As shown in Fig. 1, PNC is a water-soluble comb-like polymer of poly(VEP-co-GMA)-graft-mPEG consisting of a backbone copolymer of 5-(tert-butylperoxy)-5-methylhex-1-en-3-yne (VEP, denoted by “l”) and glycidyl methacrylate (GMA, denoted by “m”), and grafted side PEG chains (PEG denoted by “n”) (Riabtseva et al. 2012).
Synthesis and characteristics of PNC
The synthesis of the PNC was carried out as described previously (Heffeter et al. 2013; Riabtseva et al. 2012; Senkiv et al. 2014). The composition of the PNC was calculated from the results of elemental analysis and analysis of functional groups (Steyermark 1961; Voronov et al. 1996).
The PNC water solution was prepared as follows: 0.093 g of PNC was dissolved in 0.9 ml DMSO and this solution was added to 8.5 ml of saline solution (0.9% aqueous NaCl solution). Then, the solution was stirred for 1 h and sonicated for 20 s, [PNC] = 9.9 mg/ml.
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) studies of the morphology of the micelles: a transmission electron microscope JEM-200A (JEOL, Japan) was used at an accelerating voltage of 200 kV. The samples were prepared via polymer dissolution in water, as described above. Samples were prepared by spraying tested solution on a substrate using the ultrasonic dispersant UZDN-1A (Ukrrospribor Ltd, Ukraine) that produces a uniform coating on the substrates. A thin amorphous carbon film deposited on a copper grid was used as a substrate. The dispersant options used were a type UZDN-1 possessing power of 50 W and frequency of 35 kHz. A Zeiss Supra 40/40VP scanning electron microscope (Carl Zeiss Group, Germany) was also used in this study.
The size of polymer micelles was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Instruments GmbH, Stuttgart, Germany) and by photon correlation spectra, using the NIBS (Non-Invasive Back Scatter) technology at 25 °C. The samples for DLS measurements were prepared by dissolution of polymers in bidistilled water, pH 6.5–7.0, and the copolymer concentration was 1∙10−2–5∙10−2 g/ml. Three to five measurements were made for every sample (each measurement consisted of five cycles, the range between measurements was 5 min). For temperature-dependent DLS studies, the solution was allowed to equilibrate for at least 1 h prior to data collection. Zeta potential experiments were carried out with a Zetasizer Nano Particle (Malvern Instruments GmbH, Stuttgart, Germany) at a fixed temperature of 25 °C. The distributions of the hydrodynamic diameter of PNC at different temperatures were conducted on the Nano Particle Analyzer SZ-100 (HORIBA, Ltd., Kyoto, Japan) and the measurements show a dependence of hydrodynamic diameter of PNC on temperature.
Cytotoxicity in in vitro MTT assay
Human myeloid leukemia HL-60, human hepatocarcinoma HepG2 cells, and human colon carcinoma HCT116 cells were used from the Institute of Cancer Research at Vienna Medical University (Vienna, Austria). Human breast adenocarcinoma MCF-7 and human T cell leukemia Jurkat cells were obtained from the Institute of Experimental Pathology, Oncology and Radiobiology (Kyiv, Ukraine). Cells were grown in RPMI-1640 culture medium (APP, Austria) or Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Biowest, France) at the standard conditions.
The cytotoxicity was measured using colorimetric MTT assay (EZ4U, Biomedica, Austria) for assessing cell functional activity after treatment with the PNC. Cells were plated at 5000 cells/well (substrate-dependent cells) or 15,000 cells/well (suspension cells) in 100 µl in 96-well plates and allowed to incubate overnight. PNC (equivalent to the amount of polymer in complex with the anticancer drug) was added in 100 µl of culture medium and the cells were incubated for 72 h. Afterward, MTT assay was performed according to the manufacturer’s recommendations (EZ4U, Biomedica, Austria). Briefly, 20 μl of dye solution was added to 200 μl of cell culture and incubated for the next 1–4 h at 37 °C. The optical density was measured with the Absorbance Reader BioTek ELx800 (BioTek Instruments, Inc., USA) at 490 nm with 630 nm as a reference.
Trypan blue exclusion test
Cells were plated at 50,000 cells/well (substrate-dependent cells) or 500,000 cells/well (suspension cells) in 1 ml in 24-well plates and allowed to incubate overnight. PNC at different concentrations was added to the cell culture and incubated for the next 72 h. The cell number was counted in a hemocytometric Neubauer chamber after staining with Trypan blue dye (DV-T10282, Invitrogen, Life Technologies Corporation) at 0.04% final concentration. The percentage of live cells related to control was calculated as the cell number experiment/cell number control × 100%.
Data analyses
The results of MTT and Trypan blue assays are presented as the mean (M) ± standard deviation (SD) of three replications. The data were analyzed and illustrated using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA).
Evaluation of PNC toxicity in vivo
All animal studies were conducted according to the European Convention on Protection of Vertebrate Animals (Strasbourg, 1986) and corresponding Law of Ukraine (N944, 14.12.2009). The protocols of this study and experimental procedures were approved by the Ethical Committee at Lviv National Medical University (N2, 16.02.2015). The experiments were carried out on white laboratory mice 3–4 months old with body weight of 18–24 g and white Wistar rats 3–4 months old with body weight of 143–255 g. The PNC was injected daily into the peritoneal cavity. In total, 36 rats and 36 mice were used in the study. Mice were injected with the PNC at doses of 0.1, 0.3, 0.5, and 1.0 ml; in rats, the doses were 1.0, 3.0, 5.0, and 10.0 ml. The highest dose of the PNC was repeatedly administered to six rats (10.0 ml) and six mice (1.0 ml). 1 ml of the solution contained 9.9 mg of the PNC. We could not further increase the amount of applied substance because of the critical volume used for injection. Control groups of rats and mice were injected with a physiological solution of the same volume. To avoid application of too large volume of the diluted substance (20 mL/kg of body weight of laboratory rats), we injected the liquid in two steps during 30 min (Diehl et al. 2001).
Weighing of experimental animals was carried out after 5, 8, and 14 days. After administration of the PNC, laboratory animals were monitored for 14 days. The following indicators were considered: appearance, uniqueness of animal behavior, intensity and character of motor activity, assessment of food and water consumption, mass of animals, condition of fur and visible mucous membranes, respiration rate, time of occurrence and nature and severity of intoxication, as well as the time of death of the animals or their recovery (Faqi 2013, 2017).
The animals (rats) were euthanized by decapitation while under thiopental anesthesia on the 20th day. Blood was collected and used to obtain serum for determination of the activities of alkaline phosphatase (ALP; 3.1.3.1), α-amylase (3.2.1.1), γ-glutamyltransferase (GGT; 2.3.2.2), lactate dehydrogenase (LDH; 1.1.1.27), alanine transaminase (ALT; 2.6.1.2), aspartate transaminase (AST; 2.6.1.1), and creatine phosphokinase (2.7.3.2). The concentrations of total protein, glucose, urea, creatinine, and of calcium, iron, sodium and chloride ions were also measured. These parameters were measured with standard kits for an automated biochemistry analyzer (Humalyzer 3000, Germany).
Daily urine and feces were collected from treated rats, dried, and examined using thin-layer chromatography on a Sorbfil plate (Russian Federation) in acetone–dioxan (4:1) solvent mixture. The chromatographic plates were developed with iodine vapor, and pure VEP, GMA, and PEG were used as controls for the presence of specific compounds.
The results were established by colorimetric assay using the Absorbance Reader (BioTek Instruments, Inc., Winooski, VT, USA). Data analysis was performed with GraphPad Prism 6 software (GraphPad Software). Statistical analysis of the in vivo results was done using variance (MS Excel software; Microsoft Corp., Redmond, WA, USA) and Student’s t test. The difference was considered statistically significant and marked with stars for: * P ≤ 0.05.