Radionuclide imaging monitoring hypoxia tumor microenvironment improved homologous targeting probe for enhancing photodynamic therapy

Objectives: Photodynamic therapy (PDT) efficacy is often limited by the hypoxia of tumor microenvironment, while perfluorocarbon (PFTBA) has extremely high oxygen solubility and can deliver oxygen to the tumor site. In this study, we designed a novel biomimetic oxygen-delivery probe for homologous targeting and enhancing the efficacy of PDT by improving hypoxia at tumor site. Radionuclide hypoxia imaging was used to monitor the hypoxia changes.

Methods: PFTBA was uniformly mixed with HSA and ICG by ultrasound, cancer cell membranes (CCm) were obtained by differential centrifugation, and CCm-HSA-ICG-PFTBA were formed by physical extrusion. The characterization, stability properties, and protein expression of CCm-HSA-ICG-PFTBA were detected. With near-infrared (NIR) 808 nm laser irradiation, ¹O₂ and ROS were detected in vitro by indicators. In vitro quantitative PDT enhancement was evaluated. In vivo ICG fluorescence imaging was performed to show the time of CCm-HSA-ICG-PFTBA arrived at the tumor site. The tumor hypoxia was monitored by both in vivo ¹⁸F-FMISO PET imaging and ex vivo immunofluorescence staining. PDT was conducted with NIR at 24 h post-injection of CCm-HSA-ICG-PFTBA into the 4T1 tumor-bearing mice models. The state of the mice was observed every day, the body weight, the length and width of the tumor were measured. ¹⁸F-FDG PET imaging was used to monitor the tumor burden. On the 14^th day, the mice were euthanized and tumors were weighed, blood and major organs were taken for evaluating systematic toxicity.

Results: A core-shell-shell structure of CCm-HSA-ICG-PFTBA was shown, with 131 nm in hydrodynamic size. A similar protein profile was exhibited before and after membrane-coated. The stability in PBS was good for 5 days, and light stability was good till 60 h. As for in vitro ¹O₂ detection, the fluorescence intensity of the CCm-HSA-ICG-PFTBA group increased the fastest, significantly higher than that of the other groups (P<0.001). And in vitro ROS detection was the same, the CCm-HSA-ICG-PFTBA group showed the strongest green fluorescence, which means the highest ROS content. All groups without NIR exhibited negligible toxicity to 4T1 cells, while the toxicity induced by PDT in CCm-HSA-ICG-PFTBA group and HSA-ICG-PFTBA group were significantly higher than that in ICG-HSA group. In vivo ICG fluorescence imaging revealed that the best tumor signal was at 24 h post-injection. And both in vivo ¹⁸F-FMISO PET imaging and ex vivo immunofluorescence staining exhibited that tumor hypoxia was significantly improved at 24 h post-injection. As for in vivo PDT treatment, the tumor volume and weight of the CCm-HSA-ICG-PFTBA with NIR group were the smallest (P<0.01), and the tumor to muscle ratios in ¹⁸F-FDG PET imaging were significantly lower than that of other groups (P<0.05). Neither death nor significant loss in body weight among all groups was observed in 14 days. Blood chemistry tests and H&E stain suggested no obvious toxicity of CCm-HSA-ICG-PFTBA in vivo.

Conclusions: We successfully built the cancer cell membrane-coated oxygen-delivery probe CCm-HSA-ICG-PFTBA. All the in vitro and in vivo experiments confirmed the homologous targeting ability, as well as the probe could enhance the efficacy of PDT by improving the hypoxia of tumor microenvironment. The FDA-approved HSA, ICG, and PFTBA allow this probe high biocompatibility and a great chance for translation to humans. Acknowledgment: This study was supported by the National Natural Science Foundation of China (No. 81630049).

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