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Probing tumor spheroid hypoxia using red/near-infrared ratiometric O2-sensitive nanoparticles

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Probing tumor spheroid hypoxia using red/near-infrared ratiometric O2-sensitive nanoparticles Angela C. Debruyne1, Irina A. Okkelman1, Sergey M. Borisov2, Ruslan I. Dmitriev1 1Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medical and Health Sciences, Ghent University, Ghent 9000, Belgium 2Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria The role of the tumor microenvironment is paramount in supporting cell survival, promoting local invasion and metastatic dissemination. While the limited nutrient availability and hypoxia are known to be important contributors of the ‘niche’ environment and leading to metabolic cell heterogeneity, their quantitative non-destructive analysis is rather difficult. For instance, analysis of real-time oxygenation in such 3D cell cultures as spheroids and organoids, is rarely performed. To address this, we produced cell-penetrating O2-sensitive nanoparticle probes, which can enable measurements of spheroid oxygenation and consumption rate on a conventional fluorescence microscope. We used near-infrared O2-sensitive (PtTPTBPF) metalloporphyrin and red aza-BODIPY reference dyes to increase the light penetration depth and used well-known biocompatible polymer nanoparticles (Eudragit RL100 and PMMA-AA) to achieve efficient cell staining. The resulting probe, termed ‘MMIR’ (multi-modal infrared) enables measuring oxygenation gradients via fluorescence emission ratiometric measurements or the phosphorescence lifetime imaging microscopy (PLIM). To test the brightness, photostability, phototoxicity, and reproducibility of MMIR, we evaluated different reference:sensing dye (1:1, 1:0,5 and 0,5:0,5) ratio and the presence of nanoparticle charge groups. We examined multiple cell lines including HCT116, PANC-1, dental pulp stem cells, HUVEC, MCF-7, and SKOV-3. Preliminary results showed cell line-dependent staining and lower aggregation in case of negatively charged probe. No significant toxicity was found and after optimizations of the staining protocol, nanoparticle probes were used for staining 3D spheroid cultures and subsequent analysis of O2 gradients. Surprisingly, the human colon cancer HCT116 spheroids displayed an “inverted” oxygenation gradient, with elevated oxygenation at the spheroid core, compared to the cells located at the periphery. This can be explained by the presence of necrotic core and the large size of spheroids (~700 µm), which cannot consume O2. In addition, using the MMIR probe we studied how the growth medium composition can influence the oxygenation gradients. Collectively, presented ratiometric O2-sensing nanoparticle probes provide means for multi-parameter quantitative oxygenation measurements and characterization of 3D cultures. Funding: supported by the Special Research Fund (BOF) grant of Ghent University (BOF/STA/202009/003).

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MLA
Debruyne, Angela, et al. “Probing Tumor Spheroid Hypoxia Using Red/near-Infrared Ratiometric O2-Sensitive Nanoparticles.” MOLECULAR BIOLOGY OF THE CELL, vol. 34, no. 2, 2023, pp. 140–140.
APA
Debruyne, A., Okkelman, I., Borisov, S., & Dmitriev, R. (2023). Probing tumor spheroid hypoxia using red/near-infrared ratiometric O2-sensitive nanoparticles. MOLECULAR BIOLOGY OF THE CELL, 34(2), 140–140.
Chicago author-date
Debruyne, Angela, Irina Okkelman, Sergey Borisov, and Ruslan Dmitriev. 2023. “Probing Tumor Spheroid Hypoxia Using Red/near-Infrared Ratiometric O2-Sensitive Nanoparticles.” In MOLECULAR BIOLOGY OF THE CELL, 34:140–140.
Chicago author-date (all authors)
Debruyne, Angela, Irina Okkelman, Sergey Borisov, and Ruslan Dmitriev. 2023. “Probing Tumor Spheroid Hypoxia Using Red/near-Infrared Ratiometric O2-Sensitive Nanoparticles.” In MOLECULAR BIOLOGY OF THE CELL, 34:140–140.
Vancouver
1.
Debruyne A, Okkelman I, Borisov S, Dmitriev R. Probing tumor spheroid hypoxia using red/near-infrared ratiometric O2-sensitive nanoparticles. In: MOLECULAR BIOLOGY OF THE CELL. 2023. p. 140–140.
IEEE
[1]
A. Debruyne, I. Okkelman, S. Borisov, and R. Dmitriev, “Probing tumor spheroid hypoxia using red/near-infrared ratiometric O2-sensitive nanoparticles,” in MOLECULAR BIOLOGY OF THE CELL, Washington, DC, USA, 2023, vol. 34, no. 2, pp. 140–140.
@inproceedings{01GSA6H0EZMBJGYPYQ8ZTC8Z3P,
  abstract     = {{Probing tumor spheroid hypoxia using red/near-infrared ratiometric O2-sensitive nanoparticles 
Angela C. Debruyne1, Irina A. Okkelman1, Sergey M. Borisov2, Ruslan I. Dmitriev1
1Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medical and Health Sciences, Ghent University, Ghent 9000, Belgium
2Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria

The role of the tumor microenvironment is paramount in supporting cell survival, promoting local invasion and metastatic dissemination. While the limited nutrient availability and hypoxia are known to be important contributors of the ‘niche’ environment and leading to metabolic cell heterogeneity, their quantitative non-destructive analysis is rather difficult. For instance, analysis of real-time oxygenation in such 3D cell cultures as spheroids and organoids, is rarely performed. 
To address this, we produced cell-penetrating O2-sensitive nanoparticle probes, which can enable measurements of spheroid oxygenation and consumption rate on a conventional fluorescence microscope. We used near-infrared O2-sensitive (PtTPTBPF) metalloporphyrin and red aza-BODIPY reference dyes to increase the light penetration depth and used well-known biocompatible polymer nanoparticles (Eudragit RL100 and PMMA-AA) to achieve efficient cell staining. The resulting probe, termed ‘MMIR’ (multi-modal infrared) enables measuring oxygenation gradients via fluorescence emission ratiometric measurements or the phosphorescence lifetime imaging microscopy (PLIM).
To test the brightness, photostability, phototoxicity, and reproducibility of MMIR, we evaluated different reference:sensing dye (1:1, 1:0,5 and 0,5:0,5) ratio and the presence of nanoparticle charge groups. We examined multiple cell lines including HCT116, PANC-1, dental pulp stem cells, HUVEC, MCF-7, and SKOV-3. Preliminary results showed cell line-dependent staining and lower aggregation in case of negatively charged probe. No significant toxicity was found and after optimizations of the staining protocol, nanoparticle probes were used for staining 3D spheroid cultures and subsequent analysis of O2 gradients. Surprisingly, the human colon cancer HCT116 spheroids displayed an “inverted” oxygenation gradient, with elevated oxygenation at the spheroid core, compared to the cells located at the periphery. This can be explained by the presence of necrotic core and the large size of spheroids (~700 µm), which cannot consume O2. In addition, using the MMIR probe we studied how the growth medium composition can influence the oxygenation gradients. Collectively, presented ratiometric O2-sensing nanoparticle probes provide means for multi-parameter quantitative oxygenation measurements and characterization of 3D cultures.
Funding: supported by the Special Research Fund (BOF) grant of Ghent University (BOF/STA/202009/003).}},
  articleno    = {{B237/P1230}},
  author       = {{Debruyne, Angela and Okkelman, Irina and Borisov, Sergey and Dmitriev, Ruslan}},
  booktitle    = {{MOLECULAR BIOLOGY OF THE CELL}},
  issn         = {{1059-1524}},
  language     = {{eng}},
  location     = {{Washington, DC, USA}},
  number       = {{2}},
  pages        = {{B237/P1230:140--B237/P1230:140}},
  title        = {{Probing tumor spheroid hypoxia using red/near-infrared ratiometric O2-sensitive nanoparticles}},
  volume       = {{34}},
  year         = {{2023}},
}