Megafuge 16r centrifuge

The stability of the CNCs/FA-CS-FITC nanosystems was evaluated in simulated non-physiological and physiological conditions, namely at pH 2.1 (aqueous solution of 0.01 M HCl) and at pH 7.2 (PBS), for 24 h and 48 h [32 (link)]. Typically, 1 mg of CNCs/FA-CS-FITC was added to vials containing 2 mL of the two media. Then, the suspensions were placed on an orbital shaker at 37 °C in the dark for 24 h and 48 h. After these periods, the suspensions were centrifugated at 13,400 rpm during 5 min (Megafuge 16R centrifuge, Thermo Scientific, Waltham, MA, USA). The absorbance spectra of the corresponding supernatants were recorded on a Thermo Scientific Evolution 220 UV-visible spectrophotometer (Thermo Scientific, Waltham, MA, USA) to quantify the amount of the derivative that was released (calibration curve: $y=2.2591x-0.0141, R^2=0.9995$ ), concentration range of the FA-CS-FITC derivative: 0.005–0.5 mg mL⁻¹).

The pre-treated swim bladders were digested in 0.5 M acetic acid containing 2% pepsin (w/w) at a 1:40 (w/v) tissue/solution ratio. The mixture was continuously stirred at 4 °C for 24 h. After digestion, the viscous extract was filtered with two layers of cheesecloth and precipitated by adding NaCl to a final concentration of 1.2 M. The precipitate was collected by centrifuging at 16,000× g at 4 °C for 20 min using a Megafuge 16R centrifuge (Thermo Scientific Co., Waltham, MA, USA), and the resulting pellets were dissolved in 0.5 M acetic acid. This solution was purified using a dialysis membrane (14 kDa molecular weight cut-off) against distilled water for 72 h with a change of solution every 4 h. The resulting collagen was lyophilized (Free Zone 2.5 L, Labconco Corp., Kansas, MO, USA) and stored at −20 °C until further analysis.
The collagen yield was calculated based on the wet and dry weights of the raw material before and after processing, using Equation (1).
$$\mathrm{Yield}\left(\%\right)=\left[\frac{\mathrm{Wc}\left(\mathrm{g}\right)}{\mathrm{Wd}\left(\mathrm{g}\right)}\right]\times 100$$
where Wc is the weight of the lyophilized collagen and Wd is the dry weight of the initial swim bladder prior to pre-treatments.

The CNCs were functionalized with the FA-CS-FITC derivative via electrostatic assembly. Briefly, a solution of FA-CS-FITC derivative (1, 2 or 4 mg in 40 mL of 1 M acetic acid aqueous solution) was added dropwise to a suspension of CNCs (50 mg in 10 mL of ultrapure water), as summarized in Table 1. The mixture stood for 1 h under magnetic stirring in the dark. Afterwards, the suspension was centrifuged (13,400 rpm, 10 min, Megafuge 16R centrifuge (Thermo Scientific, Waltham, MA, USA)) and washed one time with 1 M acetic acid aqueous solution and three times with ultrapure water, followed by freeze-drying, which yielded solid nanosystems with an orange coloration.