Fig 1: In vivo self-assembly induced tumor accumulation of Gi-F-CAA.a GPX4 immunohistochemical staining of bladder tumor tissues and normal bladder tissues. Scale bars: 50 μm. b Fluorescence of images of the whole tumor tissues after treated with Cy labeled Gi-F-CAA and Gi-F (8 mg/kg in 100 μL PBS). Scale bars: 1 mm. c Fluorescence images of tumor tissues after treated with NBD labeled Gi-F-CAA and Gi-F-SA (8 mg/kg in 100 μL PBS). Scale bars: 20 μm. d Representative ex vivo fluorescence images of tumor and major organs (heart, liver, spleen, lung, kidney and muscle) collected after intravenous administrated with Gi-F-CAA, Gi-F and Gi-F-SA (8 mg/kg in 100 μL PBS), respectively (n = 3 mice). e Dose-dependent ex vivo fluorescence images of tumor and major organs after intravenous administrated with Gi-F-CAA (8 mg/kg, 4 mg/kg, 2 mg/kg, 1 mg/kg and 0.5 mg/kg in 100 μL PBS). f Corresponding quantitative fluorescence intensity of tumor, liver and kidney after intravenous administrated with Gi-F-CAA (n = 3 mice). g Representative time-dependent in vitro fluorescence images of EJ xenograft mice after intravenous administrated with Gi-F-CAA (8 mg/kg in 100 μL PBS) (n = 3 mice). h Representative time-dependent fluorescence images of tumor tissue lysates and plasma after intravenous administrated with Gi-F-CAA (8 mg/kg in 100 μL PBS) (n = 3 mice). i Corresponding quantitative fluorescence intensity of tumor tissue lysates and plasma after intravenous administrated with Gi-F-CAA (8 mg/kg in 100 μL PBS) (n = 3 mice). Data were expressed as mean ± SD. Source data are provided as a Source Data file.
Fig 2: Assembly enhanced binding (AEB) effect of Gi-F-CAA.a The structure of Gi-F-CAA (peptide-porphyrin conjugate decorated with acid-sensitive CAA moieties). b The structure of Gi-F (peptide-porphyrin conjugate with nanoparticle forming ability). The porphyrin parts were shown in sphere-stick mode with the carbon atoms colored in green and the other parts were shown in stick mode with the carbon atoms colored in cyan. c The self-assembly progress of Gi-F. The porphyrin parts were shown in sphere-stick mode with the carbon atoms colored in green and the other parts were shown in stick mode with the carbon atoms colored in cyan. The MD simulations begun from an initial randomly distributed system. d The self-assembled nanoparticle of Gi-F. e The hydrolysis profiles of Gi-F-CAA at pH 7.4 and 6.5 (PBS, 0.01 M) measured by HPLC (n = 3 experimental repeats). Experiment was independently repeated three times with similar results. f CAC values determined by I338/I335 ratio from pyrene as a function of concentration of Gi-F-CAA and Gi-F. g Particle size change of Gi-F-CAA and Gi-F (40 μM) in PBS (0.01 M, pH 7.4 or 6.5) measured by DLS. h Representative TEM images of Gi-F-CAA in PBS at pH 7.4 for 1 h (n = 3 experimental repeats). Experiment was independently repeated three times with similar results. i The binding mode between self-assembled nanoparticle and the GPX4mu protein. The porphyrin parts were shown in sphere-stick mode with the carbon atoms colored in green and the other parts were shown in stick mode with the carbon atoms colored in cyan. The GPX4mu protein was shown in cartoon mode and colored in magenta. j Key residues between self-assembled nanoparticle and the GPX4mu protein. k Corresponding TEM images of Gi-F-CAA at pH 6.5 incubated with protein GPX4. l Affinity binding curve of Gi-F-CAA and Gi-F with GPX4. n represents the Hill coefficient calculated from Hill plot. m The binding affinity of Gi-F-CAA to GPX4 by Microscale Thermophoresis (MST) ligand binding measurements. Kd: the dissociation constant. n The binding affinity of Gi-F to GPX4 by MST ligand binding measurements. Kd: the dissociation constant. Data were expressed as mean ± SD. Source data are provided as a Source Data file.
Fig 3: Antitumor efficacy of Gi-F-CAA against EJ xenograft tumors.a BALB/c nude mice were subcutaneously inoculated with 5 × 106 EJ cells and intravenously administrated with PBS, Gi-F (8 mg/kg in 100 μL PBS), Gi-F-SA (8 mg/kg in 100 μL PBS) and Gi-F-CAA (8 mg/kg in 100 μL PBS). b The individual tumor growth curves of mice in different treatment groups. c The average tumor growth curves of EJ xenografted mice after different treatments over 18 days (n = 6 mice). d EJ tumor tissues were harvested at 18 days after different treatments. e EJ tumor weights after different treatments over 18 days (n = 6 mice). f Body weight changes of EJ xenografted mice after different treatments over 18 days (n = 6 mice). g GPX4 activities of EJ tumor tissues after different treatments (n = 6 mice). The GPX4 activities of PBS group was normalized as 1. h Iron levels in EJ tumor tissues after different treatments by DAB-enhanced Prussian blue iron staining. Scale bar: 50 μm. i Glutathione (GSH) levels of EJ tumor tissues after different treatments (n = 6 mice). The GSH levels of PBS group was normalized as 1. j ROS levels of EJ tumor tissues after different treatments by DCFH-DA fluorescence staining. Scale bar: 50 μm. k Immunofluorescence analyses of Prostaglandin-Endoperoxide Synthase 2 (PTGS2) expression in EJ tumor tissues after different treatments. Scale bar: 50 μm. l Malonaldehyde (MDA) levels of EJ tumor tissues after different treatments (n = 6 mice). The MDA levels of PBS group was normalized as 1. P-values were performed with one-way ANOVA followed by post hoc Tukey’s test. Data were expressed as mean ± SD. Source data are provided as a Source Data file.
Fig 4: In vivo promoted ferroptosis of Gi-F-CAA by AEB effect.a Viability of EJ cells after treated with Gi-F (pH = 7.4), Gi-F-SA (pH = 7.4), NGi-F-CAA (pH = 7.4), NGi-F-CAA (pH = 6.5), Gi-F-CAA (pH = 7.4) and Gi-F-CAA (pH = 6.5) at different concentrations for 48 h (n = 3 experimental repeats). Experiment was independently repeated three times with similar results. b Confocal laser scanning microscopy (CLSM) images of EJ cells after treated with Gi-F-CAA (pH = 7.4) and Gi-F-CAA (pH = 6.5, 40 μM). Scale bar: 10 μm. c Fluorescence binding distribution images of NBD labelled Gi-F-CAA (pH = 6.5, 40 μM) and GPX4 in EJ cells. Anti-GPX4 antibody was used to label GPX4. Scale bar: 10 μm. d Intracellular glutathione peroxidase 4 (GPX4) activities of EJ cells after treated with PBS (pH = 7.4), NGi-F-CAA (pH = 7.4), Gi-F-SA (pH = 7.4), Gi-F-CAA (pH = 7.4) and Gi-F-CAA (pH = 6.5, 40 μM). The GPX4 activities of PBS group was normalized as 1 (n = 3 experimental repeats). Experiment was independently repeated three times with similar results. e Intracellular iron levels of EJ cells after different treatment by FerroOrange fluorescence staining. Scale bar: 10 μm. f Intracellular ROS levels of EJ cells after different treatment by DCFH-DA fluorescence staining. Scale bar: 10 μm. g Intracellular lipid hydroperoxide (LPO) levels of EJ cells after different treatments by C11-BODIPY staining. Scale bar: 10 μm. h Bio-TEM images of EJ cells after treated with PBS (pH = 7.4) and Gi-F-CAA (pH = 6.5, 40 μM). The red box dashed region indicates the mitochondria. Scale bar: 10 μm. Data were presented as mean ± SD. P-values were performed with one-way ANOVA followed by post hoc Tukey’s test. Data were expressed as mean ± SD. Source data are provided as a Source Data file.
Fig 5: A tumor microenvironment specifically activated peptide-ferriporphyrin conjugates (Gi-F-CAA) for ferroptosis therapy of bladder cancer by assembly enhanced binding effect.a Molecular structure and acid-responsive self-assembly behavior of Gi-F-CAA. Briefly, a GPX4 inhibitory peptide (GACNWLPLYPCPV), a peptide linker (LKLKLK) decorated with a pH-sensitive moiety (cis-aconitic anhydride, CAA) and ferriporphyrin (FeTCPP) were conjugated to produce the peptide-ferriporphyrin conjugates (Gi-F-CAA, FeTCPP-LKLKLK(CAA)GACNWLPLYPCPV). b Gi-F-CAA with a small size could easily penetrate deeply into solid tumor, which self-assembled into large nanoparticles under the acidic tumor microenvironment to improve the tumor endocytosis efficiency. Importantly, substantial inhibition of GPX4 activity was achieved by assembly enhanced binding (AEB) effect, which augmented the oxidative stress of FeTCPP-based Fenton reaction, ultimately enabling the remarkable antitumor properties by ferroptosis.
Supplier Page from Sino Biological, Inc. for Human GPX4 Protein (His Tag)