br and micelles Conjugation of curcumin to a
 and micelles [20–22]. Conjugation of curcumin to a hydrophilic polymer is another way of tackling this problem. Polymer conjugates have an advantageous drug-to-excipient ratio compared to other, mentioned above systems, what reduces their potential to induce un-desired side eﬀects. Additionally, the upscaling and production process is often less complex than for nanoparticle-based systems. Polymer conjugates often show improved bioavailability, pharmacodynamics and biodistribution . Various conjugates of curcumin have been already described in literature, including synthetic polymers (e.g. poly (ethylene glycol (PEG) [24,25] and its derivatives , poly(lactic-co-glycolic Suramin hexasodium salt (PLGA) ), albumin  and natural polysaccharides: dextran , hyaluronic acid , and carboxymethyl chitosan  and alginate . Binding curcumin to the hydrophilic macromolecule led to the increase in its solubility up to the defined degree of sub-stitution which varies depending on molecular mass, polarity and charge of the polymeric carrier. Being highly hydrophobic, curcumin
Corresponding author at: Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland. E-mail address: [email protected] (A. Karewicz).
D. Lachowicz et al.
attached to the polymeric chain introduces amphiphilic character to the hydrophilic polymer chain and tendency to form micelles. Indeed, mi-cellar aggregate formation was observed for curcumin-dextran , curcumin-hyaluronic acid  and curcumin-carboxymethyl chitosan
 bioconjugates. The average size of the aggregates was around 200–300 nm, although, as expected, it varied slightly between diﬀerent polymers used.
Sodium alginate, a natural polysaccharide obtained from the brown seaweeds, is a highly hydrophilic, negatively charged polymer, known for its biocompatibility and biodegradability. It is widely used in food industry and in biomedical applications. It has a relatively high number of carboxylic groups, what makes it an ideal polymer for curcumin conjugation, as it should allow to reach high substitution degrees while maintaining suﬃcient charge. That is expected to facilitate preparation of highly curcumin-loaded and stable micellar dispersion systems. Dey and Sreenivasan  obtained the alginate-curcumin bioconjugate and performed some preliminary in vitro studies on its cytotoxicity. The aggregates formed by the bioconjugate were, however, significantly larger than those described for dextran or hyaluronic acid. Sarika et al.
 have described galactosylated alginate-curcumin bioconjugate. The micelle aggregates formed in aqueous solution by that bioconjugate were in the range of 235 nm. The biological studies performed by these authors were limited to cytotoxicity and cellular uptake.
Here we propose an attempt to obtain the alginate-curcumin bio-conjugate system able to form micelles of the smaller size than reported before and characterized by good colloidal stability. The smaller size of curcumin-containing micellar aggregates should increase their biolo-gical lifetime and allow for their enhanced permeability and retention in tumor tissue (EPR eﬀect), increasing curcumin’s eﬃcacy as anti-cancer agent. For the synthesized conjugate we have studied the release of curcumin from its micelles crosslinked with calcium ions. The ex-tended in vitro studies on biological activity of bioconjugate micelles against several selected cancer cell lines were then performed. The protein binding of conjugate and the biocompatibility of the studied system with human blood cells were also tested. Based on these new information we have attempted to give a better insight into the possible application of the bioconjugate.
Curcumin (≥94% curcuminoid content, ≥80% curcumin), alginic acid sodium salt from brown algae (low viscosity) (AA), N,N′-dicyclo-hexylcarbodiimide (DCC, ≥99% (GC)), 4-(dimethylamino)pyridine (DMAP, ≥98% (NT) and calcium chloride were purchased from Sigma-Aldrich, Poland. Gel protein 100 assay was provided by Cormay, Poland. Spectroscopic grade methanol, dimethyl sulfoxide (DMSO) (p.a.), acetic acid, and oleic acid (p.a.) were purchased from POCH, Gliwice, Poland. Murine brain endothelial (MBE) cells were gift from R. Auerbach (University of Wisconsin, Madison, WI, USA). Murine cell lines that stably express human carcinoembrionic antigen: colon car-cinoma CT26-CEA and colon adenocarcinoma MC38-CEA were gift from Dr. Michał Bereta (Jagiellonian University, Kraków). The 4T1 mammary carcinoma (ATCC CRL-2539) was obtained from Dr. Gary Sahagian’s lab (Tufts University, Boston). Reagents used for the cell culture were provided by Lonza. Ficoll-Paque Plus used for PBMC iso-lation was obtained from GE Healthcare. The MTT reagent (3-[4,5-di-methylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) and Drabkin’s reagent were purchased from Sigma-Aldrich. Triton X 100, NaCl and ethanol were from Bioshop. Human peripheral blood from healthy donors was purchased from Regional Centre of Blood Donation and Blood Treatment in Krakow, Poland.