Andreia Ascenso

Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes

Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by “transfersomal method”, for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by “ethosomal method”. Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes and transfersomes due to the presence of both ethanol and surfactant in their composition. All these UDV were suitable for a deeper skin penetration, especially transethosomes.

Novel tretinoin formulations: a drug-in-cyclodextrin-in-liposome approach

Abstract Purpose: The aims of this experimental work were the incorporation and full characterization of the system Tretinoin-in-dimethyl-beta-cyclodextrin-in-ultradeformable vesicles (Tretinoin-CyD-UDV) and Tretinoin-in-ultradeformable vesicles (Tretinoin-UDV). Methods: The Tretinoin-CyD complex was prepared by kneading and the UDV by adding soybean phosphatidylcholine (SPC) to Tween® 80 followed by an appropriate volume of sodium phosphate buffer solution to make a 10%–20% lipid suspension. The resulting suspension was brought to the final mean vesicles size, of approximately 150 nm, by sequential filtration. The physicochemical characterization was based on: the evaluation of mean particle size and polydispersity index (PI) measured by photon correlation spectroscopy (PCS) and atomic force microscopy (AFM) topographic imaging; zeta potential (ζ-potential) and the SPC concentration determined by Laser–Doppler anemometry and an enzymatic-colorimetric test, respectively. The quantification of the incorporated Tretinoin and its chemical stability (during preparation and storage) was assayed by a HPLC at 342 nm. Results: It was possible to obtain the system Tretinoin-CyD-UDV. The mean vesicle size was the most stable parameter during experiments time course. AFM showed that Tretinoin-CyD-UDV samples were very heterogeneous in size, having three distinct subpopulations, while Tretinoin-UDV samples had only one homogeneous size population. The results of the ζ-potential measurements have shown that vesicle surface charge was low, as expected, presenting negative values. The incorporation efficiency was high, and no significant differences between Tretinoin-CyD-UDV and Tretinoin-UDV were observed. However, only Tretinoin-UDV with 20% lipid concentration formulation remained chemically stable during the evaluation period. Conclusion: According to our results, Tretinoin-UDV with 20% lipid concentration seems to be a better approach than Tretinoin-CyD-UDV, attending to the higher chemical stability.

Lycopene from Tomatoes: Vesicular Nanocarrier Formulations for Dermal Delivery

This experimental work aimed to develop a simple, fast, economic, and environmentally friendly process for the extraction of lycopene from tomato and incorporate this lycopene-rich extract into ultradeformable vesicular nanocarriers suitable for topical application. Lycopene extraction was conducted without a cosolvent for 30 min. The extracts were analyzed and incorporated in transfersomes and ethosomes. These formulations were characterized, and the cellular uptake was observed by confocal microscopy. Dermal delivery of lycopene formulations was tested under in vitro and in vivo conditions. Lycopene extraction proved to be quite safe and selective. The vesicular formulation was taken up by the cells, being more concentrated around the nucleus. Epicutaneous application of lycopene formulations decreased the level of anthralin-induced ear swelling by 97 and 87%, in a manner nonstatistically different from the positive control. These results support the idea that the lycopene-rich extract may be a good alternative to the expensive commercial lycopene for incorporation into advanced topical delivery systems.

Pickering emulsions: challenges and opportunities in topical delivery

ABSTRACT Introduction: Topical drug delivery is a challenging area with many advantages such as avoidance of first passage effect, stabilization of blood concentrations and attainment of local therapeutic effect with fewer side effects. Despite all these advantages, topical drug delivery remains limited to few molecules, since skin acts as a barrier to the delivery of many therapeutic molecules. To overcome this obstacle, a favored strategy relies on selecting suitable vehicles for dermatologic therapy, such as emulsions, gels and, more recently, nanoparticulate systems. Areas covered: Particle-stabilized emulsions, also known as Pickering emulsions, have garnered interest in recent years. Although most of the investigation on Pickering emulsions has been based on model systems with inorganic or organic solid particles, recent advances have been made regarding the application of nanocarriers, protein-based particles or cyclodextrins for this purpose. This review reports the latest advances in Pickering emulsions technical challenges, and discusses the potential benefits and drawbacks of using these formulations for topical pharmaceutical and cosmetic applications as an alternative to conventional surfactant-based systems. Expert opinion: Pickering emulsions appear as a multifunctional dosage form with endless advantages. A great deal of progress is expected in this area, which might represent a renewed vision for the pharmaceutical and cosmetic industry.

In vitro and in vivo topical delivery studies of tretinoin-loaded ultradeformable vesicles

INTRODUCTION Ultradeformable vesicles are highly promising tools to enhance the percutaneous transport of different drugs such as tretinoin across the skin barrier and also to increase the formulation stability at absorption site and reduce the drug induced irritation. METHODS Topical delivery of tretinoin-loaded ultradeformable vesicles (tretinoin-UDV) was evaluated concerning different studies, such as: the release and permeation profiles (tape stripping); skin penetration (fluorescence analysis); induced electrical changes in skin barrier properties; cytotoxicity (Trypan Blue assay) and skin irritation in in vivo conditions (Draize test). The novel formulation performance was also compared to a commercial tretinoin formulation regarding in vivo studies. RESULTS It was obtained a sustained and controlled drug release, as expected for UDV formulation. In addition, a dermal delivery was observed regarding the permeation study since it was not detected any drug amount in the receptor phase after 24h. Nile Red-UDV stained intensively mostly in the stratum corneum, corroborating the tape stripping results. Tretinoin-UDV decreased skin resistance, suggesting its ability to induce skin barrier disruption. Finally, the formulation vehicle (empty UDV) and tretinoin-UDV were not toxic under in vitro and in vivo conditions, at least, at 5×10(-3)mg/mL and 0.5mg/mL of tretinoin, respectively. CONCLUSION Tretinoin-UDV is a promising delivery system for tretinoin dermal delivery without promoting skin irritation (unlike other commercial formulations), which is quite advantageous for therapeutic purpose.

Topical Delivery of Antioxidants

Reactive oxygen species (ROS) and free radicals have been implicated in a number of diseases and disorders, and the skin, for its localization, is exposed to a large number of environmental threats. Free radical scavengers and antioxidants have thus been proposed as protective or therapeutic agents against ROS-mediated injuries. Oral treatment with several antioxidants has been reported to provide skin protection against deleterious effects of ultraviolet radiation. Topical delivery of antioxidants has increasingly gained interest and development, especially by offering better targeting to the upper skin layer. However, the topical delivery of antioxidants for dermal action is a challenging research field since the molecules are, in general, susceptible to degradation. The search for a new delivery system that, simultaneously, preserves the antioxidant stability and enhances its deposition on the skin, opened a new chapter in drug delivery design. Nanocarriers have been successful in enhancing the clinical efficiency of several drugs. More recent approaches in modulating through the skin delivery led to the development of specialized nanoparticulated systems. The first part of this article presents a review of the potential of antioxidants as pharmacological agents in ROS related diseases, with a special focus on oxidative stress implicated skin pathologies: ROS formation and natural protection against ROS toxicity, ROS-mediated skin damage and skin protection by antioxidants. In the second part of this work, we present reported formulation strategies for dermal delivery of antioxidants focusing on the nanoparticulated systems developed in recent years.

Melatonin-based pickering emulsion for skin's photoprotection

Abstract Context: Based on its antioxidant activity, melatonin was recently found to have a protection effect against photocarcinogenesis. Objective: This work aimed to develop an innovative sunscreen formulation based on the Pickering emulsions concept, stabilized by physical UV filters, modified starch and natural oils associated to melatonin as a key strategy for prevention against UV-induced skin damage. Materials and methods: For this purpose, melatonin was incorporated in Pickering emulsions that were characterized using physicochemical, in vitro and in vivo testing. Physicochemical studies included physical and chemical stability by a thorough pharmaceutical control. The possible protective effects of melatonin against UV-induced cell damage in HaCaT cell lines were investigated in vitro. The safety assessment and the in vivo biological properties of the final formulations, including Human Repeat Insult Patch Test and sunscreen water resistance tests were also evaluated. Results and discussion: These studies demonstrated that melatonin sunscreen Pickering emulsion was beneficial and presented a powerful protection against UVB-induced damage in HaCat cells, including inhibition of apoptosis. The inclusion of zinc oxide, titanium dioxide, green coffee oil and starch ensured a high SPF (50+) against UVA and UVB. Conclusion: The combination of melatonin, multifunctional solid particles and green coffee oil, contributed to achieve a stable, effective and innovative sunscreen with a meaningful synergistic protection against oxidative stress.

The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer.

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.

Chemoprevention of photocarcinogenesis by lycopene.

This review focuses on the photoprotection conferred by lycopene, one of the most potent anti‐oxidants. Lycopene has been recently proposed to play a critical role on anticarcinogenic action at different levels. The photoprotective properties of lycopene remain contradictory. Some studies point out a positive and others a negative effect in both in vitro and in vivo models. Currently, researchers recognise that crucial gaps exist in understanding the role of carotenoids as effective modulators of apoptosis, cell cycle dynamics and/or of their in vivo behaviour as cellular anti‐oxidants. The development of novel therapeutic strategies for skin disorders depends on our understanding of the molecular mechanism of UV damage on skin cells. The use of several effective phytocompounds, including lycopene, working through preventive and/or corrective pathways in the cell, may be an approach for reducing UV‐B‐generated damage.