2.4.3. Stability Index

Analytic centrifuge LUMiSizer (LUM GmbH, Dias de Sousa, Portugal), which accelerates the destabilization phenomena, was used to evaluate the simulated long-term physical stability of NLC. Briefly, the samples without prior dilution were placed in rectangular test tubes (optical path of 2 mm) and exposed to centrifugal force at 10,000 rpm, measuring 300 profiles in intervals of 10 s at 25 ◦C. These experiments allowed di fferentiation between various instability mechanisms at an accelerated rate. Extrapolated results were used to estimate dispersion shelf life in minutes. To simply assess the physical stability of NLC, the instability index was calculated by the delivered software (SepView 6.0; LUM, Berlin, Germany). The index was quantified by the clarification at a given separation time, divided by the maximum clarification, according to Ho ffmann and Schrader [37].

#### 2.4.4. Entrapment E fficiency

The drug entrapment e fficiency (EE) was determined by UV-visible spectrometry at 303 nm, using the Synergy ™ HTX Multi-Mode Microplate Reader (Biotek Instruments, Winooski, VT, USA). Briefly, NLC was placed in the dialysis bag (cuto ff 14 kDa) (Sigma-Aldrich, St. Louis, MO, USA). Then, the bags were placed in centrifuge tubes, covered with a mixture of ethanol and water 1:1 (*v*/*v*), and centrifuged for 1.5 h at 5000 rpm in centrifuge Laborzentrifugen 3K15 (Sigma, Osterode am Herz, Germany). The mixture of ethanol and water was analyzed for RSV content through the standard curve allowing the quantity of free drug to be determined. The encapsulated amount of RSV was calculated by subtracting the free amount of RSV from the total amount present in the dispersion. The measurements were performed in triplicate. The EE percentage was calculated by the Equation (1).

$$EE = \frac{(amount\ of\ initial\ RSV - amount\ of\ free\ RSV)}{amount\ of\ initial\ RSV} \times 100\tag{1}$$

## 2.4.5. Production Yield

The NLC formulations were frozen for 12 h at −80 ◦C and lyophilized at a pressure of 0.1 mbar for 24 h at −40 ◦C using a Telstar LyoQuest Freeze Dryer (Barcelona, Spain). The production yield (YNLC) was calculated by Equation (2).

$$Y\_{\rm NLS} \left( \% \right) = \frac{\left( \text{initial amount} - \text{final amount} \right)}{\text{initial amount}} \times 100 \tag{2}$$
