European Polymer Journal br X ray
European Polymer Journal 113 (2019) 208–219
2.2. X-ray photoelectron spectroscopy measurements
X-ray photoelectron spectroscopy (XPS) measurements were per-formed using a PHI 5000 Versa Probe II (ULVAC-PHI, Chigasaki, Japan) spectrometer equipped with an Al Kα radiation source (1486.6 eV). The operating pressure in the analytical chamber was less than 3 × 10−7 Pa. High resolution spectra were recorded with analyzer pass energy of 29.35 eV. A dual-beam charge neutralizer was used to compensate the charge-up eﬀect. All XPS peaks were charge referenced to the neutral
(CC) carbon C 1s peak at 284.8 eV. The spectrum background was subtracted using the Shirley method. Data analysis software from PHI MultiPak was used to calculate elemental compositions from the peak areas.
2.3. DLS and zeta potential measurements
Dynamic light scattering (DLS) and zeta potential measurements for the bioconjugate samples were performed using NANOSIZER-ZS in-strument produced by Malvern Instruments. The sample was illumi-nated with a 633-nm laser, and the intensity of light scattered at an angle of 173° was measured by an avalanche photodiode. The mea-surements were performed in water at 25 °C. The presented results are the average of three measurements. Zeta potential was measured using laser Doppler velocimetry (LDV) technique. For zeta potential de-termination, six measurements were done and the average was re-ported.
2.4. AFM and SEM measurements
Surface morphology of the obtained micelles was studied using scanning HG 9 91 01 microscopy (SEM) and atomic force microscopy (AFM). SEM observations were performed using a cold field emission scanning electron microscope (FESEM) HITACHI S-4700 equipped with a NORAN Vantage energy dispersion spectrometer.
Atomic force microscopic images were obtained using a Dimension Icon AFM microscope (Bruker, Santa Barbara, CA) working in the PeakForce Tapping (PFT) and QNM® modes with standard silicon cantilevers for measurements in the air (nominal spring constant of 0.4 N/m).
For both methods samples were prepared on previously prepared silica plates coated with poly(allylamine hydrochloride) (PAH) using a spin-coating technique. For SEM measurements they were additionally coated with graphite using sputter deposition.
2.5. Gel electrophoresis
Gel electrophoresis was performed using the electrophoresis cell Cormay S20 and the assay provided also by Cormay (Gel protein 100). Prior to the analysis the bovine serum was diluted 7-fold with the working buﬀer. To separate the proteins present in the bovine serum, five microliters of serum sample were applied to numbered sample wells on the agarose films. The pH of the samples was in the range of 7.0–7.4. The films were electrophoresed for 30 min at constant voltage of 150 V. Subsequently, the films were simultaneously fixed and stained in an amido black 10B staining solution, and then dried at 80 °C. After de-staining in acetic acid solution, the films were completely dried and scanned in a densitometer (DT-93, EMCO).
2.6. Analysis of the alginate-curcumin (AA-CUR) bioconjugate hemolytic properties in vitro
Analysis of hemolysis of human erythrocytes induced by AA-CUR was performed using the method described by Dobrovolskaia et al. . Briefly, samples of human peripheral blood were drawn from healthy subjects on sodium citrate. In the first step, plasma free he-moglobin (PFH) and total blood hemoglobin (TBH) were determined
D. Lachowicz et al.
spectrophotometrically at 540 nm using microplate reader after reac-tion with cyanmethemoglobin reagent (Drabkin’s reagent). Next, whole blood samples were diluted with DPBS to adjust total hemoglobin concentration to 10 mg/ml (diluted total blood hemoglobin, dTBH). 0.1 ml of erythrocytes suspensions (dTBH) were mixed with 0.8 ml of DPBS and (i) 0.1 ml of AA-CUR (7 mg/ml) or (ii) 0.1 ml 150 mM NaCl (negative control of hemolysis) or (iii) 0.1 ml Triton X-100 (positive control of hemolysis) and incubated at 37 °C for 3 h with gentle shaking. After incubation, samples were centrifuged (1500g × 5 min) and con-centration of free hemoglobin was determined in supernatants (sample) as described above. The level of hemolysis in blood samples treated with AA-CUR was calculated according to the following formula: %hemolysis = [Hb]sample x100% (1)
2.7. Analysis of the eﬀect of AA-CUR bioconjugate on human PBMC
Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood of healthy donors using Ficoll-Paque Plus gra-dient fractionation according to standard procedure. PBMC were cul-tivated at the density 1 × 105/well for 48 h on 96 well plate in RPMI media supplemented with 20% FBS, antibiotics and containing: (i) 15 mM NaCl (positive control of the test, referring to the percentage of dead cells in untreated PBMC), (ii) 0.35 mg/ml AA-CUR bioconjugate,