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    2020-08-18

    r> These results correspond well with observations made by Dey and Sreenivasan. They demonstrated that alginate-curcumin bioconjugate micelles had strong impact on viability of mouse L929 131438-79-4 determined after 24 h exposure [31]. In addition, Sarika and coworkers suggested the use of galactosylated alginate-curcumin micelles, analyzed me-chanism of cellular uptake and demonstrated their toxicity to HepG2 cells [32].
    We have also observed that toxicity of AA-CUR was dependent on the type of cells. The strongest cytotoxic effect of AA-CUR was observed for B16F10 and CT26-CEA cells. This phenomenon is not surprising in the light of research indicating different, often cell-dependent me-chanisms of cytotoxic curcumin activity against cancer cells [44–46].
    In the next experiment the cellular uptake of the bioconjugate by the cancer cells was confirmed and quantified. Because curcumin is characterized by an inherent green fluorescence, the internalization of bioconjugate was observed using flow cytometry analysis. CT26-CEA cells were incubated for different periods of time with mixture con-taining alginate-curcumin bioconjugate (0.088 mg/ml) and DMEM/ 10% FBS. Previous experiments (data not shown) indicated that the dose of AA-CUR was not toxic. After incubation, CT26-CEA cells were collected from plates and fluorescence intensity was determined by flow cytometer. It was observed that bioconjugate was rapidly inter-nalized by the cells within minutes of exposure and the highest micelles accumulation was detected in 1 h after treatment (Fig. 7B). Reduction in fluorescence intensity measured at that longer periods of time may result from the changes of curcumin spectral profile as it is metabolized intracellularly [47,48].
    3.5. Interaction of the AA-CUR bioconjugate with serum proteins
    Considering the possible future therapeutic applications of AA-CUR it is of great importance to study the interactions of the bioconjugate micelles with the blood plasma proteins. That is necessary to avoid the possible unwanted side-effects and to be able to take into account the effect of such interactions on bioconjugate’s pharmacokinetics. Gel electrophoresis was thus applied to study the interactions between
    Fig. 5. 2D AFM image of the surface of crosslinked AA-CUR micelles (upper part) and cross section of the nanoparticles (lower part).
    bioconjugate and the most typical proteins of the blood plasma. Bovine serum was used in these experiments.
    AA-CUR bioconjugate solution (0.4 mg/ml) was mixed with bovine serum and incubated for 4 h at 37 °C. The mixture was then centrifuged. No visible precipitate was observed. The supernatant was analyzed by gel electrophoresis and the results are presented in Table 2.
    The lack of precipitate after incubation of the serum with bio-conjugate suggests that either no aggregates are formed or they are small enough/soluble enough to stay in solution. The results of elec-trophoresis indicated that the interactions indeed occurred as, after incubation with the bioconjugate, the fraction of the free protein has decreased for all the proteins studied. Thus, one may speculate that some, not overly high, aggregation takes place, but the complexes/ag-gregates formed between AA-CUR bioconjugate and proteins are so-luble in aqueous media and thus curcumin may be transported in the form of such complexes to the cells. That effect can be beneficial. It was 
    recently observed that highly reduced interaction of the carrier system with proteins may not be optimal because limited protein adsorption is required for stealth effect, and the presence of protein corona can re-duce an unspecific uptake by the cells [49].
    3.6. Analysis of interaction of alginate-curcumin bioconjugate micelles with blood components and with endothelial cells
    Evaluation of biomaterial interaction with endothelial cells and blood components, including red cells and leukocytes, is crucial for evaluating its safety as a drug delivery system. Thus, the preliminary analysis of hemocompatibility of curcumin containing micelles was performed. First experiment was designed to check whether AA-CUR induces hemolysis of human red cells. Diluted human peripheral blood samples were incubated for 3 h with AA-CUR and then hemoglobin released from erythrocytes was measured spectrophotometrically after
    Fig. 7. Analysis of cytotoxicity and cellular uptake of AA-CUR bioconju-gate performed using different mouse cancer cell lines: (A) cell viability measurement by MTT assay, (B) mor-phology observation determined after 48 h incubation with bioconjugate, and
    (C) the uptake of bioconjugate by CT26-CEA cells, determined by the measurement of mean fluorescence intensity (MFI) of the cells with the use of flow cytometry.