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In previous studies, we developed a hydrogel formulation containing silibinin-loaded pomegranate oil nanocapsules (HG-NCSB) that had improved in vivo anti-inflammatory action in comparison to non-encapsulated silibinin. To determine skin safety and whether the nanoencapsulation influences silibinin skin permeation, NCSB skin cytotoxicity, HG-NCSB permeation in human skin, and a biometric study with healthy volunteers were conducted. The formulation of nanocapsules was prepared by the preformed polymer method while the HG-NCSB was obtained by thickening the suspension of nanocarriers with gellan gum. The cytotoxicity and phototoxicity of nanocapsules were assessed in Keratinocytes (HaCaT) and fibroblast (HFF-1) using the MTT assay. The hydrogels were characterized regarding the rheological, occlusive, and bioadhesive properties, and silibinin permeation profile in human skin. The clinical safety of HG-NCSB was determined by cutaneous biometry in healthy human volunteers. NCSB yielded better cytotoxicity results than the blank nanocapsules (NCPO). NCSB did not cause photocytotoxicity, while NCPO and the non-encapsulated substances (SB and pomegranate oil) were phototoxic. The semisolids presented non-Newtonian pseudoplastic flow, adequate bioadhesiveness, and low occlusive potential. The skin permeation demonstrated that HG-NCSB retained a higher SB amount in the outermost layers than HG-SB. In addition, HG-SB reached the receptor medium and had a superior concentration of SB in the dermis layer. In the biometry assay, there was no significant cutaneous alteration after the administration of any of the HGs. Nanoencapsulation promoted greater SB retention in the skin, averted percutaneous absorption, and made the topical use of SB and pomegranate oil safer.
Assuntos
Nanocápsulas , Punica granatum , Humanos , Silibina , Hidrogéis , Pele , BiometriaRESUMO
Abstract Fridericia caudigera and Cuspidaria convoluta (Bignoniaceae) species, which grow in the northwest of Argentina, have shown antibacterial effect against strains isolated from skin infections, and each one displayed synergism with commercial antibiotics. The aims of this work were to evaluate the antibacterial activity and toxicity of the combination of these two plant species, and to design a stable gel for topical use including the blend of extracts. The combination of extracts was evaluated for synergistic effects (chequerboard assay), genotoxicity (Ames test) and cytotoxicity (Artemia salina test). A gel was subsequently formulated with the combination of extracts using carboxymethylcellulose as a polymer. The following physico- chemical characteristics of the gel formulation: pH, viscosity, spreadability and total phenol content, as well as resistance to severe temperature changes, biological activity (diffusion in agar), in vitro permeation (Franz cells) and primary dermal irritation (Draize test) were analyzed. The combination of extracts showed a synergistic effect on pathogenic bacteria and was not toxic in the in vitro tests. The gel was stable and retained the antimicrobial activity of the original extracts. The formulation proposed in this work could constitute an alternative for primary skin infections since it proved to be safe for topical administration.
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Plantas/efeitos adversos , Artemia/classificação , Pele/lesões , Bignoniaceae/classificação , Técnicas In Vitro/métodos , Antibacterianos/farmacologia , Testes de Mutagenicidade/instrumentaçãoRESUMO
Lipid nanocarriers have a great potential for improving the physicochemical characteristics and behavior of poorly water-soluble drugs, such as aqueous dispersibility and oral bioavailability. This investigation presents a novel nanostructured lipid carrier (NLC) based on a mixture of solid lipid glycerides, fatty acid esters of PEG 1500 (Gelucire® 44/14), and an oil mix composed of capric and caprylic triglycerides (Miglyol® 812). These NLCs were developed by a simple low-energy method based on melt emulsification to yield highly encapsulating and narrowly distributed nanoparticles (~100 nm, PdI = 0.1, and zeta potential = ~-10 mV). Rhodamine 123 was selected as a poorly water-soluble drug model and owing to its spectroscopic properties. The novel NLCs were characterized by dynamic light scattering (DLS), zeta potential, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and colloidal stability. The drug release was determined through a dialysis bag and vertical Franzs' cells to provide insights about the methods' suitability, revealing similar performance regardless of their different fluid dynamics. Rhodamine 123 followed a characteristic biphasic release profile owing to the swelling of the hydrophilic polymer coating and diffusion process from the lipid core as revealed by the Korsmeyers-Peppas kinetic modeling. Moreover, to elucidate the formation and incorporation of Rhodamine 123 into the NLC core, several molecular dynamics simulations were conducted. The temperature was shown to be an important condition to improve the formation of the nanoparticles. In addition, the liquid lipid incorporation to the formulation forms nanoparticles with imperfect centers, in contrast to nanoparticles without it. Moreover, Miglyol® 812 improves hydrophobic molecule solubility. These results suggest the potential of novel NLC as a drug delivery system for poorly water-soluble drugs.
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SUMMARY Franz cells are one of the main tools to evaluate the transepithelial permeation of compounds through the performance of in vitro tests, these allows inferring the safety behavior of a compound in the skin. The objective of this research was to determine the permeation behavior of benzoic acid from complexes with polyelectrolytes (EuB 75 Cl 25 EuB 100), compared to benzoic acid without complexing, to infer its safety behavior. In a first phase, skin storage conditions were established comparatively evaluating the diffusion parameters (flow and permeation constant) and transepithelial water loss, using ears skin of freshly slaughtered pigs, stored in 1M NaCl at -2 °C for 3 days; it was worked under infinite doses conditions. Subsequently, the permeation test of two complexes between Eudragit E and benzoic acid, in comparison with benzoic acid, was carried out under finite dose conditions. The benzoic acid quantification was performed with an analytical method validated by HPLC-DAD. The results showed no significant differences showing that biological samples can be stored for 72 h under the conditions described. The permeation behavior of the complexed benzoic acid with respect to the free benzoic acid showed a better safety profile, since there was a lower permeation for the first case. These results show that complexation of benzoic acid could decrease the sensitivity reactions that it normally presents, based on the decrease in its permeation.
RESUMEN Las celdas de Franz son una de las herramientas para evaluar la permeación transe-pitelial de compuestos mediante la realización de ensayos in vitro, estos permiten inferir el comportamiento de seguridad de un compuesto en la piel. El objetivo de esta investigación fue determinar el comportamiento de permeación del ácido benzoico a partir de complejos con polielectrolitos (EuB 75 Cl 25 EuB 100) en comparación con el ácido benzoico sin complejar, para inferir su comportamiento de seguridad. En una primera fase, se establecieron las condiciones de almacenamiento de la piel comparando los parámetros de difusión (flujo y constante de permeación) y pérdida de agua transepitelial, empleando piel de orejas de cerdos recién sacrificados, almacenadas en NaCl 1M a -2°C por 3 días; se trabajó bajo condiciones de dosis infinitas. Posteriormente, se realizó el ensayo de permeación de dos complejos entre ácido benzoico y Eudragit E, en comparación con el ácido benzoico, bajo condiciones de dosis finitas. La cuantificación del ácido benzoico fue realizada con un método analítico validado por HPLC-DAD. Los resultados evidenciaron que las muestras biológicas pueden almacenarse durante 72 h en las condiciones descritas. El comportamiento de permeación del ácido benzoico complejado respecto al ácido benzoico libre demostró tener un mejor perfil de seguridad, puesto que hubo una menor permeación para el primer caso. Estos resultados demuestran que la complejación del ácido benzoico podría disminuir las reacciones de sensibilidad que normalmente este presenta, basándose en la disminución de su permeación.
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Excessive exposure to solar radiation induces injurious effects on human skin. Our previous study evidenced that protocatechuic acid (P0) and ethyl protocatechuate (P2) act against photodamage and photoaging. The present study aimed to develop solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) for topical delivery of P0 or P2, as a strategy for photoprotection. Lipid nanoparticles exhibited mean particle size, polydispersity index, zeta potential and association efficiency between 200 and 400 nm, 0.160 to 0.460, -2.2 to -5.2 mV, and 60% to 80%, respectively. The formulations were stable for 3 months when stored at 4âC and 25âC/60% RH. SLNs/NLCs-P0 showed minor cytotoxicity effects compared with SLNs/NLCs-P2, in HaCat (keratinocytes) and HFF-1 (fibroblasts) cell lines. Additionally, bare NLCs exhibited less cytotoxicity effect, compared with bare SLNs. NLCs exhibited a controlled in vitro release of P0 and P2, and were able to protect the compounds against UVB degradation. Ex vivo permeability study showed that NLCs modulated P0 and P2 retention profiles on human skin layers. Furthermore, histological analysis of skin showed that NLCs-P0 did not cause morphological alterations, while NLCs-P2 showed a potential irritation effect in the skin structure. Based on these results, NLCs were considered a potential dermatological nanocarrier for P0 delivery.
Assuntos
Portadores de Fármacos , Hidroxibenzoatos/administração & dosagem , Lipídeos/química , Nanopartículas , Protetores Solares/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Composição de Medicamentos , Estabilidade de Medicamentos , Feminino , Humanos , Hidroxibenzoatos/química , Hidroxibenzoatos/metabolismo , Hidroxibenzoatos/toxicidade , Lipídeos/toxicidade , Masculino , Permeabilidade , Pele/metabolismo , Absorção Cutânea , Protetores Solares/química , Protetores Solares/metabolismo , Protetores Solares/toxicidade , Raios UltravioletaRESUMO
This study aimed to evaluate and compare, using the methodology of Franz diffusion cells, the ketoprofen (KTP) releasing profiles of two formulations: A gel and a conventional suspension. The second aim was to show that this methodology might be easily applied for the development of semi-solid prototypes and claim proof in pre-formulation stages. Drug release analysis was carried out under physiological conditions (pH: 5.6 to 7.4; ionic strength 0.15 M; at 37 °C) for 24 h. Three independent vertical Franz cells were used with a nominal volume of the acceptor compartment of 125 mL and a diffusion area of 2.5 cm². Additionally, two different membranes were evaluated: A generic type (regenerated cellulose) and a transdermal simulation type (Strat-M®). The KTP permeation profiles demonstrated that depending on the membrane type and the vehicle used, the permeation is strongly affected. High permeation efficiencies were obtained for the gel formulation, and the opposite effect was observed for the suspension formulation. Moreover, the permeation studies using Strat-M membranes represent a reproducible methodology, which is easy to implement for pre-formulation stage or performance evaluation of semi-solid pharmaceutical products for topical or transdermal administration.
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RESUMEN En este artículo se presentan los resultados del desarrollo y validación de una metodología analítica por cromatografía líquida de alta eficiencia con detector de arreglo de diodos (HPLC-DAD) para la cuantificación de cafeína; se utilizó una columna C18 con detección UV a 273 nm y una fase móvil compuesta por agua/acetonitrilo (80:20 v/v) de modo isocrático. Las características de desempeño evaluadas permitieron comprobar que existe una adecuada selectividad, una linealidad entre 0,5 y 50 ug/mL, con una precisión expresada como RSD menor a 2%, un porcentaje de recuperación del 99,9% y unos límites de detección y cuantificación para el método de 2,99 y 2,69 ng/mL, respectivamente. El método propuesto es útil para determinar el perfil de permeación bucal de la cafeína mediante un estudio in vitro con celdas de Franz empleando membrana bucal porcina.
SUMMARY This paper presents the results of the development and validation of an analytical methodology through high performance liquid chromatography with a diode array detector (HPLC-DAD) for the quantification of caffeine, using a C18 reverse phase column with UV detection at 273 nm and a mobile phase consisting exclusively of water/acetonitrile (80:20 v/v). The evaluated performance characteristics allowed to verify that there is an adequate selectivity, a linearity between 0.5 and 50 /µg/mL, with precision expressed as RSD in less than 2%, a recovery rate of 99.9%, and limits of detection and quantification for the method of2.99 and 2.69 ng/mL respectively. The proposed method is useful for determining the oral permeation profile of caffeine by an in vitro study with Franz cells using porcine buccal membrane.
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Lipid-core polymeric nanocapsule suspensions containing adapalene and dapsone (AD-LCNC) were developed and incorporated in a Carbopol 940® hydrogel (AD-LCNC HG). A nanoemulsion (AD-NE), similarly prepared but omitting the polymer, was developed and also incorporated in a Carbopol 940® hydrogel (AD-NE HG) to evaluate the polymer effect. Physicochemical characteristics were evaluated. AD-LCNC suspensions containing 0.07% of dapsone and 0.025% of adapalene presented an average size of 194.9 ± 0.42 nm, zeta potential of -15 ± 1.2 mV and polydispersity index of 0.12 ± 0.02, using electrophoretic light scattering (n = 3). The granulometric profiles showed unimodal size distributions for AD-LCNC suspensions, demonstrating that no microscopic population is present in the formulation. No instability phenomena were observed by multiple light-scattering analysis. Photomicrographs obtained by TEM showed homogeneous- and spherical-shaped particles. The encapsulation efficiency was 99.99% for dapsone and 100% for adapalene. The pH values for AD-LCNC suspensions were 5.1 and 7.6 for AD-LCNC HG. Formulations were classified as nonirritant in the HET-CAM test. Rheological analysis demonstrated a non-Newtonian pseudoplastic profile. The in vitro skin permeation studies showed a higher amount of adapalene in epidermis (130.52 ± 25.72 ng/mg) and dermis (4.66 ± 2.5 ng/mg) for AD-NE HG. The AD-LCNC HG presented higher amount of dapsone in both the skin layers (73.91 ± 21.64 ng/mg in epidermis and 4.08 ± 0.85 ng/mg in dermis). The assay showed significant difference between AD-LCNC HG and AD-NE HG (p < 0.05), and drug was not found in the receptor medium.
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En el presente estudio se realizó la estandarización de las condiciones de operación de algunas celdas de Franz, sistema que permite realizar la evaluación de la liberación de compuestos biológicamente activos como una de sus aplicaciones. Para ello se seleccionaron y caracterizaron dos complejos obtenidos entre Eudragit® y ácido benzoico como modelos a emplear, de acuerdo con su solubilidad. Se evaluó su comportamiento de liberación en diferentes condiciones operacionales del ensayo y los resultados se evaluaron empleando el modelo de Korsmeyer-Peppas y el análisis estadístico correspondiente. El análisis de Korsmeyer-Peppas demostró que el mecanismo de liberación del ácido benzoico se ajusta a la ley de difusión de Fick, en algunos casos. Las condiciones operacionales definidas para el ensayo de liberación en las celdas de Franz fueron las siguientes: la concentración del ácido benzoico en la dispersión de complejo en el compartimento donor, 0,07%; la velocidad de agitación, 400 rpm; el volumen de muestreo, 1 mL y la frecuencia de muestreo según un esquema determinado. Se demostró la reproducibilidad del ensayo de liberación en las condiciones operacionales definidas, mediante análisis de varianza.
This paper presents the studies carried out about the standardization of the operational conditions of some Franz Cells; this system allows evaluating the drug release, among others. To this purpose, two Eudragit E- benzoic acid complexes were selected and characterized, as models due to their solubility. After that, the outstanding operational variables were established and evaluated at different levels. The delivery profiles analysis applying the Korsmeyer-Peppas model showed that the release mechanism of benzoic acid was Fick diffusion, in some cases. The operational conditions: benzoic concentration in the complex dispersion in the donor compartment, 0,07%; agitation speed, 400 rpm; volume (1 mL) and frequency sampling according to specific scheme, were determined. The delivery assay reproducibility was demonstrated in the established operational conditions by statistically analysis (ANOVA).