2.1. Cytotoxicity of Cip, PE and PE-Cip in HDF cells
2.1.1. Polyelectrolyte cell compatibility
Initially, we evaluated HDF cell viability when these cells were exposed to treatments with low (0.01% p/p, PEL) or high (0.1% p/p, PEH) concentrations of CB, SA, and SH.
The PEL concentrations did not modify the cell viability related to the control (Figure 1). In the case of SHH, the HDF cell viability was even slightly increased (15%), suggesting excellent biocompatibility properties. In agreement, Guo et al (2015)12 observed the same tendency in the mouse fibroblast line L929. The increase in HDF cell viability in the presence of SH may be related to its endogenous nature, since it is the most relevant structural element of the extracellular matrix13. In fact, HDFs have SH primary cell receptors (CD44 and RHAMM) that are involved in the modulation of complex biological functions such as cell migration and proliferation, inflammation and tumorigenicity14.
Good cell compatibility was also evident in the presence of SAH, which produced no changes in HDF cell viability. These results are not in agreement with Guo et al (2015)12, where SA 0.1% (SAH) reduced viability to approximately 70%. Such differences could be due to the cell line used (mouse fibroblast L929), to differences in viscosity or in monomeric composition (manuronic:guluronic ratio), or to combination of these. In contrast, cell viability decreased to 65% with CBH, which implied a low in vitro biocompatibility. Similar results were observed by Guo et al (2015)12 in a mouse fibroblast line L929, which could be assigned to the characteristic higher viscosity of synthetic polyelectrolytes, which generates three-dimensional structures covering living cells and hindering the arrival of culture medium nutrients, thereby reducing the metabolic activity or blocking the transport of vital molecules to cells15. However, it is important to note that this in vitro phenomenon may not necessarily occur in an in vivo condition, where the nutrition of the cells in a tissue comes from the extracellular matrix components. In fact, the healing of second-degree superficial burns treated with an hydrogel of carbomer combined with Cip and lidocaine (CbNaCipLid) has shown a fast and complete regeneration10.
2.1.2. Cell compatibility of Cip and PE-Cip complexes
The addition of Cip3 to cell cultures produced a non-cytotoxic effect (Figure 2A), whereas Cip30 treatment significantly reduced cell viability by up to 60% (Figure 2B). In this regard, it has been reported that Cip inhibits cellular functions, thus preventing the growth of mammalian cell lines. Cip also induces mitochondrial dysfunction and oxidative damage in a dose-dependent manner. However, this toxic effect can be counteracted by the exogenous incorporation of vitamin E as an antioxidant16,17.
Next, we evaluated the HDF cell viability of Cip at 3 µg/mL or 30 µg/mL in the presence of PE. No cytotoxic effect was observed when Cip 3 µg/mL was combined with PE, except for CBH-CIP3, which followed the same pattern as CBH alone. A slight cell viability reduction was observed for SAL-CIP3 and SAH-CIP3. However, these were not cytotoxic since the values were higher than 70% (Figure 2A).
Interestingly, at higher cytotoxic concentrations of Cip30, a significant increase in HDF cell viability was revealed in the presence of PEL and PEH, with the only exception being CBH−Cip30, whose non-significant increase in viability was still below 70% (Figure 2B).
The increase in HDF cell viability by PE-Cip complexes with respect to Cip alone suggests that these PE have a protective effect, which could be due to the ability of SA and SH to counteract the cellular damage produced by the reactive oxygen species arising from the presence of Cip18,19. In addition, the PE-Cip interaction can reduce free Cip concentrations in the medium20, so a combination of both mechanisms cannot be ruled out.
2.4. Wound-healing in vivo test
2.4.1. Macroscopic wound healing study
Although most non-complex burn injuries will heal spontaneously or after a conservative treatment1, several studies have shown that wounds that take more than 2-3 weeks to heal are more likely to result in scarring with functional and psychological consequences2. The management of burn wounds has a considerable influence on the time taken for the wound to heal6, so a good initial care will have a positive influence on the outcome25.
No signs of pain or general discomfort were observed in any animal during the trial, with the behavior (in terms of food and water intake and daily activity) being normal. In addition, no clinical signs of infections were observed. Figure 5 shows the wound healing results for each treatment after 7, 14, and 21 days-post burn.
On day 7, the wound area had slightly increased in the NT group, possibly due to local inflammation. However, with the exception of R-cream, the treatments with AAfilms, Cfilms, and AAhydrogel significantly reduced the wound areas faster than NT (p< 0.05). On day 14, no significant differences were observed with respect to NT, with R-cream being less effective in closing wounds than the other treatments (p< 0.05). Moreover, the R-cream group revealed a more open wound area at the trial end, even with respect to the NT group, on day 21 (p< 0.05).
All open wounds are an ideal environment for microbial colonization3,26. Thus, the early closure observed from day 7 supports a beneficial effect of the polyelectrolytes. Also, these treatments proved to be better than the standard one (Rcream), which was not effective in closing wounds at 21 days post burn. A faster closure involves cellular proliferation and migration, which is critical for restoring the barrier function and for minimizing the risk of burn wound infections4,6.
2.4.2. Microscopic wound healing study
To characterize further the healing process, the wounds were also analyzed microscopically. The epidermal evolution is shown in Figure 6, with the treatments being significantly associated with the epidermal scores (p< 0.005).
On day 7, the NT group showed an absent or discontinuous epidermis at similar frequencies, whereas all animals treated with AAfilms and AA-hydrogel already presented a discontinuous epidermis. Although the C-film group included some animals having an absent epidermis, most of these animals showed epidermal recovery to different extents. In contrast, the Rcream group revealed an absent epidermis in all cases.
On day 14, in the NT group, animals with a previous discontinuity evolved to a continuous epidermis, while those with a previously absent one evolved to a discontinuous epidermis. The AAfilms and AA-hydrogel treatment led to permanent recovery, whereas the C-film treatment achieved this to a lesser extent (on days 14 and 21). On the other hand, R-cream showed an inconstant and only partial epidermal recovery in the last 2 weeks.
On day 21, the NT group achieved complete healing, as the AAfilms and AAhydrogel had done earlier, with their effects being maintained up to the trial end. Thus, the treatments exerted an earlier and sustained epithelialization in all cases, with the polyelectrolytes accompanied by active principles being the most effective. These results are in agreement with the above-mentioned macroscopic outcomes.
In association with epithelialization, dermal regeneration is mainly carried out by the migration and proliferation of fibroblasts. In addition, collagen and some other components of the extracellular matrix are also required to close the wound efficiently. Nonetheless, a fibrotic over-response can deleteriously lead to dense skin scars. The dermis also contributes to appendage regeneration and the biomechanical properties of the regenerated skin, which affect its quality27. If the new tissue is very different to the normal skin, the functionality will be compromised25, with poor aesthetic results and psychological sequels for the patient2.
Figure 7 shows the dermal regeneration scores, with treatments being significantly associated with the scores (p< 0.05). On day 7, there were no differences among AAfilms, C-films, and AA-hydrogel, which revealed more animals with reticular recovery than the NT group or R-cream, with half of these groups still showing complete dermal disorganization.
On day 14, the AAfilms presented advanced dermal healing with scores of 4 or 5; whereas R-cream, C-films, and AA-hydrogel exerted skin recovery (scores of 3 or 4). On the other hand, the NT group showed a heterogeneous effect, with scores ranging from 1 to 4.
On day 21, the disorganized skin of the NT group evolved to recovery in the reticular and papillary layers. The R-cream group presented skin score involution, which was in agreement with the epidermal scores and macroscopic healing. In contrast, the AAfilms and AAhydrogel led to an increase in the frequency of animals with normal skin (a normal reticular dermis was found in all cases), promoting dermal remodeling with respect to groups with no treatment or the standard one.
Figure 8 shows representative photomicrographs of each experimental group, which support the results shown in Figures 7 and 8. The NT group exhibited a low definition between the reticular and papillary dermis, as well as with appendage hemorrhage occurring on day 7 post burns, which then presented cellular infiltration that evolved to a dense scar without skin appendages over the following days. Although the epidermis was continuous at the trial end, it had an atypical structure.
Concerning the R-cream group, despite this being considered the gold standard treatment for burns, it was significantly less effective than the other treatments. Moreover, wound healing was delayed compared with the NT group, with the skin recovery observed on day 14 being transient and with a later impairment on day 21 being accompanied by cellular infiltration. This is in fact a frequent problem related to the repeated application of silver sulfadiazine creams and silver-based dressings, which are deleterious to keratinocytes and fibroblasts5,28, partly due to the hydrophobic nature of the vehicle and the unspecific killing action of silver9,29,30.
The main outcome found in animals treated with AAfilms (with respect to the previous treatments) was an early epidermal and dermal healing on day 14 with appendage conservation, which then allowed the normal reticular and papillary dermis to be differentiable. Also, the epidermis was well-differentiated with clear basophilic staining and a corneal layer. The AA-hydrogel and C-films achieved similar outcomes, but to a lesser extent.
Wound healing in C-film-treated burns was superior to that found in the R-cream group, despite not having an antimicrobial in the composition. This demonstrated the beneficial healing effect of sodium alginate or sodium hyaluronate polysaccharides, which is due to several mechanisms, and is consistent with numerous scientific reports7,29,31. In this regard, the moist wound environment generated by films, as well demonstrated by the in vitro assays, was convenient for cell viability and physiology2,32. Furthermore, systems based on polymers are well-known for their ability to cool the skin surface by absorbing and dissipating the heat. This reduces the inflammatory response and limits tissue damage, resulting in faster regeneration33 and an improved well-being for the patient33–35. These benefits were increased by AAfilms, which indicated that neither ciprofloxacin nor lidocaine interfered with skin healing and could actually improve this by ciprofloxacin antimicrobial activity. This shortened evolution might prevent further development of chronic wounds and the risk of bacterial dissemination, thereby improving patient prognosis. Nevertheless, it still remains to be seen whether this system can promote faster wound healing and exhibit antimicrobial effects on an infected wound in vivo. It should be taken into account that these results were obtained in a rat burn model, whose most significant limitation is the subcutaneous panniculus carnosus muscle that facilitates skin healing by both wound contraction and collagen formation. However, this rapid wound contraction allows to study the mechanics of wound healing and to compare the performance of alternative treatments36.
The present investigation took advantage of the ionic interaction established between a polyelectrolyte and certain drugs to develop new materials with potential applications in clinic. Although both the AAfilms and AA-hydrogel promoted skin healing, the films show more advantages as wound dressings, such as their easy and noninvasive application and dose accuracy. The development or optimization of advanced dressings still represents a very active research field, with the aim being to improve skin healing in relation to specific clinical applications. Related to this, numerous new systems containing peptides, growth factors or cells25,37,38 have been proposed in the scientific literature as improved alternatives for the treatment of wounds in general, and have demonstrated the ability to increase wound healing. Compared with these antecedents, the AA-film development has revealed several advantages, such as being an economic, simple and robust manufacturing process accomplished by the rational selection of materials and the knowledge of their chemical and biological characteristics. It can thus be suggested that the design films made from biomaterials with antimicrobials and anesthetics with extended-release and a sound manufacturing process is a promising development for the improvement of burn pharmacotherapy. Moreover, from a technological and pharmaceutical point of view, the use of these biomaterials and drugs approved by regulatory health authorities constitutes a translational medicine approach with a great potential for advancing to the productive sector, with the possibility of becoming a new alternative use of the drugs.