Antioxidant Activity of Silver-containing Bionanocompositions Based on Humic Substances in Cell Culture

E E Buyko,I O Grigorieva,M V Belousov,D A Mikhalyov,I V Perminova,M V Zykova,K A Bratishko,A A Ufandeev,A V Tsupko,V V Ivanov

doi:10.33380/2305-2066-2021-10-4-46-53

Abstract

Introduction. Silver nanoparticles are promising agents for suppressing resistant strains of microorganisms and accelerating the purulent wounds healing. Oxidative stress disrupts normal wound healing processes, which leads to the formation of chronic non-healing wounds. Therefore, the determination of the ability of new wound healing agents to decrease the production of reactive oxygen species is a relevant task.Aim. The aim of the current study was to investigate the effect of silver-containing bionanocompositions based on humic substances on the basal and tert-butyl hydroperoxide-stimulated production of reactive oxygen species at the normal fibroblasts 3T3-L1 cell culture in vitro.Materials and methods. The study was carried out on 7 samples of initial humic substances and biomaterials with silver nanoparticles synthesized in the Laboratory of Natural Humic Systems, Faculty of Chemistry, Moscow State University named after M. V. Lomonosov. The intracellular production of reactive oxygen species was assessed using a 2,7-dichlorodihydrofluorescein diacetate fluorescent probe. Cells were cultured with samples for 24 h; tret-butyl hydroperoxide was used to stimulate the production of reactive oxygen species. Detection was performed fluorometrically using a microplate reader.Results and discussion. The most pronounced antioxidant activity was demonstrated by three samples of biomaterials with silver nanoparticles ultradispersed in humic substances matrices (CHS-AgNPs, CHP-AgNPs and CHE-AgNPs), which allows us to consider them as the most promising pharmaceutical agents for the treatment of purulent-inflammatory processes. The most probable mechanism of the high antioxidant activity of the studied biomaterials in relation to intracellular reactive oxygen species is the intrinsic activity of humic substances to bind reactive oxygen species, while silver nanoparticles in biomaterials catalyze the reduction processes of their interaction with reactive oxygen species.Conclusion. For the studied samples of biomaterials with silver nanoparticles ultradispersed in matrices of humic substances pronounced antioxidant activity was shown. Together with antibacterial properties, it makes it possible to consider them as potential agents for purulent wounds healing accelerating.

Highlights

Silver nanoparticles are promising agents for suppressing resistant strains of microorganisms and accelerating the purulent wounds healing
The aim of the current study was to investigate the effect of silver-containing bionanocompositions based on humic substances on the basal and tert-butyl hydroperoxide-stimulated production of reactive oxygen species at the normal fibroblasts 3T3-L1 cell culture in vitro
Cells were cultured with samples for 24 h; tret-butyl hydroperoxide was used to stimulate the production of reactive oxygen species

Summary

МАТЕРИАЛЫ И МЕТОДЫ

Объектами исследования являются 7 исходных образцов ГВ, представляющие собой как ГВ, выделенные из природного сырья (торфа) на кафедре химии СибГМУ и на химическом факультете МГУ, так и коммерческие гуминовые препараты, выделенные из угля, а также соответствующие им 7 образцов биоматериалов, содержащие наночастицы серебра (ГВ-AgNPs), синтезированные в Лаборатории природных гуминовых систем химического факультета Московского государственного университета имени М. Оценка внутриклеточной продукции АФК проводилась с использованием флуоресцентного зонда 2,7-дихлородигидрофлуоресцеиндиацетата (ДХФДА) по описанной ранее методике [23]. Клетки линии 3T3-L1 культивировали в стандартных условиях [атмосфера 5 % CO2, среда DMEM/F-12 (GibcoTM, США) + 10 % FBS (GibcoTM, США) + 2 мМ L-глутамина (GibcoTM, США) и 1 % гентамицина (GibcoTM, США)] и рассаживали на черные 96-луночные культуральные планшеты для измерения флуоресценции (1 × 104 клеток/лунку). В соответствующие лунки вносили образцы исследуемых ГВ и ГВ-AgNPs (12,5 мкг/мл) и инкубировали 24 ч. Затем клетки отмывали от образцов, в лунки вносили рабочий раствор ДХФДА (10 мкМ) (Sigma-Aldrich, США, D6883). Планшеты инкубировали в термостате 20 минут при 37 °C, отмывали от ДХФДА и для стимуляции продукции АФК вносили трет-бутилгидропероксид (t-BHP) (25 мкМ) (Sigma-Aldrich, США, B2633). Экспериментальные образцы гуминовых веществ (ГВ) и серебросодержащих бионаноматериалов на их основе (ГВ-AgNPs)

Source of the initial HS

РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ