T1/T2 Proportional Magnetic Resonance Nanoprobe Monitoring Tumor Autophagy during Chemotherapy

Abstract

Autophagy leads to cellular tolerance of the therapeutic pressure of chemotherapeutic drugs, resulting in treatment resistance. Therefore, the effective monitoring of the autophagy status of tumors in vivo and the regulating of the autophagy level are crucial for improving the efficacy of chemotherapy. In this work, we grafted nitroxide radicals onto the surface of iron oxide nanoparticles (Fe₃O₄ NPs) using dendrimer polymers, yielding Fe₃O₄-NO· NPs that are responsive to reactive oxygen species (ROS) and possess enhanced T1 and T2 signal capabilities in a magnetic resonance imaging (MRI) measurement. The ROS in tumor cells generated by autophagy quenches the nitroxide radicals, thereby weakening the T1 signal. In contrast, Fe₃O₄ NPs are unaffected by intracellular ROS, leading to a stable T2 signal. By comparing the intensity ratio of T1 to T2 in Fe₃O₄-NO· NPs, we can evaluate the in vivo autophagy status within tumors in real time. It also revealed that Fe₃O₄-NO· NPs loaded with doxorubicin (Dox) and combining the autophagy inhibitor exhibited high antitumor activity in cells and tumor-bearing mice. This system, which combines real-time monitoring of tumor cell autophagy with the delivery of chemotherapeutic drugs, provides an innovative and effective strategy for tumor treatment with potential clinical application prospects.

Keywords: autophagy; magnetic resonance imaging; nanoparticles; nitroxide radicals; reactive oxygen species.

Scheme 1

Schematic illustration of the synthesis of Fe₃O₄-NO·NPs (A) and monitoring the in vivo autophagy in tumor tissue (B).

Figure 1

TEM images of Fe₃O₄ NPs (A), Fe₃O₄-NH₂ NPs (B) and Fe₃O₄-NO· NPs (C). The size distributions of Fe₃O₄ NPs (D), Fe₃O₄-NH₂ NPs (E) and Fe₃O₄-NO· NPs (F) determined by DLS. All results were mean values of three measurements.

Figure 2

IR spectra of the Fe₃O₄-G1, Fe₃O₄-G2, Fe₃O₄-G3 NPs (A); PAMAM obtained after the hydrochloric acid dissolution of Fe₃O₄-NH₂ NPs (B) and Fe₃O₄-NH₂, Fe₃O₄-NO· NPs (C). XRD patterns of Fe₃O₄-NH₂ and Fe₃O₄-NO· NPs (D).

Figure 3

(A) The magnetic hysteresis loops of Fe₃O₄-NH₂ and Fe₃O₄-NO· NPs. (B) UV–vis absorption spectra of Fe₃O₄-NH₂ and Fe₃O₄-NO· NPs. (C) TG curves of Fe₃O₄-NH₂ and Fe₃O₄-NO· NPs. (D) EPR spectra of NO· and Fe₃O₄-NO· NPs. (E) In vitro viability of L02 cells incubated with Fe₃O₄-NH₂ and Fe₃O₄-NO· NPs. (F) In vitro viability of 4T1 cells incubated with Fe₃O₄-NH₂ and Fe₃O₄-NO· NPs.

Figure 4

(A) Images of ad-mCherry-GFP-LC3-II-transfected 4T1 cells upon the induction of autophagy with EBSS for different time intervals. (B) CLSM images of ROS in 4T1 cells upon the induction of autophagy with EBSS for different time intervals. (C) CLSM images of ROS in 4T1 cells cultured with (+NAC) or without (−NAC) NAC under normal or autophagy conditions, respectively. Green: ROS indicator H₂DCFDA in (B,C).

Figure 5

(A) T1 and T2 weighted mapping of Fe₃O₄-NO· NPs before and after H₂O₂ oxidation. (B) T1 and T2 weighted mapping of 4T1 cells co-cultured with Fe₃O₄-NO· NPs in the presence (+NAC) or absence (−NAC) of NAC under normal or autophagy conditions. T1 and T2 weighted MR images of mice bearing 4T1 tumors (circle) at 1 h (C) and 24 h (D) post-administration of Fe₃O₄-NO· NPs.

Figure 6

(A) Release of Dox from Fe₃O₄-NO·-Dox at different pH. In vitro viability of 4T1 cells 24 h post-incubation with 3-MA (B), Rapamycin (C) and Dox (D). In vitro viability of 4T1 cells 24 h post-incubation with Fe₃O₄-NO·-Dox plus 3-MA (E). In vitro viability of 4T1 cells 24 h post- incubation with Fe₃O₄-NO·-Dox plus Rapamycin (Rapa) (F).

Figure 7

(A) T1 and T2 weighted MR images of 4T1 cells at 2 h post co-culture with Rapamycin or 3-MA. (B) Fluorescence images of 4T1 cells 2 h post the coculture with PBS, Rapamycin or 3-MA.

Figure 8

(A) Body weight and (B) tumor volume profiles of 4T1 tumor-bearing mice receiving different treatments. (C) MRI images of tumor tissue 48 h post the injection of Fe₃O₄-NO·-Dox NPs + 3-MA. (D) Photos of isolated tumors of 4T1 tumor-bearing mice after 15 days of different treatments. (E) Images of tumor tissue sections stained with H&E in different groups 15 days post-treatment. (F) Images of main organs (heart, liver, spleen, lung, kidney) sections stained with H&E in different groups 30 days post-treatment.