Application of nano-silver combined with nrf2 inducer in the preparation of drugs for treating multiple myeloma

Abstract

The application provides application of nanosilver combined with an NRF2 inducer in treatment of multiple myeloma. Specifically, the nanosilver and the NRF2 inducer are combined, and in-vivo and in-vitro experiments are combined to explore the effect of the combination of the nanosilver and the NRF2 inducer on the multiple myeloma, and it is found that application of the NRF2 inducer promotes the inhibitory effect of the nanosilver on the multiple myeloma. In the application, the combination of the nanosilver and dimethyl fumarate can significantly increase the expression level of intracellular ROS, so that the multiple myeloma cells are in an over-oxidative stress state, thereby promoting cell apoptosis, which can provide a new option for future treatment of the multiple myeloma.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of nanosilver combined with NRF2 inducers in the treatment of multiple myeloma. Background Technology

[0002] Multiple myeloma (MM) is a malignant tumor caused by the unrestricted clonal proliferation of plasma cells that produce the M protein in the bone marrow. It is the second most common hematologic malignancy worldwide and can cause hematopoietic dysfunction. Patients with multiple myeloma may present with symptoms such as anemia, osteolytic lesions, hypercalcemia, abnormal renal function, and recurrent infections.

[0003] With ongoing research into the treatment of multiple myeloma, the emergence and application of new drugs and emerging treatment methods have significantly improved the cure rate, prognosis, and symptoms of the disease. Currently, first-line drugs for treating multiple myeloma include bortezomib and lenalidomide. Treatment methods include autologous stem cell transplantation, targeted therapy, and surgery. However, multiple myeloma still carries the risk of relapse, cannot be completely cured, and may even develop drug resistance, leading to reduced efficacy and a series of toxic side effects. Therefore, there is an urgent need to find new drugs and treatment methods to improve treatment outcomes and enhance patients' quality of life. Summary of the Invention

[0004] The technical problem to be solved by this invention is to propose a new method for treating multiple myeloma, which combines nano-silver and dimethyl fumarate. The combination of in vivo and in vitro experiments is used to explore the effect of nano-silver and NRF2 inducers (especially dimethyl fumarate) on multiple myeloma, aiming to provide new ideas for the treatment of multiple myeloma.

[0005] Nanoparticles (AgNPs) refer to elemental silver particles with diameters ranging from 1 to 100 nm, prepared through specific chemical or physical methods. Due to their unique physicochemical properties, they are widely used in various fields, including medicine for antibacterial purposes and treatment of breast, cervical, ovarian, and lung cancers. Smaller AgNPs typically exhibit stronger cell penetration and higher cytotoxicity. However, nanoparticles with sizes between 10 and 100 nanometers are generally considered the optimal choice for cancer treatment because particles of this size can penetrate the tumor's vascular system (EPR effect) but not the normal vascular system, thus avoiding rapid renal clearance.

[0006] Dimethyl fumarate (DMF) is a member of the fumarate family and is an α,β-unsaturated carboxylic acid ester. It is an inducer of the Nrf2 antioxidant group, activating the Nrf2 signaling pathway and enhancing the antioxidant capacity of cells. DMF is currently approved clinically for the treatment of multiple sclerosis (MS) and can also be used to treat psoriasis (PSO).

[0007] In this invention, we tested several human myeloma cell lines with 1.9 nm-scale silver nanoparticles in vitro. We then used a CCK-8 assay and flow cytometry to detect and evaluate their toxicity, and used DCFH-DA dye to detect the levels of reactive oxygen species (ROS). We also analyzed the protein level of nuclear factor (erythroid-derived 2)-like 2 (NRF2) and the expression of its downstream genes. Subsequently, we used RNA interference silencing technology and pharmacological inducers to investigate the functional role of NRF2 in the action of silver nanoparticles on MM cells. Finally, we tested the effects of silver nanoparticles and the NRF2 inducer dimethyl fumarate (DMF), alone or in combination, on the in vivo growth of MM cells in xenograft tumor model mice.

[0008] Results: Low concentrations of silver nanoparticles were found to be significantly toxic to MM cells, with significantly higher basal ROS levels compared to other cancer cells. Silver nanoparticle treatment further exacerbated ROS-induced oxidative damage and led to the accumulation of NRF2 protein in MM cells. Furthermore, we found that DMF exacerbated the toxicity of silver nanoparticles to MM cells both in vitro and in vivo.

[0009] Subsequently, verification experiments were conducted using 5nm and 30nm silver nanoparticles, and similar results were obtained.

[0010] Conclusion: Activation of NRF2 activity may exacerbate the cytotoxicity of nanosilver in MM, and the combined use of nanosilver and DMF provides a new strategy for the treatment of MM. Attached Figure Description

[0011] Figure 1 The particle size distribution curve of 1.9 nm silver nanoparticles is shown.

[0012] Figure 2 This is a microscope image of 5nm silver nanometers.

[0013] Figure 3 The particle size distribution curve of 5nm silver nanoparticles.

[0014] Figure 4 Microscopic image of 30nm silver nanometers.

[0015] Figure 5 The particle size distribution curve of 30nm silver nanoparticles.

[0016] Figure 6 The following figures illustrate the cytotoxicity of MM cells induced by low concentrations of 1.9 μm silver: (A) Cell viability of MM cell lines U266, MM1S, RPMI8226, ARP-1, and H929 after 24 h of treatment with silver nanoparticles (1–4 μg / ml); (B) Cell viability of A549, HepG2, MG63, and GC7901 after 24 h of treatment with silver nanoparticles (1–4 μg / ml); n = 4 per group. (C) Percentage of MM1S and U266 cells that apoptotic after 24 h of treatment with silver nanoparticles (1 μg / mL and 2 μg / mL, respectively) determined by flow cytometry. (D) Representative morphology of U266 and MM1S cells treated with silver nanoparticles for 12 h. Arrows indicate morphological damage. Scale bar: 50 μm. Data are expressed as mean ± standard deviation. **P < 0.01, ***P < 0.001.

[0017] Figure 7 ROS activation-mediated 1.9 nm silver nanoparticle-induced MM cell death; (A) Intracellular ROS production was measured using the DCFH-DA probe. ROS levels in MM cells (ARP-1, MM1S, and U266) and other cancer cells (MG63, A549, and HepG2) were observed under a fluorescence microscope. Scale bar: 50 μm; (B) Relative ROS levels in MM1S and U266 cells after 6 hours of silver nanoparticle treatment; Cell viability (C) and apoptosis rate (D) were assessed after silver nanoparticle treatment of MM1S and U266 cells with and without NAC (2 mM); (E) Representative morphology of U266 and MM1S cells treated with silver nanoparticles with and without NAC (2 mM), arrows indicate morphological changes. Scale bar: 50 μm **P<0.01, ***P<0.001.

[0018] Figure 8 Cytotoxicity of MM cells mediated by NRF2 accumulation mediated by 1.9 nm silver nanoparticles; (A) Western blot analysis of NRF2 accumulation in U266 cells treated with silver nanoparticles for 6 hours. (B) qPCR analysis of NRF2 target genes HO-1 and NQO-1 mRNA expression in U266 cells exposed to silver nanoparticles for 6 hours.

[0019] Figure 9NRF2 activation promoted cytotoxicity induced by 1.9 nm silver nanoparticles in MM cells; (AC) Cell viability and apoptosis rate of MM cells (MM1S, U266, and MCC1) or other cancer cells (A549 and HepG2) were measured in indicator cells treated with or without silver nanoparticles containing DMF (20 μM). (C) Cell viability was assessed in MM1S and U266 cells treated with silver nanoparticles with or without NK-252 (2 μM). (E) Relative intracellular ROS levels of U266 and MM1S cells 6 hours after silver nanoparticle treatment with or without DMF (20 μM). Data are presented as mean ± standard deviation. n = 4 per group. No significant difference was observed in ns, **P < 0.01, ***P < 0.001.

[0020] Figure 10 To investigate the synergistic inhibition of MM growth by 1.9 nm silver nanoparticles and DMF in a xenograft model; (A) Tumor volume of human U266MM xenografts in mice treated twice weekly with control solvent vehicle, silver nanoparticles (5 mg / kg body weight), and / or DMF (10 mg / kg body weight) for 11 days. (BC) Representative images (B) and weight quantification of tumor xenografts (C). (D) Kaplan-Meier survival analysis of mice treated with control solvent vehicle, silver nanoparticles, and silver nanoparticles + DMF. Statistical significance was calculated using the log-rank test. n = 8 per group. (E) Hematological analysis of mice treated with control solvent vehicle, silver nanoparticles, and silver nanoparticles + DMF. Data are presented as mean ± standard deviation. n = 8 per group. ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

[0021] Figure 11 The figure shows the effect of 5nm silver nanoparticles (Ag+DMF) on the proliferation of U266 cells, as detected by CCK-8 assay.

[0022] Figure 12 The figure shows the effect of 30nm silver nanoparticles (Ag+DMF) on the proliferation of U266 cells, as detected by CCK-8 assay.

[0023] Figure 13 The figure shows the expression of ROS in U266 multiple myeloma cells after treatment with DMF, nanosilver, and nanosilver + DMF.

[0024] Figure 14 Image showing the HE staining results of liver, kidney, and spleen sections. Detailed Implementation

[0025] To enable those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below with reference to the accompanying drawings.

[0026] laboratory animals

[0027] Forty 5-week-old neonatal severely immunodeficient mice were purchased from Silek Jingda Biotechnology Co., Ltd. The mice were housed in the animal facility of Changsha Medical College, and the environment was regularly disinfected during the experiment. All bedding and feed used in the experiment were treated with ultraviolet light, and the drinking water was single-distilled water. The mice had free access to water and food.

[0028] Cell culture

[0029] Human MM cell lines MM1S (provided by the laboratory of Central South University), H929, ARP-1 (provided by the laboratory of Central South University), RPMI8226 (CL-0546, Procell) and U266 (CL-0510, Procell) were all cultured in RPMI-1640 medium (01-100-1A, Biological Industries) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin antibiotics (P / S).

[0030] Source of materials

[0031] The 1.9nm silver nanoparticles were stored in the laboratory. The particle size distribution of the 1.9nm silver nanoparticles is shown in the figure below. Figure 1 As shown.

[0032] 5nm and 30nm silver nanoparticles were purchased from Beijing Bio-Tech Biotechnology Co., Ltd. Electron microscope images and particle size distribution maps of the 5nm silver nanoparticles are shown below. Figure 2 and Figure 3 As shown; the electron microscope image and particle size distribution map of 30nm silver nanoparticles are shown in the figures below. Figure 4 and Figure 5 As shown.

[0033] N-acetyl-L-cysteine ​​(NAC; A9165) was purchased from Sigma-Aldrich, and dimethyl fumarate (DMF; HY-17363) and NK252 (HY-19734) were purchased from MedChemExpress.

[0034] Preparation of dimethyl fumarate

[0035] Weigh an appropriate amount of dimethyl fumarate drug powder, dissolve the drug powder thoroughly with an appropriate amount of DMSO to make the concentration of the mother solution 200 mmol, and store it in a refrigerator at -80℃.

[0036] Cell viability assay

[0037] Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; C008-3, 7Sea Biotech) according to the manufacturer's instructions. (1×10⁶) 4 Cells / wells were seeded into 96-well culture plates and treated with different concentrations of nano silver and / or other compounds for 24 hours.

[0038] Apoptosis assay

[0039] MM cells were treated with a series of concentrations of nanosilver and / or other compounds for 24 hours, and then apoptosis was detected using the Annexin V-FITC / PI kit (40306ES60, Yeasen Biotech) according to the manufacturer's instructions. Apoptotic cells were analyzed using flow cytometry.

[0040] ROS-generated measurements

[0041] Intracellular ROS production was assessed using the DCFH-DA probe (D6883, Sigma). Cells were treated with 0–4 μg / mL silver nanoparticles and / or other compounds for 6 h, then washed three times with PBS, followed by incubation with 10 μM DCFH-DA at 37 °C for 20 min. Cells were then washed twice more with PBS and analyzed using a fluorescence microscope (Apotome, Zeiss) or a microplate reader (Varioskan LUX, Thermo Fisher Scientific).

[0042] Western blot analysis

[0043] MM cells were treated with 0–4 μg / mL silver nanoparticles for 6 h, then collected and lysed in RIPA lysis buffer (CW2333S, CWBio) for 30 min. Protein concentration was determined using the BCA protein assay (P0012, Beyotime). Equal volumes of protein were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated with primary antibodies: anti-β-Actin (#3700, Cell Signaling Technology) and anti-NRF2 (A11159, ABclonal). Detection was performed using HRP-conjugated secondary antibodies, and bands were visualized using an ECL detection system (Bio-Rad).

[0044] Quantitative RT-PCR

[0045] Total RNA was extracted using TRIzol reagent (Invitrogen), and cDNA was synthesized using a reverse transcription kit (Takara). The sequences of the primers used are shown in Table 1. 18S RNA was used as a reference for mRNA normalization. qRT-PCR was performed on an ABI 7500 real-time PCR system using SYBR Green PCRMaster Mix (Applied Biosystems).

[0046] Table 1List of primer sequences.

[0047]

[0048] Animals and Processing

[0049] This study was approved by the Ethics Committee of Changsha Medical University (Approval No.: 2021050) and conducted in accordance with institutional guidelines. Five-week-old male NOD / SCID mice (SJA laboratory) were used in this study. Approximately 2 × 10⁻⁶ mice were injected subcutaneously. 6 A tumor xenograft model was established using U266 cells. Tumor volume was calculated as volume (mm). 3 = (length × width) 2 ) / 2. When the tumor volume reaches approximately 100 mm 3 Mice were randomly assigned to different treatment groups (n=8 per group). Treatment consisted of twice-weekly tail vein injections of excipients, nanosilver (5 mg / kg body weight), and / or DMF (10 mg / kg body weight). Mice were euthanized when the largest tumor reached 2 cm.

[0050] Histological and immunohistochemical staining

[0051] Mice were anesthetized, and tissues were dissected and fixed in 4% formaldehyde. Samples were processed, paraffin-embedded, and cut into 5–10 μm thick sections. Sections from each group were stained with hematoxylin and eosin (H&E) for histological analysis. Immunohistochemistry (IHC) staining was performed using an anti-NRF2 antibody (A11159, 1:400, ABclonal) to detect NRF2 accumulation in tumor tissues.

[0052] Statistical analysis

[0053] Data are expressed as mean ± standard deviation and analyzed using GraphPad Prism 10. Statistical significance was determined using unpaired two-tailed Student's t-test or one-way ANOVA. Mouse survival was measured using the Kaplan-Meier method, and comparisons between groups were performed using the log-rank test. A p-value < 0.05 was considered statistically significant.

[0054] result

[0055] Low concentrations of 1.9 nm silver nanoparticles induced cytotoxicity in multiple myeloma cells.

[0056] We used the Cell Counting Kit-8 (CCK-8) to determine the cell viability of five human MM cell lines (MM1S, RPMI8226, ARP-1, U266, and H929). Cells were treated with different concentrations of 1.9 nm silver nanoparticles (1 μg / mL). Our results showed that cell viability decreased in a dose-dependent manner in all tested MM cell lines. Figure 6 A). In particular, at a concentration of 4 μg / mL, silver nanoparticles induced significant cytotoxicity, reducing the viability of ARP-1 cell lines by up to 80% ( Figure 6 A). In contrast, the same concentration of silver nanoparticles did not significantly affect the viability of other cancer cell lines, including A549 (lung cancer), HepG2 (liver cancer), MG63 (osteosarcoma), and GC7901 (gastric cancer). Figure 6 B). This suggests that MM cells have a higher sensitivity to silver nanoparticles, possibly due to differences in intrinsic oxidative stress levels.

[0057] To further elucidate the mechanism of action of silver nanoparticle-induced cytotoxicity in MM cells, we assessed apoptosis in MM1S and U266 cells using Annexin V / PI staining and flow cytometry. Treatment with 2 μg / mL silver nanoparticles significantly increased apoptosis in both cell lines. Figure 6 C). In MM1S cells, the proportion of early apoptotic cells (Annexin V+ / PI-) increased from 14.1% in the control group to 33.7%, and the proportion of late apoptotic cells (Annexin V+ / PI+) increased from 10.1% to 47.9%. Figure 6 C). Similarly, in U266 cells, the proportion of early apoptotic cells increased from 7.65% to 38.5%, and the proportion of late apoptotic cells increased from 3.75% to 25.6%. Figure 6 C). Microscopic observation showed that after 12 hours of treatment with nanosilver, MM cells exhibited significant morphological damage, including cell shrinkage and membrane blistering. Figure 6 D).

[0058] These results collectively demonstrate that low concentrations of silver nanoparticles exert significant cytotoxic effects on MM cells, primarily through apoptosis induction. The enhanced sensitivity of MM cells to silver nanoparticles highlights its potential as a targeted therapeutic strategy, warranting further investigation into its underlying mechanisms and possible clinical applications.

[0059] ROS activation-mediated 1.9 nm silver nanoparticle-induced MM cell death

[0060] First, the baseline ROS levels of various cancer cell lines were assessed using DCFH-DA dye. Our results showed that the baseline ROS levels of MM cells (ARP-1, MM1S, and U266) were higher than those of other cancer cell lines (MG63, A549, and HepG2). Figure 7 A). To further investigate the role of ROS in silver nanoparticle-induced cytotoxicity, we used a multi-functional microplate reader to measure changes in intracellular ROS levels after MM cell treatment. Silver nanoparticles significantly increased ROS levels in MM1S and U266 cells (A). Figure 7 B). This increase in ROS levels completely disappeared after the addition of the potent ROS scavenger N-acetyl-1-cysteine ​​(NAC). Figure 7 B).

[0061] Next, the protective effect of NAC against nanosilver-induced cytotoxicity was investigated. MM cells were pretreated with NAC before nanosilver treatment. CCK-8 assays showed that NAC pretreatment rescued most MM cells from the cytotoxic effects of nanosilver, manifested as a significant increase in cell viability. Figure 7 C). Flow cytometry analysis using Annexin V / PI staining showed a significant decrease in apoptosis levels. Figure 7 D). In particular, late apoptosis decreased from 63.8% to 27.6% in MM1S cells and from 20.7% to 11.4% in U266 cells. Figure 7 D). Furthermore, microscopic morphological observations showed that NAC pretreatment alleviated the morphological damage induced by silver nanoparticles. Compared with cells treated with silver nanoparticles alone, MM cells treated with NAC + silver nanoparticles exhibited less cell shrinkage and membrane blistering. Figure 7 E).

[0062] These findings suggest that ROS plays a crucial role in nanosilver-induced MM cytotoxicity. The protective effect of NAC highlights the importance of oxidative stress in mediating the cytotoxic effects of nanosilver.

[0063] NRF2 accumulation mediates 1.9 nm silver nanoparticle-induced MM cytotoxicity

[0064] To elucidate the potential mechanism by which silver nanoparticles induce MM cell toxicity, we investigated the role of nuclear factor E2-associated factor 2 (NRF2), a key regulator of oxidative stress responses. NRF2 activates the transcription of various antioxidant enzymes to mitigate stress-related toxicity. We hypothesize that NRF2 may play a crucial role in the toxic effects of silver nanoparticles on MM cells.

[0065] In our preliminary study, we examined the NRF2 protein levels in multiple myeloma (MM) cells treated with silver nanoparticles. Western blot analysis showed that silver nanoparticle treatment led to a dose-dependent accumulation of NRF2 in U266 cells (see...). Figure 8 A). This accumulation indicates that NRF2 is activated in response to silver nanoparticle-induced oxidative stress. Next, we assessed the expression levels of downstream target genes of NRF2, particularly heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1). Quantitative polymerase chain reaction (qPCR) analysis revealed a significant upregulation of HO-1 and NQO-1 mRNA levels in silver nanoparticle-treated U266 cells, indicating that NRF2 activation leads to enhanced antioxidant responses (see [link to qPCR]). Figure 8 B).

[0066] These results highlight the increased cytotoxicity observed in NRF2-deficient cells, underscoring the importance of NRF2 in regulating cellular responses to silver nanoparticles. This suggests that targeting the NRF2 pathway may enhance the therapeutic efficacy of silver nanoparticles, potentially providing a more effective treatment option for multiple myeloma cells.

[0067] NRF2 activation enhanced the cytotoxicity of 1.9 nm silver nanoparticles in MM cells.

[0068] To further investigate the role of NRF2 in mediating the cytotoxic effects of silver nanoparticles, we aimed to activate NRF2 and evaluate its impact on the sensitivity of MM cells to silver nanoparticles. Dimethyl fumarate (DMF), a known NRF2 activator approved by the FDA for the treatment of multiple sclerosis, was used to enhance NRF2 expression in MM cells.

[0069] We treated MM1S and U266 cells with DMF and evaluated the combined effect of DMF and silver nanoparticles on cell viability. Contrary to expectations, cell viability assays showed that the addition of DMF significantly increased the sensitivity of MM cells to silver nanoparticle-induced cytotoxicity. Figure 9 A). Conversely, DMF did not enhance the cytotoxic effects of silver nanoparticles in A549 (lung cancer) or HepG2 (liver cancer) cells. Figure 9 B) indicates that it has a selective effect in MM cells.

[0070] To quantify the degree of apoptosis induced by the combined therapy, we performed flow cytometry analysis on U266 cells. The results showed that late apoptosis was significantly increased when cells were treated with both nanosilver and DMF compared to treatment with nanosilver alone. Figure 9 C). Specifically, after the addition of DMF, the percentage of late-stage apoptotic cells increased from 11.9% to 29.9% (C). Figure 9C). To confirm these findings, we also treated MM cells with another potent NRF2 inducer, NK-252. The results showed that, similar to DMF, NK-252 also enhanced the cytotoxic effect of silver nanoparticles in MM cells, further validating the role of NRF2 activation in enhancing silver nanoparticle-induced cell death. Figure 9 D).

[0071] Furthermore, by measuring the increased fluorescence intensity using the DCFH-DA probe, we observed that DMF treatment promoted ROS production in silver nanoparticle-treated MM cells. Figure 9 E). This indicates that the enhanced cytotoxicity following NRF2 activation is associated with increased oxidative stress.

[0072] These results collectively demonstrate that NRF2 activation amplifies the cytotoxic effects of nanosilver in myeloma cells. Therefore, the selective sensitization of MM cells to nanosilver by NRF2 activators highlights a potential therapeutic strategy that combines NRF2 activation with nanosilver therapy to effectively target MM.

[0073] 1.9nm silver nanoparticles and DMF synergistically inhibit MM growth in xenograft model

[0074] To further investigate the efficacy of combined DMF and nanosilver therapy, we evaluated their effects in vivo using a human myeloma xenograft mouse model. U266 cells were transplanted subcutaneously into the abdomen of NOD / SCID mice (18-22 g). After establishing the xenograft model, the mice received control solvent (Vehicle), nanosilver alone, DMF alone, or a combination of both.

[0075] Nanosilver alone or in combination with DMF can significantly reduce tumor growth. Figure 10 AB) and promote mouse survival ( Figure 10 C). Combined therapy with nanosilver and DMF can synergistically reduce tumor burden, while DMF alone cannot inhibit tumor growth. Figure 10 AB). Furthermore, we observed increased NRF2 accumulation in both single and combination therapy groups. Figure 10 D), while no NRF2 accumulation was detected in the control group mice. DMF supplementation significantly increased Nrf2 protein levels. We then tested the toxic effects of nanosilver and DMF treatment on the liver, kidneys, and other major organs of mice.

[0076] We also used hematoxylin-eosin (HE) staining to assess the potential toxicity of the treatment to major organs, including the liver, kidneys, and spleen. Figure 14HE staining results showed no significant toxicity to the liver, kidneys, and spleen. Hematological parameters also indicated no abnormalities after treatment with nanosilver and DMF. Figure 10 E). These results indicate that nanosilver, alone or in combination with DMF, can effectively inhibit the growth of MM cells in vivo without causing significant toxicity.

[0077] These in vivo results demonstrate that nanosilver, especially when used in combination with DMF, effectively inhibits myeloma tumor growth without significant toxicity to the host. The enhanced antitumor activity observed when NRF2 was activated using DMF suggests a promising therapeutic strategy for multiple myeloma.

[0078] In this invention, we demonstrate for the first time that 1.9 nm AgNPS can inhibit the growth of MM cells and induce apoptosis both in vitro and in vivo. 1.9 nm AgNPS can exert cytotoxicity on MM cells at very low concentrations by inducing oxidative stress. Furthermore, we found that the NRF2 inducers DMF and NK-252 can exacerbate the cytotoxicity of 1.9 nm AgNPS-induced MM cells.

[0079] In our study, we found that myeloma cells are particularly sensitive to 1.9 nm AgNPS and exhibit higher baseline ROS levels compared to other cancer cells. Myeloma cells, derived from plasma cells, possess a unique ability to synthesize and secrete antibodies. Due to the metabolic demands of malignant proliferation, myeloma cells exhibit higher protein synthesis rates and ROS levels. A moderate increase in ROS may be beneficial for cell survival and cell growth signaling, while excessive ROS production is thought to inhibit tumor cell proliferation.

[0080] Cancer cells can utilize various mechanisms to combat oxidative stress and eliminate excess ROS. In our study, NRF2 knockdown made MM cells more sensitive to silver nanoparticles. However, the addition of DMF and NK-252 (a strong inducer of NRF2) did not decrease but rather increased the sensitivity of MM cells to AgNPS. DMF exacerbated the increase in ROS in AgNPS-treated MM cells.

[0081] Finally, we tested the inhibitory effect of AgNPS on myeloma cell growth in vivo. Simultaneously, we found that DMF (an FDA-approved drug for the treatment of multiple sclerosis and psoriasis) can synergistically work with AgNPS to inhibit myeloma growth in a xenograft model. Existing studies have confirmed that DMF can inhibit cancer cell growth in vitro at high concentrations (50–200 μM), but is not toxic to cancer cells at low concentrations (<20 μM), consistent with our results. However, low levels of DMF can amplify the toxicity of AgNPS in MM cells. Therefore, when used in combination with DMF in in vivo experiments, this strategy allows us to reduce the concentration of AgNPS to a relatively safe range. In fact, in recent years, the toxic side effects of silver nanoparticles on human health have attracted increasing attention. This combined approach will help overcome some limitations in the clinical application of silver particles and reopen the door to the use of silver particles for cancer treatment.

[0082] Validation experiments of AgNPS with different particle sizes

[0083] The combined application of 5nm and 30nm silver nanoparticles and dimethyl fumarate further inhibits the proliferation of multiple myeloma cells.

[0084] To investigate the effect of the combined application of silver nanoparticles and dimethyl fumarate on cell proliferation, we selected well-grown U266 myeloma cells and added 100 μL per well to 96-well plates for drug administration. We set up a control group, a silver nanoparticle group (using two different silver nanoparticle sizes: 5 nm and 30 nm, at concentrations of 0 μg / ml, 2 μg / ml, and 4 μg / ml), and a silver nanoparticle + dimethyl fumarate group (silver nanoparticle concentration was the same as above, and dimethyl fumarate concentration was 20 μmol). After culturing for 24 h, the proliferation of U266 myeloma cells was detected using CCK-8 at a wavelength of 450 nm (OD value). The experimental results are as follows: Figure 11 and Figure 12 As shown, the combined use of 5nm nanosilver and dimethyl fumarate has a stronger inhibitory effect on multiple myeloma cells than nanosilver alone.

[0085] The combined application of 5nm silver nanoparticles and dimethyl fumarate increases ROS levels.

[0086] To investigate the effect of the combined use of nanosilver and dimethyl fumarate on ROS in multiple myeloma cells, this experiment labeled U266 myeloma cells using the DCFH-DA fluorescent probe method, and detected the fluorescence intensity using a microplate reader. We set up a blank control group, a dimethyl fumarate group, a nanosilver group, and a nanosilver plus dimethyl fumarate group. Figure 13As shown in the experiment, the ROS level in cells was higher after the combined use of the two drugs than in the single-drug group. This means that the combined use of nano-silver and dimethyl fumarate can induce an increase in ROS levels, thereby promoting apoptosis in U266 myeloma cells.

[0087] Nanosilver increases intracellular ROS levels, which, combined with the already high basal ROS levels in multiple myeloma cells, creates an environment of excessive ROS. Dimethyl fumarate (DMF), as an agonist of Nrf2, leads to the continuous activation of Nrf2 under conditions of excessive ROS, resulting in its accumulation in the cell nucleus. Subsequently, it binds to the Klf9 promoter, and DMF further increases ROS through Klf9, thereby causing tumor cell death.

[0088] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. Application of nanosilver combined with NRF2 inducer DMF in the preparation of drugs for treating multiple myeloma, with a DMF concentration of 20 μM; The average particle size of the silver nanoparticles is 1.9–5 nm; the concentration of the silver nanoparticles is 1–4 μg / mL.

2. The application according to claim 1, characterized in that, The average particle size of the silver nanoparticles is 1.9 nm or 5 nm.

3. A drug for treating multiple myeloma, characterized in that, It includes the NRF2 inducer DMF and silver nanoparticles, wherein the concentration of DMF is 20 μM; The average particle size of the silver nanoparticles is 1.9 nm to 5 nm; the concentration of the silver nanoparticles is 1 to 4 μg / mL.

4. The drug according to claim 3, characterized in that, The average particle size of nano-silver is 1.9 nm or 5 nm.

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