Formulation of Casein Hydrogels ^†

Abstract

Protein-based hydrogels have attracted considerable interest due to their biocompatibility, nontoxic properties, biodegradability, and renewable nature, as well as their being inexpensive and easy to obtain. Hydrogel properties depend on the temperature, polymer concentration, pH, crosslinking levels, salt concentrations, and aging. Casein is a natural protein present in bovine milk (about 80%), which exists in the form of various micelles. It is composed of α-s1, α-s2, β-, and κ-casein and tends toward self-assembly. Casein-based hydrogels are suitable for use in biomedical applications. Considering their potential applications in the field of medicine, in this work, our objective is to find the best conditions for the development of a casein hydrogel with tetracaine hydrochloride as the active compound. The tetracaine hydrochloride has anesthetic properties; therefore, it would allow for a painless and comfortable treatment to be offered to the patient. Accordingly, different hydrogel formulations were proposed. The selected components were casein, glycerol, tetracaine hydrochloride, potassium carbonate, and sodium alginate. Stability and swelling tests was carried out, and apparent density, pH, and moisture content were investigated. The formulation that allowed us to obtain hydrogel with the desired properties was composed of tetracaine hydrochloride 1%, casein 2%, glycerol 50%, sodium alginate 4%, and potassium carbonate solution 18% (the percentages use the casein as the basis).

1. Introduction

Hydrogels are polymer materials with a three-dimensional network structure containing water, similar to soft tissues [1].

Biopolymer hydrogels are derived from plants and animals, and are versatile materials typically assembled from entangled or crosslinked proteins and/or polysaccharides [2]. These hydrogels are a promising class of materials as they can respond to external cues such as temperature, pH, and light [3]. In addition, they have excellent functional properties, an amphiphilic nature, and are biocompatible and biodegradable, as well as being easily renewable for other uses and showing lower toxicity in comparison with synthetic polymers [4].

Protein and polysaccharide hydrogels are generally induced via physical (heating, cooling, shear forces) and chemical (pH modulation, salt addition) induction techniques, or a mixed technique, to achieve the desired properties [5]. Currently, diverse synthesis strategies enable the preparation of functional hydrogels with a range of applications [1]. Hydrogels have numerous applications in medicine, agriculture, cosmetics, and the food industry. They are also used in personal care products, filters for water purification, and separation membranes [6].

Hydrogels can be formulated by using a wide range of polymers, including those of food origin. The benefits of hydrogels made from food-grade biopolymers include their safety, low cost, and commercial availability. One possible way of creating food-based hydrogels is through the use of caseins, alone or in combination with other food-grade polymers [7].

Four types of caseins are mainly present in bovine milk: αS1, αS2, β, and κ. These proteins adapt their structure to changes in environmental conditions and assemble to form colloidal aggregates, called casein micelles [8].

Tetracaine hydrochloride, TH, is an amphiphilic compound that also possesses colloidal properties and is one of the most-used local anesthetic drugs. Since tetracaine is a poorly water-soluble compound, it is usually formulated as tetracaine hydrochloride [9]. Considering the pharmacological properties of tetracaine, which can reduce pain caused by skin irritations from minor burns, we selected this compound for the development of a hydrogel for topical use.

In this study, different casein hydrogel formulations were prepared by changing the concentrations of the reagents. The hydrogels were characterized at the macroscopic level and their stability at different temperatures was evaluated.

2. Materials and Methods

2.1. Materials

Commercial micellar casein (CA) from bovine milk, glycerol (C₃H₈O₃) (Gly), potassium alginate [(C₁₂H₁₄CaO₁₂)_n] (PAlg), and tetracaine hydrochloride (TH) were obtained commercially. A buffer of pH 10.6 [anhydrous potassium carbonate, K₂CO₃] (AnP) was prepared using deionized water.

2.2. Preparation of a Casein Hydrogel

The preparation of hydrogels was based on the previous literature [10]. A casein solution (10 wt.%) with tetracaine was prepared by dissolving the protein in a tetracaine solution in potassium carbonate buffer (pH 10.6) at 60 °C under magnetic stirring. Then, glycerol and potassium alginate were added at different concentrations (

Table 1. Hydrogel formulations (wt. %).

Form. N°	TH (%)	CA (%)	AnP (%)	Gly (%)	PAlg (%)
1	-	9.49	85.39	4.74	0.38
2	1.86	9.31	83.8	4.66	0.37
3	1.87	9.33	83.96	4.66	0.19
4	0.1	9.5	85.3	4.70	0.40
5	0.07	7.4	88.56	3.69	0.30
6	0.07	7.1	85.41	7.12	0.28

). The pH of the mixture was adjusted to 7 with acetic acid. After mixing the mixture for 30 min and cooling at 4 °C for 1 h, the hydrogels were obtained.

2.3. Caracterization of Hydrogels

The general appearance of the hydrogels was evaluated by analyzing the physical aspect, apparent density, pH, moisture content, swelling, and stability, considering the bibliography. The composition of tetracaine in the hydrogels was obtained via analysis with UV-Vis spectroscopy.

3. Results

To obtain hydrogels of adequate consistency, different formulations were tested, changing the concentration of TH (0.07 to 1.87%), CA (7.1 to 9.5%), AnP (83.8 to 88.56%), Gly (3.69 to 7.12%), and PAlg (0.19 to 0.40%).

Figure 1 shows the obtained tetracaine hydrogels. The consistency of these formulations was evaluated at room temperature (20 °C) and at low temperature (4 °C). All formulations were initially viscous and firm; however, after 24 h, only formulations 1–4 maintained these properties. Considering that these were the best formulations, we continued with the characterization of these hydrogels.

Table 2. Hydrogel formulations (wt. %).

Form. N°	Density (%)	pH (%)	Moinsture (%)	Swelling (%)	Tetracaine (%)
1	0.51	7.1	94	5.0	0
2	0.57	7.5	92	4.5	1.9
4	0.55	7.4	93	4.5	1.8
5	0.56	7.1	91	4.2	0.2

shows the results of the quality parameters of the casein hydrogels. Considering formulation 1 as the control, the other formulations did not show significant changes in their properties following the addition of tetracaine. In all cases, the incorporated tetracaine content was in line with the theoretical amount.

The swelling ratio of all hydrogels varied from 4.2 to 5.0 after 24 h, indicating that they are suitable for topical treatment. This result is consistent with that obtained by [11].

In addition, release assays were performed in a phosphate buffer pH = 7.5, in which it was possible to evaluate that tetracaine is completely released from the hydrogels in less than one hour of testing.

4. Conclusions

In this work, casein, potassium alginate, glycerol, and tetracaine hydrochloride were used to formulate hydrogels. In these formulations, the apparent density was approximately 0.5, the pH was approximately 7, the moisture content was approximately 90%, and the swelling was approximately 4.5%. The optimal formulation was composed of tetracaine hydrochloride at 1%, casein at 2%, glycerol at 50%, and sodium alginate at 4%; therefore, it can be concluded that this combination provides the desired properties for the hydrogel. These results suggest that tetracaine hydrogel shows potential for use as an anesthetic in medicine.