## 4 Discussion  
Relaxing peptide-loaded chitosan microparticles were prepared, characterized, and evaluated. The evaluation of the physical-chemical and biological properties are important, since they are correlated with therapeutic potential effects. These microparticles exhibited good stability, and no aggregation occurred when in suspension. The positive value of zeta potential was probably 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. So, these results showed that peptide loading had no observable effect on the physical features of MPs. The relaxing peptide was successfully \begin{table}
\begin{tabular}{c c c} \hline \hline
**Formulation** & **Papp (cm/s) across TR146 Cell Layers** & **Papp (cm/s) across Caco\(-\)2/HT29 Cell Layers** \\ \hline Peptide & \(8.79\times 10^{-5}\pm 1.88\times 10^{-5}\) & \(1.24\times 10^{-4}\pm 8.96\times 10^{-6}\) \\ Guar-gum films & \(1.07\times 10^{-4}\pm 2.47\times 10^{-5}\) & \(1.11\times 10^{-4}\pm 4.83\times 10^{-5}\) \\ Chitosan microparticles & \(1.49\times 10^{-4}\pm 1.77\times 10^{-5}\) & \(4.26\times 10^{-5}\pm 2.96\times 10^{-5}\) \\ Guar-gum films with & \(1.03\times 10^{-4}\pm 3.38\times 10^{-5}\) & \(1.14\times 10^{-4}\pm 9.73\times 10^{-6}\) \\ chitosan microparticles & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Apparent permeability coefficient of relaxing peptide across TR146 and Caco-2/HT29 co-culture model. Figure 5: Cumulative relaxing peptide transport measurements across TR146 and Caco-2/HT29-MTX co-culture model in Transwell membranes expressed as peptide permeability (%, mean \(\pm\) SD). entrapped into the CH MPs with high loading efficiencies, indicating a good potential of CH MPs as a delivery system of these peptides. The relaxing peptide \(\alpha\)-casozepine has a lower isoelectric point (pH 6.56) and, therefore, a higher negative surface charge under particle formulation conditions, facilitating increased electrostatic interactions with polycationic chitosan. Association efficiencies and particles mean sizes are important indexes of the quality of the delivery systems. The process of encapsulation should generate a high yield of particles, and the levels of loading for these peptide formulations are appropriate for their potential therapeutic applications. The small sizes could enhance the absorption of buccal cells [28; 29; 30; 31]. In other studies, chitosan is widely described as bioavailable, with excellent physicochemical properties, as well as a mucoadhesive polymer that is able to increase cellular permeability and improve the bioavailability of orally administered protein drugs [28; 29; 30; 32; 33; 34]. The high AE values of loaded CH MPS are corroborated by [PERSON] and collaborators (2002) when they have prepared insulin-loaded chitosan nanoparticles with values of insulin association up to 80%, as well as [PERSON] and collaborators and other authors [35; 36; 37; 14; 38]. An FTIR analysis is proposed to investigate the interaction between substances. The regions of both amide I and amide II of relaxing peptide and relaxing peptide-loaded formulations present an overlapping, which is an indicator that there was no chemical structure modification of carried peptide during incorporation or production of the new delivery systems [4]. FTIR analysis did not indicate the formation of new covalent bonds between relaxing peptide and chitosan microparticles, guar-gum films, and guar-gum films with chitosan microparticles that could potentially modify the peptide release profile. Through the in vitro peptide release assays, it was verified that the peptide release profile from guar-gum films and chitosan microparticles and chitosan microparticles into guar-gum films were very similar, indicating that both formulations offered a slower release of carried peptide when compared to the free relaxing peptide.