3.1. Preparation and Characterization of Chitosan Microparticles
Chitosan MPs were prepared by the ionic gelation method by auto-aggregation between a positively charged amino group of chitosan and the negatively charged phosphate groups of TPP (cross-linking agent) [19
]. Chitosan MPs cross-linked with TPP have been employed in many studies for drug delivery systems because TPP is used to improve the mechanical properties and stability of CH MPs [8
]. The parameters, type of cross-linking agent and polymer were optimized in preliminary studies. The CH MPs optimized formulation was set as 40 mg chitosan (polymer) and 1.5 mg TPP (cross-linker). Figure 1
outlines the factorial design and values of the formulation parameters.
In order to achieve theoretical optimization to validate the results, the formulations of CH MPs were further assessed for mean size, polydispersity index, zeta potential and association efficiency. The obtained results were individually compared with the theoretical (predicted) values by Student t-tests. No statistically differences (P > 0.05) were found between predicted and experimental values.
After the optimization of the CH MPs, peptide-loaded CH MPs were prepared and parameters such as particle size, zeta potential, PDI, association efficiency and loading capacity were analyzed because those properties are important for therapeutic properties.
The mean size of CH MPs is dependent on both chitosan molecular weight and concentration and on TPP concentrations. Particle size can influence the biopharmaceutical properties of microparticles, their biodistribution and the particle content uptake [8
]. Particle size was the leading assessed property during formulation optimization studies, oriented towards obtaining microparticles with a mean diameter of about 2.5 µm with a reproducible size distribution. As shown in Table 1
, unloaded CH MPs presented a size of 2.544 ± 0.97 μm and peptide-loaded CH MPs had a size 2.582 ± 0.87 μm. The determined size of chitosan microparticles was in agreement with results reported in the literature [32
Peptide loading by the encapsulation method did not induced an increase in particle size when compared with empty CH MPs (Table 1
). So, the incorporation of the peptide into CH MPs did not have a significant effect on particle size.
The particle size and surface charge of MPs/NPs regulate the biodistribution and pharmacokinetic properties of the MPs/NPs in the body. Therefore, the zeta potential is another important parameter and useful indicator of the electronic charge, which can be used to predict and control the stability of colloidal suspensions or emulsions [8
]. The greater the zeta potential, the more likely the suspension is to be stable because the charged particles repel one another and thus overcome the natural tendency to aggregate. Microparticles with a zeta potential above ± 30 mV have been shown to be stable in suspension, as the surface charge prevents the aggregation of the particles [33
]. According to the results obtained (Table 1
), all the batches prepared showed a zeta potential more than + 30 mV, confirming that microspheres exhibited good stability and no aggregation in the suspension. The positive value of the zeta potential might be due to the positive charge of chitosan and the high positive zeta potential indicated that the electrostatic repulsion between particles prevented aggregation and increased their stability. The positive value of the zeta potential is important for buccal drug delivery since it can facilitate adhesion to the mucosal epithelial surface, thus prolonging the peptide release and enhancing the peptide bioavailability. The results showed that the addition of peptide has no significant effect on the microparticles zeta potential.
The PDI values of CH MPs and peptide-loaded CH MPs were around 0.5 (the index is a measure of dispersion homogeneity; values closer to zero indicate a homogeneous dispersion), indicating uniformity of particle size and monodispersity distribution, with low variability and no aggregation, as reported in the literature [30
]. If a scale from 0 to 1 is considered, a PDI lower than 0.1 might be associated with a high homogeneity in the particle population, whereas high PDI values suggest a broad size distribution or even several populations. The calculation of PDI takes into account the mean particle size, the refractive index of the solvent, the measurement angle and the variance of the distribution. So, the PDI affects the mechanical strength of the polymer and its ability to be formulated as a delivery device, and these properties may control the polymer biodegradation rate [35
Association efficiency and loading capacity are other characteristics that should be calculated for controlled delivery systems [8
]. The association of bioactive peptides with the delivery systems components conditions the delivery system success, because it can protect biomolecules against metabolic degradation and improve protein absorption into the intestinal epithelium with better bioavailability. CH MPs were successfully prepared via the ionic gelation method and ensured encapsulation of the peptide. Although the association efficiency of hydrophilic molecules is usually low, in this study we obtained high encapsulation efficiency values, similar to other studies. Table 1
shows the association efficiency and loading capacity of peptide-loaded CH MPs. The CH MPs with peptide showed an encapsulation efficiency of 76%, achieving a particle loading degree of 0.46% (n
= 3). The antihypertensive peptide was successfully entrapped into the CH MPs with a high association efficiency, indicating the good potential of CH MPs as a delivery system. The AE was optimized by varying some parameters, including the amount of chitosan and TPP concentrations. The AE and size are important indexes for evaluating the quality of delivery systems. The high AE% can improve the utilization of the peptide and a smaller size could enhance the absorption of buccal cells [36
]. Indeed, other authors [33
] have already proven that CH MPs are natural materials with excellent physicochemical properties, good carriers for encapsulating proteins, which can achieve high protein loading efficiency and protect them from degradation.
3.2. Chitosan Films Characterization
Various methods have been described in the literature as appropriate to prepare CH films for delivery systems [26
]. The solvent casting method was selected because it is the method most commonly reported in the literature due to its inherent simplicity and robustness. It is a feasible and cost-effective technique which ensures greater commercial viability.
Firstly, a CH film experimental design was performed in order to obtain optimized formulations and understand how excipients influence the mechanical characteristics of the films. Figure 2
shows the prediction profilers used in the optimization of the formulations of CH films.
By setting the desirability of dependent variables to maximum, it was possible to obtain the best possible formulations. So, the optimized formulation of the CH films was set as 0.79% (w/v) of chitosan (polymer), 6.74% (w/v) of sorbitol (plasticizer), 0.82% (w/v) of citric acid (salivary stimulator) and an ideal thickness of 136 μm.
Chitosan is commonly used for producing MPs/NPs thanks to their excellent properties (biocompatible, biodegradable, non-toxic, antihypertensive, anti-inflammatory, antimicrobial, mucoadhesive). However, the properties of these carriers can be further improved by the addition of plasticizers; for example, in this case sorbitol was used. The plasticizer molecules interposing between the polymer chains and interacting with their functional groups increased polymer chain mobility and flexibility and improved mechanical properties. Specifically, they reduced brittleness, improved flow, imparted flexibility and increased the toughness of films [17
For the preparation of the films, citric acid was also added as a saliva stimulating agent [26
]. The purpose of using citric acid is to increase the rate of production of saliva, which would aid in the faster disintegration and consequently the rapid dissolution of the film.
After optimized CH films composition, the films were produced at the same time as CH MPs. That way, the constituents of the films were added to the microparticle solution. Finally, the final solution was dried at room temperature and the optical and morphological properties were evaluated. These films were transparent, flexible and homogeneous, and their surfaces appeared to be smooth without pores and cracks [39
]. The films were thin, with a thickness ranging between 0.085 and 0.117 mm, evaluated using a digital Vernier caliper [40
]. The thickness, flexibility, elasticity and easy handling are important properties for oral films application and consumer acceptance [17
]. So, we needed to evaluate mechanical properties: the elastic modulus, to evaluate the film’s rigidity; the tensile strength, to determine the brittleness of the film; the elongation at break, to know the flexibility and elasticity. These properties needed to be investigated as they condition the film’s integrity and its performance [38
]. The Young’s modulus, tensile strength and elongation at break were measured and are shown in Table 2
The tensile strength is an important mechanical property to avoid damage (release of the carrier molecule) during post-production storage and transporting. Basically, this test is performed to measure of the maximum strength of a film to withstand applied tensile stress, and the percent elongation represents the ability of a film to stretch [41
]. In optimized CH films, the tensile strength obtained was 0.767 ± 0.091 (MPa). This result is very low when compared with the tensile strength of the pure CH film in other studies, such as 8 MPa [41
], 10.97 MPa [40
] and 98 MPa [39
]. The different results may be due to differences in chitosan type, plasticizer presence, film formation method or analytical methods used [39
Another mechanical property is the Young’s modulus, which is an indicator of the stiffness (rigidity) of the film. It is reported to offer a sharp burst release of carrier molecules. The elongation at break is an indicator of its extensibility. The Young’s modulus for CH films incorporated with peptide-loaded CH MPs are higher than CH films (Table 2
), because the Young’s modulus increased with the increase of filler content [42
], but no statistically significant differences (P
> 0.05) were found. The values obtained were low when compared with those of other oral films [43
], but the composition and the evaluation methods were not the same. The low Young’s modulus obtained in our films indicates softer networks, lower water sorption and higher solubility.
The tensile strength and elongation at break values obtained for the peptide-loaded CH MPs indicate that the incorporation of the peptide into the films did not significantly alter the tensile strength compared with that of uncoated films, corroborating the findings of Aguilar and collaborators [44
Films were also analyzed regarding disintegration capacity. Effectively, orodispersible films have a high delivery potential because when placed on the tongue, they are immediately hydrated by saliva, followed by disintegration and/or dissolution and the release of the bioactive peptide [45
]. This CH film with peptide-loaded CH MPs, when contacted with saliva solution, showed a quickly swelling (217.05 ± 122.36%) and erosion (17.25 ± 12.21%) due to the disentanglement of the loosely bound chitosan molecules, which allowed a facile diffusion of the peptide-loaded CH MPs from the matrix (see Table 3
]. The swelling of the films first increased dramatically due to the porous structure and the hydrophilicity of the CH film, indicating a strong hydration of chitosan, which facilitates the rapid mucoadhesion to the absorptive epithelia [46
]. Mucoadhesion occurs when the CH film comes in contact with buccal epithelial cells; a double layer of electrical charge forms at the interface to promote the adhesion [46
]. The data obtained in this study confirm other reports in the literature; that is, the optimized CH film provides rapid disintegration (30 s) and the release of actives when the strip comes into contact with saliva in the mouth. These results agree with the range of values indicated by the Guidance for Industry [47
3.3. In Vitro Cell Viability
In addition to the preparation and characterization, the in vitro evaluations of CH films with peptide-loaded CH MPs are important for understanding the behavior of these delivery systems in biological systems, as well as for elucidating the nature of interaction between the delivery system and tissues, i.e., the biocompatibility. Among the biocompatibility tests, cytotoxicity is preferred as it is simple, fast and has a high sensitivity. The cytotoxicity test is one of the most important methods for biological evaluation. In order to evaluate the cytotoxicity of developed formulations, the MTT assay was performed. The effect of CH films with peptide-loaded CH MPs on TR146 cells was tested in vitro.
Cytotoxicity ratios and viability were classified according to the following criteria for cytotoxicity: (a) if viability > 100%, the corresponding cytotoxicity type was class 0, indicating no toxicity; (b) if viability = 0%, the corresponding cytotoxicity type was class 5, indicating the highest toxicity; (c) 75–99%, 50–74%, 24–49% and 1–25% viability were categorized as classes 1, 2, 3 and 4, respectively [49
outlines the results obtained from the MTT assay of TR146 cells after being exposed to developed formulations for a period of 24 h. The results indicat that all experimental conditions (CH MPs; peptide-loaded CH MPs; CH films; peptide CH films; CH films with CH MPs; CH films with peptide-loaded CH MPs) assured high cell viability. Hence, the cytotoxicity could be categorized as class 1 (Figure 3
), which demonstrates that the CH films with peptide-loaded CH MPs have excellent cell biocompatibility. The results were obtained in conformity with other studies in which chitosan did not interfere with cell viability [20