1 m hydrochloric acid

Sulfated HA was dissolved in 1 M hydrochloric Acid (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 1 mg/mL in a 1.5 mL Eppendorf tube and heated on a heating block for three hours at 100 °C. Then, 20 µL of each sample was mixed with 380 µL of trichloroacetic acid at a concentration of 4% and 100 µL of barium chloride + gelatin solution (Sigma-Aldrich, Saint Louis, MO, USA). BaCl gelatin solution was prepared by dissolving gelatin in water at 70 °C with vigorous stirring at a concentration of 0.5% w/v. The gelatin solution was allowed to sit overnight, after which BaCl was dissolved at a concentration of 0.5% w/v with vigorous stirring and no heating. The solution was allowed to sit for at least 2–3 h before use and discarded and remade after 7 days. The mixture of sample and BaCl + gelatin was allowed to sit for 20 min, after which it was vortexed again and 200 µL of each sample was pipetted into a black, 96-well, clear bottom, non-binding plate and the absorbance was measured at 360 nm on a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA, USA).

Particles with varying molecular weight HA and varying degrees of sulfation were synthesized as previously described. The initial pH of the solution was measured. The pH was adjusted using 1 M hydrochloric acid (Sigma-Aldrich, Saint Louis, MO, USA) in pH increments of 0.5 and the size distribution and zeta potential were measured at each increment as previously described. Samples which went below the pH target during this process were discarded and the process was started over.

European Pharmacopoeia (Ph. Eur.) grade carvedilol (molecular mass = 406.5 g/mol) was purchased from INRESA (Barthenheim, France). The following CDs were supplied by Wacker Chemie AG (Munich, Germany): α-cyclodextrin (αCD, Cavamax W6 Pharma), β-cyclodextrin (βCD, Cavamax W7 Pharma), γ-cyclodextrin (γCD, Cavamax W8 Pharma), randomly methylated β-cyclodextrin (RAMEB, Cavasol W7 M; degree of substitution (DS) = 1.6–1.9) and hydroxypropyl-β-cylodextrin (HPβCD, Cavasol W7 HP, DS = 0.6–0.9). Hydroxypropyl-γ-cyclodextrin (HPγCD, DS = 0.6), sodium acetate, acetic acid, trisodium citrate dihydrate, citric acid monohydrate, and 1 M hydrochloric acid were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Per-2,6-di-O-methyl-β-CD (DIMEB) was synthesized as described previously [19 (link)], and its measured properties were in line with the literature data. Deuterated water was purchased from Sigma-Aldrich. The water used was MilliQ grade. All solvents used were analytical grade.