Encapsulação da vitamina c em lipossomas para o tratamento do envelhecimento cutâneo: desenvolvimento tecnológico, analítico e avaliação da performance biológica in vitro em modelos de permeação cutânea e em linhagens celulares de queratinócitos e fibroblastos
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2016-02-29
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Universidade Federal de Goiás
Resumo
Skin aging involves events that lead to the reduction of its structural integrity and loss of biological
functions. The reactive oxygen species (ROS) are potentially able to generate damage of tissues
and are related with the cutaneous photoaging. Antioxidant molecules like vitamin C (VC) are
capable of fighting these ROS. Besides, VC acts in the synthesis of collagen in the skin, the
primary protein responsible for supporting its connective tissues. However, beneficial skin effects
are only obtained when the VC is applied topically. In this work, liposomes containing VC for
topical administration were developed and characterized. For quantification of VC in different
matrixes, including pharmaceutical products, cosmetics, and porcine ear skin, a quantitative analytical method was developed and validated by high performance liquid chromatography with
diode array detection (HPLC-DAD) using ion-pair reversed phase. The developed analytical
method was capable of quantifying VC without the interference of the various components of the
pharmaceutical formulations and the endogenous compounds of the biological matrix. The diluent
chosen to extract and dilute VC was a mixture of water and methanol (4:1, v/v) acidified to pH 3.0
with phosphoric acid, with additional 0.02% sodium thiosulfate. This diluent was the most efficient
to stabilize VC compared with other pH conditions and compositions, maintaining the amount of
VC close to 100% after 10 days at 4°C. In this way, a method for quantification of VC that could
be widely used by pharmaceutical companies and research laboratories was developed. It was
precise and accurate in the evaluation of the content of VC in biological matrixes and different
pharmaceutical formulations, making it advantageous towards other methods. Liposomes with VC
were prepared by dehydration-rehydration vesicles method (DRV). Liposomes containing
phosphatidylcholine (PC) or a mixture of PC and cholesterol and other electrically charged lipids
were prepared, and liposomes with positive, negative and neutral charges were obtained. All
formulations presented mean size inferior to 200 nm and low polydispersity index (<0.2).
Encapsulation efficiency of VC was directly influenced by the amount of liposomes that were
formed. In skin permeation studies, the association of VC in the liposomes only allowed greater
retention in the dermis when negatively charged liposomes were used. After 6 hours, the
application of this formulation promoted high skin retention of VC, with an accumulation of 37.9 ±
12.02 μg/cm2 and 73.95± 23.23 μg/cm2 in the epidermis and dermis, respectively. Liposomes were
capable of increasing the flow of VC through the skin. The presence of cholesterol and negative
charge in the liposomes promoted an increase in VC flow of 4 and 7 times, respectively, when
compared to free drug (FD). The interaction of liposomes with live biological membranes was
simulated in keratinocytes (HaCat) and fibroblasts (3T3) through the analysis of cell internalization
of liposomes. For this assay, during the preparation of liposomes, fluorescent lipids were used to
label the lipid membrane (coumarin and rhodamine). After treatment, the groups treated with
negatively charged liposomal formulation presented superior fluorescence than the groups treated
with other formulations and control, suggesting a higher interaction between the negatively
charged liposomes and keratinocytes and fibroblasts. Thus, this negatively charged formulation
was compared with free VC in the cell regeneration of keratinocytes after exposure to UVA
radiation and in the production of collagen type I in fibroblasts. In both cases, the beneficial effect
was only observed when VC was encapsulated in the liposomes. Therefore, the technological
development of a liposomal formulation containing VC generated a formulation with a stability of
at least 30 days and with characteristics that favored its retention and skin flow. Besides, the
encapsulation of VC in negatively charged liposomes promoted an enhancement in the efficacy of
regeneration of keratinocytes and the synthesis of collagen in fibroblasts.
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Palavras-chave
Ácido ascórbico , Estrato córneo , Promotores de permeação , Penetração cutânea , Atividade antioxidante , Biossíntese de colágeno , 1,2-distearoil-sn-glicero-3- fosfoglicerol , Ascorbic acid , Stratum corneum , Permeation enhancers , Skin permeation , Antioxidant activity , Biosynthesis of collagen , 1,2-distearoyl-sn-glycero-3-phosphoglycerol
Citação
MAIONE-SILVA, L.. Encapsulação da vitamina c em lipossomas para o tratamento do envelhecimento cutâneo: desenvolvimento tecnológico, analítico e avaliação da performance biológica in vitro em modelos de permeação cutânea e em linhagens celulares de queratinócitos e fibroblastos. 2016. 102 f. Tese (Doutorado em Ciências da Saúde) - Universidade Federal de Goiás, Goiânia, 2016.