br iii mg ml AA
(iii) 0.5 mg/ml AA-CUR, (iv) 0.7 mg/ml AA-CUR bioconjugate. After exposition to bioconjugate, PBMC were stained with Trypan Blue and counted using Bürker’s chamber.
2.8. Viability and toxicity assays
1.49 µg/ml curcumin respectively). Cells grown in media containing 15 mM NaCl served as positive control for the test (100% viability). After 48 h incubation viability of the cells was measured by MTT assay and cell morphology was assessed using the inverted light microscope LeicaDM IL LED Fluo. Images were taken at 100× magnification with a digital camera Leica DFC450 C.
2.9. Flow cytometry analysis of AA-CUR bioconjugate uptake
CT26-CEA cells were grown overnight in 1 ml DMEM/10%FBS/an-tibiotics on 12 well plate at the density 50 × 103 cells/well. The next day, cells were incubated with 0.088 mg/ml AA-CUR bioconjugate for 2, 5, 10, 20, 30, 60 min or 2, 6, 24 h. After exposition to the AA-CUR the cells were trypsinized, and centrifuged. In order to completely remove alginate bioconjugate adsorbed to the cell membrane, additional in-cubation with trypsin was performed. This approach was previously used by us and allowed to reliably determine cellular uptake of nano-materials [34,35]. Cellular uptake of AA-CUR was then measured by flow cytometry utilizing intrinsic fluorescence of curcumin . The measurements of the mean fluorescence intensity (MFI) of the cells were done using of BD FACSCalibur flow cytometer and CellQuestPro software Becton-Dickinson. Error bars represent the mean ± SD of at least three independent experiments. Statistical significance was analyzed by the Mann-Whitney U test with the use of OriginPro 9.1 software, with European Polymer Journal 113 (2019) 208–219
P < 0.05 being considered statistical.
1H Nuclear Magnetic Resonance (1H NMR) spectra were recorded in D2O at ambient temperature on a Bruker 500 MHz spectrometer. Fourier Transformed Infrared Spectroscopy (FTIR) spectra were mea-sured using a Bruker Equinox 55 spectrophotometer with the resolution of 2 cm−1. Gel permeation chromatography (GPC) analyses were per-formed at ambient temperature using a Waters chromatographic system equipped with a set of the three aligned PL Aquagel-OH 8 µl columns and Refractive Index detector (RI 2410, Waters). The 0.1 M NaCl aqu-eous solution was used as an eluent and the flow rate was set at 1 ml/ min. UltraViolet–Visible (UV–Vis) HG 9 91 01 spectra were measured using a Hewlett–Packard 8452A diode-array spectrophotometer at ambient temperature. The critical micelle concentration (CMC) of the bioconjugate was determined by conductometric titration, based on the calibration curve - conductivity versus concentration dependence ĸ (c) obtained for a series of aqueous solutions of AA-CUR.
3. Results and discussion
The synthesis of sodium alginate-curcumin bioconjugate (AA-CUR) and the procedure to determine the curcumin content in AA-CUR are described in Supplementary Materials. AA-CUR bioconjugate was ob-tained via Steglich esterification, which allows the conversion of steri-cally demanding and acid labile substrates, in the presence of dicyclo-hexylcarbodiimide (DCC) and 4-N,N-dimethylaminopyridine (DMAP) as catalysts. First an O-acylisourea intermediate is formed between DCC and the carboxylic groups of alginate, followed by the formation of the active amide in the presence of DMAP. This allows to prevent any possible side reactions to occur. Finally the addition of the curcumin hydroxyl group to the active amide results the bioconjugate formation. The proposed bioconjugate structure is shown in the scheme in Fig. 1B.
3.1. Characterization of AA-CUR bioconjugate
To verify the successful attachment of curcumin to the alginate chain, the FTIR and 1H NMR spectroscopies were applied. The presence of the new band at 1708 cm−1 in the FTIR spectrum of AA-CUR (see Supplementary Materials, Fig. S1), which can be ascribed to the stretching vibrations of the C]O group of the ester bond created in the conjugation reaction, confirms formation of the bioconjugate. Ad-ditionally, a set of bands appeared at around 1400 cm−1, characteristic for the stretching vibrations of the CeO group of the same ester bond, as well as the new band at 1027 cm−1 originating from the ether bonds of curcumin. 1H NMR analysis of ionic polymers studied in aqueous solutions is diﬃcult and spectra usually diﬃcult to interpret, due to the significant broadening of the peaks and their overlapping. The com-parison of the spectra of alginate and the obtained bioconjugate con-firmed formation of AA-CUR. New signals, which were not present in the spectrum of the alginate, have appeared in the spectrum of the reaction product (see Supplementary Materials, Fig. S2), namely the multiplets at around 6.8 ppm and 7.9 ppm, both originating from the overlapping signals of curcumin protons observed usually for the free curcumin in the range of 6 to 8 ppm, and the singlet at around 3.2 ppm, which can be ascribed to the six ether protons of curcumin aromatic rings. The relatively intense, but narrow signal at 2.6 ppm can be as-cribed to the traces of DMSO which could not be removed by dialysis. The UV–Vis spectrum of the aqueous solution of AA-CUR revealed the band with the maximum at 406 nm, characteristic for curcumin (see Fig. 2A). The hipsochromic shift of that band in the bioconjugate in comparison to the spectrum of free curcumin in water-methanol mix-ture (90:10) was observed (see Supplementary Materials Fig. S3). In the spectrum of bioconjugate the additional band appeared at 280 nm, which could be ascribed to n →π* transition of the newly formed ester