Programmed Catalytic Therapy-Mediated ROS Generation and T-Cell Infiltration in Lung Metastasis by a Dual Metal-Organic Framework (MOF) Nanoagent

Abstract

Nano-catalytic agents actuating Fenton-like reaction in cancer cells cause intratumoral generation of reactive oxygen species (ROS), allowing the potential for immune therapy of tumor metastasis via the recognition of tumor-associated antigens. However, the self-defense mechanism of cancer cells, known as autophagy, and unsustained ROS generation often restricts efficiency, lowering the immune attack, especially in invading metastatic clusters. Here, a functional core-shell metal-organic framework nanocube (dual MOF) doubling as a catalytic agent and T cell infiltration inducer that programs ROS and inhibits autophagy is reported. The dual MOF integrated a Prussian blue (PB)-coated iron (Fe²⁺)-containing metal-organic framework (MOF, MIL88) as a programmed peroxide mimic in the cancer cells, facilitating the sustained ROS generation. With the assistance of Chloroquine (CQ), the inhibition of autophagy through lysosomal deacidification breaks off the self-defense mechanism and further improves the cytotoxicity. The purpose of this material design was to inhibit autophagy and ROS efficacy of the tumor, and eventually improve T cell recruitment for immune therapy of lung metastasis. The margination and internalization-mediated cancer cell uptake improve the accumulation of dual MOF of metastatic tumors in vivo. The effective catalytic dual MOF integrated dysfunctional autophagy at the metastasis elicits the ~3-fold recruitment of T lymphocytes. Such synergy of T cell recruitment and ROS generation transported by dual MOF during the metastases successfully suppresses more than 90% of tumor foci in the lung.

Keywords: MOF; autophagy; drug delivery; immune response; lung metastasis; nano-catalytic medicine.

Scheme 1

Schematic illustration of dual MOF catalytic activity and immune response. The dual MOF programs peroxide mimic in the cancer cells and sustain to generate ROS via the core-shell characteristics (step 1). With a high cellular uptake, chloroquine (CQ) served as an inhibitor of autophagy, and regulates the autophagy flux by de-acidifying the lysosomes, lowering the self-defense mechanism of cancer cells (step 2). The accumulation of dual MOF in lung metastasis promotes the accumulation of specific T-cell responses (CD4⁺ and CD8⁺).

Figure 1

Synthesis and characterizations of dual MOFs. (a) The schematic illustration of the synthesis process of CQ-loaded dual MOF (CQ-dual MOF). The dual MOF system was reached by coating the inner Prussian blue (PB) with an outer MIL88 as a metal source and NH₂BDC as an organic linker while. F127 pluronics and acetic acid were used for stability and tailoring the size of the MOF. (b–i) SEM and (j–m) TEM images of PB, MIL88, and dual MOFs. (n–p) Element mapping analysis of dual MOFs.

Figure 2

Characterizations of single and dual MOFs. (a) Size distribution and (b) zeta potential ofPB, MIL88, and dual MOFs. (c) XPS spectrum of all the elements and (d) Fe2p spectrum of dual MOFs, demonstrating all the iron components in the trivalent state. (e) FTIR and (f) X-ray diffraction spectrum of PB(), MIL88(), and dual MOF. Slight disappearances reflections of 2-Theta and intensities in the dual MOF were due to the shielding effect of MIL88 on PB. (g,h) Catalytic performance of control, PB, MIL88, and dual MOF.

Figure 3

(a) CLSM images of cellular uptake of B16F10 incubated with PB, MIL88, and dual MOF labeled by QDs. Blue, red, and green fluorescence represent nucleus staining with DAPI, particle staining with QDs, and cytoskeleton staining with F-actin, respectively. (b) Flow cytometry analysis of PB, MIL88, and dual MOF after 24 h of incubation in B16F10 cells. (c) Cell viability of B16F10 cells in the presence of PB, MIL88, and dual MOF solution at various concentrations. (d) Cytotoxicity of B16F10 cells incubated with CQ-loaded dual MOF at various concentrations. Quantitative significant statistical data were calculated via Student’s t-test, ** p < 0.01.

Figure 4

Autophagy activation through LC3B protein expression. (a) Confocal fluorescence images of B16F10 cells incubated with PB, MIL88, and MIL88@PB (100 µg/mL) (blue, green, and red fluorescence represent nucleus, cytoskeleton, and NP staining with DAPI, F-actin, and QD, respectively; purple fluorescence represents LC3B expression, which shows the quantity of autophagosomes), the experiment was conducted three times on three independent 6-well plates. (b) Autophagosome quantification of PB, MIL88, and MIL88@PB by image J analysis software. (c) Schematic representation showing increased LC3B expression leads to increased autophagy expression. Quantitative significant statistical data were calculated via Student’s t-test, ** p < 0.01.

Figure 5

Autophagy regulation by CQ, CQ-MIL88, and CQ-dual MOF. (a) Confocal florescence images of B16F10 cells incubated with CQ, CQ-MIL88, and CQ-dual MOF (100 µg/mL). Purple florescence represents LC3B expression, which shows the quantity of autophagosomes. Blue, green, and red fluorescence represent nucleus, cytoskeleton, and NP staining with DAPI, F-actin, and QD, respectively. The experiment was conducted for three times on three independent 6-well plates. (b) The quantification of autophagosome of B16F10 cells incubated with CQ, CQ-MIL88, and CQ-dual MOF (100 µg/mL) by image J analysis software. Quantitative significant statistical data were calculated via Student’s t-test, * p < 0.05, ** p < 0.01.

Figure 6

In vivo study of mice bearing B16F10 lung metastases treated with PB, MIL88, and dual MOF. (a) CLSM images of lung expressing CD4⁺ and CD8⁺ after treated with PB, MIL88, and dual MOF. Purple and green florescence represents CD4⁺ and CD8⁺ expressed cells. (b) Quantification of CD8 and CD4 expression by image J software. (c) The enlarged CLSM image of lung treated by dual MOF.

Figure 7

In vivo study of mice bearing B16F10 lung metastases treated with CQ-MIL88 and CQ- dual MOF. (a) CLSM images of lung metastases treated by CQ-MIL88 and CQ-dual MOF. (b) Images of dissected lung metastases treated by PBS (control), CQ-MIL88, and CQ-dual MOF. (c) Patterns of flow cytometry showing the CD4⁺ and CD8⁺ expression of T cells in lungs after various treatments. Quantitative significant statistical data were calculated via Student’s t-test, ** p < 0.01.