Streptomycin

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 macrocyclic compounds’ ability to penetrate into A549 and HuTu-80 cells was determined with a BD FACSCanto II flow cytometer. Cells were incubated for 2 h in the presence of test compounds at 37 °C and then stained with propidium iodide (PI), which selectively stains dead cells.
A549 and HuTu-80 cells were grown in DMEM (GIBCO, Waltham, MA, USA) after supplementing with 10% FBS (Corning, Inc., Corning, NY, USA), 100 units/mL penicillin (PanEco, Moscow, Russia) and 100 μg/mL streptomycin (PanEco, Moscow, Russia), at 37 °C in a humidified atmosphere with 5% CO₂. Cells were harvested and washed with fresh medium and then placed in individual sterile tubes at a concentration of 10⁵ cells/mL. After adding the test compounds to the tubes, the cell suspension was incubated for 2 h at 37 °C in the dark. Then the cell suspension was centrifuged at 2000 rpm for 5 min at room temperature, and cells were washed three times in phosphate-buffered saline (PBS, PanEco, Moscow, Russia). The cells were resuspended in 1 mL of PBS and transferred to cytometric tubes, when the samples were stained with 5 µL of PI solution (5 mg/mL), kept in the dark at room temperature for 2 min, and cytometric analysis was performed. The processing of cytometric data was carried out in the FACSDiva application.

The ability of macrocyclic compounds to inhibit the viability and proliferative activity of A549 and HuTu-80 cells was determined using the MTT assay according to [48 (link)]. Briefly, cells were grown in 96-well plates in DMEM (GIBCO, Waltham, MA, USA) after supplementing with 10% FBS (Corning Inc., Corning, NY, USA), 100 units/mL penicillin (PanEco, Moscow, Russia) and 100 μg/mL streptomycin (PanEco, Russia), at 37 °C in a humidified atmosphere with 5% CO₂ up to 80% confluence. Then, the medium in wells was replaced with a fresh medium, supplemented with test substances in the concentration range of 0.5–100 µg/mL. The volume of the culture medium in the wells was 100 µL. After 24 h of cell incubation in the presence of agents, the medium in the wells was replaced with a fresh medium containing MTT (Merck) at a concentration of 0.5 mg/mL. Cells were incubated with MTT for 3 h (HuTu-80) or 4 h (A549) at 37 °C, then the medium from the wells was aspirated and 100 µL of dimethyl sulfoxide added. Probes were incubated at 37 °C for 15 min in the dark for the formazan crystals to dissolve. The optical density of the formazan solution in the wells was measured using a reader (BioRad xMark™ Microplate Spectrophotometer, Hercules, CA, USA) at a wavelength of 570 nm. Three series of experiments were carried out with at least 8 replications for each variant in the series.

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).

Human lung adenocarcinoma A549 and H1299 and human colorectal adenocarcinoma cells DLD1 were obtained from the Collection of Cell Lines of the Institute of Cytology RAS, Russia. Mouse colon carcinoma CT-26 cells were kindly provided by Prof. G. Multhoff (Technical University of München, Germany). A549, H1299 and DLD1 cells were cultivated in DMEM and CT-26 cells were grown in RPMI-1640 media supplemented with 10% heat inactivated fetal bovine serum (FBS) (HyClone, USA), 2 mM L-glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (PanEco, Russia) in a 5% CO₂ atmosphere with 90% humidity. Viability was determined by 0.4% trypan blue exclusion.
The CT-26 luc and A549 luc cell lines were transduced with the luciferase gene using the pHIV-iRFP720-E2A-Luc [10 (link)] and pHIV-Luciferase vectors, respectively. The development of A549_shHsp70 and DLD1_shHsp70 cells was recently described [13 (link)].

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).

HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Paneco) supplemented with 2 mM L-Glutamine (Gibco), 10% fetal bovine serum (HyClone), 50 U/mL penicillin and 50 μg/mL streptomycin (both from Paneco). Transfection of HEK293 cells with H1N1 HA-encoding mRNA was performed as described previously (21 (link)) with minor modification. Briefly, HEK293 cells were plated on a 12-well plate in a density of 2×10⁵ cells per well and maintained at 37°C in 5% CO₂. The next day the medium was replaced with a fresh DMEM without antibiotics and cells were transfected by HA-mRNA using Lipofectamine 3000 reagent (Invitrogen) and Opti-Mem I Reduced Serum Medium (Gibco) in accordance with the manufacturer’s instructions. 24 h after transfection, cells were analyzed by immunocytochemical staining.