CC BY
27B-O-4
Time-resolved analysis of upconversion nanoparticles and photosensitizers fluorescence to determine the type of cell
metabolism
D.V. Pominova1'2, I.D. Romanishkin1, V.Y. Proydakova1, E.Z. Sadykova2, A.V. Ryabova12
1 - Prokhorov General Physics Institute of the Russian Academy of Sciences, Moscow, 119991, Vavilova street,
38
2 - National research nuclear university MEPHI, Moscow, 115409, Kashirskoe highway, 31 Main author email address: pominovadv@gmail.com
According to the literature data, metabolic reprogramming occurs in the process of tumor initiation and progression, which leads to reorientation of immune cells to protect the tumor and promotes proliferation, invasion, metastasis, aggressiveness and resistance of tumors to treatment [1, 2]. In this regard, one of the promising approaches to treatment today is an indirect effect on the tumor due to the reverse metabolic reprogramming of immune cells to fight the tumor.
Within the framework of this work, we investigated the possibility of recognizing various populations of macrophage cells, including tumor-associated macrophages, using time-resolved fluorescence spectroscopy. The photosensitizer Chlorin E6 ("Radachlorin", Rada-Pharma, Russia) and crystalline nanoparticles NaGdF4 co-doped with Yb3+-Er3+ rare-earth ions were used as luminescent biomarkers. The nanoparticles were synthesized using solvothermal technique [3]. Particles of a pure hexagonal phase with averaged particle size of 20 nm were obtained. Populations of macrophage cells were obtained from culture of human monocytes THP-1. Differentiation of THP-1 monocytes into adhesive growing macrophages (M0) was achieved by adding 100 nM phorbol-12-myristate-13-acetate (PMA) to the culture for 24 hours. 48 hours after incubation with PMA, human recombinant interferon gamma (IFN-y, 20 ng / ml, Sigma-Aldrich, USA) and lipopolysaccharide (LPS, 100 ng / ml, Sigma-Aldrich) were added to the medium for M1 polarization or with human recombinant interleukin-4 (IL-4, 20 ng / ml, Sigma-Aldrich, USA) and human recombinant interleukin-13 (IL-13, 20 ng / ml, Sigma-Aldrich, USA) for M2 polarization, respectively. Pro-inflammatory polarization of M1 has antimicrobial and antitumor activity, while M2 polarization, on the contrary, is anti-inflammatory and promotes tumor progression. After polarization, the cells were incubated with chlorin E6 or upconversion nanoparticles for 3, 6, and 24 hours. The cells were then washed with phosphate saline buffer and the accumulation of luminescent markers was analyzed by laser scanning microscopy (Carl Zeiss LSM 710 NLO, Carl Zeiss, Germany). After that, the cells were detached from petridish using trypsin, centrifuged, and the fluorescence lifetime from the cell sediment was measured using a streak camera (C9300-508 and C10627-13, Hamamatsu Photonics, Japan).
It was shown that the use of nanoparticles as luminescent markers provides better accumulation in macrophages due to the size of the nanoparticles. The change in the luminescence lifetime of the used markers inside the macrophages cells was registered. The possibility of determining the polarization of macrophages in vitro according to the data of time resolved fluorescence spectroscopy was demonstrated.
The study was funded by a grant from the Russian Foundation for Basic Research, project 20-0200928.
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