2.2. Permeation of Orange G into NaHA Solutions

NaHA was dissolved in albumin or HES solution and left for 24 h to solidify with gentle stirring at room temperature (25 °C). NaHA solutions were prepared at the following concentrations: (i) 0.1% (w/w) NaHA in 1.1% (w/v) albumin or HES solution; (ii) 0.2% (w/w) NaHA in 1% (w/v) albumin or HES solution; and (iii) 0.4% (w/w) NaHA in 0.8% (w/v) albumin or HES solution. In all solutions, the total concentration of solutes (i.e., NaHA plus colloids) was kept constant so as to minimize the effects of specific gravity of the solution on solute permeation. Colloid solutions without NaHA (1.2% (w/v) albumin/HES) were used as references. These NaHA concentrations fall within the range observed in the interstitial space (i.e., 0.1–1%) [1,2]. A HES concentration of 1% was chosen because this concentration falls within the range of plasma concentration in clinical settings (i.e., 1–2%) [10].

PBS solution containing 0.1 mL 0.01% (w/v) Orange G was gently poured over 1.4 mL NaHA solution in a semimicro ultraviolet (UV) cuvette (12.5 mm square; inner width, 4 mm; height, 45 mm; path length, 10 mm) (Figure 1). The amount of Orange G that permeated into NaHA solution was determined every 6 min for 20 h by measuring absorbance at 478 nm using a UV spectrometer (Model UV-1850; Shimadzu, Kyoto, Japan) (Figure 1). The temperature of the UV cuvette was maintained at 37 °C with a temperature control unit (Model TCC-100; Shimadzu, Kyoto, Japan). The top of the UV cuvette was sealed with laboratory film (Parafilm^®, Bemis Flexible packaging, Chicago, IL, USA) to prevent sample evaporation. Experiments were carried out at least in triplicate. Results were expressed as relative absorbance (i.e., relative to the absorbance value calculated assuming that Orange G is uniformly distributed into NaHA solution in the cuvette).

Experimental set-up for the measurement of solute permeation into sodium hyaluronic acid (NaHA) solution. A solution containing Orange G (0.1 mL) was poured over NaHA solution containing colloid (i.e., albumin or hydroxyethyl starch) in an ultraviolet (UV) cuvette.

For the quantitative analysis of Orange G permeation into NaHA solution, the amount of time it took for the relative absorbance of Orange G to reach 0.3 (T_0.3 in h) was determined in each NaHA solution relative to the reference solution (i.e., colloid solution without NaHA). Results were compared between NaHA solutions containing albumin and those containing HES with the unpaired t-test.