Journal: Drug Delivery

Article Title: Utilization of DOX–Fe complex and RSL3 co-loaded liposomes in ferroptosis-enhanced treatment of triple-negative breast cancer

doi: 10.1080/10717544.2025.2592412

Figure Lengend Snippet: The anticancer mechanisms of the DOX–Fe/RSL3@LIPs.

Article Snippet: Doxorubicin hydrochloride (DOX·HCl, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.); RSL3 (98%, Shanghai Macklin Biochemical Technology Co., Ltd.); Ferrous chloride (FeCl 2 ·4H 2 O, 99%, Sinopharm Chemical Reagent Co., Ltd.); Cholesterol (99%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)- 2000] (DSPE-MPEG2000, >98%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); Fetal bovine serum (FBS, special grade, Anhui Kangyuan Biotechnology Co., Ltd.); RPMI-1640 medium, trypsin digestion solution (0.25%), PBS (pH7.4) were purchased from Wuhan Servicebio Technology Co., Ltd. All other reagents are analytical grade.

Techniques:

Journal: Drug Delivery

Article Title: Utilization of DOX–Fe complex and RSL3 co-loaded liposomes in ferroptosis-enhanced treatment of triple-negative breast cancer

doi: 10.1080/10717544.2025.2592412

Figure Lengend Snippet: Synergistic ratio screening of DOX, Fe 2+ , and RSL3. (A) The changes of DOX fluorescence after the formation of DOX–Fe at various molar ratios. (B) The cytotoxicity of DOX/RSL3 at different weight ratios on 4T1 cells after 48 h of incubation. (C) Evaluation of the synergistic effects of DOX/RSL3 at different weight ratios on 4T1 cells. (**** p < 0.0001).

Article Snippet: Doxorubicin hydrochloride (DOX·HCl, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.); RSL3 (98%, Shanghai Macklin Biochemical Technology Co., Ltd.); Ferrous chloride (FeCl 2 ·4H 2 O, 99%, Sinopharm Chemical Reagent Co., Ltd.); Cholesterol (99%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)- 2000] (DSPE-MPEG2000, >98%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); Fetal bovine serum (FBS, special grade, Anhui Kangyuan Biotechnology Co., Ltd.); RPMI-1640 medium, trypsin digestion solution (0.25%), PBS (pH7.4) were purchased from Wuhan Servicebio Technology Co., Ltd. All other reagents are analytical grade.

Techniques: Fluorescence, Incubation

Journal: Drug Delivery

Article Title: Utilization of DOX–Fe complex and RSL3 co-loaded liposomes in ferroptosis-enhanced treatment of triple-negative breast cancer

doi: 10.1080/10717544.2025.2592412

Figure Lengend Snippet: Characterization of the DOX–Fe/RSL3@LIPs. (A) Particle size distribution and (B) zeta potential of DOX–Fe/RSL3@LIPs. (C) The changes of particle size and zeta potential of DOX–Fe/RSL3@LIPs in pH 7.4 PBS at 37 °C for 72 h. (D) BSA adsorption of the blank LIPs and DOX–Fe/RSL3@LIPs. In vitro drug release profiles of DOX (E) and RSL3 (F) from DOX–Fe/RSL3@LIPs in PBS containing 0.5% Tween 80 at different pH values.

Article Snippet: Doxorubicin hydrochloride (DOX·HCl, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.); RSL3 (98%, Shanghai Macklin Biochemical Technology Co., Ltd.); Ferrous chloride (FeCl 2 ·4H 2 O, 99%, Sinopharm Chemical Reagent Co., Ltd.); Cholesterol (99%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)- 2000] (DSPE-MPEG2000, >98%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); Fetal bovine serum (FBS, special grade, Anhui Kangyuan Biotechnology Co., Ltd.); RPMI-1640 medium, trypsin digestion solution (0.25%), PBS (pH7.4) were purchased from Wuhan Servicebio Technology Co., Ltd. All other reagents are analytical grade.

Techniques: Zeta Potential Analyzer, Adsorption, In Vitro

Journal: Drug Delivery

Article Title: Utilization of DOX–Fe complex and RSL3 co-loaded liposomes in ferroptosis-enhanced treatment of triple-negative breast cancer

doi: 10.1080/10717544.2025.2592412

Figure Lengend Snippet: In vitro anticancer effects of the DOX–Fe/RSL3@LIPs on 4T1 cells. (A) The viability of DOX@LIPs, DOX–Fe@LIPs, DOX–Fe/RSL3@LIPs, and RSL3@LIPs on 4T1 cells at different concentrations of DOX after incubation for 24 h (B) and 48 h (C). (D–F) The content determination of ferrous ions (D), ROS (E), and LPO (F) in 4T1 cells in each group after treatments. (** p < 0.01, * p < 0.05).

Article Snippet: Doxorubicin hydrochloride (DOX·HCl, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.); RSL3 (98%, Shanghai Macklin Biochemical Technology Co., Ltd.); Ferrous chloride (FeCl 2 ·4H 2 O, 99%, Sinopharm Chemical Reagent Co., Ltd.); Cholesterol (99%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)- 2000] (DSPE-MPEG2000, >98%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); Fetal bovine serum (FBS, special grade, Anhui Kangyuan Biotechnology Co., Ltd.); RPMI-1640 medium, trypsin digestion solution (0.25%), PBS (pH7.4) were purchased from Wuhan Servicebio Technology Co., Ltd. All other reagents are analytical grade.

Techniques: In Vitro, Incubation

Journal: Drug Delivery

Article Title: Utilization of DOX–Fe complex and RSL3 co-loaded liposomes in ferroptosis-enhanced treatment of triple-negative breast cancer

doi: 10.1080/10717544.2025.2592412

Figure Lengend Snippet: In vitro cellular uptake study of the DOX–Fe/RSL3@LIPs. (A) The CLSM images of 4T1 cells after incubation with the DOX–Fe/RSL3@LIPs suspension at different concentrations of coumarin-6 (a) and DOX (b) for 2 h. Flow cytometry analysis of the cellular uptake at different concentrations of coumarin-6 (B) and DOX (C) ( n = 3). (D) The CLSM images of 4T1 cells after incubation with the DOX-Fe/RSL3@LIPs (coumarin-6 2 μg/mL) suspension for1, 2, and 4 h. Flow cytometry analysis of the cellular uptake of coumarin-6 (E) and DOX (F) for 1, 2, and 4 h ( n = 3). (**** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05). Note: we confirm that the similarity between (A) (coumarin-6 2 μg/mL) and (D) (coumarin-6 2 h) is intentional, as this control group serves as the baseline reference for two different experiments. Concentration-dependent uptake (A): comparing different concentrations of DOX–Fe/Coumarin-6@LIP while maintaining a constant 2 h incubation time. Time-dependent uptake (D): comparing different incubation durations at fixed concentration parameters. The dual presentation enables direct visual comparison across both experimental axes while maintaining methodological consistency. This approach follows established practices for multidimensional drug uptake studies in nanomedicine research.

Article Snippet: Doxorubicin hydrochloride (DOX·HCl, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.); RSL3 (98%, Shanghai Macklin Biochemical Technology Co., Ltd.); Ferrous chloride (FeCl 2 ·4H 2 O, 99%, Sinopharm Chemical Reagent Co., Ltd.); Cholesterol (99%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)- 2000] (DSPE-MPEG2000, >98%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); Fetal bovine serum (FBS, special grade, Anhui Kangyuan Biotechnology Co., Ltd.); RPMI-1640 medium, trypsin digestion solution (0.25%), PBS (pH7.4) were purchased from Wuhan Servicebio Technology Co., Ltd. All other reagents are analytical grade.

Techniques: In Vitro, Incubation, Suspension, Flow Cytometry, Control, Concentration Assay, Comparison

Journal: Drug Delivery

Article Title: Utilization of DOX–Fe complex and RSL3 co-loaded liposomes in ferroptosis-enhanced treatment of triple-negative breast cancer

doi: 10.1080/10717544.2025.2592412

Figure Lengend Snippet: Evaluation of the antitumor efficacy of DOX–Fe/RSL3@LIPs in vivo . (A) The images of tumors harvested from 4T1 tumor xenograft mice in each group after the 15-day treatments. (B) The tumor growth curves of 4T1 tumor–bearing mice of each group during the treatment period. (C) The weights of tumors harvested from 4T1 tumor xenograft mice in each group after the 15-day treatments. (D–F) The content determination of ferrous ion (D), ROS (E), and LPO (F) in tumor tissues harvested from 4T1 tumor xenograft mice in each group after treatments.

Article Snippet: Doxorubicin hydrochloride (DOX·HCl, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.); RSL3 (98%, Shanghai Macklin Biochemical Technology Co., Ltd.); Ferrous chloride (FeCl 2 ·4H 2 O, 99%, Sinopharm Chemical Reagent Co., Ltd.); Cholesterol (99%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)- 2000] (DSPE-MPEG2000, >98%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); Fetal bovine serum (FBS, special grade, Anhui Kangyuan Biotechnology Co., Ltd.); RPMI-1640 medium, trypsin digestion solution (0.25%), PBS (pH7.4) were purchased from Wuhan Servicebio Technology Co., Ltd. All other reagents are analytical grade.

Techniques: In Vivo

Journal: Drug Delivery

Article Title: Utilization of DOX–Fe complex and RSL3 co-loaded liposomes in ferroptosis-enhanced treatment of triple-negative breast cancer

doi: 10.1080/10717544.2025.2592412

Figure Lengend Snippet: Biodistribution and safety of the DOX–Fe/RSL3@LIPs in vivo . (A) The fluorescence imaging of tumor and major organs of the mice after administration of DiR-labeled formulations at various time points in vivo . (B) The weight change curves of 4T1 tumor–bearing mice of each group during the treatment period. (C) The H&E staining images of the major organs harvested from 4T1 tumor xenograft mice in each group after treatments (**** p < 0.0001).

Article Snippet: Doxorubicin hydrochloride (DOX·HCl, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.); RSL3 (98%, Shanghai Macklin Biochemical Technology Co., Ltd.); Ferrous chloride (FeCl 2 ·4H 2 O, 99%, Sinopharm Chemical Reagent Co., Ltd.); Cholesterol (99%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)- 2000] (DSPE-MPEG2000, >98%, Shanghai Macklin Biochemical Technology Co., Ltd.); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); Fetal bovine serum (FBS, special grade, Anhui Kangyuan Biotechnology Co., Ltd.); RPMI-1640 medium, trypsin digestion solution (0.25%), PBS (pH7.4) were purchased from Wuhan Servicebio Technology Co., Ltd. All other reagents are analytical grade.

Techniques: In Vivo, Fluorescence, Imaging, Labeling, Staining

Journal: bioRxiv

Article Title: Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis

doi: 10.1101/2025.03.10.642426

Figure Lengend Snippet: Cellular proliferation rates of mPDAC1, mPDAC2, and mPDAC3 cells cultured in TIFM or TIFM + arg upon treatment with RSL3 and erastin at indicated concentrations GPX4 or erastin. Ferrostatin-1 (fer-1, 2 µM) or vehicle (DMSO) was additionally added.

Article Snippet: After seeding, cells were treated with indicated concentrations of RSL3 (Cayman Chemical, 19288), erastin (Cayman Chemical, 17754), or viFSP1 (Cayman Chemical, 39927), along with 2 μM α-ESA (Cayman Chemical, 22976) conjugated to fatty acid free BSA (Sigma, A8806) or an equivalent amount of fatty acid free BSA as vehicle control.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis

doi: 10.1101/2025.03.10.642426

Figure Lengend Snippet: Proliferation of mPDAC1 cells cultured in RPMI or TIFM when treated with α-ESA (2 µM) of vehicle (fatty acid free BSA) in tandem with (A) deprivation of cystine to fraction present in base media as indicated, (B) Erastin at indicated concentrations, or (C) RSL3 at indicated concentrations. Ferrostatin-1 (fer-1, 2 µM) or vehicle (DMSO) was additionally added.

Article Snippet: After seeding, cells were treated with indicated concentrations of RSL3 (Cayman Chemical, 19288), erastin (Cayman Chemical, 17754), or viFSP1 (Cayman Chemical, 39927), along with 2 μM α-ESA (Cayman Chemical, 22976) conjugated to fatty acid free BSA (Sigma, A8806) or an equivalent amount of fatty acid free BSA as vehicle control.

Techniques: Cell Culture

Journal: Nature Communications

Article Title: Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity

doi: 10.1038/s41467-022-31218-2

Figure Lengend Snippet: a Prophylactic vaccination model with MCA205 cells was used to assess the immunogenic potential of ferroptosis (ML162, 0.5 µM, 14 h) via their ability to induce protection against cancer tumor growth. Immunogenic (Mitoxantrone, 1 µM, 24 h) and non-immunogenic (Mitomycin C, 30 µM, 24 h) apoptosis, as well as accidental necrosis (Freeze/thaw, three cycles) conditions, were used as controls. Kaplan–Meier curves represent the % of tumor-free mice after the challenge with live cancer cells. Data from n = 3 independent experiments was analyzed by Kaplan–Meier simple survival analysis. b DAMP release from MCA205 cells stimulated with three inducers of ferroptosis: ML162 (0.5 µM), RSL-3 (0.5 µM) and Erastin (2.0 µM) as well as doxorubicin (1 µM, 24 h). Release of LDH, ATP, HMGB1 as well as cytokines CXCL1, TNF and IFN-β was analyzed at different time points after cell death induction. Data from n = 3 biologically independent samples are presented as floating bars with bounds showing the range and the center showing the mean. Data analyzed by one-way ANOVA with Dunnett’s post-hoc tests for each ferroptosis stimuli separately and two-sided t-test for doxorubicin treatment comparing the values to the untreated sample. c Analysis of calreticulin exposure on the surface of ferroptotic cells, ML162 (0.5 µM), RSL-3 (0.5 µM) and Erastin (2.0 µM). Treatment with doxorubicin (1 µM, 24 h) served as a positive control. Histograms represent the fluorescence intensity detected in non-permeabilized cells. Bounds of the bars show the range, and the center shows the mean of fold change in fluorescence intensity generated by comparing the MFI of treated cells to MFI of untreated cells. Data were generated from n = 3 biologically independent samples. One-way ANOVA, with Dunnett’s post-hoc test for each ferroptosis stimuli and two-sided t-test for doxorubicin treatment comparing the obtained values to the untreated sample d . The % of live cells exposing calreticulin during cell death. Data presented as range (box bounds) and mean (center) of n = 3 independent experiments. One-way ANOVA with Dunnett’s post-hoc test for each ferroptosis stimuli and the t-test for the doxorubicin treatment in comparison to the untreated sample.

Article Snippet: In the initial experiments, ferroptosis was induced by ML162 (#AOB1514, Aobious Inc., USA), RSL-3 (#S8155, Absource, Germany) and Erastin (#S7242, Absource) at different concentrations.

Techniques: Positive Control, Fluorescence, Generated

Journal: Nature Communications

Article Title: Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity

doi: 10.1038/s41467-022-31218-2

Figure Lengend Snippet: a Bone marrow-derived dendritic cells (BMDC) were incubated with soluble OVA in the presence of ferroptotic (ML162 0.5 µM, 14 h; RSL-3, 0.5 µM, 14 h; Erastin 2.0 µM, 24 h) or necrotic (3 cycles of freeze/thaw, FT) cancer cells. Afterwards, fluorescently labeled OVA-specific CD8 + cells, were added to the co-culture and their proliferation was assessed 72 h later. The flow cytometry dot plots present gating strategy. b Percentage of proliferating OVA-specific CD8 + T cells after co-incubation of ferroptotic and necrotic cells with BMDC. Data comes from n = 3 independent experiments and is presented as floating bars showing the range (box bounds) and mean (center line). One-way ANOVA, with Dunnett’s post-hoc test analyzing the comparison to the FT condition. c Representative histogram of OVA-specific T-cell proliferation after incubation with BMDC exposed to MCA205 cells, ferroptotic or killed by accidental necrosis (FT—freeze/thaw). d Scheme of co-culture experiments involving iGPX4KD cell line to address the importance of early events of ferroptosis in inhibiting T-cell proliferation. e Representative histograms from n = 2 independent experiments of cytotoxic T-cell proliferation after co-incubation with bone marrow-derived dendritic cells with ferroptotic cells at different stages of cell death.

Article Snippet: In the initial experiments, ferroptosis was induced by ML162 (#AOB1514, Aobious Inc., USA), RSL-3 (#S8155, Absource, Germany) and Erastin (#S7242, Absource) at different concentrations.

Techniques: Derivative Assay, Incubation, Labeling, Co-Culture Assay, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Oncogenic RAS Mutants Confer Resistance of RMS13 Rhabdomyosarcoma Cells to Oxidative Stress-Induced Ferroptotic Cell Death

doi: 10.3389/fonc.2015.00131

Figure Lengend Snippet: Oncogenic RAS genes protect against oxidative stress stimuli . RMS13 cells expressing EV, HRAS12V, KRAS12V, or NRAS12V were treated for 48 h with indicated concentrations of Auranofin (A) , RSL3 (B) , or Erastin (C) . Cell viability was determined by MTT assay and cell density by crystal violet assay; results are expressed as percentage of untreated cells. Mean + SD of three independent experiments performed in triplicate are shown; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: PI3K/mTOR inhibitor PI103 ( ) was purchased from Merck Millipore (Darmstadt, Germany), RSL3 from InterBIOScreen Ltd. (Moscow, Russia), Auranofin from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, MTT Assay, Crystal Violet Assay

Journal: Frontiers in Oncology

Article Title: Oncogenic RAS Mutants Confer Resistance of RMS13 Rhabdomyosarcoma Cells to Oxidative Stress-Induced Ferroptotic Cell Death

doi: 10.3389/fonc.2015.00131

Figure Lengend Snippet: Oncogenic RAS genes protect against ferroptotic cell death . (A,B) RMS13 cells expressing EV were treated for 48 h with indicated concentrations of RSL3 (A) or Erastin (B) in the presence or absence of 5 μM Ferrostatin-1. Cell viability was determined by MTT assay; results are expressed as percentage of untreated cells. Mean + SD of three independent experiments performed in triplicate are shown; * p < 0.05; ** p < 0.01; *** p < 0.001. (C–F) RMS13 cells expressing EV were treated for 48 h with indicated concentrations of RSL3 or Erastin. Apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei (C,D) and cell death was determined by PI staining (E,F) using flow cytometry. Mean + SD of three independent experiments performed in triplicate are shown; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: PI3K/mTOR inhibitor PI103 ( ) was purchased from Merck Millipore (Darmstadt, Germany), RSL3 from InterBIOScreen Ltd. (Moscow, Russia), Auranofin from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, MTT Assay, Staining, Flow Cytometry