3.2.5. Differential Scanning Calorimetry (DSC)

The phase state of BZ incorporated into the spray dried casein nanoparticles was analyzed using differential scanning calorimetry (DSC). The obtained thermograms are presented in Figure 5. The thermogram of casein revealed a broad endothermic peak at 84.8 ◦C, which could be attributed to the evaporation of water present in casein micelles. Benzydamine hydrochloride, on the other hand, being solid crystalline, showed a characteristic peak at 166.5 ◦C, which corresponds to its melting point. In drug-loaded samples, a gradual decrease in peak intensity was observed, with an increase in the polymer/drug ratio from 1:1 to 6:1, as shown in Figure 5C–F. It can be assumed that changes occurred in the degree of crystallinity of BZ during spray drying and the drug was partially transformed into its amorphous phase depending on the drug content in the formulated nanoparticles.

**Figure 5.** DSC thermograms of Benzydamine hydrochloride (**A**), blank casein nanoparticles (**B**), BZ-loaded casein nanoparticles of batches Cas2-Ca3-BZ-1 (**C**), Cas2-Ca3-BZ-2 (**D**), Cas2-Ca3-BZ-4 (**E**) and Cas2-Ca3-BZ-6 (**F**).

#### 3.2.6. Fourier-Transform Infrared Spectroscopy (FTIR)

The spectra for casein (Figure 6) show peaks at 1646 cm−<sup>1</sup> in the amide I region and 1530 cm−<sup>1</sup> in the amide II region, which could be assigned to the stretching of the carbonyl group (C=O) and to the symmetric stretching of N-C=O bonds, respectively. Casein shows a band at 1077 cm−1, suggesting interactions of monocationic phosphates with Na+. The peaks in the amide I and amide II regions also appear in the crosslinked nanoparticles and in the drug loaded nanoparticles. The band at 977 cm−1, attributed to bionic phosphate, has very low intensity on the casein spectrum and increased intensity in the crosslinked systems, suggesting interaction with Ca2+. Characteristic bands for stretching of aromatic C=C at 1497 cm−<sup>1</sup> of benzydamine hydrochloride can be seen in the spectra of the drug-loaded nanoparticles. The band at 1357 cm−<sup>1</sup> is attributed to C-N vibrations of the heterocyclic ring.

**Figure 6.** ATR-FTIR spectra of casein, crosslinked placebo nanoparticles and crosslinked BZ-loaded nanoparticles.

#### 3.2.7. In Vitro Drug Release

The dissolution profiles of BZ from casein nanoparticles are presented in Figure 7. The percentage of released drug during 5-h study was incomplete, varying from 73.30% (Cas2- Ca3-BZ-4) to 91.81% (Cas2-Ca3-BZ-1). Initial burst effect was observed in models Cas2-Ca3- BZ-1 and Cas2-Ca3-BZ-2 in the first 60 min, releasing more than 50% of the encapsulated drug. This was probably due to the higher drug loading and the accumulation of BZ in the periphery of the nanoparticles during the process of spray drying. The batches Cas2-Ca3- BZ-4 and Cas2-Ca3-BZ-6 demonstrated prolonged drug release over time, releasing almost 75% of the incorporated benzydamine. It was probably the greater amount of casein per unit mass in these two batches that refrained the drug from free diffusion from the particle core to the periphery, despite the larger surface area available for dissolution.

**Figure 7.** In vitro BZ release from spray dried casein nanoparticles prepared at different polymer/drug ratios (n = 3).

#### **4. Conclusions**

In this study, self-assembled casein nanocarriers were produced by nano spray drying. The process parameters were investigated, and an optimized model of blank casein nanostructures was outlined. Furthermore, four batches of BZ-loaded nanoparticles with a particle size from 135.9 nm to 994.2 nm were developed. BZ loading in the nanoparticles depended on the polymer/drug ratio. BZ was transformed from crystalline into amorphous during spray drying, which implies an increased dissolution rate. The drug release study confirmed the feasibility of the developed nanocarriers for prolonged delivery of benzydamine.

**Author Contributions:** Conceptualization, B.P. and M.M.; methodology, N.Z., S.M. and Y.U.; investigation, N.Z., B.P. and M.M.; writing—original draft preparation, N.Z.; writing—review and editing, B.P.; visualization, N.Z., Y.U. and M.M.; supervision, B.P.; project administration, M.M.; funding acquisition, M.M. and B.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors appreciate the financial support of the Bulgarian National Science Fund (BNSF) via Project KP-06-N38/3 for ensuring the chemicals and consumables.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors acknowledge Project BG05M2OP001-1.002-0005-C01 Centre of competence for Personalised Innovative Medicine PERIMED for providing access to the scientific infrastructure and sophisticated equipment for this work.

**Conflicts of Interest:** The authors declare no conflict of interest.
