CC BY
2*
ALT'23 The 30th International Conference on Advanced Laser Technologies
B-P-1
Intravital fluorescent imaging to test nanoparticles safety and distribution using liver slices
V.V. Elagin1, S.A. Rodimova1, A.M. Mozherov1, D.P. Krylov12, D.S. Kozlov12, M.V. Zyuzin3, D.S. Kuznetsova12
1 - Privolzhsky research medical university, 603005, Nizhny Novgorod, Minin and Pozharsky sq. 10/1 2 - Lobachevsky Nizhny Novgorod National Research State University, 603022, Nizhny Novgorod, Gagarina 23 3 - ITMO University, 191002, St. Petersburg, Lomonosova st., 9 e-mail: [email protected]
Despite the promising results currently achieved in stimulating liver regeneration using small bioactive molecules, there is still a problem of the lack of an effective method for their delivery with a controlled release period, their accumulation and excretion [1]. Such a technology based on complexes of nanoparticles and bioactive molecules is especially relevant due to the high risk of liver failure after liver surgery. The most promising methods for assessing the state of living liver cells seems to be multiphoton fluorescent microscopy with second harmonic generation (SHG) and time-resolved FLIM microscopy. These methods are non-invasive, label-free and allow intravital monitoring at the cellular level. In addition, the use of the model of precision-cut liver slices made it possible to screen several types of nanoparticles, excluding the individual contribution of the animal organism, simplifying the analysis and interpretation of the results. Vibrating microtome 7000 cm3-2 was used to obtain liver slices using the protocol of Pearen et al. [2], and were placed in separate wells of a 12-well plate with a standard CO2-conditioned form of DMEM supplemented with 0.1 ^m of dexamethasone and 10% FBS. Next, cultivation was carried out in DMEM medium with the addition of nanoparticles at a concentration of 50 and 100 mg/ml and incubated for 3, 24, and 48 hours. All obtained tissue explants were preincubated for 1 h in DMEM medium on an orbital shaker (90 rpm). Gold nanoparticles with a size of 100-120 nm in the form of nanorods were synthesized using a standard protocol based on seed mediated mechanism. The synthesis of SiO2 nanoparticles with a size of 20-100 nm was carried out using the sol-gel method. Polylactide (PLA) nanoparticles with a size of 100 nm are tested by single-emulsion solvent extraction. All nanoparticles were modified with a Cy 5 fluorescent label. Liver slices were stained with LysoTracker Yellow HCK-123 and Phalloidin FITC. Using multiphoton microscopy, we assessed the tissue structure of liver slices. Using FLIM, we analyzed the metabolic state of hepatocytes based on fluorescence lifetime contributions of the free and bound forms of NADH and NADPH. As a result, it was shown that SiO2 nanoparticles were practically not accumulated by liver cells and show low cytotoxicity. Gold nanoparticles showed effective accumulation in liver cells, however, its had strong cytotoxic effect on liver cells. Finally, polylactide nanoparticles accumulated most effectively in liver cells, mainly in the cytoplasm of hepatocytes. Using FLIM, we revealed low cytotoxicity of PLA nanoparticles, due to the relative contributions of fluorescence lifetimes of bound form of NADH and NADPH did not differ significantly from control values. Thus, PLA nanoparticles seem to be the most promising for further development of a strategy for stimulating liver regeneration using nanoparticles modified by bioactive molecules. Based on the obtained data, we will select the appropriate miRNAs that stimulate liver regeneration. The work was supported by the Grant from the Russian Science Foundation №23-15-00421.
[1] Liu Z., Li Y., Li W., et al. Multifunctional nanohybrid based on porous silicon nanoparticles, gold nanoparticles, and acetalated dextran for liver regeneration and acute liver failure theranostics. Advanced Materials, 30(24), 1703393, (2018).
[2] Pearen M. A., Lim H. K., Gratte F. D. et al. Murine precision-cut liver slices as an ex vivo model of liver biology. JoVE, (157), e60992, (2020).
