3.1. Characterization of Chitosan Nanoparticles
DLS measurements and transmittance analyses were performed for all the formulations, to investigate the effect of the chitosan concentration (%) and CS/TPP ratio on the formation and size of the obtained nanoparticles in acetate buffer, pH 4.5 (
Figure 1). By comparing the hydrodynamic diameters calculated for the nanoparticles prepared at different CS concentrations, it became clear that the percentage of CS markedly influences the nanoparticle size. For nanoparticles obtained with 0.1%
w/
w of chitosan, the hydrodynamic diameter was found to be in the range of 85–95 nm, while sizes of approximately 300 and 900 nm were detected for particles prepared with 0.5%
w/
w and 1%
w/
w of chitosan, respectively. The increase in size as a function of the CS concentration is well documented in the literature for nanoparticles prepared by ionic gelation [
25,
26]. On the contrary, the effect of the CS/TPP ratio is less straightforward and it required a more detailed analysis. Indeed, the size evolution nanoparticles as a function of the CS/TPP ratio displayed a typical profile at any tested concentration. Nanoparticle size remained constant in a specific range of CS /TPP ratios in a chitosan concentration dependent manner. Specifically, the range of CS/TPP ratios in which the size of the nanoparticles was similar was found to be between 10:1 and 16:1 for 0.5%
w/
w and 1%
w/
w CS concentrations and between 4:1 and 10:1 for 0.1%
w/
w CS concentration. For ratios above this range, the formation of nanoparticles generally did not occur, while for lower ratios, a progressive increase in size was observed, leading to flocculation and CS precipitation at a ratio of 2:1 for 0.5%
w/
w and 1%
w/
w of chitosan and 1:1 for 0.1%
w/
w of chitosan. To evaluate the relevance of these two specific parameters—the CS/TPP ratio and chitosan concentration (%)—on nanoparticle size, a two-way variance analysis was performed. For this purpose, 18:1, 16:1 and 14:1 ratios were not included in the analysis because they did not produce any nanoparticles. In addition, the 2:1 ratio was also excluded since large aggregates were observed and they could affect the statistical analysis, leading to false positive results. All the considered parameters (CS concentration, CS/TPP ratio and the interaction term) were statistically significant (
p value < 0.001 for CS concentration and CS/TPP ratio and 0.005 for the interaction term), but the CS concentration had a higher impact on the size (F value for CS concentration was at least 75 times higher than for the other two parameters). The effect of all the parameters considered can be easily displayed through the main effect and interaction plot (S1 and S2). A rise in CS concentration resulted in an increase of the nanoparticle size, while an opposite and limited effect was observed for CS/TPP ratios (main effect plot,
Figure S1). Interestingly, the effect of CS/TPP ratio was much more pronounced at the highest CS concentration (interaction plot,
Figure S2). Counts (Kpcs) were also recorded through DLS, as they represent a measure of the scattered light intensity received by the detector, which depends on the nature, size and concentration of the disperse system. At higher CS/TPP ratios (above 10:1 for nanoparticles containing 0.5%
w/
w and 1%
w/
w of chitosan and above 4:1 for systems containing 0.1%
w/
w chitosan), counts were constant, but at lower ratios, they progressively increased. Considering that the size of nanoparticles did not increase markedly over the different ratios (except for 2:1), it can be speculated that the increase in counts could be related to the presence of a higher number of nanoparticles at lower CS/TPP ratios. To validate this hypothesis, the transmittance was also measured. It can be observed that the increase in counts was accompanied by a decrease in the measured transmittance value of the samples. The variation in the optical properties of the samples at different CS/TPP ratios and chitosan concentrations (%) can be also seen by a visual observation of the samples. Indeed, systems at 0.1%
w/
w CS concentration are transparent (except for the 2:1), while at higher concentrations of chitosan (0.5%
w/
w and 1%
w/
w), as the CS/TPP ratio lowers, a gradual increase in the opacity of the samples can be observed (
Figure 2).
In addition to pH 4.5, CS nanoparticles in 200 mM acetate buffer were also prepared at pH 5 and pH 5.5, to evaluate the effect of pH on nanoparticles size. Results from this investigation can be found in the
supplementary materials (
Figures S3 and S4).
3.2. Rheological Analysis of HPMC/CS Hydrogels
A preliminary screening was performed, in order to investigate the HPMC concentration (%) that is required to produce hydrogels in acetate buffer, pH 4.5, showing a consistency comparable to that of a jellified commercial formulation intended for vaginal administration. For comparison, the marketed hydrogel known as Zidoval® was selected. Zidoval® is formulated using a carboxypolymethylene polymer (Carbopol 974P), that displays carboxylic groups in the backbone. The polymeric dispersion thickens and forms a hydrogel after neutralization with inorganic (e.g., sodium hydroxide) or organic (e.g., triethanolamine) bases. At this pH value, chitosan precipitation occurs, therefore, carboxypolymethylene was not suitable for the preparation of mixed chitosan hydrogels. HPMC was chosen since the formation of the hydrogels is independent from the pH, due to the absence of ionisable groups.
Hydrogel formulations prepared at 5.5%
w/
w HPMC concentration (control, CS and CS NPs 0.1%
w/
w, 0.5%
w/
w and 1%
w/
w) and Zidoval
® were compared by looking at the rheological parameter “complex viscosity” (viscosity*). Viscosity* is a measure of the viscosity of a semisolid system obtained through an oscillatory stress sweep test (
Figure 3A). Only two CS/TPP ratios (6:1 and 2:1 for 0.1%
w/
w CS and 6:1 and 12:1 for 0.5%
w/
w and 1%
w/
w CS) were selected for the preparation of HPMC/CS NP hydrogels, in accordance with DLS and transmittance results. Hydrogels at the two CS/TPP ratios had a similar appearance to that of nanoparticle dispersions. Specifically, the hydrogels prepared at lower CS/TPP ratios were more opaque with respect to those prepared at the same concentration but at higher CS/TPP ratios (
Figure S5).
In regard to the rheological results, 5.5%
w/
w HPMC hydrogel showed a viscosity* of 45 ± 3 Pa*s at 37 °C. The presence of chitosan, both as free polymer or as nanoparticles, increased the strength of the HPMC/chitosan mixed hydrogel. Particularly, a value of viscosity* comparable to Zidoval
® (75–80 Pa*s) was obtained at a concentration of 1%
w/
w chitosan, both as free polymer or nanoparticles (
Figure 3A and
Figure S6).
On the other side, the presence of chitosan did not markedly affect the viscoelastic properties of HPMC hydrogels, as we can see from the trend of the elastic (G’) or viscous (G’’) moduli (
Figure 3B and
Figure S6. However, what these analyses have pointed out is the different rheological behaviour of Zidoval
® with respect to HPMC hydrogels. Indeed, Zidoval
® can be considered a true gelling system from a rheological point of view. This is because the elastic modulus (G’) is higher than the viscous modulus (G’’) at any tested frequency (
Figure 3B). In contrast, the prepared hydrogels cannot be defined as a gel from a rheological point of view, but they can be considered as concentrated polymer dispersions. In this case, the rheological moduli (G’ and G’’) are dependent on the applied frequency and show a frequency cross point. At frequencies below this point, the viscous modulus is higher than the elastic modulus and, consequently, they behave as a liquid dispersion. On the other side, at frequencies above the cross point, the elastic modulus is higher than the viscous modulus and they behave as weak gels. As such, the negligible effect of CS on the viscoelastic properties of 5.5% HPMC hydrogels can be evaluated in terms of cross points of the rheological moduli (G’ and G’’), which were found to be in the range 3–6 Hz for all the prepared systems (HPMC, CS/HPMC and CS NPs/HPMC hydrogels).
3.3. Mucoadhesiveness Test
HPMC hydrogels containing 0.5% and 1%
w/w of chitosan, both as free polymer and as nanoparticles, were analysed in term of their mucoadhesive properties. As shown in
Table 1, a statistically significant increase (one-way ANOVA followed by Dunnett’s multiple comparisons test) in the mucoadhesiveness compared to the control (HPMC 5.5%
w/w in acetate buffer 200 mM pH 4.5) was only observed for the hydrogels containing 1%
w/w of chitosan (both as free polymer or as nanoparticles) by considering the maximum force (N) of detachment of the hydrated mucin tablet from the hydrogel surface (
p < 0.007). A slight increase in the “total work” (mJ) and the “work at the maximum force” (mJ) were observed for the 1%
w/w chitosan hydrogels, but in this case, such differences were not statistically significant with respect to the control. The results confirmed the mucoadhesive properties of HPMC. Indeed, at the concentration used, the mucoadhesion of the hydrogels is controlled by HPMC, while chitosan (as free polymer or nanoparticles) exerts a certain effect only at a concentration of 1%
w/w. The effect on mucoadhesion of lower concentrations of chitosan (as 0.5%
w/
w) can be hindered by the presence of a larger amount of the mucoadhesive polymer, HPMC (5.5%
w/
w). This assumption is supported by the fact that in hydrogels formed by polymers with less mucoadhesive properties than HPMC (e.g., Poloxamer 407) [
27], there is an effect of the presence of chitosan on mucoadhesion at a lower concentration [
23].
3.5. Antimicrobial Activity of HPMC/CS Hydrogels
Several studies have been conducted to investigate the effect of chitosan or chitosan nanoparticles on fungal growth [
29,
30,
31]. The obtained results have showed a marked antifungal activity exerted by chitosan against different fungal strains, including pathogenic species for humans as
Candida spp. [
20,
32,
33]. Fungicidal activity was demonstrated for chitosan, possibly due to membrane damage as a consequence of the interaction between protonated amino groups with negatively-charged cell surface proteins [
34]. Despite the conspicuous number of experimental works, there is still a debate regarding the greater efficacy and broader activity of nanoparticles with respect to chitosan as free polymer.
Candida albicans is the main fungus responsible for vaginal yeast infections, although less frequently co-infection with other non-albicans Candida species (
Candida glabrata,
Candida lusitaniae) can occur [
35]. The infections by species other than
Candida albicans are reported to be more resistant to common antimycotic treatments (azole drugs; e.g., fluconazole) and responsible for the recurrence of the infection [
36,
37]. In this work, a panel of eight strains of
Candida spp. (four albicans and four non-albicans) was tested, in order to investigate the anti-Candida activity of 1% chitosan as free polymer and nanoparticles, formulated both as aqueous dispersions or dispersed in a 5.5% HPMC hydrogel.
Initially, the susceptibility of the selected
Candida spp. strains to fluconazole, as an antifungal azole drug model, was studied. Most of the strains were resistant to fluconazole at the two tested concentrations (25 μg/mL and 50 μg/mL), with the exception of
C. albicans 360923, which showed inhibition diameters of 20 ± 1.25 and 28 ± 0.75 mm for fluconazole 25 μg/mL and 50 μg/mL, respectively (
Table S1), comparable to those already reported in the literature [
38].
In the present study, MIC values for chitosan were not determined since its efficacy against
Candida spp. is well-known from the literature [
32,
39]. The tested chitosan concentration (1%
w/
w) was selected as the highest that produced nanoparticles at the investigated experimental conditions. The aim was to compare the anti-Candida activity of CS (as both free polymer and nanoparticles) when dispersed into HPMC hydrogels with that of CS dispersions prepared at the same concentrations. The results obtained from the agar well diffusion method are summarized in
Table 2. The 1%
w/
w CS dispersion in 200 mM acetate buffer, pH 4.5, displayed good activity against all the tested
Candida spp. strains, with inhibition growth diameters ranging from 12 ± 0.78 mm for
C. albicans 18/01, to 13 ± 0.27 mm for
C. albicans 4940 and
C. glabrata 4955. One-percent CS nanoparticles in 200 mM acetate buffer, pH 4.5, have, instead, a different profile of activity. Indeed, 1% CS NPs were ineffective against all the tested
C. albicans strains, while showing a comparable inhibition growth effect to 1%
w/
w CS dispersion (as free polymer), against the tested non-albicans strains. By considering nanoparticles at different CS/TPP ratios, the activity of nanoparticles prepared at the ratio 12:1 was slightly higher than those prepared at 6:1 for all tested non-albicans strains. This could be related, as already reported in the literature, to the smaller particle size obtained for the systems prepared using a 6:1 CS/TPP ratio when compared to those obtained at 12:1 ratio [
19].
In regard to the HPMC-based hydrogel formulation containing 1% chitosan as free polymer, it was able to inhibit the growth of all tested
Candida spp. with a variable degree of activity. Generally, a slightly lower effect with respect to 1%
w/
w CS dispersion was observed, which could be related to the slow diffusion capacity of CS in the hydrogel matrix if compared to a buffer solution. Interestingly, hydrogels formulated with 1%
w/
w CS NPs, in contrast to 1%
w/
w CS NPs as a dispersion (which was active only on non-albicans species), showed activity against all tested albicans and non-albicans strains. Indeed, the greatest zone of inhibition was obtained with the hydrogel HPMC/CSNPs 12:1 against
C. lusitaniae 360804 (15 ± 0.57 mm), followed by
C. glabrata 4955 (12 ± 0.57 mm) and
C. albicans 18/1 (11 ± 0.25 mm), whilst lower zones of growth inhibition were observed with the hydrogel HPMC-CSNPs 6:1 (from 11 ± 0.57 mm for
C. albicans 360923 to 8 ± 0.25 mm for
C. albicans 4940). Despite CS NPs/HPMC hydrogels being active against all tested strains, a higher susceptibility was observed for non-albicans species and NPs prepared at 12:1 CS/TPP ratio. The different susceptibility among the tested strains could be explained by considering the structural differences of the cell wall among
Candida spp. The cell wall of
C. albicans, indeed, has more chitin and less adhesin molecules than that of
C. glabrata [
29,
40]. Thus, the anti-Candida activity of chitosan was more pronounced against the strains with more chitin in the cell wall. Moreover, the presence of HPMC could facilitate the penetration of chitosan in the cell wall of the strain (i.d.
C. albicans), justifying the broader range of the activity of the hydrogels containing chitosan, in comparison to CS dispersions, when analysed at the same concentration. Moreover, as for CS dispersions, the higher activity of the hydrogels prepared with CS NPs at 12:1 could be related to the smaller size.
Since common antifungal drugs are insoluble or slightly soluble in water, their formulation as hydrogels, using therapeutically-active concentrations of the drugs, is not feasible. Indeed, they are commercialized as cream for vaginal administration, instead of hydrogels. The formulation as hydrogels could be still possible using ethanol as a co-solvent to promote the solubilization of the drug. However, ethanol can act as a non-solvent for chitosan, thereby destabilizing the formulation [
41]. For these reasons, we decided to prepare CS/HPMC hydrogels loaded with metronidazole, an antiprotozoal drug, approved for the treatment of bacterial vaginosis as a 0.75%
w/
w hydrogel. Metronidazole does not have intrinsic anti-Candida activity, but hydrogels containing metronidazole are available on the market for the treatment of bacterial vaginosis. A formulation of hydrogels loaded with metronidazole in the presence of chitosan could be a strategy to formulate a jellified system with improved antimicrobial activity against both vaginal bacteria and
Candida spp. As such, metronidazole has been already formulated in other chitosan-based dosage forms, in order to address a combined effect on bacterial and yeast infections of the vaginal mucosa [
5]. Metronidazole was not encapsulated inside CS nanoparticles, but it was solubilized inside the polymeric matrix, in order to formulate a hydrogel at the same drug concentration (0.75%
w/
w) as the commercial formulations. Metronidazole solution 0.75% (in 200 mM acetate buffer, pH 4.5) and 0.75% metronidazole HPMC hydrogel, tested as controls, did not show any anti-Candida activity against all tested strains. Moreover, the addition of 0.75% to 1% CS dispersion or 1% CS/HPMC hydrogels (as free polymer or nanoparticles) did not determine any increase in the antimicrobial activity against all the tested
Candida spp. strains. These results confirmed that the presence of metronidazole did not exert any influence on the intrinsic anti-Candida effect of chitosan when both formulated as nanoparticles and dispersed into a polymeric hydrogel.