Design of experiment (DoE) as a quality by design (QbD) tool to optimise formulations of lipid nanoparticles for nose-to-brain drug delivery.

INTRODUCTION: The nose-to-brain route has been widely investigated to improve drug targeting to the central nervous system (CNS), where lipid nanoparticles (solid lipid nanoparticles - SLN and nanostructured lipid carriers - NLC) seem promising, although they should meet specific criteria of particle size (PS) <200 nm, polydispersity index (PDI) <0.3, zeta potential (ZP) ~|20| mV and encapsulation efficiency (EE) >80%. To optimize SLN and NLC formulations, design of experiment (DoE) has been recommended as a quality by design (QbD) tool. AREAS COVERED: This review presents recently published work on the optimization of SLN and NLC formulations for nose-to-brain drug delivery. The impact of different factors (or independent variables) on responses (or dependent variables) is critically analyzed. EXPERT OPINION: Different DoEs have been used to optimize SLN and NLC formulations for nose-brain drug delivery, and the independent variables lipid and surfactant concentration and sonication time had the greatest impact on the dependent variables PS, EE, and PDI. Exploring different DoE approaches is important to gain a deeper understanding of the factors that affect successful optimization of SLN and NLC and to facilitate future work improving machine learning techniques.

Lipid nanoparticles strategies to modify pharmacokinetics of central nervous system targeting drugs: Crossing or circumventing the blood-brain barrier (BBB) to manage neurological disorders.

The main limitation to the success of central nervous system (CNS) therapies lies in the difficulty for drugs to cross the blood-brain barrier (BBB) and reach the brain. Regarding its structure and enzymatic complexity, crossing the BBB is a challenge, although several alternatives have been identified. For instance, the use of drugs encapsulated in lipid nanoparticles has been described as one of the most efficient approaches to bypass the BBB, as they allow the passage of drugs through this barrier, improving brain bioavailability. In particular, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been a focus of research related to drug delivery to the brain. These systems provide protection of lipophilic drugs, improved delivery and bioavailability, having a major impact on treatments outcomes. In addition, the use of lipid nanoparticles administered via routes that transport drugs directly into the brain seems a promising solution to avoid the difficulties in crossing the BBB. For instance, the nose-to-brain route has gained considerable interest, as it has shown efficacy in 3D human nasal models and in animal models. This review addresses the state of the art on the use of lipid nanoparticles to modify the pharmacokinetics of drugs employed in the management of neurological disorders. A description of the structural components of the BBB, the role of the neurovascular unit and limitations for drugs to entry into the CNS is first addressed, along with the developments to increase drug delivery to the brain, with a special focus on lipid nanoparticles. In addition, the obstacle of BBB complexity in the creation of new effective drugs for the treatment of the most prevalent neurological disorders is also addressed. Finally, the proposed strategies for lipid nanoparticles to reach the CNS, crossing or circumventing the BBB, are described. Although promising results have been reported, especially with the nose-to-brain route, they are still ongoing to assess its real efficacy in vivo in the management of neurological disorders.

Thermosensitive in situ hydrogels of rivastigmine-loaded lipid-based nanosystems for nose-to-brain delivery: characterisation, biocompatibility, and drug deposition studies.

Acetylcholinesterase inhibitors are the most used drugs to manage Alzheimer's disease, although they show low bioavailability in the brain. In this sense, nasal administration has been considered as a promising route for the direct delivery of these drugs to the brain (nose-to-brain delivery). In this work, in situ thermosensitive nasal gels with nanostructured lipid carriers (NLC) and nanoemulsion loaded with an acetylcholinesterase inhibitor (rivastigmine- RVG) were tested. In situ gels containing optimised rivastigmine -loaded NLC and rivastigmine -loaded nanoemulsion were first characterised (size, polydispersity index - PDI, zeta potential - ZP, encapsulation efficiency - EE, loading capacity - LC, pH, osmolarity, organoleptic and morphological analysis and accelerated stability). Afterwards, rheology and texture tests and in vitro studies were conducted to evaluate mucoadhesion, drug release, biocompatibility (with nasal and pulmonary cells, respectively RPMI-2650 and Calu-3) and drug deposition in a nasal cast model. The in situ gels of rivastigmine-loaded NLC and rivastigmine-loaded nanoemulsion had a respective particle/droplet size, PDI, ZP, EE, LC, pH and osmolarity of: 114.00 ± 1.91 nm and 135.80 ± 0.50 nm; 0.45 ± 0.00 and 0.43 ± 0.02; -3.58 ± 1.62 mV and -4.06 ± 1.03 mV; 95.13 ± 0.34% and 89.86 ± 0.19%; 9.30 ± 0.03% and 8.70 ± 0.01%; 6.47 ± 0.01 and 6.451 ± 0.00; 275 ± 0.02 and 280 ± 0.00 mOsm/kg. Organoleptic analysis showed homogeneous appearance, while morphological studies demonstrated that rivastigmine -loaded NLC and rivastigmine -loaded nanoemulsion had a spherical shape. Accelerated stability studies predicted good long-term stability. Rheological and texture analysis revealed that both in situ gels showed desirable characteristics for nasal administration. In addition, suitable nasal mucoadhesion and prolonged drug release were observed. Biocompatibility studies showed low and concentration-dependent cytotoxicity in RPMI 2650 and Calu-3 cells. Nasal deposition studies revealed that 4.0% of the drug was deposited in the olfactory region for both rivastigmine -loaded NLC and rivastigmine -loaded nanoemulsion alone, while in situ gels with these lipid-based nanosystems showed 8.0% of drug deposition. The results of this study highlight the potential of using thermosensitive in situ hydrogels containing lipid-based nanosystems to improve the nose-to-brain delivery of rivastigmine, providing a promising alternative therapeutic option to advance the management of Alzheimer's disease.

Quality by design (QbD) optimization of diazepam-loaded nanostructured lipid carriers (NLC) for nose-to-brain delivery: Toxicological effect of surface charge on human neuronal cells.

Diazepam is commonly used in the management of epileptic seizures, although it has limitations that can be overcome by using formulations that are easier to administer and capable of directing the drug to the brain. In this field, it has been reported that the use of nanostructured lipid carriers (NLC) via intranasal (or via nose-to-brain) promotes the targeting of drugs to the brain, improving the effectiveness of therapy. The aim of this work was to optimize two diazepam-loaded NLC formulations for nose-to-brain delivery, one with positive surface charge and one with negative surface charge. The quality by design (QbD) approach was used to design the experiments, where the quality target product profile (QTPP), the risk assessment and the critical quality attributes (CQAs) were defined to ensure safety, efficacy and quality of the final formulations. The experiments started with the optimization of critical material attributes (CMAs), related to the ratios of lipids and emulsifiers, followed by the selection of critical process parameters (CPPs), related to the production methods of the diazepam-loaded NLC formulation (ultrasound technique and high-pressure homogenization - HPH). Afterwards, the positive surface charge of the diazepam-loaded NLC was optimized. Finally, the biocompatibility with human neuronal cells of the formulation with a negative surface charge and of the formulation with a positive surface charge was evaluated. The results of the optimization of the CMAs showed that the ratios of lipids and emulsifiers more adequate were 6.7:2.9 and 4.2:0.3 (% w,w), respectively. Regarding the CPPs, HPH was considered the most suitable production method, resulting in an optimized diazepam-loaded NLC formulation (F1C15) with negative surface charge, showing particle size of 69.59 ± 0.22 nm, polydispersity index (PDI) of 0.19 ± 0.00, zeta potential (ZP) of -23.50 ± 0.24 mV and encapsulation efficiency (EE) of 96.60 ± 0.03 %. The optimized diazepam-loaded NLC formulation (F2A8) with positive surface charge had particle size of 124.40 ± 0.84 nm, PDI of 0.17 ± 0.01, ZP of 32.60 ± 1.13 mV and EE of 95.76 ± 0.24 %. In addition, the incorporation of diazepam in NLC resulted in a sustained release of the drug. No significant changes in particle size, PDI, ZP and EE were observed for the formulation F1C15, after 3 months of storage, whereas for formulation F2A8, particle size increased significantly. Biocompatibility studies showed that the formulation F2A8 was more cytotoxic than the formulation F1C15. Thereby, we conclude that the formulation F1C15 is more suitable for targeting the brain, when compared with the formulation F2A8. From the results of these studies, it can be confirmed that the QbD approach is an adequate and central tool to optimize NLC formulations.

Lipid nanocarriers containing Passiflora edulis seeds oil intended for skin application.

Nanostructured lipid carriers (NLC) have been studied for over 20 years, constituting the second generation of lipid nanoparticles. These nanosystems were introduced to overcome the drawbacks of solid lipid nanoparticles (SLN). Passion fruit seeds oil have a high antioxidant potential and also skin whitening properties. The objectives of this work were to prepare NLC by two methods (ultrasonication and High pressure homogenization) using different solid lipids (Glyceryl Distearate, Glyceryl Dibehenate and Cetyl Palmitate) and passion fruit seeds oil as liquid lipid. The nanoparticles prepared with glyceryl distearate, using the ultrasonication method showed better characteristics, since these nanosystems presented smaller particle sizes and polydispersity index, and higher zeta potential. Besides that, these nanoparticles showed a high occlusion factor and non-irritant potential in HET-CAM assay. Based on the results obtained, it may be suggested that the prepared NLCs can be applied to the face, since they did not cause any irritation, and represent a potential strategy for further use in topical formulations with antioxidant activity.

Alginate microparticles as carriers for the UV filter 2-ethylhexyl 4-methoxycinnamate: Influence on photostability.

OBJECTIVE: The organic ultraviolet UVB filter 2-ethylhexyl 4-methoxycinnamate (EHMC) was encapsulated in microparticles (MPs) of sodium alginate and co-loaded with vitamin E (Vit.E) by an extrusion process using an aerodynamically assisted jetting (AAJ) methodology. The aim was to assess the effect of encapsulation concerning UVB filter release from the MPs and its photochemical stability. METHODS: The EHMC photostability was analysed by exposing the samples (both MPs in aqueous dispersion and incorporated in a cream preparation) during 1 h to simulated solar light. For the MPs (empty-MP, EHMC-MP and EHMC + Vit.E-MP), the morphology and size were characterized; while in the case of the encapsulated samples, the amount of EHMC-loading was determined. The release of EHMC was evaluated by adding EHMC-MP or EHMC + Vit.E-MP to 65% ethanol in water under mechanical stirring at 32°C. RESULTS: All MPs showed a homogeneous size distribution with a median of 90.5 ± 2.5 µm for EHMC-MP and 70.4 ± 1.14 µm for EHMC + Vit.E-MP. The encapsulation efficiency was 92.9% and 99.4% for EHMC-MP and EHMC + Vit.E-MP, respectively. The observed release from the MPs was lower than the dissolution of the pure UV filter. EHMC-MP and EHMC + Vit.E-MP were successfully incorporated into a cream formulation, with no evidence of phase separation or colour modification. Upon simulated light exposure, the photoisomerization/phototransformation of EHMC encapsulated in MPs and Vit.E-MP decreased as compared to free EHMC, both in aqueous dispersion and as a cream. The conformational ratio of the isomers (Z-/E-EHMC) was found to be the lowest in the presence of Vit.E. CONCLUSION: This work demonstrates that use of these alginate microparticulate carriers could enhance the effectiveness of sunscreen preparations containing this UVB filter.

OBJECTIF: Le filtre ultraviolet organique D'UVB 2-éthylhexyl 4-methoxycinnamate (EHMC) a été encapsulé dans des microparticules (MPs) d'alginate de sodium et co-chargé avec la vitamine E (Vit.E) par un processus d'extrusion utilisant une méthode de jet assisté aérodynamique (AAJ). L'objectif était d'évaluer l'effet de l'encapsulation concernant la libération du filtre UVB par les MPs et sa stabilité photochimique. MÉTHODES: La photostabilité EHMC a été analysée en exposant les échantillons (les deux MPs en dispersion aqueuse et incorporés dans une préparation de type crème) pendant 1 h à la lumière solaire simulée. Pour les MPs (MP-vide, EHMC-MP et EHMC - Vit.E-MP), la morphologie et la taille ont été caractérisées; tandis que dans le cas des échantillons encapsulés, la quantité de charge EHMC a été déterminée. Le relargage de l'EHMC a été évalué en ajoutant EHMC-MP ou EHMC - Vit.E-MP à 65% d'éthanol dans l'eau sous agitation mécanique à 32°C. RÉSULTATS: Tous les MP ont montré une distribution homogène de taille avec une médiane de 90,5 +- 2,5 µm pour EHMC-MP et de 70,4 +- 1,14 µm pour l'EHMC et Vit.E-MP. L'efficacité de l'encapsulation était de 92,9 % et 99,4 % pour EHMC-MP et EHMC - Vit.E-MP, respectivement. Le relargage observé des MPs était inférieur à la dissolution du filtre UV pur. EHMC-MP et EHMC - Vit.E-MP ont été incorporés avec succès dans une formulation de crème, sans aucune preuve de séparation de phase ou de modification des couleurs. Après exposition à la lumière simulée, la photoiomérisation/phototransformation de l'EHMC encapsulée dans les MPs et Vit.E-MP a diminué par rapport à l'EHMC libre, à la fois dans la dispersion aqueuse et la crème. Le rapport conformationnel des isomères (Z-/E-EHMC) s'est avéré le plus bas en présence de Vit.E.

Evolution of the use of antioxidants in anti-ageing cosmetics.

OBJECTIVE: Skin health and beauty are a cornerstone of general well-being in humans. Anti-ageing cosmetics are used to provide a healthy and youthful appearance. Among the different cosmetic actives, antioxidants are incorporated in anti-ageing products due to their beneficial effects in preventing and minimizing the signs of skin ageing. This work aims to understand how anti-ageing formulations changed in the past 7 years regarding pure antioxidants composition. METHODS: Data were collected from anti-ageing formulations commercialized in main stores and pharmacies in the Portuguese market. The study started on 2011 and was updated with products launched or whose composition has been renewed on 2013, 2015 or 2018. RESULTS: Ascorbic acid and tocopherol and their derivatives were consistently the most used antioxidants in anti-ageing formulations; followed by niacinamide and retinyl palmitate. Seven ascorbic acid derivatives are currently used in anti-ageing formulations while only three tocopherol derivatives were identified in this study. Several combinations of antioxidants were routinely found, mainly tocopherol (or tocopherol derivatives) with other antioxidants and tocopherol with tocopherol derivatives. We have not identified emerging antioxidants with great impact in anti-ageing formulations even though niacinamide and retinyl palmitate exhibited over 10% more usage in 2018. CONCLUSION: This insight is relevant to the cosmetic industry providing a better understanding of the scientific-based formulation of modern cosmetics and supports the need for innovative antioxidants in anti-ageing cosmetics.

OBJECTIF: La santé de la peau et la beauté constituent la base du bien-être général chez l'homme. Les cosmétiques anti-âge sont utilisés pour donner une apparence saine et jeune. Parmi les différents principes actifs des cosmétiques, des antioxydants sont incorporées dans les produits anti-âge en raison de leur effet bénéfique sur la prévention et la réduction des signes de vieillissement de la peau. Ce travail vise à comprendre comment les formulations anti-âge ont évolué au cours des 7 dernières années au niveau des antioxydants purs composition. MÉTHODES: Les données ont été recueillies sur des formulations anti-âge commercialisées dans les principaux magasins et pharmacies du marché portugais. Cette étude a commencé en 2011 et a été mise à jour avec des produits mis sur le marché ou dont la composition a été renouvelée en 2013, 2015 ou 2018. RÉSULTATS: L'acide ascorbique et tocophérol et leurs dérivés sont systématiquement les antioxydants les plus utilisés dans les formulations anti-âge; ils sont suivis de la niacinamide et du palmitate de rétinol. Sept dérivés d'acide ascorbique sont actuellement utilisés pour lutter contre le vieillissement, tandis que seulement trois dérivés de tocophérol ont été identifiés dans cette étude. Plusieurs combinaisons d'antioxydants ont été systématiquement observées, principalement le tocophérol ou les dérivés de tocophérol avec d'autres antioxydants, et le tocophérol avec des dérivés de tocophérol. Nous n'avons pas identifié de nouveaux antioxydants présentant un impact majeur dans les formulations anti-âge, même si la niacinamide et le palmitate de rétinol ont vu leur utilisation augmenter de 10% en 2018. CONCLUSION: Cette perspective est pertinente pour le secteur des cosmétiques. Elle permet de mieux comprendre la formulation scientifique des cosmétiques modernes et confirme le besoin d'innover au niveau des antioxydants utilisés dans les produits anti-âge. Les cosmétiques anti-âge sont utilisés pour donner une apparence saine et jeune. Parmi les différents actifs cosmétiques, les antioxydants sont incorporés dans les produits anti-âge en raison de leurs effets bénéfiques dans la prévention et la réduction des signes du vieillissement cutané. Ce travail vise à comprendre comment les formulations anti - vieillissement ont changé au cours des 7 dernières années en ce qui concerne la composition en antioxydants purs.

Evaluation of the biocompatibility and skin hydration potential of vitamin E-loaded lipid nanosystems formulations: In vitro and human in vivo studies.

Lipid-based nanosystems, such as nanostructured lipid carriers (NLC) and nanoemulsions (NE) have been described as promising alternatives to conventional formulations for increase skin hydration. Besides, these systems have been used as efficient vehicles for lipophilic molecules that improve skin properties (e.g. vitamin E). In this study, we performed comparative investigations between hydrogels formulations containing vitamin E-loaded NLC (HG-NLCVE) and vitamin E-loaded nanoemulsion (HG-NEVE). The experiments started with particle size measurements, which showed no significant differences between nanoparticles/nanodroplets sizes after incorporation in the hydrogel net (386 nm vs. 397 nm for HG-NLCVE and 402 nm vs. 514 nm for HG-NEVE). Afterwards, in vitro biocompatibility studies in human keratinocytes were carried out, being observed that the lipid-based nanosystems were more cytotoxic for the cells before incorporation in the hydrogel. Finally, the formulations hydration potential and sensory attributes for skin application were evaluated by in vitro occlusion tests and in vivo human experiments. The results showed that the HG-NLCVE exhibited the best occlusive properties, whereas the HG-NEVE performed a faster skin hydration effect. Furthermore, the latter was selected as the most attractive for skin application, although the HG-NLCVE was described as more suitable to obtain a long-lasting effect. This study demonstrated the in vitro and in vivo safety and hydration potential of hydrogels containing vitamin E-loaded lipid-based nanosystems. These results establish a basis to assess the cutaneous use of these systems, despite more in vivo experiments, for longer periods and in more volunteers, are required before commercialization.

Nose-to-brain delivery of lipid-based nanosystems for epileptic seizures and anxiety crisis.

Epileptic seizures and anxiety crisis are severe conditions that require fast and effective treatment, targeting the brain. Current emergency antiepiletics and anxiolytics have limited brain bioavailability, following oral, intravenous or rectal administration. This relates with the limited extent at which these drugs bypass the blood brain barrier (BBB). Thereby, the development of strategies that significantly improve the brain bioavailability of these drugs, along with a simple and safe administration by patients, attenuating and/or preventing epileptic seizures or anxiety crisis, are still a major need. In this respect, the nasal/intranasal route has been suggested as a promising strategy for drug targeting to the brain, thus avoiding the BBB. Besides, the use of lipid-based nanosystems, such as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), liposomes, nanoemulsions and microemulsions, have been demonstrating high efficiency for nose-to-brain transport. This review highlights the potential of using lipid-based nanosystems in the management of epilepsy and anxiety, by means of the nasal/intranasal route. So far, the reported studies have shown promising results, being required more in vivo experiments to further advance for clinical trials. Furthermore, toxicological concerns related to the need of evaluate the impairment on the mucociliary clearance mechanism have been pointed.

Mechanical Properties of Topical Anti-Psoriatic Medicines: Implications for Patient Satisfaction with Treatment.

Different types of topical preparations are available as anti-psoriatic medicines, semisolid formulations being the preferred dosage forms for the treatment of body lesions. The mechanical characterization of these semisolid formulations is seldom reported, although mechanical features have been recognized to play an important role in treatment acceptability and adherence. The aim of this study was to characterize the mechanical properties of semisolid topical formulations commercially available for psoriasis treatment. One complementary aim was to evaluate patient satisfaction with topical treatment and discuss the results according to the mechanical features of the dosage form. Eight ointments (O 1-8), five creams (C 1-5), one oleogel (G1), and one excipient (E1-petrolatum) were characterized for textural properties (spreadability and penetration tests) and flow behavior. Power law model was fitted to the results. A questionnaire for the assessment of satisfaction with topical medicines used for psoriasis treatment over 6 months was developed and applied to 79 psoriasis patients. All the tested formulations presented a shear-thinning behavior with power law indexes (n) lower than 1. Ointments were distinct from the other dosage forms, since they presented higher consistency coefficients (K), firmness, and adhesiveness and this was evidenced by hierarchical cluster analysis, which identified two clusters based on the mechanical properties. Cluster 1 included the ointments and petrolatum and the cluster 2 enclosed the creams and the gel. The clusters were associated with several attributes classified by patients as analyzed with Fisher's exact test. In all cases, higher satisfaction was observed for cluster 2. The knowledge obtained regarding the influence of the dosage form on the degree of satisfaction with the treatment could be helpful in supporting the selection of the dosage form in clinical practice and thus improve treatment adherence and clinical outcomes. The differences observed between the mechanical properties of the formulations studied may be also relevant to the industry, as guidance to the development of new medicines.

Patient preferences for attributes of topical anti-psoriatic medicines.

Background: Patient preferences should be considered when prescribing topical treatments to drive up adherence and improve clinical outcomes. Objective: The aim of this work was to identify the most important attributes of topical medicines for psoriasis treatment in the patients' view, and explore the sociodemographic and clinical determinants of these preferences. Methods: A questionnaire for the evaluation of the relevancy given to specific attributes of topical medicines used for psoriasis treatment was developed (PSO-TOPAP) and was applied to a total of seventy-nine patients, members of the Portuguese Association of Psoriasis (PSOPortugal) or outpatients of a dermatology unit of a central hospital. Results: Overall, attributes belonging to the formulation and application domains were greatly valued over attributes related to the container. Only a small number of patient preferences was influenced by age, gender, duration of the disease and age at first diagnosis. Limitations: Our findings need to be verified in larger and more diverse patient samples before generalization can be made. Conclusion: The insight obtained in this work can provide guidance to pharmaceutical drug product design and has also the potential to improve patient care through the acknowledgment of patient preferences in clinical practice.

Photostabilization strategies of photosensitive drugs.

PURPOSE: Several active compounds are sensitive to light, especially to the ultraviolet radiation (UV-R) leading to their degradation or modification, with lost or decrease of their biological activity. The aim of this study was to perform a systematic review regarding photostabilization strategies used on health products and perform a critical appraisal of their effectiveness. RESULTS: The bibliographic search identified 2261 results and merely 40 studies met the selection criteria. Of these, 85% referred to encapsulation strategies, 10% to antioxidants and 5% to the use of solar filters. Cyclodextrins (CD's) were the most used encapsulation systems (32.5%) followed by liposomes and lipid nanoparticles (each 17.5%), microparticles (15%) and polymeric nanoparticles (10%). The most effective were found to be liposomes and lipid nanoparticles. However, the different methodological conditions used limit the true relevance of this finding. CONCLUSIONS: A gold standard strategy suitable for all compounds cannot be proposed. Instead, case-by-case evaluation, supported on the photodegradation mechanism is recommended. Systematic studies that compare different photostabilization strategies undertaken with the same irradiation conditions are also needed.

Formulations based on solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for cutaneous use: A review.

Cutaneous use of lipid nanoparticles (solid lipid nanoparticles, SLN and nanostructured lipid carriers, NLC) has been showing promising results. These systems consist of low viscosity aqueous dispersions, being usually employed by means of semi-solid formulations with adequate consistency for skin application. This review addresses the cutaneous use of lipid nanoparticles for therapeutic and cosmetic applications. Initially, general information related to pharmaceutical semi-solid formulations is presented. Afterwards, the effects of SLN and NLC on the skin, and technological aspects related to semi-solid systems based on SLN or NLC are described. Finally, the most relevant studies related to the formulations based on SLN and NLC, for cosmetic and therapeutic applications, are reported. Notwithstanding the cutaneous use of SLN and NLC has been proposed for both local and transdermal delivery, the reported studies show promising results only for local application. In this sense, more research is required to better understanding the interaction mechanisms of lipid nanoparticles with skin lipids. Furthermore, the development of standard methods for skin experiments with nanoparticles is necessary.

In vitro cytotoxicity evaluation of resveratrol-loaded nanoparticles: Focus on the challenges of in vitro methodologies.

Assessment of toxic effects is mandatory before market placement of pharmaceutical and cosmetic products. Nanotoxicology is an emerging regulatory concern and still a challenging field. Topical application of resveratrol (RSV) has been extensively studied owing to its multi-mechanistic skin anti-aging effects. Nanoencapsulation has been suggested as a promising solution to overcome RSV stability issues. In this work RSV-loaded solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were prepared using a homogenization/sonication technique. Cytotoxicity assays were conducted with an immortalized cell line of human keratinocytes (HaCaT). For a comprehensive cytotoxicity characterization MTT and Alamar Blue® reduction assays (assessment of metabolic activity), Neutral red uptake (evaluation of lysosomal integrity), and Trypan blue (assessment of membrane integrity) were used. The results obtained with the different assays were not always concordant, as put in evidence by an adequate statistical analysis. Experimental parameters such as washing steps were found to be critical. The study is of interest because it draws attention to the importance of careful selected experimental conditions of in vitro nanotoxicological tests. Experimental protocols should be adapted taking into account nano-related features such as interference with the dyes and light dispersion/absorption properties.

Comparison between sensory and instrumental characterization of topical formulations: impact of thickening agents.

OBJECTIVE: Sensorial properties of cutaneous formulations are important in determining their acceptability by consumers. However, sensorial analysis is time-consuming and requires an available panel of trained assessors. Thus, this article aimed to study the impact of thickening agents on mechanical properties of creams and investigate how these measurements could correlate the sensory attributes using a combined instrumental-sensorial approach. METHODS: For this purpose, eight semisolid formulations were prepared, containing in their composition different thickening agents at different concentrations. These formulations were firstly microscopically observed and then assayed through textural, rheological and spreadability measurements. Textural characterization and rheological characterization were performed during six months to assess the physical stability of the studied formulations. Finally, six sensory attributes, namely firmness, adhesiveness, cohesiveness, spreadability, consistency and adhesiveness post-application, were tested by a trained panel. RESULTS: It was observed that thickening agents influence the microstructure of semisolid emulsions and also their mechanical properties. In general, an increase in the concentration of thickening agents improves the physical stability of formulations over time. Besides, textural parameters (firmness and adhesiveness), viscosity and difficulty to spread also increase. A good correlation between mechanical characterization and sensorial analysis was verified, mainly for spreadability properties. CONCLUSION: With the obtained results, it is possible to conclude that the proposed methods for mechanical characterization can be correlated with sensorial perception obtained from volunteers, representing a faster and less expensive alternative than sensory analysis.

Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line.

Toxic effects of ultraviolet (UV) radiation on skin include protein and lipid oxidation, and DNA damage. The latter is known to play a major role in photocarcinogenesis and photoaging. Many plant extracts and natural compounds are emerging as photoprotective agents. Castanea sativa leaf extract is able to scavenge several reactive species that have been associated to UV-induced oxidative stress. The aim of this work was to analyze the protective effect of C. sativa extract (ECS) at different concentrations (0.001, 0.01, 0.05 and 0.1 µg/mL) against the UV mediated-DNA damage in a human keratinocyte cell line (HaCaT). For this purpose, the cytokinesis-block micronucleus assay was used. Elucidation of the protective mechanism was undertaken regarding UV absorption, influence on (1)O2 mediated effects or NRF2 activation. ECS presented a concentration-dependent protective effect against UV-mediated DNA damage in HaCaT cells. The maximum protection afforded (66.4%) was achieved with the concentration of 0.1 µg/mL. This effect was found to be related to a direct antioxidant effect (involving (1)O2) rather than activation of the endogenous antioxidant response coordinated by NRF2. Electrochemical studies showed that the good antioxidant capacity of the ECS can be ascribed to the presence of a pool of different phenolic antioxidants. No genotoxic or phototoxic effects were observed after incubation of HaCaT cells with ECS (up to 0.1 µg/mL). Taken together these results reinforce the putative application of this plant extract in the prevention/minimization of UV deleterious effects on skin.

Photodegradation of avobenzone: stabilization effect of antioxidants.

Avobenzone is one of the most common UVA-filters in sunscreens, and is known to be photounstable. Some of the strategies used to stabilize this filter present some drawbacks like photosensitization reactions. Antioxidants are widely used as cosmetic ingredients that prevent photoageing and complement the photoprotection offered by the UV-filters preventing or reducing photogenerated reactive species. The purpose of this work was to study the effect of antioxidants in the photostabilization of avobenzone. The filter dissolved in dimethyl sulfoxide or incorporated in a sunscreen formulation was irradiated with simulated solar radiation (750 W/m(2)). The tested antioxidants were vitamin C, vitamin E, and ubiquinone. The area under the curve of the absorption spectrum for UVA range and the sun protection factor (SPF) were calculated. Vitamin E (1:2), vitamin C (1:0.5) and ubiquinone (1:0.5) were the more effective concentrations increasing the photostability of avobenzone. In sunscreen formulations, the most effective photostabilizer was ubiquinone which also promoted an increase in SPF. This knowledge is important to improve effectiveness of sunscreen formulation. Antioxidants can be valuable ingredients for sunscreens with a triple activity of filter stabilization, SPF boosting and photoageing prevention.

Characterization, sensorial evaluation and moisturizing efficacy of nanolipidgel formulations.

BACKGROUND: Nanostructured lipid carriers (NLC) have been widely studied for cosmetic and dermatological applications due to their favourable properties that include the formation of an occlusive film on the skin surface that reduces the transepidermal water loss (TEWL) and increase in water content in the skin which improves the appearance on healthy human skin and reduces symptoms of some skin disorders like eczema. OBJECTIVE: The main objective of this study was the development of semisolid formulations based NLC with argan oil or jojoba oil as liquid lipids, by addition of Carbopol®934 or Carbopol®980 as gelling agents, followed by comparison between instrumental analysis and sensorial evaluation and in vivo efficacy evaluation. METHODS: Nanostructured lipid carriers dispersions were produced by the ultrasound technique, and to obtain a semisolid formulation, gelling agents were dispersed in the aqueous dispersion. Particle size, polydispersity index and zeta potential were determined. Instrumental characterization was performed by rheological and textural analysis; the sensorial evaluation was also performed. Finally, skin hydration and TEWL were studied by capacitance and evaporimetry evaluation, respectively. RESULTS: Particles showed a nanometric size in all the analysed formulations. All the gels present pseudoplastic behaviour. There is a correspondence between the properties firmness and adhesiveness as determined by textural analysis and the sensory evaluation. The formulations that showed a greater increase in skin hydration also presented appropriate technological and sensorial attributes for skin application. CONCLUSIONS: Nanolipidgel formulations with the addition of humectants are promising systems for cosmetic application with good sensory and instrumental attributes and moisturizing efficacy.

Bacterial cellulose membranes as drug delivery systems: an in vivo skin compatibility study.

Bacterial cellulose (BC) is a highly pure form of cellulose, produced in the form of a swollen membrane by several bacteria that demonstrated to be able to modulate the skin release of model drugs. In the present study, the skin irritation potential of BC was evaluated in human subjects. BC membranes with and without glycerin (acting as plasticizer) were tested. No significant differences were observed for transepidermal water loss (TEWL) measurements in comparison with negative control, 2 and 24 h after patch removal, which is an indicator of an absence of barrier disruption. Similar results were found for erythema. Clinical scores were zero at both times for all volunteers, with the exception of five volunteers that exhibited weak reactions. BC with glycerin provided a skin moisturizing effect statistically higher than the negative control (p=0.044), which was not observed for BC alone. The good skin tolerance found after a single application under occlusion reinforces the putative interest of BC membranes as supports for drug topical delivery. Besides modifying the mechanical properties, the inclusion of glycerin results in a skin moisturizing effect which could be clinically relevant for the treatment for skin diseases characterized by dryness, such as psoriasis and atopic dermatitis.