Optimizing Thermoresponsive and Bioadhesive Systems for Local Application of Erythrosine

Abstract

Photodynamic therapy (PDT) is a light-activated chemical reaction used for the selective destruction of tissue. For this, various colorants may be applied, such as erythrosine (ERI), a dye already approved by the Food and Drug Administration (FDA) for various purposes. Although promising for PDT, ERI has a high hydrophilic profile that impacts its activity. To solve this, the combination of ERI with thermoresponsive and bioadhesive polymers may prove effective. Bio/mucoadhesive and thermoresponsive systems have attracted increasing interest in the development of novel pharmaceutical formulations for topical applications due to their ability to improve adhesion to the mucosa and prolong the residence time at the application site. In this study, systems based on poloxamer 407 (P407) in combination with cellulose derivatives (HPMC and NaCMC) were optimized, aiming at the topical release of ERI for PDT. The results demonstrated that the formulations containing low concentrations of cellulose derivatives exhibited greater adhesiveness and consistency at physiological temperature (37 °C), favoring the maintenance of the system at the application site. Regarding the gelation temperature (T_sol/gel), the formulations displayed values close to body temperature. The formulations with NaCMC showed a slightly higher T_sol/gel compared to HPMC ones, but it was adjustable by the polymer concentration. The addition of ERI influenced the mechanical and adhesive properties of the systems. In formulations containing HPMC, high concentrations of ERI increased bio/mucoadhesiveness, while in systems with NaCMC, the presence of ERI reduced this property. In both cases, the formulations maintained high consistency at 37 °C, contributing to the control of the active release at the application site. Rheological analysis revealed non-Newtonian behavior in all formulations, with greater consistency and elasticity at high temperatures. P407 was mainly responsible for the thermoresponsive transition from sol to gel, conferring desirable characteristics for topical application. Photodynamic activity was relevant in both formulations containing NaCMC and HPMC, which demonstrated greater capacity for degrading uric acid under light exposure. These systems are promising for the controlled release of drugs in photodynamic therapy, providing prolonged retention in the target tissue and maximizing the therapeutic efficacy of ERI.

1. Introduction

Photodynamic therapy (PDT) is a light-activated chemical reaction used for the selective destruction of tissue [1,2]. For this type of phototherapy, three or more factors must act simultaneously: the photosensitizer (or dye), a light source, and oxygen [3]. The transfer of energy from the excited dye to the substrate leads to the production of reactive oxygen species, which are toxic to tumor tissue and microorganisms [3,4].

Among the various dyes used in PDT, erythrosine (ERI) is a dye already approved by the Food and Drug Administration (FDA) for various purposes, such as therapeutic application and food products. This xanthene dye is characterized by maximum light absorption at wavelengths ranging from 500 to 550 nm [5,6]. This absorption is associated with consequent photochemical reactions and production of reactive oxygen species, as previously reported in the literature [7]. Because of this, this dye is widely used in photodynamic therapy with the aim of destroying tumor cells and tissues. However, ERI has some characteristics that hinder its pure action in solution, such as its high hydrophilic profile, which negatively impacts its permeation through biological membranes and consequently in reaching important organelles for a good PDT result. As a solution to this problem, the combination of biological macromolecules and thermoresponsive polymers may prove effective, increasing the retention of topically applied drugs [8].

Polymers play a crucial role in the ability to transport active ingredients, a characteristic usually linked to their physicochemical properties. Several polymer classes have been explored in different drug delivery systems, with the purpose of optimizing the administration of pharmaceutical compounds [9,10]. These systems enable control over the release of therapeutic agents, in addition to reducing toxic effects and systemic first-pass metabolism [11,12]. By definition, drug delivery systems are designed to promote the controlled and/or modified release of biologically active substances, ensuring that the drug concentration remains within the therapeutic range, ensuring greater safety and bioavailability [13].

To obtain these systems, several strategies can be used. For example, bioadhesive polymer associations for producing semi-solid systems, which aim to promote close contact between the pharmaceutical form and the application site. Thus, bioadhesion is a property that can be defined as the state in which two materials, among which at least one is of a biological nature, are kept together for a prolonged period [14,15]. When intended to increase the residence time of a drug on mucosal surfaces, these systems can be called bio/mucoadhesive [16].

The main strategies employed in the development of systems with bio/mucoadhesive properties are related to the ability of the polymer or system to adhere to the mucus or skin. To this end, the material must interact with the components of the mucus. After the first stage of contact between the formulation and the mucosa, the bio/mucoadhesive system must be able to consolidate the process through physical and chemical interactions, such as hydrogen bonds and hydrophobic, electrostatic, and ion–dipole interactions. Given their advantages and excellent characteristics for local application, bio/mucoadhesive formulations have been widely studied to improve the administration and residence time of drugs in the mucosa or target tissue [17].

Cellulose is a very abundant organic molecule, which is used in the form of its derivatives for pharmaceutical applications and is not toxic to the body [18]. Cellulose derivatives can be found in the form of methyl cellulose (MC), calcium or sodium carboxymethyl cellulose (NaCMC), ethyl cellulose (EC), hydroxyethyl cellulose (HEC), and hydroxypropyl methylcellulose (HPMC) [16]. The literature reports that cellulose derivatives present excellent bio/mucoadhesive properties and so are widely used in pharmaceutical formulations [19]. One of the reasons is related to its probable mechanism of mucoadhesion, which involves the chain interpenetration of cellulose derivatives into the biological tissues [16].

In addition to bioadhesive polymers, other polymers can constitute intelligent drug loading systems. Thermoresponsive polymers, for example, are polymers that respond to changes in temperature and can provide important properties in the development of semisolid systems [20]. When the temperature increases, a low-viscosity solution transforms into a high-viscosity system (gel) due to the reversible changes that occur in the chemical structure of the temperature-responsive polymers. In this sense, thermoresponsive polymer systems are attractive for topical and injectable drug administration since they can improve drug absorption and the stability of pharmaceutical systems [16].

Poloxamer 407 (P407) is an amphiphilic copolymer composed of blocks of ethylene oxide (EO) and propylene oxide (PO) in a chain, which has the property of forming thermoresponsive gels. At room temperature, dispersions containing this polymer are in a fluid/liquid state, transforming to the gelled state at temperatures close to body temperature [16]. Its gelation is related to its ability to form micelles with increasing temperature. The formation of micelles occurs due to the physicochemical characteristics of this polymer, forming a lipophilic core with a hydrophilic crown due to the dehydration that occurs in the EO groups. Under specific conditions of concentration (>15% (w/w) and temperature, the micellar volume reaches a cubic structure, culminating in the formation of the gel [21]. In general, P407 forms an attractive gel for drug delivery due to its low viscosity at room temperature, presenting a transition temperature for the formation of a viscous gel close to physiological temperatures. Furthermore, as it is an amphiphilic polymer, this polymer is capable of carrying both hydrophilic and hydrophobic drugs [21].

In view of the above, the combination of thermoresponsive and bio/mucoadhesive polymers, such as P407 and HPMC or NaCMC, may be an attractive alternative for the development of new pharmaceutical systems [16]. The addition of cellulose derivatives to semisolid systems containing P407 aims not only to contribute to the optimization of their structure but also to modulate the rheological properties of this polymer, ultimately obtaining formulations with excellent bio/mucoadhesive properties [16]. Previous works have studied the combination of P407 and HPMC or NaCMC in high concentrations (1.0–4.0%). However, low gelation temperatures have been observed together with impaired adhesive performance [13,16]. Moreover, for NaCMC systems, a high concentration of this negatively charged polymer demonstrated a repulsion effect on ERI when it was added as the active compound [22]. Therefore, in this work, the development of new bio/mucoadhesive and thermoresponsive polymeric systems was conducted with low amounts of cellulose derivatives, aiming at future applications in drug delivery systems. Binary polymeric mixtures of P407 and HPMC or NaCMC were studied to obtain an optimized semisolid system with appropriate mechanical, rheological, adhesive, and thermoresponsive properties for topical release of ERI, aiming at better photodynamic activity.

2. Materials and Methods

2.1. Material

Poloxamer 407 (P407), erythrosine B disodium salt (ERI), mucin (from porcine stomach, type II crude), and uric acid were purchased from Sigma-Aldrich (Sao Paulo, Brazil). HPMC K100, Methocel®, was kindly donated from Colorcon Dow Chemical Company™ (Dartford, UK). NaCMC (DS = 0.8–0.95) was purchased from Synth (Diadema, Brazil). Ultra-purified water was obtained in-house using a Milli-Q water purification system (Darmstadt, Germany). All reagents were used without purification processes.

2.2. Preparation of Polymer Systems

Binary polymeric systems were prepared by adapting the cold method described by Schmolka [23] with the concentrations of polymers and erythrosine described in

Table 1. Composition and concentration of polymers and erythrosine in the formulations.

Formulation	P407 (%, w/w)	HPMC (%, w/w)	NaCMC (%, w/w)	Erythrosine (%, w/w)
poloxHPMC 0.1	17.5	0.1	-	-
poloxHPMC 0.2	17.5	0.2	-	-
poloxHPMC 0.4	17.5	0.4	-	-
HPMC–ERI 0.1	17.5	0.4	-	0.1
HPMC–ERI 1.0	17.5	0.4	-	1.0
poloxNaCMC 0.1	17.5	-	0.1	-
poloxNaCMC 0.2	17.5	-	0.2	-
poloxNaCMC 0.4	17.5	-	0.4	-
NaCMC–ERI 0.1	17.5	-	0.1	0.1
NaCMC–ERI 1.0	17.5	-	0.1	1.0

.

The cellulose derivative (HPMC or NaCMC) was slowly added to purified water at room temperature (25 °C) under constant mechanical stirring until complete dispersion. Subsequently, P407 was added to the solution and left to hydrate at refrigerator temperature (5 °C). After 24 h, the preparations underwent slow mechanical stirring for approximately 10 min until complete system homogenization. Upon completion, the formulations were stored in the refrigerator for a minimum of 24 h before analysis [16].

For the addition of erythrosine, the active substance was first dissolved in purified water. Subsequently, the cellulose derivative (HPMC or NaCMC) and the polymer P407 were added to the solution, as previously described.

2.3. Texture Profile Analysis

The texture profile analysis (TPA) of the systems was conducted using a TA-XTplus texture analyzer (Stable Micro Systems^®, Surrey, UK) in texture analysis mode. A quantity of 16 g from each formulation was placed in a screw-capped glass jar, ensuring no air bubbles formed. In TPA mode, a deurin probe (10 mm in diameter) was compressed twice within the sample at a speed of 2 mm/s, to a depth of 15 mm, with a 15-s interval between the end of the first compression and the start of the second. The analysis was performed on at least three replicates for each sample, at temperatures of 25 and 37 °C. From the resulting force versus distance and force versus time graphs, hardness, compressibility, adhesiveness, elasticity, and cohesiveness were calculated [24,25].

2.4. Rheological Analyses

The analyses were conducted using a MARS II (Haake^®, Newington, Germany) controlled stress and shear rate rheometer, at temperatures of 25 and 37 ± 0.1 °C, with parallel plate-cone geometry of 35 mm in diameter, separated by a fixed distance of 0.105 mm. The samples were carefully placed on the lower plate, ensuring minimal shear of the formulation and allowing a rest time of one minute before each analysis.

2.4.1. Flow Rheometry

The upward and downward flow curves were obtained with a shear rate gradient ranging from 0 to 2000 s⁻¹. The shear rate gradient was increased over a period of 150 s, maintained at the maximum value for over 10 s, and then decreased over the same period (150 s), obtaining the downward curve [26].

The upward curves were analyzed using the Oswald de Waele (Equation (1)) and Casson (Equation (2)) equations, as follows, using RheoWin 4.10.0000 software:

$$\mathsf{\sigma }=K.{ẏ}^n$$

(1)

where σ = shear stress, K = consistency index, ẏ = shear rate gradient, and n = flow behavior index.

$$\mathsf{\sigma }=\sqrt[n]{{\mathsf{\sigma }}_0^n+{\left({\mathrm{ẏ}\times \mathrm{n}}_{\mathrm{p}}\right)}^n}$$

(2)

where σ = shear stress, ẏ = shear rate gradient, n = flow behavior index, σ₀ = yield value, and n_p = Casson plastic viscosity.

2.4.2. Oscillatory Rheometry

Firstly, the linear viscoelastic region (LVR) of each formulation, at each temperature (25 and 37 °C), was investigated using a stress gradient range from 0.01 to 20 Pa at a fixed frequency of 1.0 Hz. The LVR is characterized as the region where the stress and strain are directly proportional and the elastic modulus (G′) remains constant. A stress value within the LVR was selected for subsequent frequency sweep analyses.

Over a frequency range of 0.1 to 10.0 Hz, at least three replicates were analyzed for each sample [26]. The elastic modulus (G′) and the viscous modulus (G″) were analyzed using RheoWin 4.10.0000 software.

2.4.3. Determination of the Sol–Gel Transition Temperature (T_sol/gel)

Initially, the determination of the LVR for each formulation was conducted at temperatures of 5 and 60 °C. Subsequently, a temperature sweep analysis was performed, starting at 5 °C and increasing gradually to 60 °C at a rate of 10 °C/min, with a frequency of 1.0 Hz applied [26]. The gelation temperature (T_sol/gel) was determined by the crossover point of G′ and G″ along with a significant increase in dynamic viscosity.

2.5. Determination of In Vitro Mucoadhesion

Using a TA-XTplus texture analyzer (Stable Microsystems^®, Surrey, UK) in tension mode, the bio/mucoadhesive strength of all formulations was evaluated in vitro by measuring the force required to detach the formulation from a mucin disk. First, the mucin disk was prepared by compressing 250 mg of mucin powder with an automatic compressor, using a 14 mm diameter probe and a compression force of 10 tons [16].

For the analysis, the mucin disk was placed in contact with a 5% (w/w) mucin solution for 30 s, with excess liquid removed using an absorbent paper. The mucin disk was then horizontally fixed to the lower end of the analysis probe and left in contact with the sample surface for 30 s with an applied force of 0.03 N to ensure intimate contact between the mucin disk and the sample. The probe then lifted at a constant speed of 10.0 mm/s, and the force required to remove the mucin disk from the surface of the formulation was determined as the detachment force. The resulting force versus time curve was classified as the work of adhesion. For all formulations, measurements were performed with at least three replicates at 37 °C.

2.6. Determination of the Ex-Vivo Bioadhesion

For bioadhesion analysis, skin samples were obtained from the ears of young, recently slaughtered white pigs intended for human consumption. The pig ears were cleaned with cold water, and the dorsal region was dissected with the aid of a surgical scalpel.

Using a TA-XTplus texture analyzer (Stable Microsystems^®, Surrey, UK) in tension mode, the bioadhesive strength of all formulations was evaluated ex vivo as previously described in the mucoadhesion analyses. The pig skin was then horizontally fixed to the lower end of the analysis probe and left in contact with the sample surface for 30 s with an applied force of 0.03 N to ensure intimate contact between the skin and the sample. The probe was then lifted at a constant speed of 10.0 mm/s, and the force required to remove the skin from the surface of the formulation was determined as the detachment force. The resulting force versus time curve was classified as the work of adhesion. For all formulations, measurements were performed with at least three replicates at 34 °C.

2.7. Photodynamic Activity

The evaluation of the photodynamic chemical activity of the preparations was determined using a method adapted from Fischer and colleagues [27]. Approximately 15 mg of the prepared formulation was exactly weighted and added, at 25 °C, to a quartz cuvette with a 1.0 cm optical pathlength. After the addition of the formulation, it was left for 2 min until its complete gelation. Uric acid (UA) was used to evaluate singlet oxygen and the generation of other reactive oxygen species. Thus, UA was solubilized in a 50% (w/w) sodium hydroxide solution, attaining a concentration of 0.5 μmol/L. The dispersion was facilitated by subjecting the solution to an ultrasound bath for 2 min. Then, 2 mL of the previously prepared UA solution was added to the same cuvette. A spectrophotometer (UV-VIS model 1800, Shimadzu, Japan) was used to perform wavelength scans in the range of 200 to 700 nm, using water as a standard to zero the equipment. Wavelength scans were performed every 5 min, with or without a 5 min exposure to external light (LEDs with average irradiance of 0.50 mW/cm² and λmax = 525 nm). The reduction in the 295 nm band, related to UA degradation, was monitored at room temperature [8].

3. Results and Discussion

3.1. The Optimal Concentration of the Bioadhesive Polymer

As previously mentioned, the literature reports the combination of P407 and HPMC or NaCMC in elevated concentrations. However, low gelation temperatures have been observed together with restricted adhesive performance [8,13,16]. Furthermore, high concentrations of NaCMC, a negatively charged polymer, demonstrated a repulsive effect in the presence of ERI [22]. Therefore, in this work, optimized concentrations of the cellulose derivatives as well as ERI were investigated, aiming at the development of new bioadhesive and thermoresponsive polymeric systems with satisfactory properties for local application and release of ERI as a photodynamic active compound.

3.1.1. Texture Profile Analysis of Polymeric Formulations

During the development of semisolid pharmaceutical systems, the texture profile analysis (TPA) of formulations can elucidate possible behaviors during their application, as well as explain interactions between the polymers that constitute them. With TPA analysis, some mechanical characteristics can be determined, such as hardness, compressibility, adhesiveness, elasticity, and cohesiveness, parameters that will be discussed in this work.

Hardness and compressibility are relatable parameters, with hardness being the maximum force required to deform the formulation, calculated in the force versus time graph as the maximum force obtained during the first compression, and compressibility being the work required to deform the sample, calculated in the force versus time graph as the area under the curve from the beginning to the maximum force in the first compression. Therefore, as shown in the Figure 1, there was no significant difference (p-value > 0.05) in hardness and compressibility at a temperature of 25 °C with both cellulose derivatives (NaCMC and HPMC). Furthermore, at this temperature, the formulations presented low hardness and compressibility values, which is important for easier applicability and spreadability.

However, when the temperature was raised to 37 °C, the hardness and compressibility of all formulations increased significantly (p-value < 0.05). This phenomenon can be explained by the presence of the polymer P407. Due to its thermoresponsive nature, P407 can transition from a liquid solution to a gel state after reaching the gelation temperature (T_sol/gel); that is, after a certain temperature, these samples acquired behavior similar to gels. The increase in their consistency, resulting in high hardness and compressibility, was due to the gel state already being reached. Furthermore, there was no significant difference between the polymers (p-value > 0.05), except for the formulation poloxHPMC 0.1, which demonstrated significantly higher force and hardness values (p < 0.05) than the other formulations.

Adhesiveness is another parameter obtained in the TPA test and is determined by the area of the negative force in the corresponding force versus time graph during the first compression. It is described as the energy required to break the attractive interactions between the surface of the formulation and the surface of the material used in the test. So, regarding this parameter, the formulations demonstrated adhesiveness only at 37 °C (Figure 1). This can be explained by the increase in the consistency of the formulations with the rise in temperature, as samples with higher viscosities may exhibit better adhesion to surfaces. Moreover, the formulations containing HPMC showed significantly higher adhesiveness compared to those with NaCMC.

Cohesiveness can be understood as the ability of the system to reorganize after repeated compressions. Elasticity, on the other hand, is understood as the ability of the formulation to stretch and return to its original shape. Therefore, as shown in the Figure 1, the formulations showed the expected elasticity and cohesiveness for semisolids, with no significant (p > 0.05) difference between them.

3.1.2. Solution–Gel Transition Temperature Analysis

Pharmaceutical formulations containing P407 are characterized by a gelation temperature (T_sol/gel). In other words, after reaching a certain temperature, these polymeric solutions acquire gel behavior, with an increasement in viscosity as a notable characteristic. This increase in consistency can help with the bioadhesiveness of the formulation, as well as keeping the formulation in contact with the skin for longer. Therefore, it is desirable that T_sol/gel is close to skin temperature (~34 °C).

Table 2. Gelation temperatures of the formulations containing the different cellulose derivatives.

Formulation	Gelation Temperature (°C)
poloxHPMC 0.1	31.987 ± 0.055
poloxHPMC 0.2	32.293 ± 0.376
poloxHPMC 0.4	31.603 ± 0.493
poloxNaCMC 0.1	35.497 ± 0.153
poloxNaCMC 0.2	32.993 ± 0.102
poloxNaCMC 0.4	31.563 ± 0.693

provides the T_sol/gel values for the formulations containing the cellulose derivatives NaCMC and HPMC. The results indicated that for formulations containing HPMC, there was no statistically significant difference (p-value > 0.05) in T_sol/gel values as the HPMC concentration increased. This suggests that increasing the concentration of HPMC did not influence the T_sol/gel temperature.

By contrast, the formulations containing NaCMC exhibited higher T_sol/gel values compared to those containing HPMC. Additionally, in the NaCMC formulations, an increase in NaCMC concentration led to a significant decrease in T_sol/gel value (p-value < 0.05). The formulation poloxNaCMC 0.1 displayed the highest T_sol/gel value among all NaCMC formulations, making it the formulation closest to the desired temperature for topical application. Therefore, the impact of NaCMC on the T_sol/gel value appeared to be more pronounced than that of HPMC, particularly in influencing the T_sol/gel temperature based on the concentration.

3.1.3. Mucoadhesion Determination of Polymeric Formulations

Adhesion refers to the process by which two surfaces come together and adhere to one another. When one of the surfaces involved is of biological origin, the phenomenon is known as bioadhesion. In a more specific context, when this adhesion occurs on mucosal membranes, the most appropriate designation for the process is mucoadhesion [28,29]. Mucoadhesion in pharmaceutical systems offers a range of significant advantages, including prolongation of the drug’s residence time at the site of application, which contributes to the enhancement and control of its absorption. Furthermore, it provides protection to the drug against degradation resulting from the absorption environment. The ease of administration facilitated by mucoadhesion also translates into improved pharmaceutical efficacy, among other benefits [29].

The tensile strength on mucosal surfaces represents one of the most widely used methods for evaluating mucoadhesion in pharmaceutical systems [30]. In this way, the mucoadhesion of the formulations was calculated in relation to a mucin disk (Figure 2).

The graph shows that increasing the concentration of HPMC did not significantly affect (p-value > 0.05) the strength and work of mucoadhesion. However, the formulations containing NaCMC exhibited significant decreases (p-value < 0.05) in the force and work of mucoadhesion with the increase in the concentration of this polymer.

The adhesion process is complex and involves several theories to explain its mechanisms [31]. In this context, the mucoadhesion of HPMC and NaCMC polymers occurs through different mechanisms, which are influenced by both the polymer concentration and their interaction properties with mucosal surfaces [16].

Regarding HPMC, its bio/mucoadhesive properties are frequently associated with the entanglement of polymer chains and their physical interlocking with mucus, as research suggests that HPMC does not form direct bonds with mucin [16,32]. This entanglement is facilitated in less concentrated formulations, which have low viscosity, allowing the polymer to spread more easily over the mucosal surface and enhancing the interaction between polymer chains and mucin. At high concentrations, the increased viscosity can hinder this interpenetration, potentially negatively impacting the mucoadhesion process [16,33]. However, the results indicated that there was no significant difference in bio/mucoadhesive performance with the increase in HPMC concentration, despite literature reports suggesting that variations in the concentration of this polymer can significantly impact bio/mucoadhesive properties [16]. The discrepancy between these results may be attributed to the fact that the concentrations evaluated in the present study showed only a small variation, which may not have been sufficient to generate significant differences in the bio/mucoadhesive properties. Therefore, it can be concluded that the limited spacing between the HPMC concentrations used (0.1%, 0.2%, and 0.4%) may have contributed to the absence of detectable effects in the analyzed formulations.

Alternatively, NaCMC is distinguished by its ability to form strong hydrogen bonds with mucin. However, for mucoadhesion to be effective, these interactions need to be complemented by physical interlocking with mucus. In more diluted systems, the lower viscosity facilitates better interpenetration of NaCMC polymer chains with mucin, enhancing the establishment of these bonds [11,32]. This explains why, with the decrease in NaCMC concentrations, improved bio/mucoadhesive performance was observed.

In this way, based on the performance observed in the previous experiments, one formulation from each cellulose derivative was selected for further studies. Therefore, the formulations poloxHPMC 0.4 and poloxNaCMC 0.1 were chosen for the subsequent experiments and the incorporation of erythrosine.

3.2. Effect of Erythrosine

3.2.1. Texture Profile Analysis of Polymeric Formulations with Erythosine

Among the selected formulations containing NaCMC and HPMC, ERI additions were made. To evaluate the effect of this addition, two concentrations of ERI were evaluated: 0.1 and 1% (w/w). Therefore, TPA of the formulations containing ERI was performed, as shown in Figure 3.

Initially, when analyzing the influence of the addition of ERI on the hardness of the formulations, it was observed that the incorporation of erythrosine resulted in a significant reduction in hardness (p < 0.05) for the formulations containing both polymers, NaCMC and HPMC, when evaluated at 25 °C. However, when increasing the temperature to 37 °C, the addition of ERI did not promote significant changes in the hardness of the formulations (p > 0.05).

Furthermore, increasing the ERI concentration from 0.1 to 1.0% did not result in significant changes in the samples (p > 0.05) at both temperatures (25 and 37 °C). The formulations containing HPMC maintained higher hardness values than those containing NaCMC, as previously observed, and the addition of ERI did not influence this behavior.

The same behavior was observed in relation to compressibility. The addition of ERI reduced the compressibility of the formulations at 25 °C; however, at 37 °C, there was no significant influence (p > 0.05). Furthermore, the increase in the ERI concentration did not significantly alter the characteristics of the formulations.

The formulations exhibited adhesiveness only at 37 °C, a behavior attributed to the increased consistency observed after reaching the gelation temperature. The first observed phenomenon was that the addition of ERI to the formulations reduced their adhesiveness in both HPMC- and NaCMC-containing formulations. This effect may have been related to the reductions in hardness and compressibility caused by the addition of ERI, since it is known that the consistency of a material influences its ability to adhere to surfaces [26]. Regarding the increase in ERI concentration from 0.1% to 1%, no significant difference was observed in the NaCMC-containing formulations. However, in the HPMC-containing formulations, the increased ERI concentration resulted in higher significant adhesiveness (p = 0.00017).

Regarding elasticity, no significant changes were observed with the addition of ERI, nor were there significant differences between the different concentrations of ERI. Concerning cohesiveness at 25 °C, the formulations containing NaCMC exhibited greater cohesiveness compared to those containing HPMC. Furthermore, increasing the ERI concentration in the HPMC-containing formulations significantly reduced the cohesiveness (p-value = 0.002). By contrast, for the NaCMC-containing formulations, no significant difference in cohesiveness was observed between the different ERI concentrations (p = 0.261). At 37 °C, the cohesiveness of the HPMC-containing formulations significantly decreased with the addition of ERI (p-value = 0.002), while no significant difference was observed for the NaCMC-containing formulations.

3.2.2. Solution–Gel Transition Temperature Analysis

The analysis of the gelation temperatures revealed subtle differences between the formulations containing HPMC and NaCMC, as well as between the different ERI concentrations (

Table 3. Gelation temperature (T_sol/gel) of the formulations containing erythrosine (ERI).

Formulation	Tsol/gel (°C)
HPMC–ERI 0.1	32.037 ± 0.067
HPMC–ERI 1.0	32.760 ± 0.096
NaCMC–ERI 0.1	33.547 ± 0.482
NaCMC–ERI 1.0	33.890 ± 0.052

). For the formulations containing HPMC, an increase in gelation temperature was observed as the ERI concentration increased from 0.1% to 1.0%; however, this increase was not statistically significant (p-value = 0.253). Similarly, in the formulations containing NaCMC, the gelation temperature also increased as the ERI concentration increased from 0.1% to 1.0%. Nonetheless, this increase was also not statistically significant (p = 0.951), suggesting that the ERI concentration had a low impact on the gelation thermal characteristics of both NaCMC- and HPMC-based formulations.

When comparing the HPMC- and NaCMC-based formulations, it was consistently observed that the gelation temperatures of the NaCMC formulations were higher than those of the HPMC formulations, regardless of the ERI concentration. The addition of ERI to the HPMC formulations resulted in a progressive increase in gelation temperature. This behavior may be attributed to interactions between erythrosine and HPMC, which could alter the dehydration dynamics of the polymer during the gelation process, slightly delaying gelation. In the case of the NaCMC formulations, the addition of ERI caused a significant reduction in the gelation temperature. The presence of erythrosine may have modified intermolecular interactions within NaCMC, facilitating the sol–gel transition at slightly lower temperatures, as previously reported somewhere [16].

Overall, the HPMC-containing formulations consistently exhibited lower gelation temperatures than the NaCMC-containing formulations, both in the absence and presence of erythrosine. Additionally, the addition of erythrosine produced opposite effects on the polymers: an increase in the gelation temperature for HPMC and a slight decrease for NaCMC.

3.2.3. Mucoadhesion Determination of Polymeric Formulations with Erythosine

An investigation was also conducted to evaluate the impact of adding ERI at concentrations of 0.1% and 1.0% on the adhesive capacity of the formulations (Figure 4).

Initially, when ERI was incorporated into the formulations, the observed effects varied depending on the type of polymer and the dye concentration. In the formulations containing HPMC, the addition of 0.1% ERI resulted in a bio/mucoadhesive strength without significant difference (p = 0.999) compared to the formulation without ERI, suggesting that, at low concentrations, ERI does not significantly affect adhesion. However, increasing the concentration to 1.0% led to a significant increase in bio/mucoadhesive strength (p-value = 0.0345). This finding suggested that, at high concentrations, ERI may enhance adhesive interactions, possibly due to changes in the matrix structure or the formation of additional interactions between the formulation and the mucosa.

Conversely, in the formulations containing NaCMC, the addition of ERI exhibited an opposite effect. The inclusion of 0.1% ERI reduced the bio/mucoadhesive strength, and even with an increase in concentration to 1.0%, the bio/mucoadhesive strength remained lower than that of the formulation without ERI. These results suggested that, unlike the formulations with HPMC, the presence of ERI negatively interfered with the adhesion of the NaCMC-based formulations, possibly due to unfavorable chemical interactions or alterations in the physical properties of the system, since both NaCMC and ERI are in their anionic form, repelling each other. Furthermore, the impact of increasing the ERI concentration depended on the type of polymer. Therefore, the formulations containing HPMC exhibited a positive effect on mucoadhesion with higher ERI concentrations, while the NaCMC-based formulations showed limited recovery in adhesion, with values remaining below those observed in the absence of ERI.

Regarding the work of mucoadhesion, the addition of ERI had distinct effects on each polymeric system. In the formulations containing HPMC, the inclusion of 0.1% ERI did not result in a significant increase in the work of mucoadhesion. However, when the ERI concentration was increased to 1.0%, the work of mucoadhesion was significantly high, indicating a positive relationship between erythrosine concentration and the adhesive capacity of this system, consistent with the observations from the bio/mucoadhesive strength analysis. By contrast, in the formulations containing NaCMC, the addition of ERI appeared to reduce the work of mucoadhesion, although the reduction was not statistically significant.

Overall, the impact of ERI on the work of mucoadhesion was shown to depend on the type of polymer used. In the formulations containing HPMC, the addition of ERI increased the mucoadhesion as the concentration increased. Conversely, in the formulations containing NaCMC, ERI initially decreased the mucoadhesion, although high concentrations may have the potential to improve the formulation’s performance.

3.2.4. Bioadhesion Determination

The bioadhesion strength of the formulations containing the cellulose derivatives HPMC (poloxHPMC) and NaCMC (poloxNaCMC) showed relevant differences (Figure 5).

The formulation with NaCMC exhibited greater bioadhesion strength and work compared to the formulation containing HPMC. The addition of ERI to the formulations showed a variable impact on bioadhesion, depending on both the cellulose derivative used and the ERI concentration. The inclusion of ERI into the formulations containing HPMC led to a slight improvement in the bioadhesion strength and work at both concentrations, when compared to HPMC (poloxHPMC). This behavior suggested that ERI may have functioned as an auxiliary agent, promoting additional intermolecular interactions with the skin. However, increasing the erythrosine concentration from 0.1% to 1% did not result in a significant increase.

The bioadhesion strength was drastically increased in the formulation with NaCMC and 0.1% ERI, reaching the highest value observed in the graph. However, when the erythrosine concentration was increased to 1%, there was a reduction in both the strength and work of bioadhesion. The results showed that NaCMC was superior to HPMC in terms of adherence to the skin, both in strength and work values, especially when combined with 0.1% ERI. The mechanism of adhesion in the skin and mucosa were suggested to be different, with the anionic species of both NaCMC and ERI presenting a positive impact. Therefore, erythrosine was demonstrated to improve the adhesiveness of the formulations to porcine skin; however, its effectiveness depended on the type of polymer used and the concentration applied.

3.2.5. Rheometry

Continuous Shear (Flow) Rheology

Through continuous shear rheometry, it is possible to analyze some parameters, such as ease of administration and time-dependent recovery after application [34,35]. This research not only examined the rheological response of the formulations under a wide range of continuous stresses but also provides an understanding of the interactions between the polymers that make up the system. This understanding is crucial to selecting the most appropriate formulations for specific clinical uses, considering the stress variations to which these formulations are exposed during procedures such as production, packaging, storage, unpacking, and use. This understanding, by anticipating and preventing the breakdown of the polymer structure, directly contributes to the stability and therapeutic efficacy of the product [34].

The flow curves of the poloxHPMC and poloxNaCMC formulations without and with ERI are presented in Figure 6 and the parameters are presented in

Table 4. Flow parameters obtained by continuous shear rheological analysis of the formulations at temperatures of 25 and 37 °C ^a.

Formulations	n (Dimensionless)		K (Pa.s)		σy (Pa)		Hysteresis Area (Pa/s)
	25 °C	37 °C	25 °C	37 °C	25 °C	37 °C	25 °C	37 °C
HPMC–ERI 0.1	1.016 ± 0.014	0.209 ± 0.011	0.188 ± 0.020	94.513 ± 8.171	7.475 ± 0.913	210.167 ± 15.701	−56,646.667 ± 8575.834	−20,633 ± 12,827.611
HPMC–ERI 1.0	1.040 ± 0.007	0.193 ± 0.012	0.153 ± 0.013	101.433 ± 8.402	7.867 ± 4.032	205.567 ± 9.730	−47,403.333 ± 2220.480	−7396.667 ± 8754.705
NaCMC–ERI 0.1	0.974 ± 0.020	0.184 ± 0.002	0.215 ± 0.034	106.700 ± 1.652	0.183 ± 0.185	216.600 ± 7.618	−37570 ± 11,556.003	−2472.583 ± 2224.258
NaCMC–ERI 1.0	0.976 ± 0.013	0.181 ± 0.001	0.210 ± 0.020	103.533 ± 2.409	0.151 ± 0.121	197.067 ± 4.314	−35,903.333 ± 4947.447	7656.600 ± 10,166.537
poloxHPMC	0.901 ± 0.034	0.184 ± 0.005	0.397 ± 0.091	115.167 ± 5.900	2.127 ± 1.533	227.133 ± 3.365	−21,354.667 ± 10427.377	−30,576.667 ± 9452.335
poloxNaCMC	0.950 ± 0.013	0.194 ± 0.003	0.232 ± 0.016	88.583 ± 3.593	0.420 ± 0.157	185.467 ± 10.433	−21,230 ± 4497.699	−1192.133 ± 8213.354

^a Result expressed as mean ± standard deviation of three experiments performed independently. Where n is the flow behavior index, K is the consistency index, and σ_y is the yield value.

.

In analyzing the flow curves of the formulations without ERI, it is possible to see that both the poloxHPMC and poloxNaCMC formulations showed a decrease in tension with increasing shear. In addition, the flow behavior index (n) showed values lower than 1 (n < 1), indicating that the formulations were non-Newtonian materials with shear thinning curves at both temperatures of 25 and 37 °C. This result was in accordance with the literature, which indicated that binary systems containing poloxamer with HPMC or NaCMC exhibit shear thinning behavior [13]. Furthermore, there was no significant difference (p-value > 0.05) in the n value between the formulations with NaCMC and HMPC.

At 25 °C, the formulations presented a flow behavior index (n) close to one, characteristic of Newtonian materials. This characteristic close to Newtonian behavior at 25 °C can be explained by the presence of P407. Monomeric systems composed of P407 tend to exhibit Newtonian behavior at room temperature [13,21]. Thus, considering that in the formulations developed in this study P407 was the major polymer (17.5%), while cellulose derivatives (HPMC at 0.4% and NaCMC at 0.1%) were present in small proportions, the behavior of the formulations predominantly reflected the properties of P407. However, the formulations were still classified as non-Newtonian formulations because, despite the proximity of the flow behavior index to the value of one, it remained slightly lower, indicating shear thinning behavior.

At 37 °C, it was possible to observe a decrease in the flow behavior index of both formulations, attributed to the presence of P407, which was responsible for the induced gel behavior after reaching the gelation temperature.

Regarding the consistency index (K) at 25 °C, both formulations presented low consistency, which suggested they would be easy to apply. Furthermore, there was no significant difference (p = 0.091) in the consistency index values between the formulations containing NaCMC and HPMC at this same temperature.

However, with the increase in temperature to 37 °C, it was possible to observe an increase in the consistency index caused by reaching the gelation temperature. At this temperature, the system containing HPMC presented significantly (p-value = 0.007) greater consistency than the system containing NaCMC. Increased consistency at body temperature may be beneficial during application, as it contributes to bioadhesion and may modulate the release of the active ingredient at the application site [36].

The yield value (σ₀) value corresponds to the minimum stress required to initiate the flow of a material [26]. Furthermore, it has been noted that as a material’s consistency increases, so does the force necessary to start its flow. Therefore, when examining Table 4, it is noted that the increase in temperature considerably increased the yield value of the formulations, accompanying the increase in consistency after reaching the gelation temperature. Only at the temperature of 37 °C was there a significant difference (p-value = 0.022) between the formulations with NaCMC and HPMC. These higher yield values of the formulations stand out for their ability to remain at the application site, as they are systems capable of withstanding in vivo movements, such as mucociliary movement, clearance, and chewing [35,37].

When shear rate is applied, pharmaceutical systems may show changes in viscosity over time. Thus, in rheogram analyses, on the outward curve (increased shear), the samples may present different viscosities when compared to the return curve (decreased shear), causing a hysteresis area. This area, when positive, is called thixotropy, occurring when the material presents lower viscosities over time; however, when negative, the phenomenon is called rheopexy, resulting in materials with high viscosities on the return curve of the rheograms [31].

In analyzing the rheograms of the formulations in Figure 6 and the values of the hysteresis area in Table 4, it is possible to observe that both formulations (poloxNaCMC and poloxHPMC) presented rheopexy at both temperatures (25 and 37 °C). There was a significant difference between the formulations in the hysteresis area only at 37 °C (p-value = 0.015), with the poloxHPMC formulation presenting a larger hysteresis area, possibly due to its greater consistency, which required a longer time for the polymer chains to realign and return to the initial viscosity.

The continuous rheological analysis of the formulations containing erythrosine revealed important differences in the flow behaviors of the samples. The flow behavior index (n), which reflects the degree of pseudoplasticity, was significantly influenced by the polymers used, the analysis temperature, and, in low extension, by the erythrosine concentration.

The addition of ERI significantly influenced the flow behavior index of the formulations containing HPMC at 25 °C, causing Newtonian behavior of the formulations in both ERI concentrations. This indicated that, at low temperatures, the molecular interactions within the formulations containing HPMC maintained a relatively linear structure under shear. On the other hand, the formulations with NaCMC presented slightly low values, indicating pseudoplastic behavior at both ERI concentrations. Furthermore, the addition of ERI did not significantly influence the change in the behavior of these formulations at 25 °C.

By contrast, at 37 °C, the n values for all formulations were significantly reduced, mainly due to the influence of the thermoresponsive characteristic of P407, demonstrating a pseudoplastic material characteristic. Furthermore, the addition of ERI did not significantly alter the flow behavior index of the formulations containing NaCMC or HPMC.

Furthermore, the ERI concentration did not significantly alter the flow behavior of the formulations at both temperatures. In general, the formulations containing NaCMC exhibited greater shear strength than those containing HPMC. This behavior may be advantageous for applications requiring greater rheological control under shear conditions, such as during topical application or in controlled release systems. On the other hand, the formulations with HPMC demonstrated low shear sensitivity at 25 °C, which may favor some industrial applications.

Regarding the consistency index, at 25 °C, the addition of ERI did not influence the behavior of the formulations and there was no significant difference between the formulations containing NaCMC or HPMC.

On the other hand, at 37 °C, the addition of ERI significantly influenced the formulations, but in diverse ways. While the addition of ERI decreased the viscosity of the formulations containing HPMC, those containing NaCMC showed an increase in consistency. Furthermore, at this temperature, there was a considerable increase in the viscosity of all formulations when compared to 25 °C, mainly due to the presence of P407 after reaching the gelation temperature.

In summary, the formulations with NaCMC and ERI presented a high consistency index, suggesting greater potential for applications in which viscosity plays a crucial role, such as in controlling the release of active ingredients. The influence of erythrosine concentration was insignificant at both temperatures.

Regarding the yield value, at 25 °C, the formulations containing HPMC presented high yield values compared to the formulations containing NaCMC. Furthermore, the addition of ERI significantly impacted the formulations, but in diverse ways. In the formulations with HPMC, the addition of ERI resulted in an increase in the yield value, while in the formulations containing NaCMC, there was a reduction in these values.

At 37 °C, the yield strength values increased dramatically, regardless of the polymer type or erythrosine concentration, influenced by the presence of P407. Among the polymers, the difference in yield strength values observed at 25 °C was reduced when the temperature was increased to 37 °C, indicating that the structural effect of poloxamer at elevated temperatures overrode the individual characteristics of HPMC and NaCMC. Despite this, the formulations with HPMC maintained a slight superiority in yield strength values.

Overall, the polymer type had a significant impact on the rheological behavior at 25 °C, with the HPMC formulations showing higher initial flow resistance. However, at 37 °C, the rheological properties were largely dominated by the thermoreversible behavior of poloxamer, with high yield values in all formulations. The erythrosine concentration had a limited influence in both conditions, with the greatest impact observed in the HPMC formulations at 25 °C.

Oscillatory Rheology

Oscillatory rheology studies determine the viscoelastic properties of materials by subjecting the systems to low shear stresses with sinusoidal movements [13,34]. In this way, properties such as the elastic (G′) and viscous (G″) moduli are obtained, enabling the simulation of the behavior of formulations in physiological environments [13].

The elastic modulus (G′) is defined as the energy stored and recovered per deformation cycle applied to the system, and the viscous modulus (G″) represents the viscosity of the material [34]. Thus, the G′ and G″ values of the formulations determined at temperatures of 25 and 37 °C are shown in Figure 7.

Furthermore, good elasticity of a formulation can significantly contribute to retention at the application site and release control [38,39]. Figure 7 shows that the poloxHPMC formulation exhibited viscoelastic properties at both temperatures evaluated (25 °C and 37 °C), characterized by higher values of the elastic modulus than the viscous modulus (G′ > G″), a typical property of gels [34]. However, this system showed greater frequency dependence at the temperature of 25 °C.

On the other hand, the poloxNaCMC system demonstrated elastoviscous behavior (G″ > G′) at 25 °C, indicating characteristics closer to polymer solutions [34]. In these systems, there is enough time for the polymer chains to unravel and flow under the action of oscillations, resulting in a predominant viscous modulus in relation to the elastic modulus [34]. However, when the temperature was raised to 37 °C, it was observed that the poloxNaCMC system acquired viscoelastic characteristics. This behavior (G′ > G″) suggested that, at 37 °C, the time available for the disentanglement of the polymer chains was insufficient, promoting a predominance of the elastic modulus over the viscous one. Furthermore, the system response became less frequency dependent, reflecting a more stable structure and typical gel characteristics [34]. This behavior was mainly explained by the presence of P407.

The incorporation of ERI had a significant impact on the rheological behavior of the formulations containing HPMC, as shown in Figure 7. Specifically, these formulations exhibited a clear dependence on the frequency applied during the analysis, with their behavior being predominantly viscous at lower frequencies, which then gradually transitioned to a state characterized by predominantly elastic behavior as the frequency increased. By contrast, when evaluating the formulations containing NaCMC, under the same temperature conditions of 25 °C, no significant differences were observed in their rheological behavior regardless of the presence of ERI.

Conversely, at the elevated temperature of 37 °C, all of the tested formulations demonstrated a clear predominance of G′ over G″, which is a typical characteristic of gel-like materials. Moreover, the addition of ERI, regardless of its concentration, did not produce any observable alterations in the rheological behavior of the formulations under these higher temperature conditions.

Based on the results observed, it is possible to conclude that the incorporation of ERI had a more significant influence on the viscoelastic properties of the formulations containing HPMC when analyzed at a temperature of 25 °C, that is, before these formulations reached the gelation state, characteristic of thermoresponsive conditions. On the other hand, the formulations that used NaCMC as the main component demonstrated a low sensitivity to the presence of ERI at the concentrations evaluated, presenting only minimal changes in their viscoelastic behavior.

The formulations containing 1% ERI (HPMC-ERI 1.0 and NaCMC-ERI 1.0) were selected for the analysis of photodynamic activity, as they presented better characteristics compared to those containing 0.1%. These formulations demonstrated greater strength and mucoadhesion performance. In addition, the gelation temperature of these formulations presented values closer to body temperature, which facilitates application under physiological conditions. For these reasons, formulations containing 1% ERI were considered the most suitable for the subsequent analysis of photodynamic activity.

3.3. Determination of Photodynamic Activity

Figure 8 presents graphs that evaluate the potential photodynamic activity of the formulations containing 1% ERI. The absorbance spectra were constructed in the range of 250 to 350 nm, to highlight the peak at 295 nm, which is characteristic of uric acid. The corresponding controls, called Control (HPMC) and Control (NaCMC), represent the formulations subjected to analysis without exposure to external light, serving as a reference for evaluating the dependence of the photodynamic process.

The HPMC and NaCMC formulations showed progressive reductions in absorbance at 295 nm with increasing exposure time to light, indicating effective degradation of UA mediated by photodynamic activity. This result reflected the ability of ERI to function as a photosensitizer, generating reactive oxygen species (ROS), especially singlet oxygen, in response to light, which promotes the oxidation of UA. These results were in good agreement with the literature, which already showed appropriate values of quantum singlet oxygen yield for ERI in water (0.62) and P407 dispersions (0.64) [8,40]. The comparison among the formulations suggested that NaCMC may present a faster reduction in UA absorbance compared to HPMC (NaCMC with 0.573–0.272, and HPMC with 0.799–0.432). However, many parameters may have impacted the reduction in the UA peak, including the different release rates of ERI in each system.

The controls, in which there was no exposure to light, showed stability in absorbance over the time, confirming that UA degradation occurred exclusively due to the photodynamic process induced by light. The study was also performed in systems in the absence of the drug, showing the maintenance of the absorption of UA, either in the presence or absence of light (Figure 9). This behavior reinforced the importance of ERI as a photosensitizer and showed that light was the determining factor for the generation of ROS and subsequent degradation of the substrate.

In general, the results indicated that both systems evaluated could degrade uric acid, presenting great efficiency in photodynamic activity. The type of cellulose derivative was demonstrated to impact the capacity of the production of ROS, possibly due to differences in the interaction of the polymer with ERI and in the diffusion of the ROS generated. These results are important for characterizing the systems developed in this work, which can be applied in PDT to optimize the efficacy of the treatment. Moreover, the systems presented improved T_sol/gel and mechanical and adhesive properties.

4. Conclusions

The developed bio/mucoadhesive and thermoresponsive systems, containing P407 combined with cellulose derivatives (HPMC and NaCMC) in reduced concentrations, displayed promising characteristics for topical application, especially in photodynamic therapy using ERI as a photosensitive agent. The systems containing 0.4% HPMC and 0.1% NaCMC demonstrated effective thermoresponsive transition, with gelation temperatures closer to skin temperature (~34 °C), facilitating application and retention at the target site. The formulations containing HPMC showed greater mucoadhesiveness and consistency at 37 °C, favoring their use in topical applications that require greater contact with the mucosa. The formulations with NaCMC, in turn, exhibited gelation temperatures that were adjustable by their concentration. The incorporation of ERI influenced the adhesive and mechanical properties of the systems in a differentiated manner. At high concentrations, ERI improved the bio/mucoadhesiveness of the formulations and showed gelation temperatures closer to physiological ones. The rheological tests revealed non-Newtonian behavior, with increased consistency and elasticity at physiological temperatures, characteristics attributed to P407. Regarding photodynamic activity, both formulations with 1% ERI showed greater efficiency, standing out as good options for potential use in photodynamic therapy. The superiority of the bioadhesive performance of NaCMC suggests that this formulation has promising potential for pharmaceutical applications, such as PDT with ERI, mainly for skin applications. Meanwhile, HPMC was demonstrated to be more appropriate for mucosal applications. Considering future applications of the optimized systems, further studies are necessary for their full implementation. These may include the ERI release profile; studies of permeation into the skin and mucosa; in-depth in vitro and ex vivo studies assessing the biocompatibility, cytotoxicity, and photodynamic efficiency of the formulations on relevant tissue models; in vivo testing and conducting preclinical trials to evaluate the formulations’ safety, effectiveness, and pharmacokinetics in animal models; stability studies; scalability and manufacturing validation; regulatory evaluation; and clinical trials.