Liquid crystalline nanodispersion functionalized with cell-penetrating peptides improves skin penetration and anti-inflammatory effect of lipoic acid after in vivo skin exposure to UVB radiation.

In this study, the development and the performance of a new targeted liquid crystalline nanodispersion (LCN) by the attachment of cell-penetrating peptides (CPP) onto their surfaces to improve skin delivery of lipoic acid (LA) were evaluated. For that, the synthesis and characterization of this new platform as well as its spatiotemporal analysis from in vitro and in vivo topical application were explored and extensively discussed in this paper. The TAT or D4 peptides were chosen as CPP due to specific target strategies by the charge grouping on the skin surface or target the overexpressed epidermal growth factor receptor (EGFR) of cell membrane of keratinocytes, respectively. Thus, the nanoparticle characterization results when taken together suggested that designed LCNs maintained their hexagonal phase structure, nanoscale particle size, and low polydispersity index even after drug, lipopolymers, and peptide additions, which are proved to be favorable for topical skin delivery. There were no statistical differences among the LCNs investigated, except for superficial charge of LCN conjugated with TAT which may have altered the LCN zeta potential due to cationic charge of TAT amino acid sequence compared with D4. The cumulative amounts of LA retained into the skin were determined to be even higher coming from the targeted LCNs. Moreover, the exogenous antioxidant application of the LA from the LCNs can prevent ROS damage, which was demonstrated by this study with the less myeloperoxidase (MPO) activity and decrease in cytokine levels (TNF-alpha and IL-1ß) generated by the oxidative stress modulation. Together, the data presented highlights the potential of these targeted LCNs, and overall, opens new frontiers for preclinical trials.

The prominence of the dosage form design to treat ocular diseases.

The eye is susceptible to various diseases commonly difficult to treat. To overcome the barriers imposed by this organ for required drugs penetration, technological strategies have been implemented to ocular formulations. Among them are the use of temperature or electric stimuli and the development of nanoparticles. The objective of this review is to present the main barriers to ocular drug delivery and to discuss strategies used in the development of ocular dosage forms, primarily for topical delivery, to increase the local bioavailability of drugs, target their delivery and increase patient compliance. Results obtained in the last years related to the topical administration of liposomes, dendrimers, iontophoresis, among other nanoparticulate systems focused on ophthalmic delivery, will be addressed. Finally, some clinical trials and marketed formulations that use nanotechnology to topically treat eye diseases will be presented.

In Vitro TyRP-1 Knockdown Based on siRNA Carried by Liquid Crystalline Nanodispersions: an Alternative Approach for Topical Treatment of Vitiligo.

PURPOSE: Vitiligo is a skin disease characterized by depigmentation and the presence of white patches that are associated with the loss of melanocytes. The most common explanation for the cause of this condition is that it is an autoimmune condition. TyRP-1 is involved in melanin pigment synthesis but can also function as a melanocyte differentiation antigen. This protein plays a role in the autoimmune destruction of melanocytes, which results in the depigmentation, characteristic of this disease. In this study, we evaluated liquid crystalline nanodispersions as non-viral vectors to deliver siRNA-TyRP-1 as an alternative for topical treatment of vitiligo. METHODS: Liquid crystalline nanodispersions were obtained and characterized with respect to their physical-chemical parameters including size, PdI and zeta potential, as well as Small Angle X-ray Scattering and complexing to siRNA. The effects of the liquid crystalline nanodispersions on the cellular viability, cell uptake and levels of the knockdown target TyRP-1 were evaluated in melan-A cells after 24 h of treatment. RESULTS: The liquid crystalline nanodispersions demonstrated adequate physical-chemical parameters including nanometer size and a PdI below 0.38. These systems promoted a high rate of cell uptake and an impressive TyRP-1 target knockdown (> 80%) associated with suitable loading of TyRp-1 siRNA. CONCLUSIONS: We demonstrated that the liquid crystalline nanodispersions showed promising alternative for the topical treatment of vitiligo due to their physical parameters and ability in knockdown the target protein involved with autoimmune destruction of melanocytes.

Evaluation of critical parameters for in vitro skin permeation and penetration studies using animal skin models.

In vitro skin permeation/penetration studies may be affected by many sources of variation. Herein, we aimed to investigate the major critical procedures of in vitro skin delivery studies. These experiments were performed with model drugs according to official guidelines. The influence of skin source on penetration studies was studied as well as the use of a cryopreservation agent on skin freezing evaluated by transepidermal water loss, electrical resistance, permeation/penetration profiles and histological changes of the skin. The best condition for tape stripping procedure was validated through the evaluation of the distribution of corneocytes, mass of stratum corneum (SC) removed and amount of protein removed using finger pressure, a 2kg weight and a roller. The interchangeability of the tape stripping procedures followed by the epidermis and dermis homogenate and the micrometric horizontal cryostat skin sectioning methods were also investigated, besides the effect of different formulations. Noteworthy, different skin sources were able to ensure reliable interchangeability for in vitro permeation studies. Furthermore, an increased penetration was obtained for stored frozen skin compared to fresh skin, even with the addition of a cryoprotectant agent. The best method for tape stripping was the finger pressure followed by the addition of a propylene glycol solvent leading to better SC removal. Finally, no significant difference was found in skin penetration studies performed by different methods suggesting their possible interchangeability.

Poly-epsilon-caprolactone nanoparticles enhance ursolic acid in vivo efficacy against Trypanosoma cruzi infection.

Despite affecting millions of people worldwide, Chagas disease is still neglected by the academia and industry and the therapeutic option available, benznidazole, presents limited efficacy and side effects. Within this context, ursolic acid may serve as an option for treatment, however has low bioavailability, which can be enhanced through the encapsulation in polymeric nanoparticles. Therefore, herein we developed ursolic acid-loaded nanoparticles with poly-ε-caprolactone by the nanoprecipitation method and characterized them for particle size, zeta potential, polydispersity, encapsulation efficiency, morphology by scanning electron microscopy and thermal behavior by differential scanning calorimetry. Results indicated that an appropriate ratio of organic phase/aqueous phase and polymer/drug is necessary to produce smaller particles, with low polydispersity, negative zeta potential and high drug encapsulation efficiency. In vitro studies indicated the safety of the formulation against fibroblast culture and its efficacy in killing T. cruzi. Very importantly, the in vivo study revealed that the ursolic acid-loaded nanoparticle is as potent as the benznidazole group to control parasitemia, which could be attributed to improved bioavailability of the encapsulated drug. Finally, the toxicity evaluation showed that while benznidazole group caused liver toxicity, the nanoparticles were safe, indicating that this formulation is promising for future evaluation.

Development and characterization of novel 1-(1-Naphthyl)piperazine-loaded lipid vesicles for prevention of UV-induced skin inflammation.

1-(1-Naphthyl)piperazine (1-NPZ) has shown promising effects by inhibiting UV radiation-induced immunosuppression. Ultradeformable vesicles are recent advantageous systems capable of improving the (trans)dermal drug delivery. The aim of this study was to investigate 1-NPZ-loaded transethosomes (NPZ-TE) and 1-NPZ-loaded vesicles containing dimethyl sulfoxide (NPZ-DM) as novel delivery nanosystems, and to uncover their chemopreventive effect against UV-induced acute inflammation. Their physicochemical properties were evaluated as follows: vesicles size and zeta potential by dynamic and electrophoretic light scattering, respectively; vesicle deformability by pressure driven transport; rheological behavior by measuring viscosity and I-NPZ entrapment yield by HPLC. In vitro topical delivery studies were performed in order to evaluate the permeation profile of both formulations, whereas in vivo studies sought to assess the photoprotective effect of the selected formulation on irradiated hairless mice by measuring myeloperoxidase activity and the secretion of proinflammatory cytokines. Either NPZ-TE or NPZ-DM exhibited positive results in terms of physicochemical properties. In vitro data revealed an improved permeation of 1-NPZ across pig ear skin, especially by NPZ-DM. In vivo studies demonstrated that NPZ-DM exposure was capable of preventing UVB-induced inflammation and blocking mediators of inflammation in mouse skin. The successful results here obtained encourage us to continue these studies for the management of inflammatory skin conditions that may lead to the development of skin cancers.

RNAi mediated IL-6 in vitro knockdown in psoriasis skin model with topical siRNA delivery system based on liquid crystalline phase.

Gene therapy by RNA interference (RNAi) is a post-transcriptional silencing process that can suppress the expression of a particular gene and it is a promising therapeutic approach for the treatment of many severe diseases, including cutaneous disorders. However, difficulties related to administration and body distribution limit the clinical use of small interfering RNA (siRNA) molecules. In this study, we proposed to use nanocarriers to enable siRNA application in the topical treatment of skin disorders. A siRNA nanodispersion based on liquid crystalline phase and composed of monoolein (MO), oleic acid (OA) and polyethylenimine (PEI) was developed and its physicochemical properties, efficiency of complexation and carrier/siRNA stability were assessed. Subsequently, cell viability, cellular uptake, in vitro skin irritation test using reconstructed human epidermis (RHE) and in vitro IL-6 knockdown in psoriasis skin model were evaluated. The results showed that the liquid crystalline nanodispersion is a promising topical delivery system for administration of siRNA, being able to overcome the limitations of the route of administration, as well those resulting from the characteristics of siRNA molecules. The formulation was effective at complexing the siRNA, presented high rate of cell uptake (∼90%), increased the skin penetration of siRNA in vitro, and did not cause skin irritation compared with Triton-X (a moderate irritant), resulting in a 4-fold higher viability of reconstructed human epidermis and a 15.6-fold lower release of IL-1α. A single treatment with the liquid crystalline nanodispersion carrying IL-6 siRNA for 6h was able to reduce the extracellular IL-6 levels by 3.3-fold compared with control treatment in psoriasis skin model. Therefore, liquid crystalline nanodispersion is a suitable nanocarrier for siRNA with therapeutic potential to suppress skin disease-specific genes. This study also highlights the applicability of reconstructed skin models in pharmaceutical field to evaluate the performance of delivery systems without the use of animal models.

Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors.

Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection. These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 × 4 mm, 5 µm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71%. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 µg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 µg/mL, respectively, for HPLC/EC for the added LA concentration (6.67 µg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 µg/mL, respectively, for HPLC/ELS for the added LA concentration (10 µg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.

Inhibitory effect of GB-2a (I3-naringenin-II8-eriodictyol) on melanogenesis.

ETHNOPHARMACOLOGY RELEVANCE: GB-2a is a I3-naringenin-II8-eriodictyol compound isolated from Garcinia gardneriana (Planchon & Triana) Zappi, a plant used in folk medicine for the treatment of skin disorders. AIM OF STUDY: In the search for new depigmenting agents, this study was carried out to investigate the in vitro effects of GB-2a isolated from G. gardneriana (Planchon & Triana) Zappi in B16F10 melanoma cells. MATERIALS AND METHODS: The effects of GB-2a were evaluated through determination of melanin biosynthesis in B16F10 melanoma cells in comparison with the reference drug kojic acid (500µM). In parallel, the GB-2a effect was assessed in a cell viability assay. Mushroom tyrosinase activity assays were conducted to verify the effect of this enzyme. In order to ascertain the nature of enzyme inhibition on tyrosinase, kinetics analysis of the GB-2a was performed with L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) substrates. RESULTS: The results showed that GB-2a biflavonoid significantly inhibited the melanin content, without reducing cell viability. GB-2a also showed a strong antityrosinase activity in the mushroom tyrosinase assay. GB-2a inhibited the tyrosinase activity, exerting a mixed inhibition. For the L-tyrosine substrate the inhibition was in non-competitive mode and for L-DOPA it was in uncompetitive mode. CONCLUSION: GB-2a biflavonoid promoted inhibition on tyrosinase activity and reduced melanin biosynthesis in B16F10 cells, which suggests great potential for medical and cosmetic uses as a depigmenting agent.

Advances in the bioanalytical study of drug delivery across the skin.

The study of a drug's dermal penetration profile provides important pharmaceutical data for the rational development of topical and transdermal delivery systems because the skin is a broadly used delivery route for local and systemic drugs and a potential route for gene therapy and vaccines. Monitoring drug penetration across the skin and quantifying its levels in different skin layers have been constant challenges due to the detection limitations of the available techniques, as well as the inherent interference in this tissue. This review explores and discusses several bionalytical methods that are indispensable tools to study drugs across the skin. In addressing the main topic, we structure the review highlighting the skin as an important route of drug administration and its structure, skin membrane models most used and its properties, in vitro and in vivo assays most used in the study of drug delivery to the skin, the techniques for processing the skin for subsequent analysis by bioanalytical methods that have a theoretical and practical approach showing its applicability, limitations and also including examples of its use. This review has a comprehensive approach in order to help researchers design their experiments and update the applicability and advances in this area of expertise.

Effect of a Garcinia gardneriana (Planchon and Triana) Zappi hydroalcoholic extract on melanogenesis in B16F10 melanoma cells.

ETHNOPHARMACOLOGY RELEVANCE: Garcinia gardneriana (Planchon and Triana) Zappi (Clusiaceae) is popularly called "bacopari" in southern Brazil. The leaves of this plant are traditionally used to treat skin disorders. AIM OF STUDY: This study evaluated the effects of a hydroalcoholic extract of Garcinia gardneriana leaves (HEGG) on B16F10 murine melanoma cells in order to search for new depigmenting agents. MATERIALS AND METHODS: The effects of HEGG were assessed in melanin content assays in B16F10 melanoma cells compared with the reference drug kojic acid (500mM). Melanin content was measured after spontaneous melanogenesis, UVB-induced melanogenesis and melanogenesis induced by α-MSH. At the same time, cell viability assays were conducted. Intracellular and mushroom tyrosinase activity assays were employed to evaluate the effect of HEGG on tyrosinase activity. RESULTS: HEGG decreased the level of melanin under all three experimental conditions of melanin content evaluation without reducing cell viability. In intracellular tyrosinase assays, the enzyme's activity was reduced about 19% with extract concentrations ranging 0.1-10µg/mL. In the mushroom tyrosinase activity assay a maximal inhibition of 35% (1000µg/mL) was observed. CONCLUSION: These results suggest that HEGG inhibition relates to its tyrosinase activity. Therefore, the hydroalcoholic extract of Garcinia gardneriana shows great potential for use as a depigmenting agent in hyperpigmentation disorders.