3.1. Polymeric Systems
Previously characterized binary polymer blends [23
] were selected to evaluate the robustness of the mucoadhesive methods. These systems, composed of P407 and poly(acrylic acid) derivatives possess different rheological and mechanical properties. These polymers contribute in a unique way to the formulation properties. With a temperature increase, P407 increases the viscosity of the system and the other decreases it (C971P, C974P, or PCB). Although other polymers as natural polymers (i.e., gelatin and agar) and synthetic polymers (i.e., poly(vinylpyrrolidone) and poly(vinyl alcohol)) were already widely used to investigate the mucoadhesion process, the selected thermorresponsive blends, besides being very well characterized as a complex system, can be a challenge for different mucoadhesion analyses [23
]. The carbomers C971P and C974P are mucoadhesive polymers crosslinked with allyl-ethers of pentaerythritol, displaying a concentration ranging from 56% to 68% of carboxylic groups in the chain. However, C974P displays a higher crosslinking degree than C971P. PCB is also a high-molecular-weight polymer of polyacrylic acid, cross-linked with divinylglycol or polyalkenyl ethers, displaying a large number of carboxylic groups (COOH) on the molecular chain, and known by its strong mucoadhesive properties [23
]. The P407 monopolymeric formulation displays higher consistency at body temperatures than the blends containing the same amount of this thermogelling polymer [12
]. Rheological, texture, and mucoadhesion studies have been performed to select the most suitable mucoadhesive semi-solid systems of each blend in previous studies (Table 2
The interaction parameter evaluates the adhesive interaction between polymers and can select optimal formulations for pharmaceutical and biomedical application [29
]. The gelation temperature and adhesiveness are also important for performance testing of thermogelling systems to ensure that they solidify on application and are retained on the mucosa. The P407/C971P (20/0.20%, w
), P407/C974P (15/0.25%, w
), and P407/PCB (15/0.25%, w
) system were selected based on previously-published information [23
]. A gelation temperature range near the body temperature, between 25 and 37 °C, is considered appropriate for pharmaceutical systems [2
Methods for analyzing the performance (e.g., mucoadhesion) should be investigated for accuracy, precision, specificity, linearity, and range [36
]. In this work, some of these characteristics (precision by repeatability and robustness) were studied to evaluate the importance of standardizing the conditions of method. The robustness of an analytical method is defined by ICH guidelines as a measurement of resistance against small variations of the analytical parameter. Thus, it is possible to verify the robustness of the method by demonstrating its reliability during the normal use [36
]. In this study, the methods to investigate the mucoadhesive characteristic were kept constant and the semi-solid formulations were changed to consider the formulations as a parameter or independent variable. Even known the temperature is not the same at 37 °C for different routes, and diseases are able to change the body temperature (e.g., fever), 37 °C was kept through the studied methods. Since most mucosal routes demonstrate this mean temperature. In addition, the selected thermoresponsive systems maintain their physicochemical profile with small variation of temperature (30–38 °C) and gel in this temperature range.
3.2. Analysis of the Detachment Force
The detachment force is an in vitro method widely used to analyze the mucoadhesive properties of the most dosage forms. By this method, it is possible to measure the Wadh
, which is based on the area under a force-distance curve obtained during the detachment process, as well as the Fadh
, defined as the maximum force to separate an adhesive surface from a mucous substrate [1
]. There is not a standard methodology to this determination [37
], so not only the choice of the technique, but also the choice of membrane (mucus or other mucosal types) and instrument parameters are critical to investigate the mucoadhesive properties of drug delivery systems. The limited use of the human mucosa means that ex vivo animal models are required, but these require validation and an understanding of intra-species variability. Moreover, the process of mucus/mucosa preparation can modify the physicochemical properties and results in an altered structure when compared to a fresh mucosa [37
]. To begin this process, a standardization of this method using mucin disks and oral porcine mucosa for the three semi-solid systems was proposed.
The results of the detachment force method (Figure 1
and Table S1
) demonstrate the reproducibility of the method with a coefficient of variation (or relative standard deviation), lower than 5% for mucin disks and lower than 12% for porcine oral mucosa, with the variation believed to be a result of the mucosa’s natural variability [33
]. Previous studies have demonstrated differences among mucin from different sites. Likewise, the variability between individuals and mucosa thickness may also affect results on ex vivo mucosa [16
]. In a previous investigation using mucin disks, polymeric blends containing P407/C971P, P407/C974P, or P407/PCB showed the same values of the mucoadhesion force, when compared by this method, supporting its reliability between laboratories [23
The P407/C971P formulation demonstrated the greatest mucoadhesive strength on mucin disks, with the rank order P407/C971P > P407/PCB > P407/C974P. Binary polymeric systems composed of P407 and poly(acrylic acid) derivatives have the availability of the carboxylic groups present in the mucoadhesive polymer decreased. It is believed that interactions between P407 and the mucoadhesive polymer reduces the possibility of interaction between the poly(acrylic acid) derivate and the mucin. However, C971P has higher carboxyl concentration, when compared with C974P and PCB, which allows the P407/C971P system to interact with mucin groups even with the P407 interaction [2
]. Moreover, P407/C971P demonstrated in TPA analysis higher values of hardness, cohesiveness, and compressibility, which contribute with the higher values of mucoadhesion observed with mucin disk. Using porcine buccal mucosa as a substrate gave a different rank-order of mucoadhesion: P407/C974P > P407/PCB > P407/C971P. The PCB with an intermediate amount of free carboxyl groups had in the two cases, also the intermediate mucoadhesive force. Furthermore, C974P, a more cross-linked material with a lower amount of hydroxyl groups, demonstrated similar results using mucin disk and animal mucosa. It is believed that the polymer has greater attractive forces among its polymeric chains, which can be also observed ahead by the high cohesiveness value, in TPA analysis. Creating a more cohesive system, with reduced mucin interaction, macroscopic differences were not observed with the substrate variation [19
The discrepant answers observed between the results obtained with the two substrates for the P407/C971P system can suggest this formulation uses hydration to establish adhesive bonds between mucin and polymeric blend [1
]. The C971P polymer exhibits lower cross-linking degree allowing higher swelling in presence of water. Therefore, in a low-hydration surface, such as the mucin disk, this system does not swell and displays a greater response than in high hydrated surface as the animal mucosa [43
]. The water concentration also influences the tensile strength method [42
], and a super hydration state of some polymers can reduce their mucoadhesive performance [43
]. Thus, the selection of the mucosal surface needs to consider the composition and consistency of the formulation analyzed, where the most consistent systems (with high mechanical resistance) have better response using mucin disks.
To compare in vitro and in vivo tests, the same condition of analysis should be used [17
]. However, using the same parameters, significant differences were observed between analyses performed with isolated mucin and ex vivo mucosa, particularly in the P407/C971P system. Furthermore, the Wadh
response, related to elasticity and plasticity, demonstrated a significant difference between the three polymeric systems. On the other hand, for the Fadh
response, significant difference was not found between the binary blends (p
> 0.05). Therefore, the Wadh
is suggested as a better response for the detachment force method and, when calculated as an N·mm unit, it can be converted to Joules, showing the energy dispending at the separation process of the adhesive surfaces.
In order to optimize this method, the responses of the P407/PCB polymeric blend were evaluated (Table 3
) under the variation of the instrumental parameters in a factorial design.
The Pareto diagrams (Figure 2
) shows the estimated effect of each factor and their interactions. A factor and an interaction are considered to influence the response only if the estimated effect is significant, i.e., p
< 0.05. It was found that variation of substrate, force, probe velocity rate, and contact time influenced the Fadh
. Applied force had the highest positive influence, and the substrate had a negative influence on this response of mucoadhesive force. For the Wadh
, only the contact time and some interactions demonstrated positive influence on the response. Speed and the change of the substrate from mucin disk to animal mucosa had the greatest negative contribution.
The surface response plots (Figure 3
) demonstrate that the largest Fadh
was obtained using the mucin disk, 0.1 N of contact force, 120 s of contact time, and a speed of 10 mm/s. On the other hand, for the Wadh
), the highest value was observed using mucin disk, 0.1 N, 1 mm/s, which was time independent. The surface was adjusted to the experimental data by multiple adjusted correlation coefficient values R2adj
. Thus, through the regression analysis, it was observed that 93.98% of the variation in the Fadh
response is explained with this model (R2adj
= 0.9398). Moreover, 96.08% of the variation in the Wadh
response is explained with this model (R2adj
= 0.9608). Therefore, the method can estimate the Fadh
and the Wadh
according to these parameters.
Additionally, the desirability of the method was evaluated for both responses, which represents the combination of factors required to obtain a better response [49
]. Figure 5
shows the desirability response for the Fadh
results, which is 85% of the maximum value experimentally obtained (1.147 N).
About the Wadh
B), the desirability was at 92% of the maximum experimental value (1.019 N.mm). Moreover, the substrate variation from the mucin disk to the animal mucosa reduced both results for the studied semi-solid formulation. It was also observed that the Fadh
increases when changing the levels from minimum to maximum for time, force, and speed parameters. Prior studies had demonstrated the increase of contact time between formulation and substrate surface increases the interaction between mucin and polymer chains; thus, higher values of adhesion are obtained [43
]. About the Wadh
, changing the levels from minimum to maximal for force and speed parameters, the response is greater as well. On the other hand, the time was not relevant to this answer. The statistical analysis suggests that it is not possible to compare results present in the literature, which use different substrates and parameters, for the tensile strength method. Thus, it is clear the necessity of standardization of this method, to have real comparisons among mucoadhesive profiles of different formulations and polymeric systems.
The desirability cannot show the proximity to real values, which characterizes a limitation of the in vitro methods. Nevertheless, this analysis allows a standardization of this method using both responses, since the higher value is obtained using these parameters. Therefore, for semi-solid formulations containing P407 and poly(acrylic) derivate, it can be suggested that the use of mucin disk, a force of 0.1 N, 120 s of contact time, and 10 mm/s probe speed will obtain better Fadh measurement. Using a mucin disk, 0.1 N of force, 30 s of contact time, and a probe speed of 10 mm/s can be used to obtain a better Wadh response.
Therefore, with the information obtained, it will be possible in future studies to select and validate the best conditions to perform comparisons.
3.3. Rheological Methods
Rheological analysis represents an in vitro model which can predict the in vivo behavior of adhesive formulations and also investigate their structural interactions. The mucoadhesion process is a phenomenon which combines different interaction types and the rheological method is used to evaluate these interactions between mucin and polymeric system [3
]. The mucoadhesion process is characterized when the rheological response of a polymer-mucin mixture is higher than the isolated contributions, giving rise to an “interaction parameter” [8
There are different ways to use the rheological analysis in studying mucoadhesion. It can be performed by consistency measurement over a range of shear rates or by monitoring the viscoelastic properties [27
]. Whilst rheology is preferred as a secondary technique, it is often used to measure changes in viscosity or elastic behavior [2
]. However, a lack of understanding about the meaning of the interaction parameter further confounds the great variability of results in the literature. Therefore, a standardization of the interpretation of results from the rheological analysis was proposed for the three semi-solid systems using the continuous shear analyses to evaluate the mucoadhesive properties (Table 4
The addition of mucin to the polymeric system demonstrates reduced consistency of the P407/C971P and P407/C974P systems, with a positive interaction parameter for the PCB-containing system. Consistency is a measure of internal friction and the presence of mucin appears to reduce interaction within the formulation. Comparing with the data in Figure 1
A, it can be seen that P407/C971P had the greatest force of adhesion, and a very large, negative, interaction parameter. It is suggested that a negative parameter for the consistency index could be a result of interaction between mucin and mucoadhesive polymer, which reduces bridging of poloxamer micelles in the formulation, lowering the overall consistency. It is also possible that the mucin glycoproteins affect the micellization behavior in an unpredictable manner. On the other hand, the P407/PCB polymeric system mixed with mucin exhibited a consistency index greater than the mucin and polymeric systems isolated. This is evidence of a strong interaction between the polymers and mucin, forming a highly cohesive system.
As already described in the literature, there are disadvantages in the use of flow rheometry as a method to evaluate mucoadhesive properties. The continuous shear analysis is a destructive test, then the mucin–polymer interactions can be disrupted, and it is not often possible to observe the mucoadhesion phenomenon [8
]. This study exposes the limitations of the continuous flow rheological method to evaluate the mucoadhesive profile of semi-solid systems, and it is best used to understand mechanistic aspects.
For oscillatory rheology, the synergism between polymer and mucin is employed to evaluate mucoadhesion. This technique has advantageous characteristics, such as being a non-destructive method which simulates the formulation behavior during application. Recently, Jones and co-workers [52
] reinforced the correlation between viscoelasticity and mucoadhesion, using linear regression [8
]. The adhesive interactions in the polymeric blends were observed by the elastic modulus (G
′) analysis of the mixture as a function of frequency. When the G
’ of the mixture is higher than the isolated polymer and mucin contributions, ∆G
′ > 0, rheological synergism occurs [2
]. The observed and calculated values of G
′ moduli were obtained at 10.0 Hz, since in this oscillatory frequency the systems are forced at higher oscillatory intensity.
The elastic, or “storage” modulus measures the storage and recovered energy at each deformation cycle, reflecting the solid component of a viscoelastic material [47
]. The elastic modulus will demonstrate higher values if a sample is predominantly elastic, i.e., highly structured. In contrast, the loss modulus (G
′′) demonstrates the lost energy at each cycle, and will be higher when the sample is predominantly viscous. The frequency sweep at the linear viscoelastic region allows the three-dimensional structure of the sample to be preserved throughout analysis. The type of cross-linked structure can be revealed in the oscillations, where a small effort is exerted at each frequency. The oscillatory analysis allows the differentiation of the physical entanglements and secondary bonds, since in low frequencies the polymeric physical entanglements can be separated, while, secondary bonds remain fixed [46
]. The results are displayed in Table 5
The viscoelastic response of the systems suggests the entanglement and secondary bonds (hydrogen bonds) between mucoadhesive polymers and mucus glycoproteins for the P407/C974P and P407/PCB binary systems, since the establishment of secondary bonds results in the increase of the elasticity into the formulations. Furthermore, the difference observed on the rheological interaction parameter of the three formulations can be attributed to the structural differences of these polymers [46
Formulations which show high viscosity have demonstrated to suffer low clearance and, consequently, they remain longer at the action site [55
]. However, despite having higher viscosity, the P407/C971P platform displayed rheological antagonism, demonstrating that the addition of mucin did not increase overall interaction in the system. Edsman & Hägerström had already demonstrated positive values (rheological synergism) for low concentrations of cross-linking polymers, while high concentration of them results in negative values. In this sense, the evaluation of the G
’ gives positive values for weakly hydrogels and strongly cross-linked hydrogels will demonstrate a negative interaction parameter [20
The system composed of P407 and C971P has the higher polymeric concentration when compared with the P407/C974P and P407/PCB formulations. Large concentrations of the polymers into the mixture added mucin, as well as the high viscosity of this system reduces the availability of the solvent, which makes the interpenetration of the polymer and mucin chains difficult, since them flexibility and mobility are reduced [47
Oscillatory rheometry is a valid and sensitive method to evaluate interactions in polymer-mucin mixtures, and although the P407/C974P and P407/PCB formulations had demonstrated similar answers between these methods, a direct comparison with the standard tensile method was not possible. Moreover, it was observed that the results depend more on the polymer concentration than of the polymers chemical structure, since P407/C974P and P407/PCB systems exhibited similar results. In this way, to use the oscillatory rheology as a method to evaluate the mucoadhesive profile of semi-solid formulations, a prior cohesiveness analyses must be done.
3.4. Texture Profile Analysis
TPA is a quick and common analytical methodology. It can be used for the mechanical characterization of semi-solid pharmaceutical dosage forms and aid understanding of structure. The results of this analysis allow easy identification of the physicochemical interactions among the components of the formulations and also allow prediction of the behavior of these systems under different analytical conditions, and during use in pharmaceuticals. The values obtained by this technique are: hardness, elasticity, adhesiveness, cohesiveness, and compressibility [57
The development of pharmaceutical dosage forms for topical application requires formation of a target profile. These formulations need to be easily removed from the packing to have good spreadability, bio/mucoadhesion, and adequate viscosity in order to facilitate retention and thus patient compliance with the treatment. Moreover, for mucoadhesive systems the resistance to the natural defense processes needs to be considered [34
To be easily applied, a semi-solid needs to demonstrate low values for hardness and compressibility, since these values indicate how easy it is to remove the formulation from the packing, as well as the spreadability and removal of the product at the desirable site. However, very low values of them can impair the retention of the formulation. Ideally, semi-solid systems must demonstrate high values of adhesion to ensure retention. In addition, high values of elasticity aid retention, because the systems have a tendency to return to their structure. On the other hand, high values to the cohesiveness parameter are also desirable [2
In order to obtain pharmaceutical systems with acceptable mechanical characteristics, ensuring in vivo retention and therapeutic efficiency, the texture profile properties of the formulations must be studied. As previously mentioned, the chosen systems have already demonstrated themselves to be favorable pharmaceutical systems, about the TPA properties [23
]. In addition to studying mechanical properties, it may be used to evaluate the mucoadhesion profile of semi-solid systems by the analysis of the polymer-mucin interaction.
Comparing the results of polymeric systems with and without mucin (Table 6
) there was a decrease in the hardness, compressibility and adhesiveness. The cohesiveness had increased values for two of the three systems, and the elasticity was unaltered. The P407/C971P system demonstrated higher values of hardness, compressibility and adhesiveness than P407/C974P and P407/PCB, probably because its polymeric concentration is also greater. In this sense, this formulation displayed also superior hardness, compressibility and adhesiveness responses. However, the decrease of this parameter after mucin addition was more significant, since the C971P presents a lower degree of crosslinking; therefore, it has a greater number of free carboxyl groups for interaction with the mucin, which promotes a more intense disintegration of this blend when compared with those containing other adhesive polymers (C974P and PCB). Similar to rheological analysis, it appears that polymer–mucin interactions may not provide constructive synergism in these systems.
The absence of change in the elasticity of the systems, even with the mucin addition, reflects the results obtained in the oscillatory rheology where the viscoelastic property was retained. Furthermore, the decrease of hardness and compressibility in the added mucin systems agrees with the flow rheology and demonstrates the loss of internal friction in these systems after mucin addition. The cohesiveness of the P407/C974P blend with mucin was superior to the two other blends. Probably, it is due to the C974P chemical structure, since it is a poly(acrylic acid) derivative with a higher degree of cross-linking, providing a more cohesive system, and the increase of the attractive force in the formulation is more dramatic.
The interaction parameters of the responses obtained by TPA were calculated between the polymeric blends and mucin solution (Figure 6
); this is an important value to investigate the mucoadhesive profile of the semi-solid preparations. All polymer blends showed a negative interaction parameter, i.e., the mixture with mucin in all the cases obtained lower values when compared with the pure polymeric systems. In this way, the results suggest the interaction between mucoadhesive polymer and mucin, which promotes mechanical changes.
The adhesiveness interaction parameter is a result of interaction between the formulation and polycarbonate probe. Therefore, negative values for mucoadhesive formulations are expected. Considering a higher adhesive polymer-mucin interaction, there are fewer free carboxyl groups to interact with the probe, and the adhesiveness of the mixture on the probe is lower when compared with the mixture with added mucin. Thus, as a new way to measure mucoadhesive properties of semi-solid systems, the analysis of the adhesiveness interaction parameter can be simple and accessible with fast execution. Moreover, a correlation was observed between this method and the tensile strength method using the mucin disks, since the adhesive profile of the three evaluated systems demonstrated a similar ranking evaluation.