Mucoadhesive Properties of the Chitosans
In order to predict the mucoadhesive properties of the chitosans used to produce hybrid electrospun Ch/P fibers, interactions of chitosans and chitosans/phospholipids with mucins were investigated through changes in size, zeta potential and turbidity of the samples, as a first step.
Turbidity measurements were used to monitor the interactions of chitosans with mucins and phospholipids and the mixtures of chitosans/phospholipids with mucins. Individual solutions of chitosans, phospholipids and mucins were used as controls.
The absorbance of the individual chitosans didn’t vary significantly from sample to sample as shown in
Figure 2. The addition of mucin to the chitosans increased the measured absorbance due to the interactions between components (
Figure 2). This increase is assumed to be related with the formation of chitosan-mucin nano complexes as reported elsewhere [
7,
31]. As illustrated in the
Figure 2, all the samples exhibited a higher turbidity after the addition of mucin. Accordingly, all chitosan-based formulations interacted with mucin. However, the variations in the magnitude of the interactions, suggest differences in the mucoadhesive properties of the formulations. Ch 3 displays slightly higher absorbance (1.78) than Ch 1 (
p < 0.05), potentially due to the slightly higher Mw which favors more interaction mucin-Ch. Ch 4 was expected to exhibit the highest absorbance due the the higher interactions of mucin–chitosan as result of lower DA. However, this was not observed. Indeed, the lower absorbance of Ch 4 comparatively to Ch 3 is not statistically significant.
The addition of phospholipid increased the absorbance in all the Ch samples reaching the maximum of 0.53 for S2 due to the higher content of phospholipid present in the sample. S1 and S3 have similar absorbance values of 0.27 as those chitosans have similar DA, thus similar level of interactions with phospholipids would be expected. S4 has lower absorbance (0.22) comparatively to S3 (
p < 0.02). However, upon the addition of mucin, an abrupt increase of the absorbance to 1.71, 1.74, 1.79 and 1.91 were observed for S1, S2, S3 and S4, respectively, confirming again the interactions of mucin–chitosan/phospholipids. The presence of phospholipids did not affect the interaction of mucin–chitosans and confirm the expectations in terms of binding affinity of chitosan–mucin, being the S4 the most favorable to interact with mucin due to the lowest DA, followed by S3 and S1. The combination of phospholipids with chitosan have been documented as powerful mucoadhesive carriers for the delivery of drugs [
32], highlighting the potential of combining these types of molecules for mucoadhesive applications.
The presence of higher content of phospholipid (S2) led to a lower increase in absorbance from 0.53 to 1.73 comparatively to S1 (0.279 to 1.713), suggesting that such an increase in phospholipid content from 1:1 to 1:3 (Ch:P) did not favor the increase in interactions of mucin–Ch/P.
Zeta potential measurements are a common method to investigate the mucoadhesive properties of several biopolymers [
31,
33]. Mucin has negative charges, with zeta potential of approximately −5 mV, thus the positive surface charges of chitosan-based formulations are expected to interact strongly with mucin (
Figure 3). Several studies have been reporting the negative zeta potential of mucins, around −7.9 mV (PGM) [
31],which tends to vary with the concentration of mucin and pH.
Chitosans, on the other hand, have a positive surface potential due to the protonation of the amino groups at pH 3.5. The zeta potential for chitosan 1, 3, 4 was found to be of 41.62 ± 4.6; 39.66 ± 5.8 and 47 ± 3.9 mV, respectively (
Figure 3). The highest zeta potential observed for Ch 4 is related to the lowest DA. Chitosan 1 and 3 did not exhibit significantly different zeta potential. The addition of mucin led to the decrease of the zeta potential to 13.475 ± 0.6, 18.65 ± 1.9 and 17.3 ± 2.6 mV for samples 1, 3 and 4, respectively. This observation confirms that chitosan binds onto the mucinous aggregates, thus changing their zeta potential towards negative values. The addition of phospholipids was observed to slightly increase the zeta potential; however, these changes in zeta potential were not statistically different. Similarly to Ch–M mixtures, the addition of mucin to
Ch/P led to the decrease of zeta potential to 10.4 ± 0.4, 13.0 ± 1.3, 22.4 ± 2.2 and 5.9 ± 2.9 mV. For most of the Ch formulations the decrease in zeta potential was more notorious in the presence of phospholipids and significant differences were found for between groups (
p < 0.02). Therefore, this data suggests that the binding affinity to mucin follows the order S4 > S1 > S3 > S2.
Figure 4 shows the intensity weighted particle size distributions of the solutions as examined by dynamic light scattering (DLS). In our study, three major populations appeared in the intensity weighted size distribution of the hydrodynamic diameter (D
H) for mucin (M) with local maxima 87.12 ± 29 nm, 949.3 ± 272.3 nm and 5302 ± 321.4 nm (
Table 1). Similar size distributions with three main populations were also found by Patil and co-workers [
34]. Those authors pointed out that the multiple peaks must be associated with the presence of non-bound impurities or linkers between mucin molecules often present in commercially available mucins [
34].
Chitosans on the other hand display two main populations, which vary according the Mw and DA, as can be seen in
Figure 4 and
Table 1.
The addition of mucin to the chitosans resulted in the increase of the hydrodynamic diameter (
Table 2), as result of the binding of mucin with chitosan, as discussed previously in terms of turbidity and zeta potential (
Figure 2 and
Figure 3). However, this increase in D
H was more notorious for samples 1 and 4.
The inclusion of phospholipid within chitosan solution was also observed to increase the D
H of the samples (
Table 3). Furthermore, with exception for S1, only one population of particulates was observed for
Ch/P samples. The increased size of the mixture suggested the formation of chitosan/phospholipid complexes, as a result of their interactions as reported in a previous study [
28].
The addition of mucin to
Ch/
P solutions was observed to shift the main populations to 1647 ± 650, 859.99 ± 25, 888.2 ± 321.3 and 855.6 ± 291.3 nm for samples S1, S2, S3 and S4 respectively (
Table 4). This increase in the hydrodynamic diameter has been previously reported as result of the binding of mucins with the biopolymeric formulation [
7,
31,
34], confirming the mucoadhesive properties of
Ch/
P formulation.