br apparent curcumin content with decreasing bioconjugate concentration were
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 Sphingosine-1-phosphate (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).
the PDI values increased, implying that the more complex (e.g. layered) structures appeared in the system .
Micellar structures formed by the AA-CUR bioconjugate were not mechanically strong and stiﬀ enough to allow their imaging by scan-ning electron microscopy. Thus we have reinforced them by the cross-linking process. Alginate can be physically cross-linked upon addition
of the calcium ions, due to the reorganization of the polymeric chains around the calcium ions and the formation of so-called egg-box struc-tures. A separate experiment was performed in order to adjust the op-timal calcium chloride concentration for the crosslinking process, using a series of solutions containing the same amount of the polymer and decreasing concentration of calcium chloride. Prior to the crosslinking