**1. Introduction**

Over recent decades there has been a growing scientific interest towards the use of naturally occurring materials for drug delivery purposes. This is mainly due to their numerous advantages over synthetic materials, namely biocompatibility, biodegradability, and low immunogenicity [1]. Moreover, natural materials produce non-toxic metabolites, unlike synthetic polymers which can be contaminated with unreacted toxic monomers and crosslinkers [2]. Due to their specific structure and corresponding features, naturally occurring materials such as polysaccharides and peptides are widely used for the formulation of micro- and nanoparticulate drug delivery systems. These carriers can provide controlled and targeted release, thus improving the therapeutic performance of the encapsulated drug and minimizing the risk of side effects [3]. Amongst the potential biopolymers, proteins are preferred as natural drug delivery systems due to the relatively easy preparation processes and production of well-defined structures, which enables surface modification and may provide modified and targeted release [1]. Among proteins, casein (CAS) is considered a suitable biopolymer for the preparation of nanoparticulate drug delivery systems due to its structural and physicochemical characteristics [2].

Casein is a collective term used to define a family of calcium (phosphate)-binding phosphoproteins commonly found in mammalian milk [4]. Casein from bovine milk

**Citation:** Zahariev, N.; Marudova, M.; Milenkova, S.; Uzunova, Y.; Pilicheva, B. Casein Micelles as Nanocarriers for Benzydamine Delivery. *Polymers* **2021**, *13*, 4357. https://doi.org/10.3390/ polym13244357

Academic Editors: Ariana Hudita and Bianca Galˇ a¸ˇteanu

Received: 24 November 2021 Accepted: 10 December 2021 Published: 13 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

is composed of four peptides, namely αs1, αs2, β, and k, which differ in the content of amino acids, phosphorus, and carbohydrates, but they are all amphiphilic in nature [5]. Cysteine amino acid residues that allow the formation of disulfide bonds are found only in the polypeptide chains of k-casein. In general, the peptide surface is negatively charged due to phosphorylation [1]. The lack of secondary structures because of the proline-rich amino acid sequence [6] and the tendency for binding amorphous calcium phosphate cause electrostatic, hydrogen, and hydrophobic interactions, leading to self-assembly of the casein peptides into stable agglomerates known as casein micelles [7]. The inner part of the micelle is composed of αs1, αs2, and β caseins, whereas the outer layer that stabilizes the micelle contains glycosylated k-casein [8]. Casein micelles exhibit pH-dependent behavior. Their structure tightens when the negative surface charge of casein molecules decreases, and expands with increasing surface charge, which leads to electrostatic repulsion between the molecules [9–11]. Given the amphiphilic properties and pH-dependent behavior of casein, and its ability to participate in hydrophobic and hydrophilic interactions, it is clear why this biopolymer has found a place in scientific research as a potential nanoparticle drug delivery carrier.

Various methods have been reported for the preparation of casein nanoparticles for drug delivery, including pH-shifting [12], high pressure homogenization [13–18], electrostatic complexation [19], solvent displacement [20], emulsification solvent evaporation [21], and spray drying [22–24]. Nano spray drying, a variation of the established spray drying technology used to convert liquids into solid powders, is a relatively new technique adopted for the preparation of nanosized drug delivery systems. The method is based on the use of a revolutionary sprayer developed by the Swiss Büchi Labortechnik AG, which is equipped with a piezoelectric vibrating spray mesh head, allowing the formation of fine droplets, which are dried and electrostatically collected [25]. As a result, spherical submicron structures of particle size below 1000 nm with improved biopharmaceutical behavior are obtained [26–33]. Although spray drying of proteins has been reported in numerous scientific papers [34–37], no data on nano spray drying of casein have been found in the literature. The technology was therefore a research challenge. For the present study, benzydamine hydrochloride (BZ) was used as a model drug.

Benzydamine hydrochloride is a nonsteroidal anti-inflammatory drug with local anesthetic and analgesic properties for pain relief and treatment of inflammatory conditions of the mouth and throat such as oral mucositis, postoperative sore throat and mucosal ulcers. The mechanism of the anti-inflammatory effect of benzydamine has not yet been fully understood. According to Quane et al. [38], the anti-inflammatory activity of benzydamine may be due to its membrane-stabilizing or inhibitory effect of the synthesis of TNF-α. Unlike NSAIDs, which have acidic properties, benzydamine is a weak base, highly lipidsoluble in its unionized form [39].

According to Beckett et al. [40] and Bickel et al. [41], only a limited amount of weak, basic, lipid-soluble drugs is absorbed into buccal tissue via mouthwash application. The small degree of absorption into buccal tissue is confirmed by the poor systemic availability (5%) [42]. To enhance absorption and thus bioavailability, benzydamine hydrochloride was incorporated into nanoparticles. Due to the specific binding properties and pHdependent drug release, casein is considered a promising biopolymer for the preparation of benzydamine loaded casein nanoparticles.

The aim of the present work was to optimize the process parameters of the nano spray drying technique for the formulation of BZ-loaded casein nanoparticles. Furthermore, an investigation of the effect of process variables on structural and morphological characteristics and release behavior was conducted.

#### **2. Materials and Methods**

Benzydamine hydrochloride (Mw 345.87 g/mol), sodium caseinate (from bovine milk) and CaCl2·2H2O (Mw 147.01 g/mol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.

#### *2.1. Preparation of Blank Casein Nanoparticles and BZ-Loaded Casein Nanoparticles*

The blank casein nanoparticles where prepared via coacervation followed by spray drying using nano spray dryer Büchi B-90 (Büchi Labortechnik AG, Flawil, Switzerland), as previously reported by Gandhi et al. [2]. A certain amount of sodium caseinate was dissolved in 100 mL deionized water, previously adjusted to pH 2 with 1M hydrochloric acid. Then, the crosslinking agent CaCl2·2H2O (2 μL/mL) was added dropwise to the casein solution under high-speed homogenization at 25,000 rpm (Miccra MiniBatch D-9, MICCRA GmbH, Heitersheim, Germany) for 15 min and casein micelles were produced. The obtained nanosuspension was then stirred on a magnetic stirrer at 500 rpm for 30 min) to allow effective crosslinking of casein molecules. Finally, the suspension was spray dried using nano spray dryer Büchi B-90 under the following predetermined conditions: mesh size of 4.0 μm, inlet temperature 40 ◦C, solution feed rate 50%, spray intensity 70%, drying gas speed 120 L/min, pressure 30 nbar. To study the effect of different formulation variables on the produced particles, 32 full factorial design was applied. Nine batches of formulations were prepared at varied protein and crosslinker concentrations (Table 1).

**Table 1.** Composition and characteristics of blank casein nanoparticles (n = 3, PDI (polydispersity index), Dv10, Dv50 and Dv90 (10, 50 and 90% of the total volume of particles, respectively, are with size below the indicated value).


BZ-loaded casein nanoparticles were prepared following the methodology described in the previous paragraph. Briefly, protein aqueous solution (1.5% *w*/*v*) was prepared by dissolving a certain amount of sodium caseinate in deionized water, previously acidified to pH 2 using 1N hydrochloric acid. Then, BZ was added to the solution, followed by protein crosslinking with CaCl2·2H2O (2 μL/mL) at a stirring rate of 25,000 rpm. The procedure continued as previously described. Four batches of drug-leaded formulations were developed at varied drug–polymer ratios. The composition of the batches is presented in Table 2.

**Table 2.** Composition of BZ-loaded casein nanoparticles of various batches.

