2.2.3. SEM Analysis

Imaging of the obtained CAS nanoparticles was performed using scanning electron microscopy (Prisma E SEM, Thermo Scientific, Waltham, MA, USA). The samples were loaded on a copper sample holder and sputter coated with carbon followed by gold using vacuum evaporator (BH30). The images were recorded at 15 kV acceleration voltage at various magnifications using DBS (back-scattered electrons) detector.

#### 2.2.4. FTIR Spectroscopy

The samples were evaluated for drug/polymer interactions by Fourier transformed infrared spectroscopy (FTIR). The spectra were collected using a Nicolet iS 10 FTIR spectrometer (Thermo Fisher Scientific, Pittsburgh, PA, USA), equipped with a diamond attenuated total reflection (ATR) accessory, operating in the range from 600 cm−<sup>1</sup> to 4000 cm−<sup>1</sup> with a resolution 4 nm and 16 scans. The obtained spectra were analysed with OMNIC® software package (Version 7.3, Thermo Electron Corporation, Madison, WI, USA).

#### 2.2.5. Differential Scanning Calorimetry (DSC)

Thermal analysis of the CAS nanoparticles was performed using DSC 204F1 Phoenix (Netzsch Gerätebau GmbH, Selb, Germany) based on the heat flux principle and cooled with a an intracooler. An indium standard (Tm = 156.6 ◦C, ΔHm = 28.5 J/g) was used for the temperature and heat flow calibration. The samples were hermetically sealed in aluminum sample pans. An empty pan, identical to the sample pan, was used as reference. The measurements were performed under argon atmosphere at a heating rate of 10 ◦C/min.

#### 2.2.6. In Vitro Drug Release

In vitro release study was carried out by diffusion using dialysis bag. The dialysis membrane (Sigma, MWCO 12,000 Da) was cut into equal pieces (6 × 2.5 cm2) and soaked in distilled water for 24 h before use. An accurately weighed amount of nanoparticles (equivalent to 10 mg BZ) was dispersed in 2 mL of PBS buffer (pH = 7.4) and transferred into the dialysis bag. Each bag was placed into a beaker containing 20 mL dissolution media (PBS buffer, pH 7.4) and kept on an electromagnetic stirrer at 50 rpm and 37 ± 0.5 ◦C. Samples of 2 mL were taken at predetermined time intervals and replaced with equivalent volume of fresh media. The samples were then filtered (0.45 μm Chromafil® syringe filter, Macherey-Nagel, Düren, Germany) and analyzed for drug content as mentioned above. Mean results of triplicate measurements and standard deviation were reported.

#### **3. Results and Discussion**

#### *3.1. Synthesis and Characterization of Blank Casein Nanoparticles*

Casein concentration and the crosslinking agent (CaCl2·2H2O, Mw = 147.01 g/mol) concentration were varied at three different levels according to the applied 3<sup>2</sup> full factorial design. Three different concentrations of casein solution were used: low concentration 0.05% (1), medium concentration 0.1% (2) and high concentration 0.15% (3). The concentration of the crosslinking agent was also set at three levels: low concentration 0.5 M (1), medium concentration 1.0 M (2) and high concentration 1.5 M (3). The other process parameters were kept constant as described in the Materials and Methods section. The dependent variables were particle size and production yields. The composition of the obtained casein nanoparticles with different formulation variables is shown in Table 1.

Nine batches of blank casein nanoparticles were obtained by spray drying technique. Mean particle sizes (Dv50, Table 1) varied in a wide range between 74.5 nm and 4.19 μm. The smaller particles showed extremely high degree of aggregation, leading to the formation of larger clusters, as evidenced from the scanning electron micrographs. Analysis of Dv10 was found to be far more representative for the particle size range of the formulated structures. According to the results, a tendency for reduction of the particle size was observed when the crosslinker concentration increased. According to the data published in the literature [42], the larger the amount of the crosslinker, the stronger the packing of the structure and the denser the micelle, resulting in particles of a smaller size range. Our results confirmed this relation, but the impact was not significant. As for the aggregation, clusters of nanoparticles occurred within each of the three groups of batches and no dependences could be outlined. For that reason, the combined effect of the two factors—the concentration of casein and the crosslinker—on the particle size was investigated, and the plot is shown in Figure 1. Since our goal was to produce particles of the smallest possible size, the batches revealing practically no or little degree of aggregation were considered for further investigation (samples prepared at casein concentration 1.5% and crosslinker concentration of 0.5 M proved to be unsatisfying).

Production yields, on the other hand, gradually increased when higher casein concentrations were used. The yields obtained varied in the range from 35.04% to 64.80%. Production yields were determinative for the selection of optimum models for drug loading and further investigation, therefore the combined effect of the two variables on the yields was studied. The plot is presented in Figure 2. The highest values were obtained when 1.5 M calcium chloride solution was used. Among all the formulated batches, Cas2-Ca3 (casein 1%, calcium chloride 1.5 M) was determined to be optimal in terms of production yield and desired particle size range.

**Figure 1.** 3D plot representing the impact of the concentration of the polymer and the crosslinking agent on the mean particle size of blank casein spray dried nanoparticles.

**Figure 2.** 3D plot representing the impact of the concentration of the polymer (%) and the crosslinking agent (M) on the particles production yields.

#### *3.2. Synthesis and Characterization of BZ Loaded Casein Nanoparticles*

BZ-loaded CAS nanoparticles were prepared via coacervation method, followed by spray drying. In order to investigate the effect of polymer and drug concentration over the production yield, particle size, surface morphology, drug entrapment efficiency and release behavior, four batches of drug-loaded nanoparticles were prepared based on the optimized formulation of blank nanoparticles (sample Cas2-Ca3, prepared at 1.0% casein concentration, 1.5 M CaCl2·2H2O) and varying the polymer/drug ratio (1:1, 2:1, 4:1, 6:1) (Table 2). The results of the study are summarized in Table 3.

#### 3.2.1. Drug Loading and Entrapment Efficiency

Drug loading of the developed BZ-loaded casein nanoparticles varied in a wide range from 16.02% to 57.41%. A tendency for decrease in drug loading was observed with increase of polymer/drug ratio, which was not surprising regarding the amount of polymer used for the formulation of the model particles. Entrapment efficiency was substantial, varying from 76.23% to 78.82% for the samples prepared at 2:1, 4:1 and 6:1 ratio. Significantly lower entrapment efficiency was determined for the sample Cas2-Ca3-BZ-1, prepared at 1:1 polymer/drug ratio. It could be suggested that the polymer had a limited capacity to incorporate drug molecules during nanoparticles formulation. For the above sample, the amount of the polymer was probably not sufficient to entrap and retain the drug and form a stable structure.

Our hypothesis was confirmed by morphological analysis of the samples using scanning electron microscopy (Figure 4). The lack of clearly defined nanostructures in model Cas2-Ca3-BZ-1 was evidenced by the obtained scanning electron micrographs in contrast to the other samples. In addition, the larger amount of BZ in this sample probably led to displacement of calcium phosphate and disruption of micellar integrity. The phenomenon has been observed in other studies and has been thoroughly described in the literature [43]. With an increase of the polymer/drug ratio from 1:1 to 2:1, a double increase of the EE was observed (Table 3). Higher amounts of casein led to more efficient incorporation of benzydamine in the nanoparticles, which is probably due to the enhanced hydrophobic effect favoring micellar solubilization of the drug [19]. A further increase in the polymer/drug ratio (4:1 and 6:1) did not lead to a significant change in the drug entrapment efficiency.

#### 3.2.2. Production Yield

Production yields were high, ranging from 58.23% to 74.71% except for the batch produced at 1:1 polymer/drug ratio (34.61%). The increase in the amount of casein in the formulations, relative to BZ, led to a slight reduction of production yields, which was probably due to the enhanced viscosity of the feeding suspension, which made it difficult to pass through the spray mesh. On the other hand, batch Cas2-Ca3-BZ-1, although expected to provide the highest yield, refuted our suggestions. A possible explanation for this could be the disruption of micellar integrity due to displacement of calcium phosphate and the formation of precipitate prior to spray drying [44].

**Table 3.** Characteristics of the spray dried BZ-loaded casein nanoparticles (n = 3). DL = drug loading, EE = entrapment efficiency.


#### 3.2.3. Particle Size and Size Distribution

Particle size and size distribution were analyzed by dynamic light scattering and the results are presented in Table 3 and Figure 3. The median particle size ranged from 135.9 nm to 994.2 nm with a clear tendency for size reduction with increase in casein concentration. Bimodal particle size distribution was observed in batch Cas2-Ca3-BZ-1, suggesting a high aggregation tendency. However, no clearly distinguished structures were observed under scanning electron microscope, corresponding to the results obtained for production yield, drug loading and entrapment efficiency. Probably, nanoparticle formation could not be accomplished at 1:1 polymer/drug ratio, whereas the samples prepared at 2:1, 4:1 and 6:1 polymer/drug ratio were clearly distinguished and less cohesive, with minimal degree of aggregation.

**Figure 3.** Dynamic light scattering histograms of BZ loaded casein nanoparticles of batches Cas2- Ca3-BZ-1 (**A**), Cas2-Ca3-BZ-2 (**B**), Cas2-Ca3-BZ-4 (**C**) and Cas2-Ca3-BZ-6 (**D**).

#### 3.2.4. Surface Morphology

Surface morphology evaluation of the four batches of nanoparticles was performed using scanning electron microscopy. The micrographs are presented in Figure 4. Three different patterns of surface morphology were observed: rough spherical particles, wrinkled spherical particles and wrinkled irregularly shaped particles. A tendency towards increased surface roughness was observed with raising casein concentrations. Irregular, wrinkled, fragmented, and highly aggregated structures with an average particle size of about 994 nm were observed at drug/polymer ratio 1:1 (Figure 4A, batch Cas2-Ca3-BZ-1). It is well known that inlet temperature plays a key role in the spray drying process, significantly affecting the surface morphology of the dry particles. According to Both et al. [45], spray drying at high inlet temperatures generally results in the formation of less wrinkled particles with a large, hollow core. Therefore, it could be assumed that the higher viscosity of the feeding suspension together with the low inlet temperature (40 ◦C) might be associated with increased stickiness and subsequent agglomeration of these particles. As for the other three batches of nanoparticles, they all had a rounded shape and a wrinkled surface. In addition, as the concentration of the polymer raised and the percentage of drug diminished relative to the casein content, the rugosity degree of particles increased. It could be assumed that the lower drug content per unit mass led to the formation of loose matrix structures. Upon drying, these structures shrink, leading to the formation of smaller particles with multiple surface invaginations. Our hypothesis was confirmed by particle size analysis.

**Figure 4.** SEM micrographs of BZ-loaded casein nanoparticles of batches Cas2-Ca3-BZ-1 (**A**), Cas2- Ca3-BZ-2 (**B**), Cas2-Ca3-BZ-4 (**C**) and Cas2-Ca3-BZ-6 (**D**) at 25,000× magnification.
