Oxygen tank for synergistic hypoxia relief to enhance mitochondria-targeted photodynamic therapy

Abstract

Background: Mitochondria play an essential role in cellular redox homeostasis maintenance and meanwhile serve as an important target for organelle targeted therapy. Photodynamic therapy (PDT) is a promising strategy for organelle targeted therapy with noninvasive nature and highly spatiotemporal selectivity. However, the efficacy of PDT is not fully achieved due to tumor hypoxia. Moreover, aerobic respiration constantly consumes oxygen and leads to a lower oxygen concentration in mitochondria, which continuously limited the therapeutic effects of PDT. The lack of organelle specific oxygen delivery method remains a main challenge.

Methods: Herein, an Oxygen Tank is developed to achieve the organelle targeted synergistic hypoxia reversal strategy, which not only act as an oxygen storage tank to open sources and reduce expenditure, but also coated with red blood cell membrane like the tank with stealth coating. Within the oxygen tank, a mitochondrion targeted photosensitizer (IR780) and a mitochondria respiration inhibitor (atovaquone, ATO) are co-loaded in the RBC membrane (RBCm) coated perfluorocarbon (PFC) liposome core.

Results: Inside these bio-mimic nanoparticles, ATO effectively inhibits mitochondrial respiration and economized endogenous oxygen consumption, while PFC supplied high-capacity exogenous oxygen. These Oxygen modulators reverse the hypoxia status in vitro and in vivo, and exhibited a superior anti-tumor activity by mitochondria targeted PDT via IR780. Ultimately, the anti-tumor effects towards gastric cancer and colon cancer are elicited in vivo.

Conclusions: This oxygen tank both increases exogeneous oxygen supply and decreases endogenous oxygen consumption, may offer a novel solution for organelle targeted therapies.

Keywords: Artificial red blood cells; Mitochondrial respiratory inhibition; Organelle targeted therapy; PDT; Synergistic oxygen modulation; Tumor hypoxia.

Scheme 1

Schematic illustration of the design, synergistic hypoxia reversal function, and therapeutic functions of the Oxygen Tank. a) Design illustration of the Oxygen Tank. On the one hand, the biomimetic coating of the Oxygen Tank is similar to the stealth coating of a battle tank; On the other hand, the Oxygen Tank opened source and reduced the expenditure of oxygen as a gas tank. b) Oxygen Tank reduced oxygen consumption by mitochondria respiration inhibition and increased oxygen supply by PFC to achieve synergistic hypoxia regulation. c) Such synergistic hypoxia reversal and Mt-PDT strategy simultaneously supplied exogenous oxygen and inhibited endogenous oxygen consumption to manipulate the tumor hypoxia microenvironment, and ultimately attack the mitochondria of tumor cells

Fig. 1

Characterization of the Oxygen Tank. a Schematic illustration of the design and synthesis of the Oxygen Tank. b Hydrodynamic diameters of AIP. Inset shows photographs and TEM images of these NPs. The scale bars are 100 nm. c Zeta potential of AIP NPs and Oxygen Tank. d Result of SDS-PAGE analysis to test whether similar protein brands exhibited in Oxygen Tank compared to RBCm. e UV-vis spectra of the free liposome, PFC, ATO, and IR780. f UV vis spectra of the Oxygen Tank and AIP NPs in PBS. g UV-vis spectra of Oxygen Tank in different concentrations (0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, and 6 μg/ml)

Fig. 2

Synergistic hypoxia reversal by oxygen supplied and consumption reduced. a The uptake behavior of the Oxygen Tank by AGS cells in different time points (1 h, 2 h, 4 h, and 8 h) is determined by flow cytometry. b Quantification result of the flow cytometry evaluation. c Schematic illustration of instrument for dissolved oxygen measurement. d Oxygen release curves of different NPs (PBS, RBCm@AIP NPs, and Oxygen Tank). e The dissolved oxygen curves of the culture medium within AGS cells after different treatments (PBS, IP NPs, AIP NPs, and Oxygen Tank). f Confocal fluorescence images of Hif-1α staining of AGS cells after different treatments (PBS in hypoxia condition, IP NPs in hypoxia condition, AIP NPs in hypoxia condition, Oxygen Tank in hypoxia condition, and PBS in normoxia condition). The scale bar is 20 μm

Fig. 3

Prolonged circulation and mitochondria-targeted ability of Oxygen Tank. a Schematic illustration of the prolonged circulation by RBCm-coating NPs. b The clearance rate of the Oxygen Tank is determined by flow cytometry. RAW264.7 cells were treated with the Oxygen Tank or AIP for different lengths of time (0 h, 1 h, 2 h, and 4 h). c Subcellular localization of Oxygen Tank compared to lysosome and mitochondria trackers determined by CLSM. The scale bar is 10 μm. PC means the Pearson Correlation coefficient. d. Colocalization analysis of Oxygen Tank in AGS cells with lysosome tracker. e Colocalization analysis of Oxygen Tank in AGS cells with mitochondria tracker. f Mitochondria potential evaluation by JC-1 after AGS cells treated with PBS, ATO, IP NPs, and Oxygen Tank NPs. Red signals (JC-1 aggregates) suggested a normal polarized mitochondrial membrane. Green signals (JC-1 monomers) suggested an abnormal depolarized mitochondrial membrane. The scale bar is 50 μm

Fig. 4

Amplified PDT by synergistic hypoxia reversal strategy. a Schematic illustration of the synergistic hypoxia reversal strategy, which both increased oxygen supply and decreased oxygen consumption and ultimately enhanced PDT efficacy. b and c CLSM images and fluorescence intensity quantification of ROS generation in AGS cells after different treatments (PBS, PBS with laser, Oxygen Tank, IP NPs with laser, AIP NPs with laser, and Oxygen Tank with laser). Green fluorescence stained by H2DCFHDA depicted ROS. The scale bar is 100 μm. d CLSM images of AGS cells after different treatments (PBS, PBS with laser, Oxygen Tank, IP NPs with laser, AIP NPs with laser, and Oxygen Tank with laser) determined by Calcein-AM/Propidium iodide double stain kit. Viable cells were stained green with Calcein-AM, and dead cells were stained red with PI. The scale bar is 100 μm. e Relative cell viability determined by CCK-8 kit (n = 4). Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

In vivo anti-tumor effect of the Oxygen Tank. a Schematic illustration of the PDT treatment (1 W cm − 2, 30s) after tail vein injection (200 uL, 100 μg/mL IR780, n = 6). b and c Body weight and tumor volume curves. d Weight of tumors. e Hif-1α staining tumor sections. The scale bar is 50 μm. f Photographs of the H&E and TUNEL staining of the AGS-bearing mice in different treatments (PBS, PBS with laser, Oxygen Tank, IP NPs with laser, AIP NPs with laser, and Oxygen Tank with laser). The scale bars are 100 μm. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, while N.S. means Not Significant

Fig. 6

Potential long-term in vivo biosafety analysis of Oxygen Tank (200uL, 100μg/mL IR780, tail vein injection). a H&E staining images of major organs of the AGS-bearing mice in different treatments (PBS, PBS with laser, Oxygen Tank, IP NPs with laser, AIP NPs with laser, and Oxygen Tank with laser). The scale bar is 200 μm. b-e Hematology assay of the AGS-bearing mice in different treatments (PBS, PBS with laser, Oxygen Tank, IP NPs with laser, AIP NPs with laser, and Oxygen Tank with laser). b. WBC: white blood cell; c. LYM: lymphocytes; d. RBC: red blood cell; PLT: platelets. f-g Serum biochemical assay of the AGS-bearing mice in different treatments (PBS, PBS with laser, Oxygen Tank, IP NPs with laser, AIP NPs with laser, and Oxygen Tank with laser). f BUN: blood urea nitrogen; g Scr: serum creatinine. Data are demonstrated as mean ± SD and analyzed by the one-way ANOVA method (n = 3). N.S. means Not Significant