Biopolymers for targeted drug delivery have been explored in many studies to develop the ideal dermatological biomedical platform. Carrageenans are sulfated biocompatible polysaccharides with prominent relevance in medicinal chemistry, pharmaceutical applications, and biotechnological research [
48]. The
k-car sulfate groups and F127 unimers offer polar and apolar environments for Chls monomerization. Furthermore, the combination of these polymers confers viscosity and adequate interfacial tension to avoid the coalescence processes of the CO droplets. The following analyses highlight the physical, rheological, microbiological, and curative aspects of phytotherapeutic gels (Phy-gels), with potential uses in human and veterinary medicine.
3.1. Physical and Chemical Aspects
The Phy-gels were homogeneous, opaque and did not separate phases throughout the experiment. The
Spirulina sp. had a high concentration of Chls
a and
b (and their derivatives compounds)
, with an intense Q-band in the red region, between 645 and 695 nm (
Figure 5A). The high concentration of chlorines showed photodynamic activity, a fact verified by the degradation of uric acid (UA, chemical probe) after activating the Chls compound. UA had a band at 294 nm and no bands in the visible region. The reduction of around 40% at 294 nm was related to the formation of reactive oxygen species formed after the light-activated Chls undergoes the process of inter-system conversion [
8,
9]. As shown in
Figure 5B, the molecular oxygen in the ground state has a triplet state (
3∑
g). It has two low-lying singlet excited states, the
1∆
g (first excited state) and
1∑
g (second excited state), which differ in their electronic configuration of the π-antibonding orbitals. The transition from the
1∆
g state to the
3∑
g state is forbidden, leading to a long lifetime for the
1∆
g state [
49]. On the other hand, the second excited state (
1∑
g) is short-lived, due to its permitted transition to the
1∆
g state. Most PDT compounds produce singlet oxygen (type II mechanism), which can oxidize the nearby biological species, such as lipids, amino acids, and DNA, due to the high reactivity of the unoccupied
from singlet oxygen. Type I mechanisms also occur (forming O
2•−, •OH, H
2O
2), but with lower quantum yields. In the UA assays, all formed reactive oxygen species allowed its degradation, ensuring spirulina’s potential as a PS source in PDT. In addition to the decay of the UA band, the Chls absorptions were reduced by 24% due to photobleaching—chlorophyll degradation caused by radical species formed in PDT mechanisms [
50,
51].
After checking the in vitro photodynamic potential, the
Spirulina sp. was combined with
C. reticulata to obtain Phy-gels (
Figure 6). Carrageenan is a known healing agent that acts as a structuring agent [
52,
53]. Pluronic
® F127 has been added in small amounts so that its unimers act as monomerization agents for hydrophobic PS, as previously shown [
8,
42]. Furthermore, F127 acts as a promoter of cutaneous permeation of C. reticulata, as we have recently shown [
38].
The emulsions can be expressed by Gibbs free energy (ΔG), described by ΔG = ΔAγ-TΔS, being γ, the droplet interfacial tension, and ΔS, the entropy. The physical gel stability indicates that the cross-linked polymer (k-car) and the F127 polymeric surfactant reinforced the droplet copaiba oil interface, preventing coalescence in short periods. The smaller the droplet size, the better the thermodynamic stability the system acquires, according to Laplace’s law [
54,
55]. Therefore, the employment of a significant amount of energy to obtain small droplets is essential during manufacturing. The microstructure analysis of Phy-CO-Chl gel showed well-delimited interfaces and an accumulation of F127 on the drop’s surface (the lighter region in
Figure 6). For the Phy-CO-Chl emulgel, 50% of the droplets were up to 10 μm in size [
56]. We previously showed that Chls [
8] and copaiba oil [
38] have enhanced skin permeation with F127 micelles, reaching the dermis after 30 min of topical administration.
3.2. Mechanical and Rheological Properties
The rheological and textural studies can estimate the suitability of the gel platforms under industrial manufacture and also in environmental and physiological body conditions [
38]. The data from the textural mechanical analysis are displayed in
Table 3.
The hardness values reflect the force required to produce deformation in the semi-solid system. The gels exhibited a reduction in this parameter with the CO and
Spirulina sp. addition (
p < 0.05), due to the influence on the polymeric chain interactions [
10]. The adhesiveness property may indicate the potential interaction of formulation with the skin after administration. The Phy-gels showed variations without statistical significance (
p > 0.05) for this parameter. However, all systems proved to be an adhesive interface, an essential feature for wound closure at joints and other moving parts. The compressibility showed significant reduction (
p < 0.05), with Chl components and variations without statistical relevance after CO addition (
p > 0.05). Cohesiveness and elasticity are related to the ability of the system to restructure after successive deformations. For these parameters, no significant changes were verified with the variation of the formulation’s composition (
p > 0.05). Furthermore, all gel formulations showed property values that were similar to those of commercial products consolidated in the market, as previously reported (
Table 4) in other studies about k-car gels [
57,
58].
After knowing the gel’s mechanical properties, the flow rheograms were investigated. Rheological studies can characterize the flow behavior of a formulation and allow the understanding of the structuring and interactions between the components of the gel. The continuous flow rheograms are shown in
Figure 7.
The flow rheograms (
Figure 7A, B) showed the non-linear relationship between the applied shear stress (τ) and the shear rate (velocity gradient in reciprocal time units—ẏ). In this process, the viscosity coefficient represents the proportionality relationship between the τ and the ẏ. Its values (angular coefficient) were higher at lower oscillatory frequencies and became lower as the shear rate progressed. The viscosity reduction shown in
Figure 7 resulted in an
n exponent (flow behavior index) <1, and the gel can be classified as a non-Newtonian and pseudoplastic system [
8,
61,
62]. This conduct can be explained by the high entanglement and friction of the
k-car and F127 chains in the resting state, which allow higher apparent viscosity. However, the application of tension leads to the alignment of the polymer chains, which reduces viscosity. Its behaviors are typical of semi-solid systems [
8,
63]. The incorporation of
Spirulina sp. (Phy-Chl and Phy-CO-Chl, as shown in
Figure 7B) reduced the overall viscosity of the system; data concordant with structural change showed in the textural parameters (
Table 3), which may be linked to reduced interaction between the polymer and polysaccharide chains. The CO effect, which remains restricted to the micellar core as a droplet, increased the global viscosity of the Phy-CO gel, an effect already verified for
C. reticulata-loaded in semi-solid systems [
38]. The application of shear stress forces to the Phy-CO gel allows the unraveling of chains and reduces the droplet size to the critical rheological point, which reduces overall viscosity [
38,
64,
65,
66]. Most of the flow rheograms showed no hysteresis area, except the Phy-CO gel, which presented little thixotropy, due to the oil droplets. The Phy-CO-Chl gel showed rheopexy indicative (negative hysteresis area), due to the finite time nature of the analysis.
The Ostwald–de Waele and Casson equations modeled the upward curves for statistical analyses. A flow behavior index (
n) below unity was obtained (
n from 0.32 to 0.40 values for all systems, as shown in
Table 5) and showed an increase with the drug incorporation (
p < 0.05). Furthermore, the consistency index (
K) and the yield value (σ
y) decreased up to 50% with the Chls addition, and up to 19% with the CO incorporation (
p < 0.05). The σ
y values indicated the need for a prior force to start the lamellar flow. The data obtained for this parameter were typical of semi-solid systems and indicated an adequate capacity of the gel to remain at the site of action, in addition to the preventive capacity to destroy the structure of the emulsion (coalescence of the droplets) [
67]. Campanholi et al. (2022) [
38] showed complex rheograms from
Copaifera reticulata Ducke in drug-delivery systems for topical use (blends of F127 and carbopol C934P). The complex nature and difficulty of upward curve modeling were attributed to the complexity of the gel structure, which suffered constant effects of the droplet redistribution during shear.
The thermoresponsive nature of the
k-car was also subjected to oscillatory analysis (
Figure 8). The CO and Chls incorporation into Std-gel led to an increase of the G’ (elastic) and G” (viscous) moduli at all temperatures, a behavior already reported for carrageenan [
68]. In addition, the systems showed viscoelastic behavior (G’ > G”), with a pronounced viscosity reduction above 45 °C. Therefore, the thermo-responsive properties of these compositions can be advantageous in the gel-preparation step, which can be more easily obtained at 45 °C. However, with the administration process, the temperature variation (7 °C) does not substantially change the behavior of the formulation, as shown in
Figure 8B.
Regarding oscillatory rheometry, an analysis within the linear viscoelastic region (fixed stress values ranging from 2.5 to 8 Pa, depending on the formulation) was employed at skin temperature (32 °C) (
Figure 9). This region allows infinitesimal deformation, and the biopolymer chains are kept close to equilibrium. Thus, the oscillatory responses showed molecular-level interaction information. The viscoelastic properties were verified by obtaining the G’ and G” moduli. Their relationship, expressed as G”/G”, showed viscoelastic behavior when the values were less than one. In this condition (G”/G’ < 1 ), there was a predominance of elastic interactions and of reversible nature [
69].
The viscoelastic behavior was dependent on composition and frequency. The CO and Chls incorporation (Phy-CO and Phy-Chl) did not significantly affect the G’ modulus (p > 0.05). On the other hand, the Phy-CO-Chl obtention increased the G’ modulus in a pronounced way, especially at the highest frequencies (p < 0.05). The G” modulus showed slight increases in the following order: Std-gel, Phy-CO, Phy-Chl, and Phy-CO-Chl. Nevertheless, the increases were not statistically significant (p > 0.05). The viscous moduli increase was of greater magnitude than the elastic moduli increase, thus enhancing the plastic characteristic of the formulations. An elastic deformation nature may be related to drug/copolymer interaction, while plastic deformations may be related to changes in the helical conformation of the k-car biopolymer. The dynamic viscosity was reduced with small movements (low oscillatory frequencies). However, above 6 Hz, the variation was less pronounced. The gel composition did not cause significant changes in the dynamic viscosity (p > 0.05).
The tangent loss, expressed as tan δ (G”/G’), decreased with the oscillatory frequency increase (
p < 0.05) for all systems. The enhanced viscoelasticity followed the complexity of the matrix composition, where Phy-CO-Chl showed tan δ of 0.259, a nearly two-fold increase compared with that of Std-gel. All formulations had tan δ values lower than one (
Figure 9D), and were classified as viscoelastic systems. This is an essential property when considering drug-delivery systems, as it suggests an appropriate retention of these formulations at the application site [
63,
69,
70,
71,
72,
73,
74].
3.3. Photodynamic Inactivation of Staphylococcus Aureus Bacterial
Wound healing involves three major phases: inflammatory reaction, cell proliferation, and remodeling. First, the inflammatory process occurs with vasoconstriction, hemostasis, and inflammatory mediator release. Then, the granulation tissue is triggered, with fibroblast proliferation and angiogenesis effects. At this point, opportunistic pathogens, such as
Staphylococcus, infect small dermal lesions and invade subcutaneous tissue [
46,
75,
76]. Therefore, inactivation studies of these pathogens are fundamental to evaluate the healing and antibiotic potential of the drug. The antibacterial activity of Phy-gels against
S. aureus is presented in
Figure 10.
Bacterial cell incubation with gels led to statistically significant count reductions of the colony-forming units (CFU) (
p < 0.05). The Std-gel showed bacterial potential, with a countdown of 41%, a similar result to that previously reported [
77]. The Chls incorporation further reduced the count of viable cells count (
p < 0.05). The Phy-Chl activity without light showed a 30% CFU reduction, and this value increased to 37% after light. The CO addition into the matrix gel increased the potential of the formulation, allowing the total elimination of all viable cells in the light and in the dark. This pronounced increase was probably related to the bactericidal behavior of the CO and its influence on the Chls additional monomerization capacity. The interface-defined droplets can solubilize hydrophobic PS in their microdomains, a known behavior for emulgels with hydrophobic compounds [
67]. The higher rate of monomers increases the number of molecules able to undergo intersystem conversion, so that singlet oxygen (the PDT protagonist) is formed.
Although in vitro treatment suggested efficiency in the absence of light for the Phy-CO-Chl gel, photodynamic treatment is essential, because Chls inhibit the resistance effect on pathogens by generating singlet oxygen. At the same time, CO acts as a wound-healing agent [
9,
37,
38,
44,
78]. We have previously shown severe damage that low concentrations of Chls can cause to the bacteria wall, making its efficiency in PDT unquestionable [
9]. Moreover, the literature reports that red wavelength LED significantly enhanced the skin graft score, which increased the transforming growth factor beta (TGF-β) protein expression and the density of collagen fibers [
79]. Therefore, at the same time as the drugs act, red LED enhances the dermo–epidermal junction and modulates the expression proteins related to tissue repair [
79].
The combined mechanical, rheological, and photo-antimicrobial benefits encouraged in vivo studies on animals with wound lesions.
3.4. Photodynamic Treatment: In Vivo Assays in Rabbits
The benefits of carrageenan and copaiba oil in wound healing are already known [
48,
80,
81,
82], but not widely reported with the addition of Chls.
The lesion shrinkage is shown in
Table 6. The values, measured at 7 day intervals, were expressed in percentages, considering the first and last day of the treatment. The mean shrinkage value of the wounds treated with the formulations varied between 59.5% and 100%.
The lesion contraction effect between the groups treated with Phy-gels was statistically similar (
p > 0.05), but different from that of the iodine treatment (
p < 0.05). For lesions submitted to the control treatment, 80% showed an increase in lesion severity, and only 20% had their lesions reduced by up to 37%. Although the phytotherapeutic treatments (light and dark) were equivalent, the PDT reduced the pathogen load without generating bacterial resistance effects, which inhibited the systemic level of infection or the development of chronic wounds [
7,
9,
10].
The worsening of the clinical condition of the iodine-treated animals can be seen in the macroscopic images of the rabbits’ feet and their respective histologies (
Figure 11).
The histology in
Figure 11 showed a similarity between the histology of the iodine-treated (
Figure 11B) and the untreated animals (
Figure 11C). Injuries tissue (
Figure 11A), vasodilatation, and migration of inflammatory cells to the lesion region, due to the attraction of chemopathic molecules, can be verified (
Figure 11B and
Figure 11C). Furthermore, there was an increase in space between the connective tissue cell fibers, due to the increased interstitial fluid resulting from edema, as described by other authors [
80,
83].
The treatment with Phy-gel was more effective than that of the control. In all cases treated with Phy-gel, the healing progression was concordant with the initial degree of wound healing.
The Phy-CO formulation showed almost complete healing by the 21st day. However, the epithelium was still thick, with a thin keratin layer. The dermis was in a phase of organization, while the hair follicles were organized, with signs of hair emergence [
46]. However, as seen in the
Figure 12, healing was not complete, and inflammatory cells were still found in specific segments, with blood containing leukocytes accumulating (
Figure 12). The benefits of CO as a healing and anti-inflammatory agent have already been reported in oral or topical administration [
25].
The treatment with Phy-Chl (dark) favored healing, which was maintained on 21
st day with only slight edema (
Figure 12). The treated lesion tissue showed intact epithelium, with reorganized dermis and hair follicles. Moreover, fur began to develop and emerge. There are few studies on the healing capacity of Chls [
84,
85], with a few occurring in the 1950s. The results presented here support the potential for combining Chls and k-car. The Phy-Chl treatment showed more promise than Phy-CO for the tested treatment interval.
Phy-CO-Chl (
Figure 13) treatment showed edema at the end of treatment, with the presence of inflammatory cells in the dermis region, broken follicles, and the presence of blood accumulation due to the effect of vasodilation. The treatment variations (all Phy-gels in dark conditions) were linked to deep lesions and animal management, as the animals were kept in cages during treatment, which made healing difficult due to the constant friction of the feet in the cages. Overall, the treatments showed benefits of the Phy-gels (without the use of PDT) for healing, even if the benefits were incomplete (an increase in the frequency of dosage or treatment time could lead to complete healing). On the other hand, the treatment with iodine worsened the condition of all animals. The results obtained in the dark were similar to those reported by Plefh et al. (2021) [
46], who evaluated 21 day treatments with a fluid gel containing 2% of clove powder (
Syzigium aromaticum), as antimicrobial, antioxidant, and anti-inflammatory properties on wounds (pododermatitis) of rabbits.
PDT use in pododermatitis cases aims to reduce the pathogen load. Furthermore, the short lifetime of singlet oxygen does not allow antimicrobial resistance [
7,
9,
86]. The activation of the photochemical and photophysical mechanisms was possible due to the overlap of the chlorophyll absorption spectrum (Q-band at 670 nm) with the emission from the red source (660 nm). The evolution of the lesions submitted to PDT is shown in
Figure 14.
Phy-Chl+PDT showed superior healing signs, with the regeneration of the dermal papillae and the formation of a thick keratin layer. Furthermore, the treatment favored the regeneration of the epithelium, which was intact on the last day of treatment. Similarly, the Phy-CO-Chl+PDT exhibited a process of reepithelization in addition to the formation of fur. Furthermore, the presence of a crust was verified in the lesion region (according to the 21st day photograph, as shown in
Figure 14), indicating that the healing process was still in progress [
48,
80]. The improvement in the results is linked to the tissue-recovery capacity stimulated by the red light, which is associated with the reduction of the pathogen loads that cause infection (
Figure 10). In addition, the benefits of using red light in the reepithelialization process are known, with light being a treatment mechanism (photobiomodulation to improve immune system function) [
87,
88,
89].