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.

Pharmacokinetic study of AmB-NP-GR: A new granule form with amphotericin B to treat leishmaniasis and fungal infections.

Amphotericin B (AmB) has been the gold standard to treat systemic fungal infections. The use of AmB is restricted to hospitals because it poses several risks, mainly risks related to its high nephrotoxicity. Given the importance of this drug in medicine, new therapeutics and AmB formulations with nanotechnological improvements are required and could bring many benefits to patients. A new drug formulation with gastro-resistant coated granules has been proposed. The lipid-based system containing AmB was produced and used as raw material in the granulation/coated process. The new developed formulation (AmB-NP-GR) was characterized by optical microscopy, granulometry, and atomic force microscopy (AFM) after disintegration test. AmB-NP-GR showed granular shape, with most granules measured between 250 and 500 µm (37 ± 7% w/w). The AFM images indicated that the granule formulation should disintegrate in the intestine, to release the lipid-based carriers, making them available for absorption and allowing them to reach the blood circulation. The developed formulation was administered to rats in a single dose of 4.0 or 8.0 mg kg-1 and the pharmacokinetics was studied. The samples were analyzed by liquid chromatography coupled to mass spectrometry. Before the pharmacokinetic studies were conducted, the bioanalytical method was validated according to the EMA guideline and all evaluated parameters agreed with this guideline. The pharmacokinetic results showed that Cmax was similar for both doses and that tmax was reached at 4-12 h for dose of 4.0 mg kg-1 and 4 h for dose of 8.0 mg kg-1. The half-life related to the dose of 8.0 mg kg-1 increased significantly compared to the dose of 4.0 mg kg-1 (an increase of more than 3 times). In addition, the mean residence time related to the dose of 8.0 mg kg-1 was 4 times higher than for the lower dose. The clearance value showed to be higher for the lower dose. Together, these results provide important conclusions for experimental design of other in vivo safety and efficacy studies of AmB-NP-GR.

In vitro leishmanicidal effect of Yangambin and Epi-yangambin lignans isolated from Ocotea fasciculata (Nees) Mez.

Introduction: Yangambin and epi-yangambin are the main lignans found in Louro-de-Cheiro [Ocotea fasciculata (Nees) Mez], a tree native to the Atlantic forests of northeastern Brazil whose leaves and bark are widely used in folk medicine. The present study investigated the leishmanicidal and immunomodulatory effects of both lignans in in vitro models of infection by Leishmania amazonensis or Leishmania braziliensis, both etiological agents of Cutaneous Leishmaniasis in Brazil. Methods: Bone marrow-derived mouse macrophages were infected with L. amazonensis or L. braziliensis and then treated for 48 h at varying concentrations of yangambin or epi-yangambin. Results: Yangambin and epi-yangambin were found to reduce the intracellular viability of either Leishmania species in a concentration-dependent manner, with respective IC50 values of: 43.9 ± 5 and 22.6 ± 4.9 µM for L. amazonensis, compared to IC50 values of 76 ± 17 and 74.4 ± 9.8 µM for L. braziliensis. In this context, epi-yangambin proved more selective and effective against in vitro infection by L. amazonensis. However, both lignans were found to distinctly modulate the production of inflammatory mediators and other cytokines by macrophages infected by either of the Leishmania species evaluated. While yangambin increased the production of IL-10 by L. braziliensis-infected macrophages, both compounds were observed to lower the production of NO, PGE2, IL-6 and TNF-α in both Leishmania species. Discussion: The present results serve to encourage the development of novel studies aimed at screening natural bioactive compounds with the hope of discovering new therapeutic options for the treatment of Cutaneous Leishmaniasis.

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.

Yangambin and Epi-yangambin Isomers: New Purification Method from Ocotea fasciculata and First Cytotoxic Aspects Focusing on In Vivo Safety.

Ocotea fasciculata presents yangambin (YAN) and its isomer epi-yangambin (EPI-YAN) as major lignans, which are employed as the plant markers for quality control purposes and as potential pharmacological compounds. However, a gap between the pure isomers and safety and efficacy protocols is faced by the scientific community. In this context, this work aimed to report (i) a new and advantageous purifying process in a semi-preparative scale for YAN and EPI-YAN isolation from Ocotea fasciculata, and (ii) an in vitro cytotoxicity study to estimate, for the first time, the LD50 values of the isolated epimers, as well as the influence of albumin concentration in cell culture medium. The best condition for epimers isolation was achieved in normal-phase liquid chromatography. The lignan fraction (LF), previously obtained from the plant ethanolic extract, was purified yielding 17% and 29% of YAN and EPI-YAN, respectively. The in vitro study demonstrated that YAN and EPI-YAN were safe, and only at the highest concentration studied, a decrease on cell viability was observed. The estimated LD50 value was higher than 1612 mg/kg for both epimers. The LF, on the other hand, demonstrated an estimated LD50 of 422 mg/kg. Lignan cytotoxicity studies also evidenced that the higher cell viability was related to the higher concentration of fetal bovine serum as a source of albumin in medium. This is the first time the LD50 and safety of the isolated epimers were estimated, opening up great perspectives of success in in vivo studies.

Development, characterization and pre-clinical trials of an innovative wound healing dressing based on propolis (EPP-AF®)-containing self-microemulsifying formulation incorporated in biocellulose membranes.

The considerable role of pristine bacterial cellulose membranes (BC) as ideal dressings have been widely demonstrated to treat wounds and burns. Nevertheless, drawbacks regarding antimicrobial spectrum and frequent dressing replacement are still present. Based on this, the present work proposes an innovative dressing by incorporating a technological self-microemulsifying formulation (SMEF) encapsulating propolis (BC/PP). BC/PP was fully chemically and biologically characterized employing in vitro and in vivo models. Antimicrobial studies demonstrated BC/PP high efficiency against both gran-negative and gran-positive bacteria. Release studies evidenced propolis markers sustained release for up to 7 days. In vivo wound healing activity was assessed by wound healing rate, anti-inflammatory and tissue formation events and the results evidenced the pro-inflammatory activity of BC/PP, which could promote improved healing results. To conclude, BC/PP presented an outstanding antibacterial activity in vitro with weekly replacement and promotion of healing, offering, for the first time, a broad-spectrum biomembrane potential to treat infected wounds.

Evaluation and Comparison of Wound Healing Properties of an Ointment (AlpaWash) Containing Brazilian Micronized Propolis and Peucedanum ostruthium Leaf Extract in Skin Ulcer in Rats.

Several previous studies have demonstrated improved wound healing associated with natural-based formulations. Therefore, the purpose of this study was to assess the efficacy of a topical formulation containing both a Brazilian micronized propolis extract and a Peucedanum ostruthium leaf extract for the treatment of wounds created by surgical punch in rats. The study was conducted for 14 days and animals were treated as follows: gauze group (G), polyethylene glycol base ointment (Control), AlpaWash (an ointment containing a Brazilian micronized propolis extract and Peucedanum ostruthium leaf extract [Treatment]), and polysporin (one of the most commonly used topical antibiotic ointments, based on bacitracin zinc and polymyxin B sulfate [Reference Standard]). In general, the results demonstrated that ointments, due to occlusiveness and the ability to maintain moisture under the damaged area, offered improvements when compared to lesions without any treatment. Additionally, the presence of phenolic and flavonoid compounds, as well as antioxidants and antimicrobials, offered improved stimulation and could accelerate wound healing. The Control, Treatment, and Reference Standard groups were able to close the lesion, as measured by the wound healing rate determination and follow-up photographs. However, AlpaWash and Polysporin presented some additional benefits- anti-inflammatory activity, measured using myeloperoxidase and histological count, as well as fibroplasia and hydroxyproline production, suggesting that skin with a better quality could be formed following these two treatments. Therefore, based on the current concern of antibiotic overuse in wound healing, the emergence of multi-resistant organisms and the decrease in newer antibiotics, AlpaWash is considered a prominent formulation to be employed in wound-healing applications.

Method validation and nanoparticle characterization assays for an innovative amphothericin B formulation to reach increased stability and safety in infectious diseases.

Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-effective treatments of various infectious diseases as they may increase drug bioavailability, control drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0±11.8nm and Zeta Potential -42.5±1.5mV suggestive of important stability. DSC/TGA and FT-IR assessments suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were developed and validated according to regulatory agencies in order to fast and assertively determine AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability studies. The quantification method for AmB in NLC suspension presented linearity in 5.05-60.60µgmL-1 range (y=0.07659x+0.05344) and for AmB in receptor solution presented linearity in 0.15-10.00µgmL-1 range (y=54609x+263.1), both with r≥0.999. EE% was approximately 100% and according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46h. In the meantime, a micellar AmB solution demonstrated an instability pattern after 7h of contact with the medium. Degradation and release studies under acid conditions (infectious condition) firstly depicted a prominent degradation of AmB (raw-material), with 20.3±3.5% at the first hour, reaching 43.3±7.0% after 7h of study. Next, particles faster disruption in acid environment was evidenced by measuring the NLC size variation by DLS and by the loss of the bluish sheen, characteristic of the nanostructured system macroscopically observed. Finally, safety studies depicted that NLC-AmB presented reduced toxicity in fibroblast cells, corroborating with AmB aggregated form study. Therefore, an innovative AmB formulation was fully characterized and it is a new proposal for in vivo investigations.

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.

Validation of a RP-HPLC-DAD Method for Chamomile (Matricaria recutita) Preparations and Assessment of the Marker, Apigenin-7-glucoside, Safety and Anti-Inflammatory Effect.

Chamomile is a medicinal plant, which presents several biological effects, especially the anti-inflammatory effect. One of the compounds related to this effect is apigenin, a flavonoid that is mostly found in its glycosylated form, apigenin-7-glucoside (APG), in natural sources. However, the affectivity and safety of this glycoside have not been well explored for topical application. In this context, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC-DAD) method to quantify APG in chamomile preparations. Additionally, the safety and the anti-inflammatory potential of this flavonoid were verified. The RP-HPLC-DAD method was developed and validated with linearity at 24.0-36.0 µg/mL range (r = 0.9994). Intra- and interday precision (RSD) were 0.27-2.66% and accuracy was 98.27-101.21%. The validated method was applied in the analysis of chamomile flower heads, glycolic extract, and Kamillen cream, supporting the method application in the quality control of chamomile preparations. Furthermore, the APG safety was assessed by MTT cytotoxicity assay and mutagenic protocols and the anti-inflammatory activity was confirmed by a diminished TNF-α production showed by mice macrophages treated with APG following LPS treatment.

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.

Evaluation of Mucoadhesive Gels with Propolis (EPP-AF) in Preclinical Treatment of Candidiasis Vulvovaginal Infection.

Vulvovaginal candidiasis is the second cause of vaginal infection in the USA. Clinical treatment of C. albicans infections is routinely performed with polyenes and azole derivatives. However, these drugs are responsible for undesirable side effects and toxicity. In addition, C. albicans azole and echinocandin resistance has been described. Propolis is a bee product traditionally used due to its antimicrobial, anti-inflammatory, and other properties. Therefore, the present work aimed to evaluate different propolis presentations in order to evaluate their in vitro and in vivo efficacy. The methodologies involved antifungal evaluation, chemical analysis, and the effects of the rheological and mucoadhesive properties of propolis based gels. The obtained results demonstrated the fungicide action of propolis extracts against all three morphotypes (yeast, pseudohyphae, and hyphae) studied. The highest level of fungal cytotoxicity was reached at 6-8 hours of propolis cell incubation. Among the based gel formulations developed, the rheological and mucoadhesive results suggest that propolis based carbopol (CP1%) and chitosan gels were the most pseudoplastic ones. CP1% was the most mucoadhesive preparation, and all of them presented low thixotropy. Results of in vivo efficacy demonstrated that propolis based gels present antifungal action similar to clotrimazole cream, suggesting that future clinical studies should be performed.

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.