Antimelanoma effect of manool in 2D cell cultures and reconstructed human skin models.

Melanoma is the most aggressive and lethal type of skin cancer, characterized by therapeutic resistance. In this context, the present study aimed to investigate the cytotoxic potential of manool, a diterpene from Salvia officinalis L., in human (A375) and murine (B16F10) melanoma cell lines. The analysis of cytotoxicity using the XTT assay showed the lowest IC50 after 48 h of treatment with the manool, being 17.6 and 18.2 µg/ml for A375 and B16F10, respectively. A selective antiproliferative effect of manool was observed on the A375 cells based on the colony formation assay, showing an IC50 equivalent to 5.6 µg/ml. The manool treatments led to 43.5% inhibition of the A375 cell migration at a concentration of 5.0 µg/ml. However, it did not affect cell migration in the B16F10 cells. Cell cycle analysis revealed that the manool interfered in the cell cycle of the A375 cells, blocking the G2/M phase. No changes in the cell cycle were observed in the B16F10 cells. Interestingly, manool did not induce apoptosis in the A375 cells, but apoptosis was observed after treatment of the B16F10 cells. Additionally, manool showed an antimelanoma effect in a reconstructed human skin model. Furthermore, in silico studies, showed that manool is stabilized in the active sites of the tubulin dimer with comparable energy concerning taxol, indicating that both structures can inhibit the proliferation of cancer cells. Altogether, it is concluded that manool, through the modulation of the cell cycle, presents a selective antiproliferative activity and a potential antimelanoma effect.

Effect of natural primer associated to bioactive glass-ceramic on adhesive/dentin interface.

OBJECTIVES: This study evaluated the effect of propolis associated with Biosilicate on the bond strength (BS) and gelatinolytic activity at the adhesive/dentin interface. METHODS: Occlusal cavities were prepared in 320 human molars. Half of them were submitted to cariogenic challenge. All the teeth were separated into eight groups (n = 20): Control - Adhesive System (Single Bond Universal, 3 MESPE); CHX - 0.12 % Chlorhexidine; Bio - 10 % Biosilicate; P16 - Propolis with low levels of polyphenols; P45 - Propolis with high levels of polyphenols; CHX Bio - CHX + Bio; P16 Bio - P16+Bio; P45 Bio - P45+Bio. The adhesive was applied (self-etch mode) after treatments. Restorations (Filtek Z350, 3 MESPE) were performed and samples sectioned into sticks, separated and stored in distilled water at 37 °C for 24 h, 6 months and 1 year. Microtensile BS (0.5 mm/min) was tested and analyzed (2-way ANOVA, Bonferroni's Test, p < .05 and Weibull analysis). Fracture patterns (VH-M100, Keyence) and adhesive interfaces (SEM, EVO-MA10, ZEISS and TEM, JEM-1010, JEOL) were observed; and biodegradation and in situ zymography performed. RESULTS: P16 presented the highest BS values on sound dentin after 6 months. In caries-affected dentin (CAD), the association of treatments promoted the highest BS after 24 h. Sound dentin obtained significantly higher Weibull modulus than CAD. SEM displayed resin tags in P16, P45 and association of treatments. TEM showed good interaction between adhesive and dentin. According to the in situ zymography and biodegradation assay all natural primers reduced the gelatinolytic activity. P45 presented the lowest biodegradation and enzymatic activity. CONCLUSIONS: Propolis and the association of treatments promoted the highest bond strength results and preserved the dentin. All the experimental groups exhibited low gelatinolytic activity. CLINICAL SIGNIFICANCE: Propolis and the association of treatments with Biosilicate could preserve the dentin substrate and improve the longevity of composite restorations.

Evaluation of in vitro and in vivo Efficacy of a Novel Amphotericin B-Loaded Nanostructured Lipid Carrier in the Treatment of Leishmania braziliensis Infection.

BACKGROUND: Leishmaniasis is a neglected disease, and the current therapeutic arsenal for its treatment is seriously limited by high cost and toxicity. Nanostructured lipid carriers (NLCs) represent a promising approach due to high drug loading capacity, controlled drug release profiles and superior stability. Here, we explore the efficacy of a unique pH-sensitive amphotericin B-loaded NLC (AmB-NLC) in Leishmania braziliensis infection in vitro and in vivo. METHODS AND RESULTS: AmB-NLC was assessed by dynamic light scattering and atomic force microscopy assays. The carrier showed a spherical shape with a nanometric size of 242.0 ± 18.3 nm. Zeta potential was suggestive of high carrier stability (-42.5 ± 1.5 mV), and the NLC showed ~99% drug encapsulation efficiency (EE%). In biological assays, AmB-NLC presented a similar IC50 as free AmB and conventional AmB deoxycholate (AmB-D) (11.7 ± 1.73; 5.3 ± 0.55 and 13 ± 0.57 ng/mL, respectively), while also presenting higher selectivity index and lower toxicity to host cells, with no observed production of nitric oxide or TNF-α by in vitro assay. Confocal microscopy revealed the rapid uptake of AmB-NLC by infected macrophages after 1h, which, in association with more rapid disruption of AmB-NLC at acidic pH levels, may directly affect intracellular parasites. Leishmanicidal effects were evaluated in vivo in BALB/c mice infected in the ear dermis with L. braziliensis and treated with a pentavalent antimonial (Sb5+), liposomal AmB (AmB-L) or AmB-NLC. After 6 weeks of infection, AmB-NLC treatment resulted in smaller ear lesion size in all treated mice, indicating the efficacy of the novel formulation. CONCLUSION: Here, we preliminarily demonstrate the effectiveness of an innovative and cost-effective AmB-NLC formulation in promoting the killing of intracellular L. braziliensis. This novel carrier system could be a promising alternative for the future treatment of cutaneous leishmaniasis.

Physicochemical characterization by AFM, FT-IR and DSC and biological assays of a promising antileishmania delivery system loaded with a natural Brazilian product.

The control and treatment of Leishmaniasis, a neglected and infectious disease affecting approximately 12 million people worldwide, are challenging. Leishmania parasites multiply intracellularly within macrophages located in deep skin and in visceral tissues, and the currently employed treatments for this disease are subject to significant drawbacks, such as resistance and toxicity. Thus, the search for new Leishmaniasis treatments is compulsory, and Ocotea duckei Vattimo, a plant-derived product from the biodiverse Brazilian flora, may be a promising new treatment for this disease. In this regard, the aim of this work was to develop and characterize a delivery system based on solid lipid nanoparticles (SLN) that contain the liposoluble lignan fraction (LF) of Ocotea duckei Vattimo, which targets the Leishmania phagolysosome of infected macrophages. LF-loaded SLNs were obtained via the hot microemulsion method, and their physical and chemical properties were comprehensively assessed using PCS, AFM, SEM, FT-IR, DSC, HPLC, kinetic drug release studies, and biological assays. The size of the developed delivery system was 218.85±14.2 nm, its zeta potential was -30 mV and its entrapment efficiency (EE%) was high (the EEs% of YAN [yangambin] and EPI-YAN [epi-yangambin] markers were 94.21±0.40% and 94.20±0.00%, respectively). Microscopy, FT-IR and DSC assays confirmed that the delivery system was nanosized and indicated a core-shell encapsulation model, which corroborated the measured kinetics of drug release. The total in vitro release rates of YAN and EPI-YAN in buffer (with sink conditions attained) were 29.6±8.3% and 34.3±8.9%, respectively, via diffusion through the cellulose acetate membrane of the SLN over a period of 4 h. After 24 h, the release rates of both markers reached approximately 45%, suggesting a sustained pattern of release. Mathematical modeling indicated that both markers, YAN and EPI-YAN, followed matrix diffusion-based release kinetics (Higuchi's model) with an estimated diffusion coefficient (D) of 1.3.10(-6) cm(2)/s. The LF-loaded SLNs were non-toxic to murine macrophages (20-80 µg mL(-1) range) and exerted a prominent anti-leishmanial effect (20 µg mL(-1)). These data suggest this new and well-characterized lipid nanoparticle delivery system safely and effectively kills Leishmania and warrants further clinical investigation.

Effect of iontophoresis on topical delivery of doxorubicin-loaded solid lipid nanoparticles.

The combination of iontophoresis with solid lipid nanoparticles (SLNs) for targeting drug delivery to the epidermis has not been explored. The goal of this paper was to study the influence of iontophoresis on the penetration of doxorubicin (DOX) delivered in SLNs (DOX-SLNs). We measured the contribution of electroosmotic flow to the transport of DOX, and the accumulation of DOX in the stratum corneum (SC) and in the viable epidermis was determined. In addition, we evaluated the cytotoxicity of DOX-SLNs against skin cancer cells. Iontophoresis of unloaded SLNs decreased the electroosmotic flow by a factor of 5 and increased the skin resistance. Nevertheless, iontophoresis of DOX-SLNs increased DOX delivery to the viable epidermis, with 56% of all DOX penetrating this skin layer. Only 26% of the drug was retained in the SC. In contrast, passive delivery retained 43% of DOX in the SC and 26% in the viable epidermis. DOX-SLNs increased DOX cytotoxicity against melanoma cells by 50%. These results suggest the use of DOX-SLN iontophoresis in the topical treatment of skin cancer.

Antimicrobial Brazilian Propolis (EPP-AF) Containing Biocellulose Membranes as Promising Biomaterial for Skin Wound Healing.

Among remarkable discoveries concerning propolis, such as antifungal, antiviral, and antioxidant activities, its anti-inflammatory, and mainly its antibacterial, properties deserve special attention when skin wound healing is concerned. Based on this and knowing the distinctive performance of bacterial (BC) membranes on wound healing, in this work it is proposed to demonstrate the potent antimicrobial activity and wound healing properties of a novel propolis containing biocellulose membrane. The obtained propolis/BC membrane was able to adsorb propolis not only on the surface, but also in its interstices demonstrated by scanning electron microscopy, X-ray diffraction, Fourier transform infrared (FT-IR) spectroscopy, and thermogravidimetric assays. Additionally, the polyphenolic compounds determination and the prominent antibacterial activity in the membrane are demonstrated to be dose dependent, supporting the possibility of obtaining propolis/BC membranes at the desired concentrations, taking into consideration its application and its skin residence time. Finally, it could be suggested that propolis/BC membrane may favor tissue repair in less time and more effectively in contaminated wounds.

Evaluation of the Potential of Brazilian Propolis against UV-Induced Oxidative Stress.

This study investigated the potential use of topically and orally administered propolis extracts to prevent UV irradiation-induced oxidative stress in skin. The results illustrated that green propolis extract (GPE) contained greater amounts of polyphenols, coumaric acid, drupanin, baccharin and artepillin C than did brown propolis extract (BPE). GPE showed higher antioxidant activity than BPE when the IC(50) (concentration that caused 50% inhibition) values were compared. Interesting, the oral treatment of hairless mice demonstrated a recovery of 30.0% for GPE and 22.8% for BPE with respect to UV irradiation-induced GSH depletion. The topical pretreatment of animals with both propolis extract solutions recovered around 14.0% of the depleted GSH. However, the employed treatments did not inhibit the increase of cutaneous proteinase secretion/activity caused by irradiation. These findings indicate that despite differences in composition and antioxidant properties, GPE and BPE both successfully prevent UV-induced GSH depletion in vivo and are both promising antioxidant systems against oxidative stress in skin. Based on these findings, complementary studies should be performed to enhance our understanding of the protective effects of propolis extracts in skin.

Development of nitrosyl ruthenium complex-loaded lipid carriers for topical administration: improvement in skin stability and in nitric oxide release by visible light irradiation.

The prominent nitric oxide (NO) donor [Ru(terpy)(bdqi)NO](PF(6))(3) has been synthesized and evaluated with respect to noteworthy biological effects due to its NO photorelease, including vascular relaxation and melanoma cell culture toxicity. The potential for delivering NO in therapeutic quantities is tenable since the nitrosyl ruthenium complex (NRC) must first reach the "target tissue" and then release the NO upon stimulus. In this context, NRC-loaded lipid carriers were developed and characterized to further explore its topical administration for applications such as skin cancer treatment. NRC-loaded solid lipid nanoparticles (SLN) and nanostructured lipid carriers were prepared via the microemulsification method, with average diameters of 275+/-15 nm and 211+/-31 nm and zeta potentials of -40.7+/-10.4 mV and -50.0+/-7.5 mV, respectively. In vitro kinetic studies of NRC release from nanoparticles showed sustained release of NRC from the lipid carriers and illustrated the influence of the release medium and the lyophilization process. Stability studies showed that NO is released from NRC as a function of temperature and time and due to skin contact. The encapsulation of NRC in SLN followed by its lyophilization, significantly improved the complex stability. Furthermore, of particular interest was the fact that in the NO photorelease study, the NO release from the NRC-loaded SLN was approximately twice that of just NRC in solution. NRC-loaded SLN performs well enough at releasing and protecting NO degradation in vitro that it is a promising carrier for topical delivery of NO.