Mitochondria-Targeted Artificial "Nano-RBCs" for Amplified Synergistic Cancer Phototherapy by a Single NIR Irradiation

Abstract

Phototherapy has emerged as a novel therapeutic modality for cancer treatment, but its low therapeutic efficacy severely hinders further extensive clinical translation and application. This study reports amplifying the phototherapeutic efficacy by constructing a near-infrared (NIR)-responsive multifunctional nanoplatform for synergistic cancer phototherapy by a single NIR irradiation, which can concurrently achieve mitochondria-targeting phototherapy, synergistic photothermal therapy (PTT)/photodynamic therapy (PDT), self-sufficient oxygen-augmented PDT, and multiple-imaging guidance/monitoring. Perfluorooctyl bromide based nanoliposomes are constructed for oxygen delivery into tumors, performing the functions of red blood cells (RBCs) for oxygen delivery ("Nano-RBC" nanosystem), which can alleviate the tumor hypoxia and enhance the PDT efficacy. The mitochondria-targeting performance for enhanced and synergistic PDT/PTT is demonstrated as assisted by nanoliposomes. In particular, these "Nano-RBCs" can also act as the contrast agents for concurrent computed tomography, photoacoustic, and fluorescence multiple imaging, providing the potential imaging capability for phototherapeutic guidance and monitoring. This provides a novel strategy to achieve high therapeutic efficacy of phototherapy by the rational design of multifunctional nanoplatforms with the unique performances of mitochondria targeting, synergistic PDT/PTT by a single NIR irradiation (808 nm), self-sufficient oxygen-augmented PDT, and multiple-imaging guidance/monitoring.

Keywords: nanoliposomes; nanomedicine; perfluorooctyl bromide; photodynamic therapy; photothermal therapy.

Scheme 1

Schematic illustration of the theranostic functions of as‐synthesized NIR‐responsive “Nano‐RBCs,” including targeting cancer cells and mitochondria via loaded IR780, self‐sufficient oxygen supply by PFOB core for enhanced PDT/PTT and the guidance/monitoring by multimodal imaging including CT, PA, and FL imaging.

Figure 1

a) Schematic illustration of synthetic procedure of PFOB@LIP‐IR780. b) TEM image of PFOB@LIP‐IR780 (scale bar: 2 µm). c) Size distribution of PFOB@LIP‐IR780 as measured by DLS. d) Absorbance spectra of IR780, PFOB@LIP, PFOB@LIP‐IR780 as recorded by UV–vis–NIR spectrophotometer. e) UV–vis–NIR absorbance spectra of IR780 at elevated concentrations. f) The relative absorbance intensity of IR780 in UV–vis–NIR spectrum at the wavelength of 780 nm. g) Oxygen‐concentration changes after addition of PFOB@LIP‐IR780 and LIP‐IR780 into deoxygenated water.

Figure 2

a) Temperature changes of pure water and PFOB@LIP‐IR780 aqueous suspension at different IR780 concentrations under photoirradiation (808 nm, 1.0 W cm⁻²). b) Temperature changes of PFOB@LIP‐IR780 aqueous suspension at different power densities of 808 nm laser with fixed IR780 concentration (100 µg mL⁻¹). c) Concentration‐dependent ¹O₂ generation of PFOB@LIP‐IR780 and LIP‐IR780 under normoxic and hypoxic conditions as irradiated by 808 nm (1.0 W cm⁻², 5 min). (Values are means ± s.d., n = 3, *P < 0.05.) d) Time‐dependent ¹O₂ generation of PFOB@LIP‐IR780 under normoxic condition as photoirradiated by 808 nm laser (1.0 W cm⁻²). The concentration of IR780 was 15 µg mL⁻¹.

Figure 3

a) In vitro CT contrast images and CT values of PFOB@LIP‐IR780 at different concentrations. b) In vivo CT images of tumors on 4T1‐tumor‐bearing mice after i.v. injection of PFOB@LIP‐IR780 as recorded at different time points. The top row shows black and white images, and bottom row represents the pseudo‐colored images. c) Changes of CT‐signal intensities within tumor regions at corresponding time points. (Values are means ± s.d., n = 3.)

Figure 4

a) In vitro PA contrast images and PA values of PFOB@LIP‐IR780 at different IR780 concentrations. b) In vivo PA images of tumors in 4T1 tumor‐bearing mice after i.v. injection of PFOB@LIP‐IR780 at different time points. c) Changes of PA‐signal intensities within tumor regions at corresponding time points. (Values are means ± s.d., n = 3.)

Figure 5

a) In vivo NIR fluorescence images of tumors in 4T1‐tumor‐bearing mice after i.v. injection of PFOB@LIP‐IR780 at different time points. b) Changes of fluorescence signal intensities within tumor regions at corresponding time points. c) Ex vivo fluorescence images of major organs and tumor dissected from mice 24 h postinjection of PFOB@LIP‐IR780. d) Quantitative biodistribution of PFOB@LIP‐IR780 in mice as determined by the average FL intensities of organs and tumors. (Values are means ± s.d., n = 3.)

Figure 6

a) Intracellular uptake of PFOB@LIP‐IR780 as observed by CLSM after various intervals of incubation. The scale bars are 50 µm. b) Flow‐cytometry analysis of intracellular uptake of PFOB@LIP‐IR780 and PFOB@LIP labeled with DiI. c) PFOB@LIP‐IR780 colocalized with mitochondrial and lysosome trackers as observed by CLSM. The scale bars are 20 µm. d) Enhanced ROS production by PDT@O₂ in 4T1 cancer cells. Confocal images (green fluorescence indicates positive staining for ROS stained with DCFH‐DA) and flow‐cytometry analysis of ROS generation by different treatments. The scale bars are 100 µm.

Figure 7

a) Relative cell viability of 4T1 cells after incubation with PFOB@LIP‐IR780 or LIP‐IR780 followed by various treatments. (Values are means ± s.d., n = 5, *P < 0.05.) b) Confocal images of CAM and PI costained 4T1 cells after coincubation with PFOB@LIP‐IR780 (C _IR780 = 4 µg mL⁻¹) for 4 h followed by various treatments. The scale bars are 50 µm.

Figure 8

a) Immunofluorescent images of tumor slices stained by the hypoxia probe. The scale bars are 50 µm. b) Quantitative analysis of tumor hypoxia areas. c) In vivo oxyhemoglobin saturation of tumor in 4T1‐tumor‐bearing mice after i.v. injection of PFOB@LIP‐IR780 at different time points. Tumor oxygenation was detected by PA imaging in oxyhemoglobin mode. d) Quantification of oxyhemoglobin saturation at tumor sites by measuring the ratios of oxygenated hemoglobin (λ = 850 nm) and deoxygenated hemoglobin (λ = 750 nm). (Values are means ± s.d., n = 3, *P < 0.05.)

Figure 9

a) Photographs of 4T1 tumor‐bearing mice of six groups taken during 18 d period after various treatments. b) Photographs of tumors dissected from mice of six groups after various treatments. c) Tumor growth curves of six groups after various treatments. d) Weight of tumors 18 d post various treatments. (Values are means ± s.d., n = 5, *P < 0.05.)

Figure 10

a) H&E staining, TUNEL staining, and immunochemical staining of PCNA on tumor sections from 4T1 tumor‐bearing mice after various treatments. b) H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of 4T1 tumor‐bearing mice after different treatments. The scale bars are 50 µm.