Chondroitin-Sulfate-A-Coated Magnetite Nanoparticles: Synthesis, Characterization and Testing to Predict Their Colloidal Behavior in Biological Milieu

Biopolymer coated magnetite nanoparticles (MNPs) are suitable to fabricate biocompatible magnetic fluid (MF). Their comprehensive characterization, however, is a necessary step to assess whether bioapplications are feasible before expensive in vitro and in vivo tests. The MNPs were prepared by co-precipitation, and after careful purification, they were coated by chondroitin-sulfate-A (CSA). CSA exhibits high affinity adsorption to MNPs (H-type isotherm). We could only make stable MF of CSA coated MNPs (CSA@MNPs) under accurate conditions. The CSA@MNP was characterized by TEM (size ~10 nm) and VSM (saturation magnetization ~57 emu/g). Inner-sphere metal–carboxylate complex formation between CSA and MNP was proved by FTIR-ATR and XPS. Electrophoresis and DLS measurements show that the CSA@MNPs at CSA-loading > 0.2 mmol/g were stable at pH > 4. The salt tolerance of the product improved up to ~0.5 M NaCl at pH~6.3. Under favorable redox conditions, no iron leaching from the magnetic core was detected by ICP measurements. Thus, the characterization predicts both chemical and colloidal stability of CSA@MNPs in biological milieu regarding its pH and salt concentration. MTT assays showed no significant impact of CSA@MNP on the proliferation of A431 cells. According to these facts, the CSA@MNPs have a great potential in biocompatible MF preparation for medical applications.

The pristine Na2CSA and the H-ion exchanged CSA (H-CSA) were subjected to thermal analysis (Fig. S1). The Na2CSA and the H-CSA have a weight loss of ~12% and ~20%, respectively, up to 170°C caused by the evaporation of adsorbed water. The degree of sulfonation of Na2CSA was calculated as 0.95, from its ash content (Na2SO4) and the amount of evaporated water. Figure S1. TG curves of the pristine Na2CSA and the synthesized H-CSA samples.
We intended to characterize the dissociation degree of CSA polyelectrolyte. The primary result of potentiometric acid-base titrations is the sum of net proton consumption in all probable processes such as hydrolysis, for example, occurring in parallel with the protonation or deprotonation of the functional groups (e.g., −COOH ↔ −COO − + H + or -SO3H ↔ -SO3 -+ H + ).
The primary net proton consumptions of Na2CSA (Fig. S2) are in the positive range under pH ~6, i.e., protons are consumed by some alkaline species present in the solution of untreated CSA. We have experienced similar effect of base contamination in some non-monocationic forms of clays [1,2]. To get a well-defined initial state, we had to prepare a hydrogen-form of CSA (see in Materials section). The net proton consumption curves of H-CSA (Fig. S2) are in good agreement with the expected tendencies, i.e., the negative values even at the lowest pH indicate a small proton deficit on CSA due to dissociation of -COOH, which significantly rises up to pH ~7.
The net proton consumption curves show a characteristic ionic strength-dependence, i.e., the degree of dissociation of functional groups is influenced by not only the pH but also the salt concentration. The pH-dependent dissociation curves of polyelectrolytes are typically different from that of their monomers [3]. The functional groups are close to each other in a polyelectrolyte, so in the dissociated form, they generate an electrostatic field along the chain of the polyanion. The dissociation of the next proton from the already negatively charged chain is electrostatically hindered. Simultaneously, the ions of the indifferent electrolyte distribute in the local field; thus, they have a charge-screening effect proportional to the ionic strength. This explains the characteristic ionic strength-dependence of polyelectrolytes, i.e., at fixed pH, the degree of dissociation increases with the increasing ionic strength.
The value of the net proton consumption of H-CSA at the fully deprotonated range is ~ -2.05 mmol/g (Fig. S2), which corresponds to one mole of deprotonated functional group on one mole of repeating unit considering its molar mass (459 g/mol). During the hydrogen-form preparation, the pH of Na2CSA solution was set to ~1. The strongly acidic sulfate groups can be fully deprotonated even at this pH [40,41], so only the protonation/deprotonation of the -COOH/-COOgroups could be measured in the course of the titration. The evaluation of the Na2CSA's measurement data became possible by comparing the net proton consumption curves of Na2CSA and H-CSA, and assuming the hydrolysis of the sodium salt of CSA: Na2CSA + H2O ↔ NaHCSA + Na + + OH -. On the basis of this, Na2CSA releases ~0.95 mol OHper one mole of repeating units.

Characterization of colloidal stability
The critical coagulation concentration (CCC) of a naked and three CSA-coated MNP dispersions was determined by DLS method. The average hydrodynamic diameter (ZAve) was measured at 25±0.1 °C as a function of time at different salt concentrations (c) (see the kinetic curves of three systems in Figure S4). The initial slope of the kinetic curves is proportional to the coagulation rate (kactual) at the given salt concentration (see the slope values in the right columns in Figure S4). The coagulation rate is small at sufficiently small salt concentrations and increases with increasing salt concentration until reaching a maximum value and remains constant in the fast coagulation regime (kfast). The stability ratio (W) can be calculated as the ratio of the initial slopes belonging to the fast and slow coagulation for each salt concentration, i.e., Wactual=kfast/kactual.
The salt tolerance of the samples is given as the critical coagulation concentration (CCC) obtained from the stability ratio (log10W) vs. electrolyte concentration (log10 c) functions (see Fig. 6.) as the intersection point of the fast (Wactual~1) and slow (Wactual>1) coagulation regimes (marked with red arrows in Fig.6.).