ABSTRACT
Introducción: Las nanoemulsiones son excelentes sistemas de transporte y entrega de fármacos. La ftalocianina de aluminio clorada (PcAlCl) en terapia fotodinámica constituye una alternativa de tratamiento en leishmaniasis cutánea. Objetivo: Determinar la difusión y retención en piel humana de la PcAlCl contenida en una nanoemulsión (nano-PcAlCl) para su optimización en formulaciones tópicas. Materiales y métodos: Se prepararon y caracterizaron fisico-químicamente dos formulaciones (nano-PcAlCl y solución-PcAlCl) y sus vehículos sin-PcAlCl. La permeación se determinó en ensayos en celdas de difusión de Franz y la retención por el método de la cinta adhesiva. La concentración de PcAlCl fue determinada fluorométricamente (nM/cm2). Biopsias de piel fueron analizadas histotécnicamente. Resultados: El tamaño promedio, el potencial Z y el índice de polidispersión de la nano-PcAlCl en agua fue de 132,9 nm, -19,23 y 0,14 y diluida en PBS fue 125,33 nm, -13,69 y 0,139. Las concentraciones de PcAlCl se mantuvieron estables. La PcAlCl no atravesó la piel y fue retenida en sus capas, en estrato córneo y epidermis+dermis con valores de 44,17 nM y 8,48 nM postratamiento con nano-PcAlCl, y 96,90 nM y 9,80 nM postratamiento con solución-PcAlCl. Esta última promovió mayor retención en estrato córneo y ambas formulaciones promovieron similar retención en epidermis+dermis. Se observó desprendimiento del estrato córneo y fragmentación del colágeno. Conclusión: La PcAlCl no atravesó la piel, se retuvo en estrato córneo y epidermis+dermis. Se sugiere realizar ensayos de permeación utilizando piel humana desprovista de estrato córneo y ensayos de distribución en animales con leishmaniasis cutánea.
Introduction: The nanoemulsions constitute excellent drug delivery systems for carrying and delivering active drugs. Chloroaluminum phthalocyanine (ClAlPc) in photodynamic therapy constitutes an interesting alternative in cutaneous leishmaniasis treatment. Objective: To determine the diffusion and retention of ClAlPc contained in a nanoemulsion (nano-ClAlPc) in human skin membranes for optimization of topical formulations. Materials and methods: Two formulations (ClAlPc-nano- and ClAlPc-solution) and vehicles without ClAlPc were prepared and physicochemical characterized. The permeation was tested in Franz-diffusion cells and the retention by the tape stripping method. ClAlPc concentration was determined fluorometrically (nM/cm2). Skin biopsies were analyzed by histologic technics. Results: The ClAlPc-nano average size, zeta potential and polydispersity index diluted in water was 132.9 nm, -19.23 and 0.14 and diluted in phosphate-buffer-saline was 25.33 nm, -13.69 and 0.139. ClAlPc maintains its stability in each formulation. ClAlPc was unable to pass completely through the skin; it was retained in the different skin layers. A ClAlPc retention in stratum corneum and epidermis+dermis was observed with values of 44.17 nM and 8.48 nM after ClAlPc-nano treatment and 96.90 nM and 9.80 nM after ClAlPc-solution treatment. The ClAlPc-solution promoted greater retention in stratum corneum and both formulations showed similar ClAlPc-retention in epidermis+dermis. Histological changes as stratum corneum detachment and collagen-fragmentation were observed. Conclusion: ClAlPc was not able to cross completely the skin, it was retained in stratum corneum and epidermis+dermis Human permeation test using skin membranes without stratum corneum, and distribution assays in cutaneous leishmaniasis-infected animals, are suggested.
Introdução: as nanoemulsões são excelentes sistemas de transporte e entrega de fármacos. A ftalocianina de alumínio clorada (PcAlCl) em terapia fotodinâmica constitui uma alternativa de tratamento em leishmaniose cutânea. Objetivo: determinar a difusão e retenção em pele humana da PcAlCl contida em uma nanoemulsão (nano-PcAlCl) para sua otimização em formulações tópicas. Materiais e métodos: se prepararam e caracterizaram fisico-quimicamente duas formulações (nano-PcAlCl e solução-PcAlCl) e seus veículos sem-PcAlCl. A permeação determinou-se em ensaios em celas de difusão de Franz e a retenção pelo método da fita adesiva. A concentração de PcAlCl foi determinada fluorometricamente (nM/cm²). Biopsias de pele foram analisadas histotecnicamente. Resultados: o tamanho médio, o potencial Z e o índice de polidispersão da nano-PcAlCl em água foi de 132,9 nm, -19,23 e 0,14 e diluída em PBS foi 125,33 nm, -13,69 e 0,139. As concentrações de PcAlCl mantiveram-se estáveis. A PcAlCl não atravessou a pele, foi retido em suas capas. A PcAlCl foi retida em estrato córneo e epiderme+derme com valores de 44,17 nM e 8,48 nM pós-tratamento com nano-PcAlCl e 96,90 nM e 9,80 nM pós-tratamento com solução-PcAlCl. A solução-PcAlCl promoveu maior retenção em estrato córneo e ambas as formulações promoveram similar retenção em epiderme+derme. Observou-se desprendimento do estrato córneo e fragmentação do colágeno. Conclusão: a PcAlCl não atravessou a pele; reteve-se em estrato córneo e epiderme+derme. Sugere-se realizar ensaios de permeação utilizando pele humana desprovida de estrato córneo e ensaios de distribuição em animais com leishmaniose cutânea.
Subject(s)
Humans , Solutions , Therapeutics , Histological Techniques , Leishmaniasis, Cutaneous , Dermis , Epidermis , MethodsABSTRACT
Una formulación tópica en leishmaniasis cutánea debe garantizar la permeación y acumulación del compuesto en la dermis, sitio donde se encuentran los macrófagos infectados con Leishmania. Se determinó la difusión y retención de PcAlCl contenida en una nanoemulsión (nano-PcAlCl) y en solución-PcAlCl y su distribución en ratas Wistar, con piel sana o lesionada quirúrgicamente. La nano-PcAlCl se preparó por el método de emulsificación espontánea y la solución-PcAlCl en mezcla DMSO: Tween 80: agua tipoI; la estabilidad se determinó espectrofotométricamente. La difusión se determinó utilizando celdas de Franz y la retención en el estrato córneo (EC) y la epidermis-dermis (E+D) por "tape stripping". Las formulaciones mantuvieron las características espectroscópicas del compuesto. La PcAlCl no difundió al medio receptor. Después de 12 y 24 horas la retención del compuesto en EC con nano-PcAlCl fue 99,73 y 79,40nM y con solución-PcAlCl 66,73 y 57,01nM; en E+D la retención con nano-PcAlCl fue 40,94 y 62,49nM y con solución-PcAlCl 81,92 y 50,28nM. En EC la nano-PcAlCl registró mayor retención y en E+D la solución-PcAlCl, registró mayor retención a las 12 horas y la nano-PcAlCl a las 24 horas. Se observó desprendimiento y disminución en las capas del epitelio. El tratamiento con nano-PcAlCl retuvo el compuesto en EC y E+D de los animales con valores de 6,43nM y 33,18nM con piel sana y 0,99nM y 17,08 con piel lesionada; valores menores de 1,92nM fueron detectados sólo en pulmón. El tratamiento con solución-PcAlCl retuvo el compuesto en EC y E+D de los animales con valores de 31,86 y 202,40nM con piel sana y 5,93 y 63,83nM con piel lesionada; se encontraron valores de 2,36nM y 20,33nM en los pulmones y valores entre 1,58 y 5,80nM en otros órganos. La piel lesionada presentó regeneración del epitelio, desprendimiento de queratina, infiltrado celular y neovascularización. La PcAlCl retenida en EC y E+D indicó la eficacia de las formulaciones para penetrar el EC y retener el compuesto en las capas viables de la piel. El tratamiento con nano-PcAlCl permeó el EC, se retuvo en E+D y no presentó distribución hacia órganos.
A successful formulation contained aluminum phthalocyanine chloride (ClAlPc) able for cutaneous leishmaniasis (CL) ensures the skin permeation and retention of the compound in dermis, a place where macrophages infected with Leishmania are located. The aim of this study was to determine the ClAlPcex vivo permeation and retention in Wistar rat skin and its distribution in animals with healthy skin and surgically damaged skin. Two formulations were prepared: nanoemulsion (ClAlPc-nano) and solution (ClAlPc-solution). The diffusion was determined using Franz diffusion cells with Wistar rat skin as membrane after 12 and 24 hours of testing. Retention in stratum corneum (SC) and epidermis plus dermis (E+D) was determined by the tape stripping method and organic solvent extraction. The distribution was performed after the topical application of formulations analyzing the CLAlPc concentration in the skin layers and organs by fluorometry. The results were expressed as nM/mg of tissue of ClAlPc. Simultaneously, skin biopsies were taken in order to determine their histological characteristics. ClAlPc formulations were effective in maintaining compound solubility and spectroscopic characteristics. ClAlPc was unable to pass completely through the skin; its skin retention was related with the used formulation, time of assays and type of skin layer. After 12 and 24 hours, the SC retention induced by ClAlPc-nano treatment was 99.73 and 79.40nM and by ClAlPc-solution was 66.73 and 57.01nM. In E+D the retention induced by ClAlPc-nano was 40.94 and 62.49nM and by ClAlPc-solution was 81.92 and 50.28nM. In SC, ClAlPc-nano showed greater retention after 12 and 24 hours, while in E+D, ClAlPc-solution was greater retained at 12 hours and ClAlPc-nano at 24 hours. After the permeation assays, detachment and reduction in epithelial layers were observed in skin membranes. In Wistar rats, ClAlPc-nano placed on healthy skin retained the compound in E+D and SC with values of 33.18nM and 6.43nM and placed on damaged skin retained in E+D in SC with values of 17.08nM and 0.99nM respectively; no organs distribution occur, except in lung where values of 0.82nM in animals with healthy skin and 1.92nM in animals with damaged skin were observed. ClAlPc-solution induced retention of 31.86 in SC and 202,40nM in E+D placed on healthy skin and 5.93 in SC and 63.83nM E+D placed on damaged skin; concentrations in lung with values of 2,36nM after application in animals with healthy skin and 20,33nM in animals with damaged skin were observed. In addition, ClAlPc-solution promoted, after application on damaged skin, distribution of ClAlPc to other organs with values between 1.58 and 5.80nM. In animals without skin injury, normal histologic skin anatomy were observed after treatment. In contrast, epithelial regeneration, detachment of keratin, loss of cellular differentiation, cellular infiltration and neovascularization were observed in animals with skin injury. The efficacy of the formulation to penetrate the SC and be retained in the viable skin layers was demonstrated. The in vivo ClAlPc-biodistribution was related on its concentration and skin state. A nanoemulsion contained ClAlPc, represents a good system of drug delivery because induce both the SC penetration and E+D retention, without systemic organ distribution even when presented tissue repair and regeneration. A ClAlPc-nano prepared in this work constitutes a good formulation for the topical application of ClAlPc in LC treatment.