Development and characterization of topical delivery systems based on liquid crystals for siRNA in gene therapy

A terapia gênica por interferência de RNA (RNAi) trata-se de um processo de silenciamento pós-transcricional capaz de suprimir a expressão de um determinado gene. A RNAi é uma proposta terapêutica promissora para o tratamento de muitas doenças severas que ainda não possuem cura ou terapias bem definidas. Porém, é necessário o desenvolvimento de sistemas de liberação clinicamente adequados, seguros e eficazes para se viabilizar essa nova terapêutica, uma vez que obstáculos na administração e distribuição in vivo comprometem o uso clínico dos siRNAs (small interfering RNA). Paralelamente, a liberação tópica de siRNAs surge como uma alternativa promissora para o tratamento de patologias cutâneas. Neste contexto, a presente pesquisa teve como objetivo o desenvolvimento de um sistema de liberação baseado em nanotecnologia para a liberação tópica de siRNAs, visando introduzir a terapia gênica como nova abordagem para o tratamento de patologias cutâneas. Como sistema de liberação, foram desenvolvidas nanodispersões líquido-cristalinas aquosas, compostas por monoleína (MO), um lipídeo polar biocompatível, associadas ou não com ácido oléico (AO). Foram incorporados a esses sistemas os adjuvantes catiônicos polietilenoimina (PEI) e oleilamina (OAM) para obtenção das nanodispersões. Dentre as nanodispersões aquosas desenvolvidas, foram escolhidas as preparações com as menores concentrações de adjuvantes catiônicos, a saber: MO e OAM a 0,4%, MO e PEI a 0,4%, MO, AO e OAM a 2,5% e MO, AO e PEI a 1,0%. Estas formulações apresentaram: reduzido tamanho médio das partículas, baixa polidispersividade, valores de potencial zeta positivos (característica interessante para interação com as moléculas de siRNA que apresentam carga negativa), baixa citotoxicidade in vitro e foram capazes de complexar o siRNA na concentração final de 2,5 ?M. A análise de difração de raios X caracterizou a fase líquido-cristalina desses sistemas como hexagonal, exceto a nanodispersão MO e PEI a 0,4% que foi caracterizada como uma mistura de fase hexagonal e cúbica. As nanodispersões obtidas foram capazes de aumentar a penetração cutânea de siRNA in vitro. Face aos resultados obtidos, podemos concluir que as formulações desenvolvidas são sistemas de liberação de base nanotecnológica promissores para administração tópica de siRNA para o tratamento de patologias cutâneas na terapia gênica.Gene therapy by RNA interference (RNAi) is a post-transcriptional silencing process that can suppress the expression of a particular gene. The RNAi is a promising therapeutic approach for the treatment of many severe diseases that have no cure or well-defined treatments. However, the development of clinically appropriate, safe and effective delivery systems is necessary to enable this new therapy, since obstacles in the in vivo administration and distribution committed the clinical use of siRNAs (small interfering RNA). In addition, the topical delivery of siRNAs appears as a promising alternative for the treatment of cutaneous pathologies. In this context, this research aimed to develop a delivery system based on Nanotechnology for the topical delivery of siRNAs, aiming to introduce gene therapy as a new approach for the treatment of skin disorders. As a delivery system, liquid-crystalline nanodispersions, composed by monoolein (MO), a polar biocompatible lipid, associated or not with oleic acid (OA) were developed. The cationic adjuvants polyethylenimine (PEI) and oleylamine (OAM) were incorporated into these systems to obtain the nanodispersions. Among the aqueous nanodispersions developed, preparations with lower concentration of cationic adjuvant were chosen, these consisting of: MO and OAM at 0.4%, MO and PEI at 0.4%, MO, OA and OAM at 2.5% and MO, OA and PEI at 1.0%. These formulations presented: reduced average particle size, low polydispersity, positive values of zeta potential (an interesting feature for interacting with the siRNA molecules that have a negative charge), low cytotoxicity in vitro and they were able to complex the siRNA at a final concentration of 2.5 ?M. The X-ray diffraction analysis characterized the liquid crystalline phase of these systems as hexagonal, except the nanodispersion MO and PEI at 0.4% which was characterized as a mixture of cubic and hexagonal phases. The nanodispersions obtained were able to increase the skin penetration of siRNA in vitro. With the results obtained, we can conclude that the formulations developed are delivery systems based on nanotechnology, promising for topical administration of siRNA for the treatment of cutaneous diseases in gene therapy

Liquid crystalline nanodispersions as a siRNA carrier in the topical treatment of psoriasis: Evaluation in in vitro and in vivo models

A terapia gênica por interferência de RNA (RNAi) é um processo de silenciamento gênico pós-transcricional onde moléculas de small interfering RNA (siRNA) específicas são capazes de suprimir a expressão de um determinado gene. Atualmente, é uma abordagem terapêutica promissora para o tratamento de muitas doenças graves, incluindo doenças cutâneas. No entanto, dificuldades relacionadas à administração e à biodistribuição limitam a utilização clínica dos siRNAs. Neste contexto, foi proposto o uso de um nanocarreador a base de cristais líquidos para viabilizar a aplicação de siRNAs no tratamento tópico da psoríase. A nanodispersão líquido-cristalina (NLC) desenvolvida, composta por monoleína, ácido oleico, polietilenoimina e fase aquosa (8:2:1:89, p/p/p/p), foi capaz de superar as barreiras da via de administração tópica, bem como as limitações resultantes das características das moléculas de siRNA. A NLC foi capaz de complexar o siRNA, protege-lo por 24 h da degradação enzimática e liberá-lo de forma intacta. A NLC promoveu uma alta taxa de captação celular do siRNA em fibroblastos e macrófagos. Sua aplicação tópica pode ser considerada segura para a pele, pois manteve a viabilidade da epiderme humana reconstruída acima de 50% e a quantidade de IL-1? liberada foi inferior a 60 pg/mL. A NLC apresentou eficácia em promover a liberação funcional do siRNA nos modelos in vitro avaliados. No modelo de pele humana reconstruída psoriática, reduziu os níveis de IL-6 (~ 70%) após um único tratamento por 6 h e, os níveis do RNAm IL6 (~ 50%) após o tratamento por 3 dias consecutivos. Em macrófagos, reduziu os níveis do RNAm Tnf em 40% após o tratamento concomitante com LPS por 24 h e, em 60% após o tratamento por 24 h pós-estímulo prévio com LPS. A eficácia foi ainda maior com o pré-tratamento por 24 h seguido do estímulo com LPS, que normalizou os níveis do RNAm Tnf. O tratamento tópico com a NLC veiculando siRNA Tnf foi eficaz em reduzir significativamente os níveis do RNAm Tnf nos modelos in vivo de inflamação cutânea aguda induzida por TPA (~ 60%) e de psoríase induzida por imiquimode (~ 90%) avaliados. No modelo in vivo de psoríase, o tratamento tópico com a NLC veiculando siRNA Tnf também reduziu significativamente a atividade da mieloperoxidade (~ 65%) e a espessura da epiderme (~ 70%) em comparação aos grupos controle. Também foi eficaz na melhora fenotípica dos animais, reduzindo o rubor, descamação, acantose e o número de ataques de coceira, resultados da redução do processo inflamatório decorrente da ação efetiva das moléculas de siRNA Tnf. A NLC também promoveu a penetração cutânea das moléculas de siRNA nas camadas mais profundas da pele, in vivo. Face aos resultados obtidos, pode-se concluir que a NLC é uma estratégia relevante para a administração tópica de siRNAs, que mostrou potencial terapêutico para suprimir genes específicos relacionados a doenças de pele.Gene therapy by RNA interference (RNAi) is a post-transcriptional gene silencing process in which specific small interfering RNA (siRNA) molecules can suppress the expression of a particular gene. Currently, is a promising therapeutic approach for the treatment of many severe disorders, including skin diseases. However, difficulties related to the administration and biodistribution limit the clinical use of siRNAs. In this context, a nanocarrier based in liquid crystal has been proposed to enable the application of siRNAs in the topical treatment of psoriasis. The liquid crystalline nanodispersion (LCN) developed, composed by monoolein, oleic acid, polyethylenimine and aqueous phase (8:2:1:89, w/w/w/w), was able to overcome the barriers of topical administration route, as well as limitations resulting from the characteristics of the siRNA molecules. The LCN was able to complex the siRNA, protect it for 24 h from enzymatic degradation and release it in an intact form. The LNC promoted a high cellular uptake of siRNA in fibroblasts and macrophages. Its topical application can be considered safe to the skin since viability of the reconstructed human epidermis remained above 50% and the amount of IL-1? released is less than 60 pg/mL. The LCN showed efficacy in promoting functional release of siRNA in in vitro models evaluated. In psoriatic reconstructed human skin model, it reduced the IL-6 levels (~ 70%) after a single treatment for 6 h and the mRNA IL6 levels (~ 50%) after treatment for 3 consecutive days. In macrophages, it reduced the mRNA Tnf levels in 40% after the concomitant treatment with LPS for 24 h and, in 60% with treatment for 24 h after prior stimulation with LPS. The efficiency was higher with the pretreatment for 24 h followed by stimulation with LPS, that normalized the RNAm Tnf levels. Topical treatment with NLC carrying siRNA Tnf was effective in reduce significantly the mRNA Tnf levels in in vivo models of skin acute inflammation induced by TPA (~ 60%) and of psoriasis induced by imiquimod (~ 90%) evaluated. In the in vivo model of psoriasis, topical treatment with the NLC carrying siRNA Tnf also significantly reduced activity of myeloperoxidase (~ 65%) and the epidermis thickness (~ 70%) compared to control groups. It has also been effective in animal phenotypic improvement, reducing redness, desquamation, acanthosis and the number of itch attacks, results of the reduction in inflammatory process obtained by the effective action of siRNA Tnf molecules. The LCN also promoted the penetration of siRNA molecules into the deeper layers of the skin in vivo. With the results obtained, we can conclude that the LCN is a relevant strategy for topical administration of siRNAs, which showed therapeutic potential to suppress specific genes related to skin diseases

An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs

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 0.1 M 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

Fluorometric quantification of protoporphyrin IX in biological skin samples from in vitro penetration/permeation studies

A fluorometric analytical method was developed for quantification of protoporphyrin IX (PpIX) in skin samples and receptor phase solution after in vitro cutaneous penetration/permeation studies. Analytical conditions used were: excitation and emission wavelengths: 400 nm and 632 nm; bandwidth: 0.5 nm; excitation and emission slits: 10/10. PpIX was recovered from two different layers of skin, the stratum corneum (SC) and the epidermis plus dermis ([E+D]), by vortex homogenization, probe and bath sonication, using DMSO as an extraction solvent. The detection and quantification limits were 0.002 and 0.005 &#956;g/mL, respectively. The assay was linear from 0.005 - 0.5 &#956;g/mL. The within-day and between-day assay precision and accuracy in DMSO and receptor phase solution were each studied at the two concentration levels 0.04 and 0.2 &#956;g/mL, and 0.01 and 0.08 &#956;g/mL, respectively. The coefficients of variation and deviation from the theoretical values were lower than 5%. The skin recovery of PpIX from SC and [E+D] layers using two different concentrations (0.5 and 1.0 &#956;g/mL) were all above 90.0%. The method described has potential application to in vitro penetration/permeation studies of PpIX using porcine skin as a biological membrane model.<br>Um método analítico por espectrofluorimetria foi desenvolvido para quantificar a protoporfirina IX (Pp IX) em amostras de pele e fase receptora após a realização de testes in vitro de penetração/permeação cutâneas. As condições analíticas utilizadas foram: comprimentos de onda de excitação e emissão: 400 nm e 632 nm; largura de banda: 0,5 nm; fendas de excitação e emissão: 10/10. A PpIX foi extraída de amostras de estrato córneo (EC) e da epiderme sem estrato córneo + derme ([E+D]) através da agitação em vórtex e sonicação por haste e banho, utilizando-se o DMSO como solvente extrator. O limite de detecção e quantificação foram, respectivamente, de 0,002 e 0,005 &#956;g/mL. O método mostrou-se linear da faixa de 0,005 - 0,5 &#956;g/mL. A precisão e exatidão intra e inter-ensaio em DMSO e na fase receptora foram validadas utilizando-se duas concentrações distintas, respectivamente, de 0,004 e 0,2 &#956;g/mL, e 0,01 e 0,08 &#956;g/mL. Os valores de coeficiente de variação e o desvio do valor teórico foram inferiores a 5%. A recuperação da PpIX das camadas da pele (EC e [E+D]) utilizando-se duas concentrações distintas (0,5 e 1,0 &#956;g/mL) foram todas acima de 90,0%. O método descrito pode ser utilizado para determinação da PpIX após estudos de penetração/permeação cutânea in vitro utilizando pele de porco como modelo de membrana

Genomic epidemiology reveals how restriction measures shaped the SARS-CoV-2 epidemic in Brazil

Abstract Brazil has experienced some of the highest numbers of COVID-19 infections and deaths globally and made Latin America a pandemic epicenter from May 2021. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of local virus transmission dynamics. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and an adjacent country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed, the absence of effective restriction measures led to the local emergence and international spread of Variants of Concern (VOC) and under monitoring (VUM), including the Gamma (P.1) and Zeta (P.2) variants. In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring and providing a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies