Multifunctional nanoplatform as nano-inducer of ferroptosis for targeted recognition and imaging-guided therapy of metastatic prostate cancer

Abstract

Metastasis prostate cancer (PCa) precision detection and effective treatment remain significant challenge in clinic. Ferroptosis brought promising therapeutic strategy for the treatment of metastatic PCa, effectively inducing ferroptosis in PCa cells represents key to improve therapeutic efficacy. Herein, we developed a multifunctional nanoplatform Fe/Au nanodots-bombesin (FGN-BBN) as the ferroptosis nano-inducer to generate large amount of ROS to induce ferroptosis through an "open-source throttling" strategy for targeted imaging-guided therapy of metastatic PCa. On the one hand, FGN-BBN serves as an efficient biomimetic nanozyme and photothermal agent, exhibiting great POD-like activity and generating abundant reactive oxygen species (ROS) via photothermal-enhanced chemodynamic therapy (CDT) to induce ferroptosis, which is achieving "open source" aspect. On the other hand, FGN-BBN exhibit GPx-like activity that depletes overexpressed glutathione (GSH) within the tumor microenvironment, thereby preventing the neutralization of ROS and achieving the "throttling" effect. Furthermore, bombesin facilitates targeted delivery of the nanozyme to metastatic PCa cells, synergistically enhancing ferroptosis activity. In terms of diagnosis, FGN-BBN possesses targeted recognition capabilities and enables multimode bioimaging including fluorescence (FL), computed tomography (CT), and magnetic resonance imaging (MRI), allowing for the "visualization" of tumor localization and real-time imaging-guided therapy. In summary, the multifunctional nanoplatform integrates multienzyme activity, targeted recognition, multimodal imaging, photothermal therapy, and CDT to induce high-efficiency ferroptosis, offering an effective theranostic strategy for metastatic PCa.

Keywords: Ferroptosis nano-inducer; Metastasis prostate cancer; Multi-mode imaging; Nanozyme; Tumor targeting.

Graphical abstract

Scheme 1

Schematic illustration of the synthesis of FGN-BBN and its application in GRPR-targeted FL/CT/MRI multimodal imaging and combined PTT-Ferroptosis therapy for bone metastatic PCa.

Fig. 1

Structural, morphological, and compositional characterization of FGN-BBN. (a) ¹H NMR spectrum of SH-PEI and PEI. (b) TEM image and (c) size distribution histogram of the FGN. Scale bar: 20 nm (d) TEM image and (e) size distribution histogram of the FGN-BBN. Scale bar: 20 nm (f) UV–vis absorption spectrum of FGN-BBN, FGN and BBN. (g) FT-IR spectra of FGN, BBN and FGN-BBN. High-resolution XPS spectra of (h) Au 4f and (i) Fe 2p of FGN-BBN.

Fig. 2

Evaluation of the photothermal performance and catalytic activity of FGN-BBN. (a) Temperature elevation curves and (b) infrared thermal images of PBS (control) and FGN-BBN under 808 nm laser irradiation (2.0 W/cm²) over time. (c) Temperature profiles of FGN-BBN under 808 nm laser irradiation at varying power densities (1.0, 1.5, 2.0, and 2.5 W/cm²). (d) Photothermal stability of FGN-BBN assessed over three on/off laser irradiation cycles. (e) UV–Vis absorbance spectra of MB after reaction with FGN-BBN and different concentrations of H₂O₂. (f) Absorbance of MB after treatment with FGN-BBN under 808 nm laser irradiation at varying power densities. (g) ESR spectra under various reaction conditions with DMPO as a spin trap agent. (h) GSH depletion by FGN-BBN.

Fig. 3

In vitro FL, CT and MR imaging properties of FGN-BBN. (a) Excitation and emission spectra of FGN-BBN. (b) Emission spectra of FGN and FGN-BBN. Inset, photographs of FGN-BBN solution under UV lamp. (c) FL intensity of FGN-BBN at different pH values. (d) FL stability of FGN-BBN in the presence of high concentrations (200 mM) of interfering ions (K⁺, Na⁺, NH₄⁺, and Ca²⁺). (e) CT signal intensity of FGN-BBN at varying concentrations. Inset: corresponding CT images. (f) Relative MR signal intensity of FGN-BBN at different concentrations. Inset: corresponding T1-weighted MR images.

Fig. 4

In vitro GRPR targeting specificity assays of FGN-BBN. (a). FCM analysis of cellular uptake of FGN and FGN-BBN in RM-1 and 3T3 cells at 4 h and 12 h. (b). Quantification analysis of FCM results for RM-1 cells. (c). Quantification analysis of FCM results for 3T3 cells. (d) CLSM visualization of FGN and FGN-BBN uptake in RM-1 cells at 12 h. Scale bar: 100 μm. (e) CLSM visualization of FGN and FGN-BBN uptake in 3T3 cells at 12 h. Scale bar: 100 μm. (f). Semi-quantitative analysis of CLSM results in RM-1 cells. (g). Semi-quantitative analysis of CLSM results in 3T3 cells. ∗∗∗p < 0.001.

Fig. 5

Multi-mode imaging (FL/CT/MR) of FGN-BBN in PCa bone metastasis. (a) In vivo FL images of different timepoints (0, 1, 2 and 3 h) following intravenous injection of FGN and FGN-BBN. (b) Quantitative analysis of FL intensity in tumor region. (c) CT images at different timepoints (0, 0.5, 1, 1.5, 2, 2.5, and 3 h) post-injection of FGN and FGN-BBN. (d) Quantitative analysis of CT intensity. (e) T1-weighted MR images at different timepoints (0, 1, 2 and 3 h) post-injection of FGN and FGN-BBN. (f) Quantitative analysis of MRI signal intensity in tumor site. ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 6

In vitro cytotoxicity and anti-tumor effect of FGN-BBN. (a) Viability of 3T3 fibroblast cells after 24 h co-incubation with GN, FGN, or FGN-BBN. (b) GN, FGN, and FGN-BBN induced cell death in PCa cells. (c) Synergistic cell-killing effect of FGN-BBN on RM-1 cells via PTT and ferroptosis under NIR irradiation. (d) Live/Dead cell staining of PCa cells following different treatments. Scale bar: 100 μm. (e) FCM analysis of apoptosis in RM-1 PCa cells treated with Ctrl, GN, FGN, FGN-BBN, FGN + NIR, and FGN-BBN + NIR. (f) Semi-quantitative analysis of live/dead cell staining. (g) Quantification of apoptotic cells from FCM results. ∗∗∗p < 0.001.

Fig. 7

Detection of ROS generation and LPO after different treatments. (a) FL imaging of intracellular ROS levels in various treatment groups using DCFH-DA staining. Scale bar: 200 μm. (b) FL imaging of lipid peroxidation using BODIPY C11 staining. Scale bar: 20 μm. (c) Semi-quantitative analysis of ROS FL intensity. (d) Semi-quantitative analysis of LPO levels based on BODIPY FL. ∗∗∗p < 0.001.

Fig. 8

Assessment of ferroptosis-associated biomarkers. (a) Quantitative analysis of intracellular GSH levels following different treatments. (b) Quantitative analysis of LPO levels using MDA assay after different treatments. (c) Western blot analysis for GPX4 protein expression in various treatments group. (d) Cell viability assay with Fer-1 rescue experiment. ∗p < 0.05, ∗∗p < 0.01, ∗p < 0.001.

Fig. 9

GRPR targeted photothermal with ferroptosis therapy effect and security evaluation of FGN-BBN in vivo. (a) Photothermal images of mice after intravenous injection of PBS or FGN-BBN under 808 nm laser irradiation at different time points. (b) Schematic diagram showing the establishment of PCa tibial metastasis model and nano-system treatment. (c) Tumor images for different groups. (d) Relative tumor volume curve. (e) Tumor weight after the anti-tumor experiment. (f) Body weight curve. (g) H&E-stained tumor tissue and immunohistology stained (Ki67, Caspase-3, and TUNEL). Scale bar: 100 μm (h) Semi-quantities analysis of apoptosis area, Ki67 positive percentage, Caspase-3 positive percentage and TUNEL positive percentage. Data are presented as mean ± SD (n = 5), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.