L glutamine

Murine glioma GL261 cells were cultured in DMEM medium (PanEco, Moscow, Russia) supplemented with 4.5 g/L glucose (PanEco, Moscow, Russia), 2 mM L-glutamine (PanEco, Moscow, Russia), 100 μM sodium pyruvate (Thermo Fisher, Waltham, MA, USA), 100 units/mL penicillin (PanEco, Moscow, Russia), 100 μg/L streptomycin (PanEco, Moscow, Russia), and 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA). At the end of the exponential growth phase, the cells were detached with trypsin-versine solution (1:3), centrifuged at 1000 rpm for 3 min, and reseeded at a density of 5 × 10⁵ cells/mL and a multiplicity of seeding of 1:10. Incubation was maintained at 37 °C under 5% CO₂ in humidified air in a Binder C150 incubator (BINDER GmbH, Tuttlingen, Germany). All the experiments were performed after the third passage [21 (link)].

The sterilized rS1/9 microparticles were placed into 24-well plates, and 1 mL of cell culture medium was added to each well. The culture growth medium contained Neurobasal medium A (Gibco, United States) containing 2% serum-free supplement NeuroMax (PanEco, Russia) and 0.5 mM L-glutamine (PanEco). Microparticles were incubated for 1 week at 37°C and 5% CO₂. Then, medium with microparticles was transferred to sterile microcentrifuge tubes and centrifuged at 16,000 g for 30 min. The supernatant was collected and used immediately in the tests.

For preparing RC-containing MPs (RC MPs) as a source of PS was used a concentrate for the infusion solution “Radachlorin^Ò” manufactured by RADA-PHARMA (Moscow, Russia), comprising 3.5 mg/mL of a mixture of sodium salts of chlorin e6, chlorin p6 and purpurin 5. Poly(lactic-co-glycolic acid) 65:35 copolymer (PLGA) RESOMER RG 653 H was purchased from Evonik Industries AG (Essen, Germany); span 80, Polysorbate 20, light mineral (paraffin) oil, 1,3-diphenylisobenzofuran (DPBF) were from Sigma (Saint Louis, MO, USA); polyvinyl alcohol 18-88 was from Merck (Darmstadt, Germany); methylcellulose A4M was from Ashland (Covington, KY, USA); DMEM12 medium, penicillin, streptomycin, L-glutamine, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were from PanEco (Moscow, Russia); fetal bovine serum was from Hyclone (Chicago, IL, USA). Methylene chloride, acetonitrile, isopropyl alcohol, n-hexane, n-heptane, as well as the reagents used for preparing buffer solutions, were qualified as chemically pure or pure for analysis and were obtained from Chimmed (Moscow, Russia). Experiments were conducted using purified water obtained by a reverse osmosis UVOI 1812C6 system (MEDIANA FILTER, Moscow, Russia).

HaCaT (immortalized non-tumorigenic human keratinocytes) (Table S1), A431 (human epidermoid carcinoma cells), human keratinocytes (passage 2 and passage 3), and HeLa (human adenocarcinoma cells) were kindly provided by the Cell Culture Collection for Biotechnological and Biomedical Research, Koltsov Institute of Developmental Biology, Russian Academy of Sciences. The cells were grown in DMEM (Dulbecco Modified Eagle’s Medium; PanEco, Moscow, Russia) (HaCaT and A431) or DMEM/F-12 (human keratinocytes and HeLa) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 2 mM L-glutamine (PanEco), 80 μg/mL gentamicin (Belmedpreparaty, Minsk, Belarus), and 0.2% Defined Keratinocyte-SFM Growth Supplement (Thermo Fisher Scientific) (only for human keratinocytes) under standard conditions (37 °C, 5% CO₂). The cells were collected from the surface of a plastic flask with a 1:3 mixture of trypsin solution (PanEco) and Versene (0.2% EDTA in phosphate buffer) (PanEco) and plated on glass cover slips or on Petri dishes with glass bottom and grown for 48 h. Then, GA or DTT was added, and cells were incubated for 24 h.

Cells were obtained from human glioblastoma tissues after tumor resection. Cell culture G01 was chosen for this research as it exhibits high expression of the CD133 cell surface marker. This culture was obtained by washing cells in Versen solution (Paneco, Russia), incubation with 0.25% trypsin-EDTA (Ethylenediaminetetraacetic acid) solution (Gibco, UK) at 37°C for 40 min, and disaggregation from tissue followed by centrifugation at 1,000 rpm for 5 min. Cells were cultivated in DMEM/F12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12) medium (Gibco, UK) containing 1% L-glutamine (Paneco, Russia), 10% FBS (Fetal Bovine Serum) (Thermo Scientific, USA), and 1% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Sigma-Aldrich, USA) at 37°C with a humidified atmosphere of 5% CO2. Passaging was performed at 80% confluency, for which cells were washed in PBS (Phosphate Buffered Saline) (Gibco, UK) and incubated with a 0.25% trypsin-EDTA solution. After trypsin inactivation by fresh medium, cells were centrifuged at 1,000 rpm for 5 min, the supernatant was removed, fresh medium was added, and the cells were resuspended and plated into cultural flasks. The cell count was defined using Trypan Blue 1:1.

To study polyplex transfection kinetics, human embryonic kidney 293 (HEK293) cells were used. To investigate the transfecting ability of the released polyplexes and the GAS influence on cell osteodifferentiation mesenchymal stem cells (MSCs), derived from rat adipose tissue, 3–4 passages were used [58 ]. Cells were incubated in growth medium: DMEM/F12 (PanEco, Moscow, Russia), 10% fetal bovine serum (FBS, PAA Laboratories, Etobicoke, ON, Canada), 0.584 mg/mL L-glutamine (PanEco, Moscow, Russia), 5000 u/mL streptomycin (PanEco, Moscow, Russia), and 5000 u/mL penicillin (PanEco, Moscow, Russia) in Petri dishes under standard culture conditions (37 °C, 5% CO₂).