Intracellular marriage of bicarbonate and Mn ions as "immune ion reactors" to regulate redox homeostasis and enhanced antitumor immune responses

Abstract

Background: Different from Fe ions in Fenton reaction, Mn ions can function both as catalyst for chemodynamic therapy and immune adjuvant for antitumor immune responses. In Mn-mediated Fenton-like reaction, bicarbonate ([Formula: see text]), as the most important component to amplify therapeutic effects, must be present, however, intracellular [Formula: see text] is strictly limited because of the tight control by live cells.

Results: Herein, Stimuli-responsive manganese carbonate-indocyanine green complexes (MnCO₃-ICG) were designed for intracellular marriage of bicarbonate and Mn ions as "immune ion reactors" to regulate intracellular redox homeostasis and antitumor immune responses. Under the tumor acidic environment, the biodegradable complex can release "ion reactors" of Mn²⁺ and [Formula: see text], and ICG in the cytoplasm. The suddenly increased [Formula: see text] in situ inside the cells regulate intracellular pH, and accelerate the generation of hydroxyl radicals for the oxidative stress damage of tumors cells because [Formula: see text] play a critical role to catalyze Mn-mediated Fenton-like reaction. Investigations in vitro and in vivo prove that the both CDT and phototherapy combined with Mn²⁺-enhanced immunotherapy effectively suppress tumor growth and realize complete tumor elimination.

Conclusions: The combination therapy strategy with the help of novel immune adjuvants would produce an enhanced immune response, and be used for the treatment of deep tumors in situ.

Keywords: Immune activator; Manganese immunotherapy; Orthotopic liver cancer; Redox homeostasis; Self-supplying intracellular ions.

Scheme 1

Schematic representation shows that intracellular marriage of bicarbonate and Mn ions as “immune ion reactors” to regulate redox homeostasis and antitumor immune responses for synchronous enduring orthotopic cancer treatment. In the acidic environment, MnCO₃-ICG are decomposed to release ions (Mn²⁺, ${\text{HCO}}_3^{-}$) in situ that regulates intracellular redox homeostasis and antitumor immune responses. The self-supplying ${\text{HCO}}_3^{-}$ escalates tumor oxidative stresses by catalyzing Mn-based Fenton-like reaction to efficiently generate hydroxyl radicals (·OH); the released ICG amplifies tumor oxidative stress by photothermal process; and Mn²⁺ ions as one of adjuvants enhance the immune response

Fig. 1

Preparation and characterization of the MnCO₃-ICG Complexes. A TEM imaging of MnCO₃-ICG complexes. B STEM mapping analysis of MnCO₃-ICG. C TEM images of MnCO₃-ICG after biodegradation in neutral (pH 7.4) and acidic (pH 5.8) PBS for different durations: 0.5, 1, 2, 4 and 8 h. D T₁-weighted images of MnCO₃-ICG complexes after incubation within different buffer solutions. E Time-dependent T₁ intensity change of MnCO₃-ICG complexes based on region of interest (ROI) analysis on images from panel D. F Ultrasound images of the generation of CO₂ from MnCO₃-ICG complexes in acidic buffer solution (pH 5.8 + H₂O₂) for different time. G Ultrasound signal intensity of the generation of CO₂ based on based on ROI analysis on images from panel F. H The decline of absorption peak of MB after MnCO₃-ICG incubated at different buffer solutions. I The ESR spectra to detect ·OH produced by MnCO₃-ICG, using DMPO as the spin trapper. J Temperature elevation of H₂O and different concentrations of MnCO₃-ICG ([ICG]: 18, 36 and 72 μg/mL) suspensions under continuous irradiation (808 nm, 0.5 W/cm², 5 min). K Photothermal heating and natural cooling cycles of ICG, Mn-ICG and MnCO₃-ICG ([ICG]: 72 μg/mL, 808 nm, 0.5 W/cm²)

Fig. 2

The Efficiently Induce Tumor Cell Death by MnCO₃-ICG. A Cell uptake of 4T1 cells incubated with MnCO₃-ICG at different time (scale bar, 50 μm). B Flow cytometric and quantitative analyses of internalization of MnCO₃-ICG at different time. C CLSM evaluation on the lysosomal escape of MnCO₃-ICG. The blue, green, and red colors indicate cell nucleus, ICG, and lysosome, respectively (scale bar, 50 μm). D CLSM imaging of the MnCO₃-ICG penetration after different treatment (pH 6.5 with laser or pH 7.4 without laser) in MCSs (scale bar, 200 μm). E Corresponding fluorescence profiles in D. F Calcein-AM and PI co-stained 4T1 cells with different concentration of MnCO₃-ICG with or without 808 nm laser (0.5 W/cm², 5 min) irradiation, the green and red fluorescence indicate live cells and dead cells. G Cell viabilities of 4T1 cells measured by MTT assays, after incubating with different concentration of MnCO₃-ICG with or without 808 nm laser (0.5 W/cm², 5 min) irradiation. H Calcein-AM and PI co-stained 4T1 MCSs after different treatment for three days (PBS, MnCO₃-ICG with [Mn]: 20 μg/mL, 50 μg/mL without and with laser irradiation (808 nm, 0.5 W/cm², 5 min)). I Flow cytogram representing apoptosis assay based on Annexin V-FITC and propidium iodide staining of 4T1 cells after treatment with different therapeutic groups. J Early apoptosis (V + /PI −) and (K) late apoptosis (V + /PI +) of the 4T1 cells after treatment with different therapeutic groups

Fig. 3

In vitro Oxidative Stress Generation of MnCO₃-ICG. A Intracellular ·OH generation detected by DCFH-DA probe (scale bar, 30 μm). B Flow cytometry data for DCFH-DA probe treated with Group 1: PBS, Group 2: MnCO₃-ICG ([Mn]: 5 μg/mL), Group 3: MnCO₃-ICG ([Mn]: 10 μg/mL), Group 4: MnCO₃-ICG ([Mn]: 20 μg/mL). C CLSM observation on the intracellular distribution of lipoperoxides in 4T1 cells after incubation with PBS and MnCO₃-ICG for 24 h. The red fluorescence is the lipid ROS in cells and membranes after the staining with BODIPY-C11 (scale bar, 50 μm). D Flow cytometric and quantitative analyses of lipid peroxidation. E CLSM observation on the changes in the mitochondrial membrane potential of 4T1 cells after incubation with different concentration of MnCO₃-ICG. The blue, red, and green colors indicate cell nucleus, and JC-1J-aggregates and monomer, respectively (scale bar, 50 μm). F ΔΨ_m was assessed by detecting the red fluorescence and green fluorescence via flow cytometer. G LDH release assay after incubation with different concentration of MnCO₃-ICG (***p < 0.001)

Fig. 4

The Distribution of the MnCO₃-ICG Complexes on 4T1 Tumor-bearing Mice. A Time course of blood levels of MnCO₃-ICG levels following intravenous injection. B In vivo MRI images and (D) fluorescence images of BALB/C tumor-bearing mice taken at different time points after injection of MnCO₃-ICG. C Quantification analysis of MRI signal change in tumor/muscle based on region of interest (ROI) analysis on images from panel B. E Quantification analysis of the tumor ratio of fluorescence signal change in tumors based on ROI from panel D. F Biodistribution of Mn (% injected dose (ID) Mn per gram tissue) in main tissues and tumors after intravenous administration of MnCO₃-ICG. G Thermal images and (H) real-time temperature curve of BALB/C tumor-bearing mice treated with MnCO₃-ICG and 808 nm laser (0.5 W/cm²) irradiation

Fig. 5

In vivo therapeutic efficacy and immunity response of the MnCO₃-ICG complexes on 4T1 Tumor-bearing Mice. A Therapy approach for tumor-bearing mice. B Tumor growth curves of BALB/C tumor-bearing mice after various treatments (n = 5) (**p < 0.01; ***p < 0.001). C Final tumor weights of BALB/C tumor-bearing mice exposed to different formulations after the different treatment (*p < 0.05; **p < 0.01; ***p < 0.001). D Hematoxylin & eosin (H&E)-stained tumor sections from BALB/C tumor-bearing mice after various treatments (scale bar, 100 μm). E The T₂-MR imaging and digital photos of mice through the treatment period (group 5 and group 6). F IL-6, IL-1α and TNF-α in serum obtained from immunized mice. Flow cytometric analyses of the populations of (G) matured DC cells and (H) CD8 + T cells and CD3 + T cells in splenocytes of mice immunized after the different treatment

Fig. 6

The Antitumor Efficiency for Hepatocellular Carcinoma (Hep 1–6) Cells and Orthotopic Hepatic Tumors. A Flow cytogram representing apoptosis assay based on Annexin V-FITC and propidium iodide staining of 4T1 cells after treatment with different therapeutic groups. B In vivo T₁/T₂ MRI images for orthotopic hepatic tumor-bearing mice taken at different time points after injection of MnCO₃-ICG. Quantification analysis of MRI signal change in C tumor/noise and (D) tumor/liver based on region of interest (ROI) analysis on images from panel B. E Relative luminescence intensity changes based on the bioluminescence images (BLI). F BLI changes after different treatments duration of therapy. G Photographs of representative tumors and (H) H&E-stained tumor sections taken from tumor-bearing mice after various treatments (scale bar: 200 μm)