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  • br Fig The structure of

    2020-08-18


    Fig. 1. The structure of curcumin (A) and bioconjugate AA-CUR (B). (C), Schematic illustration of the micelle formed by conjugate AA-CUR; (D), Network structure scheme of the micelle from bioconjugate AA-CUR crosslinked by Ca2+ ions.
    group [37,38], thus further confirming the effective conjugation.
    The effectiveness of the purification process and the dispersity of the product was evaluated by GPC. The chromatogram obtained for AA-CUR showed one band, thus providing additional proof of the successful conjugation. The observed band was narrower than that for the un-modified alginate and appeared at lower retention time, confirming sufficient purification of the product, its lower dispersity and increased size of the chain due to its modification with curcumin (see Fig. 2B). It is, however, not possible to calculate the exact molecular weight of such polysaccharide derivative based on the typical low dispersity standards (e.g. dextran or pullulan). The reason is that the introduction of the hydrophobic groups to the polyelectrolyte chain results in the sig-nificant change of conformation, accompanied by a decrease in the hydrodynamic volume of the macromolecule. The resulting GPC chro-matogram is a superposition of the effects related to the increase in the polymer chain weight due to modification and to the decrease in hy-drodynamic volume, what makes it difficult to use it for accurate cal-culation of degree of curcumin substitution. Thus, the curcumin content in AA-CUR bioconjugate was determined from electronic HG-9-91-01 spectra. However, as curcumin shows a tendency to aggregate, with aggregates’ absorption spectrum differing significantly from that of monomer [39], the possible influence of the aggregation process on determination of its content in bioconjugate was considered. A series of bioconjugate solutions were tested and the changes in the measured 
    apparent curcumin content with decreasing bioconjugate concentration were analysed. The result obtained for the concentration range where no more changes in the determined result were registered (0.05 mg/ml) was assumed as the actual curcumin content in the bioconjugate, and it was estimated as 45 mg in 1 g of the bioconjugate.
    Additionally, surface chemical bonds of alginate and its derivative were determined with the wide scan XPS. As shown in Fig. 3, the binding energy (B.E.) peaks at 283.4, 284.9 and 286.5 eV can be as-signed to CeC, CeO and C]O, respectively [40,41]. The relative content ratio of C to O (C/O) in the bioconjugate AA-CUR is higher than that in alginate, due to the presence of the curcumin in obtained ma-terials. Furthermore, the relative content of C]O group to CeO group in the bioconjugate is slightly higher than in alginate, which also con-firms the presence of curcumin in the bioconjugate structure (see Supplementary Materials, Table S2).
    AA-CUR is well soluble in water up to 7 mg/ml. Due to the am-phiphilic character of the bioconjugate it shows tendency to aggregate in aqueous solutions resulting in micelle formation. In order to study this phenomenon in more detail, the aqueous solutions of the conjugate were analyzed using conductometry, as well as by the dynamic light scattering (DLS) and zeta potential measurements. The critical micelle
    Fig. 2. (A) UV–Vis spectra of alginate (blue) and AA-CUR bioconjugate (red), (B) GPC chromatograms for alginate and AA-CUR bioconjugate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    concentration (cmc) was determined based on conductometric titration, as presented in Fig. 4A. The precise value of cmc was calculated to be 0.654 mg/ml. DLS measurements were carried out to confirm the AA-CUR micelle formation. Based on the obtained results (see Supplementary Materials, Table S1 and Fig. S4) one may conclude that in the low concentration range the bioconjugate forms aggregates of ca. 160 nm and the system is characterized by relatively large dispersity (polydispersity index, PDI, above 0.3). When the polymer concentration
    increases, a slight increase in the aggregate diameter is observed, ac-companied by the decrease in PDI. This suggests the reorganization of the aggregate structure and formation of stable micelles. The lowest PDI value was observed at c(AA-CUR) = 0.6 mg/ml, which is in good agree-ment with the cmc value determined by conductometry. The average zeta potential value measured for the micelles was ς = −53 [mV]. Its relatively high value ensures the colloidal stability of their aqueous dispersion. At the concentrations above 0.6 mg/ml, both: the size and
    Fig. 4. (A) Dependence of conductivity of the aqueous solution of the AA-CUR bioconjugate on its concentration, (B) SEM image of AA-CUR micelles cross-linked with calcium ion (top) and the result of SEM/EDX analysis of the same sample (bottom).