The topical treatment of melanoma consists of the application of drugs directly on the skin cancer lesion and can be considered to initial stages of cancer, small injuries, and large areas in which surgical management can deform, such as the face. This option also can be adopted in association with others, such as localized radiotherapy [3]. In clinical aspects, topical management represents a treatment that is less invasive, localized, and easy to apply. However, this approach is hampered by the skin barrier conferred by the stratum corneum, which can often limit drug penetration into deeper skin layers [31].
The 5-FU is applied topically to treat skin cancer lesions as commercial products such as Efurix® (Valeant Pharmaceuticals), Fluoroplex® (Allergan), and Carac® (Dermik Laboratories). However, the application of this drug is related to several adverse effects [32] that can be overcome by delivering this drug through nanocarriers [33–35].
In this work, we proposed an NLC aiming for biocompatibility with skin and also other important features such as stability, low toxicity behavior, efficient encapsulation and release of drugs, and strong protective effect on the incorporated drugs [36]. Moreover, because of this lipid composition and positive surface charge, this NLC is capable of a co-loading of lipophilic drug, as 5-FU, and gene, as siRNA BCL-2. The characterization of NLC (Table 1) showed particle size values around 250 nm and 0.2 of PdI, and they are in accordance with literature [37–39]. AFM images showed a morphology suggesting a spherical shape (Fig. 1-A) with size in agreement with DLS and NTA analysis. The presence of siRNA, which has a negative charge, as expected, neutralized the zeta potential of NLC. Sato et al. (2018) emphasized the importance of neutralized residual charges of cationic lipid by siRNA on the toxicity decreases [40]. In the present work, this neutralization was related to the complexation process (Fig. 1-B) that occurs between siRNA and NLC containing DOTAP. The anionic competition using heparin [17, 41, 42] resulted in an efficient decomplex, where the siRNA band was intact without drag bands indicating that no degradation of siRNA occurred. In other words, this result indicates that the siRNA can be released intact inside the cell.
Concerning the co-administration of 5-FU, it is important to know the release and penetration profile of this drug. Figure 2-A indicated a Higuchi kinetic (Supplementary Table 2) that means a modified release following Fick's Law. The Higuchi kinetic model characterizes controlled release systems, such as nanoparticles. The release of 5-FU, as well as the skin permeation and retention profile, are important studies to demonstrate that NLC is appropriate for topical use. Praça et al. (2018) studied the critical parameters of skin permeation studies in several types of skin [27] and following their work, we perform the in vitro skin studies in porcine ear skin, considering all parameters, such as thickness and viability.
In order to reach the full extent of the skin, it is necessary to overcome stratum corneum (SC), the skin barrier to penetration of substances into the skin [43, 44]. 5-FU is a small molecule (130.077 g/mol), and this drug penetrates the skin layers probably by preferred paths such as pores, glands, and hair follicles crossing to the bloodstream without difficulties [8]. An association with a lipid-based nanoparticle can increase the retention in the skin layers and also guarantee that 5-FU reaches to blood as minimally as possible for adequate topical performance, being released sustained during the time and localized [16].
The permeation of 5-FU was minimal (Fig. 2-B), and its retention (Fig. 2-C) demonstrates greater retention in viable epidermis and dermis. The NLC 5-FU presented 5-FU Cmax in EP + D about 25 µg/cm2, three-fold than in SC (Table 2). The horizontal sections (Fig. 2-D) showed 5-FU in the viable epidermis, about 20 to 30 µg/cm2. These results evidenced that 5-FU was higher in EP, demonstrating the capability to overcome the SC and, which is in line with other papers for topical 5-FU to skin cancer [33, 34]. The confocal images (Fig. 2-E) were in line with the quantitative analysis that showed a fluorescence related to Alexa 647 (NLC with Alexa 647 siRNA), mostly in EP.
Although porcine skin is widely accepted for studies of skin products, the present work evaluated the behavior of the proposed NLC in human skin, using an ex vivo model, which maintains the ideal conditions for the tissue to remain alive during the time [28].
The ex vivo results indicated that NLC provided 5-FU skin delivery about 60% of the applied dose in 72 hours and, the 5-FU was detected in the culture medium only after 96 hours of application (Fig. <link rid="fig3">3</link>-A and 3-B). The siRNA retention in the skin layers is also evidenced by the confocal images (Fig. 3-C).
The in vitro and ex vivo results are in accordance, and they indicated a NLC distribution and consequently, 5-FU and siRNA releases preferential in the epidermis. Further, the NLC conferred protection to natural SC flaking, suggesting a lipid film formation. The SC flaking is a natural behavior that occurs over the time of skin culture, previously observed by authors [45]. Therefore, to our knowledge, this is the first time that lipid film formation by lipid nanoparticles was in the hOSEC model (Fig. 3-D).
Beyond the skin studies, cell toxicity and uptake of the NLC was studied (Fig. 4). The NLC and Lipofectamine 2000® did not show uptake difference in A375 cells, both presented uptakes around 90% in 4 hours. However, smaller uptake percentages were observed in HaCaT than A375.
The IC50 values indicated that NLC increased the 5-FU toxicity compared to 5-FU solution, probably due to the more effective cell internalization provided by the nanocarrier. Moreover, tumoral cells (A375) were more sensitive than non-tumoral (HaCaT). It is important to highlight that this is first time that 5-FU is applied to melanoma in combination with siRNA for Bcl-2 and this combination showed highly sensibility in A375, presenting a combination index (CI) < 1.0 for the NLC with 5-FU combined with Bcl-2 siRNA for both cell lines.
Overexpression of anti-apoptotic Bcl-2 family members is a hallmark of many neoplasms including melanoma, and the Bcl-2 siRNA has been investigated to treat this skin cancer [46]. In this paper, we evaluated the knockdown of the Bcl-2 by western blot (Fig. 5-A) and ELISA (Fig. 5-B), which showed relevant decrease (silencing) of Bcl-2 protein in 24 hours for 5-FU solution and NLC 5-FU and, in 48 hours, for treatments containing Bcl-2 siRNA. Both multifunctional treatments (Multifunctional NLC-a and -b) inhibit the Bcl-2 translation, showed synergic effect (Bcl-2 levels tended to zero) between 5-FU and Bcl-2 siRNA 100 pM (Multifunctional NLC-b). qRT-PCR (Fig. 5-C) exhibited the same pattern for the multifunctional treatments, reinforcing the synergic effect.
Our findings indicate that 5-FU enhances the reduction of Bcl-2 levels, and additionally, treatments incorporating siRNA are more effective in decreasing Bcl-2 levels compared to those relying solely on 5-FU. To further elucidate the effects of Bcl-2 inhibition by siRNA, we examined the impact on other members of the Bcl-2 protein family and related genes. Our analysis revealed that Bcl-2 knockdown significantly influences both anti-apoptotic and pro-apoptotic proteins. Notably, the co-delivery of 5-FU and Bcl-2 siRNA via NLCs led to increased levels of BAK and BAX, indicating a pronounced apoptotic response. These findings underscore the potential of our NLC formulation as a potent option for the treatment of cutaneous melanoma, demonstrating its capability to effectively trigger apoptosis.