*2.2. Characterization*

2.2.1. Production Yield, Drug Loading and Entrapment Efficiency

The production yields of the nanoparticles from different batches were calculated using the weight of the spray dried nanoparticles with respect to the initial quantity of the drug and polymer, according to the following equation:

$$\text{Production yield } (\%) = \frac{\text{Spy dry dried nanopparticles (mg)}}{\text{Drug (mg)} + \text{Polymer (mg)}} \tag{1}$$

Drug loading and drug entrapment efficiency of BZ-loaded CAS nanoparticles were determined spectrophotometrically. BZ-loaded nanoparticles were dispersed into 10 mL previously acidified at pH 2 deionized water and were stirred for 60 min until complete swelling of casein micelles occurred, allowing BZ extraction in the aqueous medium. Then, the blend was centrifuged at 5000 rpm and filtered (0.22 μm, Chromafil®, Macherey-Nagel, Düren, Germany). Drug concentration was determined after proper dilution using an Evolution 3000 Pro UV/Visible spectrophotometer (Thermo Scientific, Waltham, MA, USA) at a wavelength of 306 nm. The drug loading (DL) was calculated according to Equation (2), and entrapment efficiency (EE) was calculated according to Equation (3):

$$\text{DL} \left( \% \right) = \frac{\text{Amount of drug in the formulation}}{\text{Total amount of nanopparticles}} \times 100 \tag{2}$$

$$\text{EE } (\%) = \frac{\text{Actual drug content}}{\text{Theoretical drug content}} \times 100\tag{3}$$

#### 2.2.2. Particle Size Analysis, Size Distribution and Zeta Potential

Particle size of the obtained CAS nanoparticles was determined by dynamic light scattering method using Nanotrac particle size analyzer (Microtrac, York, PA, USA). The system is equipped with 3 mW helium/neon laser at 780 nm wavelength and measures the particle size with noninvasive backscattering technology, performing particle size analysis in the range of 0.8 nm to 6.5 μm. The equipment allows determination of ζ-potential in the range from −200 mv to +200 mV. The samples were prepared by dispersing a small amount of dry nanoparticles in purified water, and the dispersions (refractive index 1.33, average viscosity 0.87 ± 0.05 cP) were stirred on a magnetic stirrer and then analysed for particle size and zeta potential. All the measurements were performed at 25.0 ◦C at 20-s intervals and were repeated three times.
