RESUMEN
With the increasing number of studies proposing new and optimal delivery strategies for the efficacious silencing of gene-related diseases by the local administration of siRNAs, the present review aims to provide a broad overview of the most important and latest developments of non-viral siRNA delivery systems for local administration. Moreover, the main disease targets for the local delivery of siRNA to specific tissues or organs, including the skin, the lung, the eye, the nervous system, the digestive system and the vagina, were explored.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Administración Tópica , Animales , Sistemas de Liberación de Medicamentos/instrumentación , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Low-level laser therapy (LLLT) has been widely used in the treatment of the stomatognathic system dysfunction; however, its biological effect remains poorly understood. This study evaluated the effect of LLLT (GaAlAs, 780 nm, 20 J/cm(2), 40 mW) on masseter muscle of HRS/J mice after different numbers of laser irradiations (three, six, and ten) for 20 s in alternate days. Three experimental groups were defined according to the number of laser irradiations and three control groups (n=5) were used. On the third day after the last irradiation, all animals were killed and the masseter muscle was removed and processed for the following analysis: (a) transmission electron microscopy, (b) zymography, (c) immunohistochemistry for vascular endothelial growth factor (VEGF) and VEGFR-2. The results showed: (a) with six laser applications, a dilation of T tubules, and sarcoplasmic reticulum cistern, increased pinocytosed vesicles in the endothelium; with ten laser applications, few pinocytic vesicles in the endothelium and condensed mitochondria. (b) Under the conditions of this study, the synthesis of other matrix metalloproteinases was not observed, only the MMP-2 and -9. (c) After ten laser irradiations, immunostaining was observed only for VEGFR-2. We conclude that after six laser applications, ultrastructural changes may facilitate the Ca(+2) transfer to cytosol and increase the fluid transport from one surface to another. The ultrastructural changes and no immunostaining for VEGF with ten applications may decrease the metabolic activity as well as damage the angiogenic process, suggesting that an effective number of laser applications may be less than ten, associating to this therapy a better cost-benefit.
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Terapia por Luz de Baja Intensidad , Músculo Masetero/efectos de la radiación , Animales , Humanos , Masculino , Músculo Masetero/metabolismo , Músculo Masetero/ultraestructura , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Pelados , Microscopía Electrónica de Transmisión , Modelos Animales , Trastornos de la Articulación Temporomandibular/radioterapia , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pharmaceutical films are polymeric formulations used as a delivery platform for administration of small and macromolecular drugs for local or systemic action. They can be produced by using synthetic, semi-synthetic, or natural polymers through solvent casting, electrospinning, hot-melt extrusion, and 3D printing methods, and depending on the components and the manufacturing methods used, the films allow the modulation of drug release. Moreover, they have advantages that have drawn interest in the development and evaluation of film application on the buccal, nasal, vaginal, and ocular mucosa. This review aims to provide an overview of and critically discuss the use of films as transmucosal drug delivery systems. For this, aspects such as the composition of these formulations, the theories of mucoadhesion, and the methods of production were deeply considered, and an analysis of the main transmucosal pathways for which there are examples of developed films was conducted. All of this allowed us to point out the most relevant characteristics and opportunities that deserve to be taken into account in the use of films as transmucosal drug delivery systems.
RESUMEN
We developed poly-ε-caprolactone (PCL)-based nanoparticles containing D-α-tocopherol polyethylene glycol-1000 succinate (TPGS) or Poloxamer 407 as stabilizers to efficiently encapsulate genistein (GN). Two formulations, referred to as PNTPGS and PNPol, were prepared using nanoprecipitation. They were characterized by size and PDI distribution, zeta potential, nanoparticle tracking analysis (NTA), GN association (AE%), infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). PNTPGS-GN exhibited a particle size of 141.2 nm, a PDI of 0.189, a zeta potential of -32.9 mV, and an AE% of 77.95%. PNPol-GN had a size of 146.3 nm, a better PDI than PNTPGS-GN (0.150), a less negative zeta potential (-21.0 mV), and an AE% of 68.73%. Thermal and spectrometric analyses indicated that no new compounds were formed, and there was no incompatibility detected in the formulations. Cellular studies revealed that Poloxamer 407 conferred less toxicity to PCL nanoparticles. However, the percentage of uptake decreased compared to the use of TPGS, which exhibited almost 80% cellular uptake. This study contributes to the investigation of stabilizers capable of conferring stability to PCL nanoparticles efficiently encapsulating GN. Thus, the PCL nanoparticle proposed here is an innovative nanomedicine for melanoma therapy and represents a strong candidate for specific pre-clinical and in vivo studie
Asunto(s)
Genisteína/farmacología , Nanopartículas/análisis , Melanoma/tratamiento farmacológico , Tamaño de la Partícula , Análisis Espectral/clasificación , Rastreo Diferencial de Calorimetría/métodos , Cromatografía Líquida de Alta Presión/métodosRESUMEN
Abstract Currently, mucosal vaccine administration has stood out as an easier and non-invasive application method. It can also be used to induce local and systemic immune responses. In the COVID-19 pandemic context, nasal and oral vaccines have been developed based on different technological platforms. This review addressed relevant aspects of mucosal vaccine administration, with emphasis on oral and nasal vaccinations, in addition to the importance of using nanotechnology-based delivery systems to enable these strategies.
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Vacunas/análisis , Vacunación/efectos adversos , Nanotecnología/instrumentación , Inmunidad/inmunologíaRESUMEN
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 (â¼90%), 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.
Asunto(s)
Interleucina-6/genética , Modelos Biológicos , Psoriasis/genética , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Técnicas de Silenciamiento del Gen , Humanos , Técnicas In VitroRESUMEN
The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 µM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA.
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Celulitis (Flemón)/prevención & control , Técnicas de Transferencia de Gen/efectos adversos , Polietileneimina/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Tratamiento con ARN de Interferencia/efectos adversos , Sustancias Viscoelásticas/química , Animales , Celulitis (Flemón)/inducido químicamente , Celulitis (Flemón)/inmunología , Celulitis (Flemón)/patología , Femenino , Geles , Glicéridos/efectos adversos , Glicéridos/química , Inyecciones Subcutáneas , Ratones Endogámicos BALB C , Monoglicéridos/efectos adversos , Monoglicéridos/química , Polietileneimina/efectos adversos , Propilenglicol/efectos adversos , Propilenglicol/química , ARN Interferente Pequeño/efectos adversos , ARN Interferente Pequeño/química , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Solubilidad , Tejido Subcutáneo/efectos de los fármacos , Tejido Subcutáneo/inmunología , Tejido Subcutáneo/patología , Sustancias Viscoelásticas/efectos adversos , Viscosidad , Agua/análisisRESUMEN
The ability of small interfering RNAs (siRNAs) to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. However, the largest remaining hurdle for the widespread use of this technology in skin is the lack of an effective delivery system. The aim of the present study was to evaluate nanodispersed systems in liquid crystalline phases that deliver siRNA into the skin. The proposed systems present important properties for the delivery of macromolecules in a biological medium, as they are formed by substances that have absorption-enhancing and fusogenic effects; additionally, they facilitate entrapment by cellular membranes due to their nano-scale structure. The cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based systems in different concentrations, and after dispersion in aqueous medium, liquid crystalline phase nanodispersions were obtained and characterized by their physicochemical properties. Then, in vitro penetration studies using diffusion cell and pig ear skin were carried out to evaluate the effect of the nanodispersions on the skin penetration of siRNA; based on these results, the nanodispersions containing MO/OA/PEI/aqueous phase (8:2:5:85, w/w/w/w) and MO/OA/OAM/aqueous phase (8:2:2:88, w/w/w/w) were selected. These systems were investigated in vivo for skin penetration, skin irritation, and the ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models. The results showed that the studied nanodispersions may represent a promising new non-viral vehicle and can be considered highly advantageous in the treatment of skin disorders; they were effective in optimizing the skin penetration of siRNA and reducing the levels of the model protein GAPDH without causing skin irritation.
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Cristales Líquidos , Nanopartículas , ARN Interferente Pequeño/administración & dosificación , Absorción Cutánea , Aminas/química , Animales , Femenino , Técnicas de Silenciamiento del Gen , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Glicéridos/química , Masculino , Ratones , Ratones Pelados , Polietileneimina/química , ARN Interferente Pequeño/farmacocinética , Piel/metabolismo , PorcinosRESUMEN
ABSTRACT Transdermal nicotine patches have been used in smoking cessation therapy, suggested for the treatment of skin disorders with eosinophilic infiltration and have been found to improve attention performance in patients with Alzheimer's disease and age-associated memory impairment. However, skin irritation with extended patch use is still a problem. The aim of this work was to develop a simple to prepare liquid crystalline system containing vitamin E TPGS that would be able to control nicotine delivery and reduce irritation and sensitization problems. The liquid crystalline phases were macroscopically characterized by visual analysis and examined microscopically under a polarized light microscope. Topical and transdermal delivery of nicotine were investigated in vitro using porcine ear skin mounted on a Franz diffusion cell. Nicotine skin permeation from the developed cubic phase followed zero-order kinetics (r = 0.993) and was significantly enhanced after 12 h when compared to the control formulation (nicotine solution) (p < 0.05) (138.86 ± 20.44 and 64.91 ± 4.06 μg/cm2, respectively). Cubic phase was also able to target viable skin layers in comparison to control solution (8.18 ± 1.89 and 2.63 ± 2.51 μg/cm2, respectively). Further studies to evaluate skin sensitization and irritation are now necessary.
RESUMO Adesivos transdérmicos de nicotina são utilizados para cessação de fumar, tratamento de problemas de pele com infiltração de eosinófilos e para melhorar a atenção em pacientes com doença de Alzheimer e enfraquecimento da memória associada à idade. No entanto, a irritação da pele com o uso prolongado dos adesivos ainda é um problema. O objetivo deste trabalho foi desenvolver sistema líquido cristalino (SLC) de preparo simples contendo vitamina E TPGS capaz de controlar a liberação de nicotina e reduzir os problemas de irritação cutânea. Os SLCs foram caracterizados por análise visual e microscopia de luz polarizada. As administrações tópica e transdérmica de nicotina foram investigadas in vitro utilizando pele de orelha de porco em célula de difusão de Franz. A permeação da nicotina veiculada pela fase cúbica desenvolvida seguiu cinética de ordem zero (r = 0,993) e foi significativamente maior do que o controle (solução de nicotina) após 12 h (p < 0,05) (138,86 ± 20,44 e 64,91 ± 4,06 µg/cm2, respectivamente). A fase cúbica também promoveu aumento da penetração de nicotina nas camadas viáveis da pele quando comparado ao controle (8,18 ± 1,89 e 2,63 ± 2,51 µg/cm2, respectivamente). Estudos futuros para avaliar a sensibilização e irritação da pele são necessários.
Asunto(s)
Vitamina E/análisis , Nicotina/farmacocinética , Piel/lesiones , Parche TransdérmicoRESUMEN
OBJECTIVE: This study aims to analyze the effects of low-level laser therapy (LLLT) on the oxidative activity and the expression/activity of metalloproteinases of the masseter muscle. BACKGROUND DATA: Currently in dentistry LLLT has been used on patients with muscular disorders, such as the temporomandibular disorders (TMDs) but its effect at the cellular level has not been fully elucidated. METHODS: Thirty male Wistar rats divided into 6 groups (n=5) received 10 laser irradiations (780 nm, 5 mmW, CW laser, illuminated area 0.04 cm(2), power density 125 mW/cm(2)), with different energy densities (group I-0; group II-0.5; group III-1.0; group IV-2.5; group V-5.0; and group VI-20 J/cm(2)). Muscles were processed for nicotinamide adenine dinucleotide diaphorase (NADH) and sucinate dehydrogenase (SDH) activities and zymography. The photomicrographs were evaluated by the point counting method using a test system and ImageJ software; and by the ANOVA statistical test. The proteinases' secretion/activity was qualitatively analyzed by zymography. RESULTS: LLLT significantly increased (p<0.05) masseter muscle oxidative metabolism shown by the increased area of intermediary fibers in the NADH (groups IV, V, and VI) and SDH (group V) reactions. The same metabolic pattern was observed among the groups in both reactions. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymography detected only the MMP-2 expression/activity for the untreated-control group (group I). The exposure to LLLT increased the activity of MPP-2 in group VI and the activity of MMP-9 in all groups exposed to different energy densities of laser irradiation (groups II, III, IV, V, and VI). CONCLUSIONS: Thus, LLLT stimulated the oxidative metabolism and the expression of matrix metalloproteinase (MMPs) of the masseter muscle, which may indicate a matrix remodeling process. However, group VI did not show the best results for oxidative metabolism, probably indicating that the dosage they were given was high for this protocol.
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Terapia por Luz de Baja Intensidad/métodos , Músculo Masetero/efectos de la radiación , NAD/metabolismo , Succinato Deshidrogenasa/metabolismo , Análisis de Varianza , Animales , Respiración de la Célula , Electroforesis en Gel de Poliacrilamida , Masculino , Músculo Masetero/enzimología , Fotomicrografía , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
To be effective against the oxidative damages induced by UVB irradiation in the skin, the drug needs to release from the formulation in which it was incorporated and reach the skin layers where the ROS are generated. Thus, it is very important the development of a robust and sensitive methodology to extract and quantify in different skin layers the antioxidant agent delivered from topical formulations. Therefore, in the present work suitable methods to extract and quantify quercetin in skin samples and receptor phase after in vitro penetration studies were developed. The results demonstrated that the recovery from two different layers of skin, the SC and [E+D], using two different methods of quantification (DPPHò assay and HPLC, respectively), was 93.8 percent when the quercetin spiked dose was 50 µg/mL, 100.4 percent when it was 100 µg/mL and 89.9 percent for 250 µg/mL and the average recovery of the quercetin extraction from receptor phase when dichloromethane was used as extractor solvent was 96 percent. These results demonstrate that the described methods have a potential application to in vitro skin penetration studies of quercetin, since it showed to be accurate and sensitive.
Para ser efetiva contra os danos oxidativos induzidos pela radiação UVB na pele, é necessário que o ativo seja liberado da formulação na qual foi incorporado e alcance as camadas da pele onde são geradas as EROS. Desta forma, torna-se de grande importância o desenvolvimento de métodos eficazes e sensíveis para extrair e quantificar, nas diferentes camadas de pele, o agente antioxidante liberado de formulações tópicas. No presente trabalho foram desenvolvidos métodos adequados para extrair e quantificar a quercetina em amostras de pele e na fase receptora após estudos de penetração cutânea in vitro. Os resultados demonstraram que a recuperação das camadas de pele, EC e [E+D], quando do uso de duas diferentes metodologias de quantificação (ensaio de DPPHò e CLAE, respectivamente), foi de 93,8 por cento quando aplicada uma dose de 50 µg/mL de quercetina, 100,4 por cento para 100 µg/mL e 89,9 por cento para 250 µg/mL e a recuperação média da extração da quercetina da fase receptora, quando do emprego de diclorometano como solvente extrator, foi de 96 por cento. Tais resultados demonstram que os métodos descritos têm grande potencial de aplicação em estudos de penetração in vitro já que apresentaram exatidão e sensibilidade.