br reaction with Drabkin s
reaction with Drabkin’s reagent. As demonstrated in Fig. 8B, AA-CUR micelles were not toxic for human red cells. No red cells aggregation and bioconjugate-induced hemolysis was observed. Low toxicity of AA-CUR micelles was also confirmed in the studies that involved human
PBMC isolated from peripheral blood of healthy donors and mouse primary brain endothelial cells MBE (Fig. 8C).
Viability and morphology of MBE cells (Fig. 8C) was not aﬀected by incubation with various doses of micelles. At the same time PBMC cells
Protein composition of the bovine serum after incubation without and with AA-CUR.
Protein fractions Albumins [%] α1 Globulins [%] α2 Globulins [%] β Globulins [%] γ Globulins [%]
Fig. 8. Interaction of AA-CUR bioconjugate with human blood components and mouse endothelial cells: (A) analysis of morphology and viability of human PBMC incubated with bioconjugate for 48 h, (B) hemolysis of human erythrocytes after treatment with bioconjugate for 3 h and (C) morphology and viability analysis of mouse MBE cells incubated for 48 h with bioconjugate.
were sensitive (decrease in viability at about 30% compared to control cells, Fig. 8A) only to AA-CUR used at the highest concentration (0.7 mg/ml). AA-CUR used at two diﬀerent concentrations: 0.5 mg/ml and 0.35 mg/ml had no eﬀect either on PBMC viability or morphology, while its application at concentrations: 0.35 mg/ml and even at 0.175 mg/ml significantly reduced the viability of all murine cancer cell lines studied. That observation indicates that normal human and mouse cells are less sensitive to AA-CUR than mouse cancer cell lines. That is in line with previously published data obtained for free as well as for encapsulated curcumin .
The bioconjugate of sodium alginate and curcumin was successfully obtained in a simple one-step synthesis. In the aqueous media the bioconjugate can form Amyloid Beta-Peptide1-40 already at the concentrations slightly above 0.2 mg/ml, although the most regular, uniform micelles can be observed at the concentration of 0.6 mg/ml, when the cmc for AA-CUR
is reached. That AA-CUR aqueous dispersion shows high colloidal sta-bility (ζ = −53 mV). The AA-CUR micelles have an average diameter of 200 nm, which is an optimal size for the biological applications. The content of curcumin in the bioconjugate was established spectro-photometrically, in the concentration range assuring that there was no influence of the curcumin aggregation process, as 45 mg/g of bio-conjugate. Promising curcumin release profiles, with the prolonged release for at least 5 h, were achieved for the bioconjugate micelles cross-linked with calcium ions. The biological studies have shown that AA-CUR did not induce red cells aggregation or hemolysis. The lack of significant toxicity was also confirmed by the experiments done on human PBMC isolated from peripheral blood of healthy donors and mouse primary brain endothelial cells. Thus, we report here, for the first time, on the interaction between AA-CUR bioconjugate and cells isolated from human blood. The dose dependent sensitivity of all tested cancer cells to the bioconjugate was observed. The highest cytotoxicity of the studied micelles was observed at 0.7 mg/ml concentration of AA-CUR. At that concentration viability of cancer cells decreased by ca.
D. Lachowicz et al.
80%. It was also shown that normal cells are far less sensitive to AA-CUR than mouse cancer cell lines. The simplicity of synthesis, excellent physicochemical characteristics of the AA-CUR micelles and their aus-picious biological properties make that bioconjugate a promising anti-cancer drug system.
Conflict of interest
Authors declare no conflict of interest.
Faculty of Biochemistry, Biophysics and Biotechnology and Faculty of Chemistry of Jagiellonian University are members of the Leading National Research Centers (KNOW) supported by the Ministry of Science and Higher Education. The work was supported by the NCN grant DEC-2013/09/N/ST5/02488.
Data availability statement
The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.
Appendix A. Supplementary material
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M. Nowakowska, S. Zapotoczny, Biocompatible and fluorescent superparamagnetic iron oxide nanoparticles with superior magnetic properties coated with charged polysaccharide derivatives, Colloids Surf. B Biointerfaces. 150 (2017) 402–407, https://doi.org/10.1016/j.colsurfb.2016.11.003.