Marriage of black phosphorus and Cu2+ as effective photothermal agents for PET-guided combination cancer therapy

The use of photothermal agents (PTAs) in cancer photothermal therapy (PTT) has shown promising results in clinical studies. The rapid degradation of PTAs may address safety concerns but usually limits the photothermal stability required for efficacious treatment. Conversely, PTAs with high photothermal stability usually degrade slowly. The solutions that address the balance between the high photothermal stability and rapid degradation of PTAs are rare. Here, we report that the inherent Cu2+-capturing ability of black phosphorus (BP) can accelerate the degradation of BP, while also enhancing photothermal stability. The incorporation of Cu2+ into BP@Cu nanostructures further enables chemodynamic therapy (CDT)-enhanced PTT. Moreover, by employing 64Cu2+, positron emission tomography (PET) imaging can be achieved for in vivo real-time and quantitative tracking. Therefore, our study not only introduces an “ideal” PTA that bypasses the limitations of PTAs, but also provides the proof-of-concept application of BP-based materials in PET-guided, CDT-enhanced combination cancer therapy.

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Wei Tao, Ming-Rong Zhang, Lu Wang, Steven H. Liang Apr 21, 2020 BD FACSCalibur was used to collect and Flowjo (7.6) was used to analyze flow cytometry data. The radioactivity in cells and organs was measured by a1490 Wizard automatic gamma counter. TEM data was collected using HITACHI (HT7700). AFM data was collected using Bruker Dimension Ico (MDTC-EQ-M16-01). The hydrodynamic size was measured using a Malvern Zetasizer Nano-ZS (ZEN3600). Raman data were collected using Horiba Jobin -Yvon Lab Ram HR VIS HR-Raman microscope. XPS data was recorded on ESCALAB 250 (Thermo Fihser). UV-Vis-NIR data were measured using the Agilent Cary 8454 UV-Visible spectrophotometer. EPR data was acquired in a Bruker EMXPlus-10/12 system. The infrared thermographic and temperatures of tumor/cell were recorded by a FLIR E6 infrared camera (Arlington, VA). PET images were collected using Inveon PET scanner (Siemens Medical Solutions). Fluorescent cell imaging was performed on KEYENCE BA-X700 (all-in-one Fluorescence Microscope). MTT data was collected using Bio-Rad Mode 680 microplate reader.
Analysis of flow cytometry data was performed with Flowjo v7.6 software. Graph Pad Prism 8.0 and Origin 9.0 were used for data statistics and statistical significance calculation. Microsoft Excel 2016 was used for biodistribution and tumor size analysis. PET images were analyzed using ASIPro VM version 6.3.3.0 software (Siemens Medical Solutions). Zetasizer Nano software v3.30 for analyses of particle size. TEM data was analyzed using Gatan-DigitalMicrograph-3.9.

October 2018
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The authors declare that the data supporting the findings of this study are available with the papers and its Supplementary Information. Additional data and source files are available from the corresponding author upon reasonable request.
No statistical methods were used to predetermine the sample sizes. The number of animals in each group (n = 5-6) was determined according to previous studies cited in our manuscript. The size of each sample is in close agreement with those studies already published and with the need for statistical analysis to discuss the degree of differences and measure the variability of these in vivo data. In previous studies we determined this sample size is sufficient to ensure reproducibility. For all cell based assays, all experiments were performed for more than 3 biological independent samples in triplicate. For PET imaging study, at least three times independent experiments were performed in three biological independent mice each time. For blood biochemical, H&E staining, and hematology studies, three mice for each sample were used based on previous studies cited in our manuscript. This size is sufficient because no significant difference was observed between biological replicates.
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All the experimental findings were replicated with the number of replicates, animals and variation shown by n and SD. All experimental findings were replicated successfully using biological replicates on different days. For PET imaging study, all the control and experimental groups were repeated at least three times.
PET imaging study: animals were distributed randomly into different groups. Therapeutics study: animals were inoculated with tumor cell suspensions and after randomly distributed into the control and experimental groups. Each specific treatment was administrated to animals according to established schedules and regimens. For blood biochemical, H&E staining, and hematology studies, genetically identical animals were randomly allocated into different groups. Each specific treatment was administrated to animals according to established schedules and regimens. The blood samples and organs were collected from the corresponding animals without randomization process.
Investigators were blinded when grouping tumor bearing mice, measuring tumor size, performing biodistribution and PET imaging study. Investigators for TEM and AFM characterization are blinded to the samples. During the experiments designed to evaluate anti-tumor efficacy, animals were inoculated with tumor cells and randomly divided into control and treatment groups. The investigators were not blinded during these pre-clinical proof-of-concept studies based on combinatorial schemes. Specifically, the researcher who administrates the drugs into animals was blinded to the drugs and animals. The researcher in charge of IR laser irradiation is not blinded to the groups because of predesigned protocol should be applied to different groups. For blood biochemical, H&E staining, and hematology studies, the researchers were blinded to group allocation during data collection and analysis. For cell based FACS and fluorescence imaging, the researchers who analysis the samples are independent to who collected the samples. For other experiments, the investigators were blinded to group allocation during data collection and/or analysis.
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ATCC used morphology, karyotyping, and PCR based approaches to confirm the identity of human cell lines and to rule out both intra-and interspecies contamination. Also, the cell line were frequently checked by their morphological features.
All cells were negative for mycoplasma.
No commonly misidentified cell line were used.
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6 to 8-week-old male Balb/c nude mice, C3H/HeJ mice, and male C57BL/6J mice (purchased from Japan SLC, Shizuoka, Japan) were used in this study.
No wild animals were used in this study.
This study did not involve samples collected from the fields.
All animals received humane care, and the Animal Ethics Committee of the National Institute of Radiological Sciences approved all the animal experiments. All experiments were carried out according to the recommendations of the Committee for the Care and Use of Laboratory Animals, National Institute of Radiological Sciences.

nature research | reporting summary
October 2018 Note that full information on the approval of the study protocol must also be provided in the manuscript.

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