running water for 15-30min (to neutralize the acidity and make the nucleus appear bright blue), stain with 0.5% eosin aqueous solution for 3min, and rinse briefly with distilled water for 30s (to prevent over-staining).

[0074] (4) Gradient ethanol dehydration: 80% → 95% → anhydrous ethanol I → anhydrous ethanol II (5 min each), clearing: xylene I → xylene II (5 min each), sealing: drop neutral resin, cover with a coverslip (avoid air bubbles), and air dry to cure.

[0075] (5) Obtain images using a NanoZoomer S60 scanner.

[0076] Figure 2 The BE results showed that the use of arsenene nanoparticles alone or in combination with β-elemene molecules could effectively inhibit the growth of gliomas in mice and significantly prolong the survival of mice.

[0077] 4. ELISA test (1) Coating: Dilute the capture antibody with coating buffer, add 100 μL to each well, and incubate overnight at 4°C.

[0078] (2) Washing: After discarding the liquid, wash 3 times with PBST, soaking for 1-2 minutes each time, and then pat dry.

[0079] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at 37°C for 1 h. Wash the plate 3 times to remove excess blocking agent and prevent nonspecific binding.

[0080] (4) Sample addition and incubation: Add 100 μL of standard / sample to each well, and set 3 replicates to reduce error. Incubate at 37℃ for 2 h, cover with a membrane to prevent evaporation, and shake to promote binding. Wash thoroughly 5 times and pat dry.

[0081] (5) Dilute the enzyme-labeled detection antibody (HRP label) with diluent, add 100 μL to each well, incubate at 37°C for 1 h, and wash the plate 5 times.

[0082] (6) Color development and termination: Add 100 μL of TMB substrate to each well and develop color at 37°C in the dark for 30 min (a blue gradient is visible to the naked eye); add 100 μL of stop solution to each well, and the blue color will turn yellow.

[0083] (7) OD value measurement: Read the ELISA reader at 450nm within 15 minutes after termination, plot the fitting curve of standard concentration (X) and OD value (Y) (four-parameter logistic regression), infer the concentration from the curve based on the OD value, and correct it in combination with the sample dilution factor.

[0084] Figure 2 The ELISA results in F showed that, compared with the control group, the expression of immune-related cytokines in the serum of mice in the treatment group was increased to varying degrees, indicating that the use of arsenene nanoparticles alone or in combination with β-elemene molecules can activate the anti-tumor immune effect in mice to varying degrees.

[0085] 5. Flow cytometry detection (1) After blood was collected from mice in anticoagulant tubes, the mice were sacrificed and their spleen and brain tumor tissue were collected.

[0086] (2) Cut fresh tumor tissue into small pieces with surgical scissors, add enzyme digestion buffer, and shake digest at 37°C for 4 hours; grind the spleen in a tube containing Hanks buffer and grind it gently with a rubber grinding stick.

[0087] (3) Mouse blood, spleen and digested tumor suspension were filtered through a 70 μm nylon filter, the filter was rinsed with Hanks buffer, and the filtrate was collected.

[0088] (4) Centrifuge at 4℃ (300g, 5min), discard the supernatant, add 1–2mL of red blood cell lysis buffer and incubate on ice for 5min, then centrifuge after PBS termination.

[0089] (5) Discard the supernatant, resuspend the cell pellet in PBS containing 2.5% FBS, and then count the cells.

[0090] (6) Surface antigen staining: stain cells (2×10⁻⁶) with the surface antigen. 6 The sample was resuspended in 100 μL of PBS containing 2.5% FBS, and live / dead cell dye was added. The mixture was incubated in the dark for 20 min. A pre-titrated antibody mixture (such as CD45, CD3, CD4, CD8, CD11b, etc.) was added and the mixture was incubated on ice in the dark for 10 min.

[0091] (7) Wash twice with staining buffer (300g, 5min) to completely remove unbound antibodies. Resuspend the cells in 500μl of buffer and perform flow cytometry.

[0092] like Figure 3 , 4 Results 5 showed that, compared to the control group, the CD8 levels in the blood, spleen, and tumor tissue of mice in the treatment group were significantly higher. + The proportions of T cells, NK cells, and DC cells increased to varying degrees, while the proportion of M2 macrophages decreased, consistent with the ELISA results. This indicates that the use of arsenene nanoparticles alone or in combination with β-elemene molecules can activate anti-tumor immune effects in mice to varying degrees.

[0093] 6. Masson staining (1) Mouse brain tissue sections were soaked in xylene twice, 15 min each time, to completely dissolve the paraffin. Gradient ethanol dehydration: anhydrous ethanol I → anhydrous ethanol II → 95% ethanol → 80% ethanol → 70% ethanol (5 min each), rinsed with running water for 5 min to remove ethanol residue.

[0094] Standard Procedure for Paraffin Sectioning (2) Mordant treatment (enhancing fiber coloring): After soaking in Bouin solution (picric acid + formaldehyde + glacial acetic acid) at room temperature overnight, rinse with running water until the yellow color fades completely.

[0095] (3) Nuclear counterstaining: Mayer hematoxylin staining for 3 min, rinse with running water, differentiate with 1% hydrochloric acid ethanol for a few seconds, and then return to blue with warm water for 5 min.

[0096] (4) Collagen / muscle fiber color separation: After 10 min of treatment with Ponceau red and magenta dye solution, wash with weak acid water (0.2% glacial acetic acid) for 1 min, treat with 1% phosphomolybdic acid for 10 min (differentiating collagen fibers), then wash with weak acid water for 1 min, and treat with aniline blue dye solution for 5 min (collagen fibers), then wash with weak acid water.

[0097] (5) Dehydration and sealing: 95% ethanol → anhydrous ethanol (10 seconds each), clear: xylene I → II (2 min each), neutral resin for sealing.

[0098] (6) Obtain images using a NanoZoomer S60 scanner.

[0099] like Figure 6 As shown, the accumulation of arsenene nanoparticles in the body can cause certain cardiotoxicity, leading to an increase in the degree of myocardial fibrosis and the expression of myocardial injury markers, as well as a decrease in heart weight. However, in the treatment group where arsenene nanoparticles were combined with β-elemene molecules, β-elemene played a protective role in the heart and reduced the cardiotoxicity caused by arsenene nanoparticles.

[0100] 7. Construction of a mouse model of lung cancer brain metastasis (1) Anesthesia and fixation: Anesthetize the mice by intraperitoneal injection of 4% chloral hydrate (200μL / 20g). Fix the mice in a supine position, elevate the neck, shave and disinfect (iodine tincture → 75% ethanol).

[0101] (2) Exposing the carotid artery: Make a longitudinal incision of 1-1.5 cm along the midline of the neck, and bluntly dissect the subcutaneous tissue and sternocleidomastoid muscle. Dissect the blood vessels to expose the common carotid artery (CCA) and its branches (external carotid artery ECA, internal carotid artery ICA), avoiding damage to the vagus nerve (which can lead to respiratory arrest).

[0102] (3) Vascular occlusion: The proximal end is ligated and fixed with sutures, and sutures are prepared at the distal end for rapid ligation after injection. Insert the needle between the two ligations and inject Lewis-Luciference cell suspension (containing 1×10⁻⁶ cells). 5 After injecting (one cell), wait 30 seconds, slowly withdraw the needle, and quickly ligate the distal end.

[0103] (4) Wound treatment: Rinse the surgical field with saline, suture the muscle and skin in layers with 6-0 sutures, disinfect with iodine solution twice, and keep warm at 40℃ until the patient wakes up.

[0104] A mouse model of lung cancer brain metastasis was randomly divided into four groups: PBS group, 5 mg / kg arsenene nanoparticles (AsNDs@PEG group), 40 mg / kg β-elemene (ELE group), and a combination of arsenene nanoparticles and β-elemene (Combo group). Drug administration was performed via tail vein injection. Arsenene nanoparticles were injected every 3 days after tumor implantation, and β-elemene was injected every 2 days after tumor implantation. Mice were sacrificed on day 21, and samples were collected for analysis and statistical data. The combination of arsenene nanoparticles and β-elemene was a combination of the individual drugs mentioned above.

[0105] The mouse lung cancer brain metastasis animal model constructed using the methods described above was used to detect relevant indicators. The experimental results are as follows: Figure 7 As shown, the use of arsenene nanoparticles alone or in combination with β-elemene molecules can effectively inhibit the brain metastasis of lung cancer cells in mice, while there is no significant difference in the weight changes of the mice. At the same time, compared with the control group, the expression of immune-related cytokines in the serum of mice in the treatment group was increased to varying degrees, which is consistent with the ELISA results in the mouse orthotopic glioma animal experiment. This further illustrates that the use of arsenene nanoparticles alone or in combination with β-elemene molecules can significantly activate the anti-tumor immune effect in mice and exert an anti-tumor effect.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of arsenene nanoparticles and / or β-elemene in the preparation of products for treating brain tumors.

2. The application as described in claim 1, characterized in that, The brain tumors include gliomas and brain metastases from lung cancer.

3. Application of β-elemene in the preparation of products that reduce the toxicity of arsenic nanoparticles.

4. The application as described in claim 3, characterized in that, The toxicity caused by the arsenene nanoparticles refers to the cardiotoxicity caused by arsenene nanoparticles in the treatment of brain tumors.

5. The application as described in any one of claims 1-4, characterized in that, The dosage of the arsenene nanoparticles is 3-7 mg / kg, and the dosage of the β-elemene is 30-50 mg / kg.

6. The application as described in any one of claims 1-5, characterized in that, The arsenene nanoparticle formulation includes arsenic nanodots and a DSPE-PEG layer coating the surface of the arsenic nanodots.

7. The method for preparing the arsenene nanoparticle formulation according to any one of claims 1-6, characterized in that, The process includes the following steps: arsenic powder, antioxidant, and N-methylpyrrolidone are mixed and sonicated; a first centrifugation is performed to remove large particles that have not been peeled off; a second centrifugation is performed to collect the arsenic nanoparticle precipitate; DSPE-PEG2000 or DSPE-PEG2000-cy5.5 is co-incubated with the arsenic nanoparticles and ultrasonically broken up; after rotary evaporation, arsenene nanoparticles are formed by thin-film hydration.

8. The preparation method according to claim 7, characterized in that, The ratio of arsenic powder, N-methylpyrrolidone, and antioxidant is 0.2-1.5g:15-25mL:100-200mg; the mass ratio of arsenic nanodots to DSPE-PEG2000 or DSPE-PEG2000-cy5.5 is 1:0.5-5.

9. The preparation method according to claim 7, characterized in that, The parameters of the hybrid ultrasound include: power 125-175W, ice bath temperature ≤20℃, working time 8-12s / pause 3-7s, and total time 25-35h.

10. The preparation method according to claim 7, characterized in that, The first centrifugation was performed at a speed of 4500-5500 rpm, and the second centrifugation was performed at a speed of 11500-12500 rpm.

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