Nanopartículas de quitosana contendo inibidores de integrinas como potenciais sistemas de liberação para terapia e diagnóstico por imagem em câncer
Carregando...
Data
2016-05-24
Autores
Kiill, Charlene Priscila [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os glioblastomas multiformes (GBMs) são os tumores mais invasivos do sistema nervoso central (SNC) e sua invasividade está relacionada com a interação entre glicoproteínas da matriz extracelular com integrinas presentes nas células tumorais. As integrinas α5β1, αvβ5, e αvβ3 são fortemente expressas em GBMs, e podem ser utilizadas como moléculas alvo na terapia e no diagnóstico por imagem dos tumores do SNC. Os inibidores de integrinas cRGDfV, cRGDfK e vicrostatin têm sido estudadas no tratamento de GBMs pois inibem as integrinas αvβ3, αvβ5 e α5β1, e, assim, exibem potentes propriedades antitumorais. Entretanto, a aplicação de proteínas terapêuticas é limitada freqüentemente pela baixa estabilidade em fluídos fisiológicos e a baixa penetração através das membranas biológicas. Para contornar estes problemas, tem˗se buscado novos sistemas de liberação de fármacos e rotas alternativas de administração, como por exemplo, a via nasal. Assim, este trabalho teve como objetivo a encapsulação dos peptídeos cRGDfV e cRGDfK em nanopartículas de quitosana, pelo método de gelificação ionotrópica, empregando o planejamento fatorial 23. Este sistema baseado em quitosana promoveu um aumento da mucoadesão e podem auxiliar o acesso dos peptideos cRGDfV e cRGDfK ao SNC pela via nasal. Outra estratégia utilizada nesse trabalho foi a utilização das nanopartículas de quitosana escolhidas no planejamento fatorial para serem modificadas com PEG por meio de interações supramoleculares e também por interações covalentes com metoxil polietilenoglicol e maleimida polietilenoglicol hidroxissucimida. As nanopartículas modificadas por interações covalentes foram funcionalizadas através do acoplamento do peptídeo cRGDfK na superfície das nanopartículas, a fim de promover um aumento do direcionamento das nanopartículas para o ambiente tumoral por meio de interações específicas com as integrinas superexpressas nos GBMs. Também foram desenvolvidas nanopartículas teranósticas de carboximetilquitosana com agente de contraste gadolínio˗dietilenotriamina pentaacetato meglumina por interações eletrostáticas, as quais foram modificadas por interações covalentes para o acoplamento da desintegrina vicrostatin na superfície das nanopartículas. Todas as formulações obtidas foram caracterizadas por estudos de distribuição granulométrica, potencial zeta, microscopia eletrônica de varredura e de força atômica, ressonância magnética nuclear, calorimetria exploratória diferencial, espectroscopia de dicroísmo circular e infravermelho por transformada de Fourier. Nos ensaios de citotoxicidade in vitro com células de GBMs foi possível observar que ao encapsular os peptídeos cRGDfV e cRGDfK nas nanopartículas elas são menos citotóxicas comparadas nanopartículas com o peptídeo cRGDfK está na sua superfície. As nanopartículas com cRGDfK na superfície levaram a uma diminuição significativa da viabilidade celular em comparação às nanopartículas em que os peptídeos estão encapsuladas. Nos ensaios de captação e internalização por citometria de fluxo e microscopia confocal, as nanopartículas de quitosana acopladas com cRGDfK e as nanopartículas teranósticas de carboximetilquitosana acopladas com vicrostatin apresentaram interação com as integrinas, levando a uma maior internalização das nanopartículas pelas células de GBMs. No caso das nanopartículas teranósticas, pode ocorrer um aumento de gadolínio nos GBMs facilitando o diagnóstico por imagem. Portanto, todos as nanopartículas desenvolvidas podem ser futuramente exploradas como uma plataforma a fim de auxiliar no tratamento e no diagnóstico por imagem dos GBMs.
Glioblastoma multiforme (GBMs) is the most invasive tumor from central nervous system (CNS) and its invasiveness is related to interaction between glycoproteins of extracellular matrix and integrins present in tumor cells. Integrins α5β1, αvβ5, e αvβ3 are overexpressed in GBMs and they could be used as target molecules in the treatment and diagnostic imaging of the tumors from CNS. The disintegrins cRGDfV, cRGDfK and vicrostatin have been studied for the treatment of GBMs because they inhibit the integrins αvβ3, αvβ5, and α5β1, and, therefore, they show potent anti˗tumor properties. However, the administration of therapeutic proteins is limited by their low stability in physiological fluids and low penetration in biological membranes. To overcome these problems, drug delivery systems have been developed and alternative route of administration, such as nasal administration, has been used. Thus, the aim of this study was the development of chitosan nanoparticles, employing factorial design, by the means of ionotropic gelification and the encapsulation of disintegrins cRGDfV e cRGDfK. The chitosan˗based drug delivery system increased the mucoadhesion, which could promote the blood˗brain barrier permeation of disintegrins cRGDfV and cRGDfK. Another strategy aimed in this work was the use of chitosan nanoparticles selected by factorial design to be modified with PEG by supramolecular interactions and also by covalent interactions with methoxy˗polyethylene glycol and maleimide˗polyethylene glycol˗hydroxisuccinimide. The modified nanoparticles by covalent interactions were functionalized through linkage of disintegrin cRGDfK on nanoparticles’ surface in order to improve the targeting of nanoparticles to tumor environment by specific interactions with the integrins overexpressed in the GBMs. Theranostic carboxymethylchitosan nanoparticles containing the contrast agent gadolinium˗diethylenetriamine˗pentaacetate˗meglumine were also developed; these nanoparticles was developed by eletrostatic interactions, and then they were modified by covalent interactions to attach the desintegrin vicrostatin on the surface of these nanoparticles. All formulations obtained were characterized by size distribution, zeta potential, scanning eletron microscopy, atomic force microscopy, nuclear magnetic resonance, differential scanning calorimetry, circular dichroism and Fourier transform infrared spectrocopy. In in vitro cytotoxicity assays using GBMS cells, it was possible to observe the differences in the cell viability when the desintegrins cRGDfV and cRGDfK were encapsuled into nanoparticles and when the desintegrin cRGDfK is on the surface of nanoparticles. The nanoparticles with cRGDfK on the surface presented a significative decrease in cell viability as compared to nanoparticles which the disintegrins are encapsulated. In the uptake and internalization assays by flow citometry and confocal microscopy, the cRGDfK attached to chitosan nanoparticles and vicrostatin attached to the theranostic carboxymethylchitosan nanoparticles showed a specific interaction with the integrins, leading to higher uptake of nanoparticles by GBMs cells, and, thus, promoting an anti˗tumor effect due to integrins inhibition. In the case of theranostic nanoparticles, an increase in uptake of gadolinium could happen and it could facilitate the diagnosis by image. The nanoparticles developed can be exploited as a platform in order to assist in the treatment and diagnosis by image of the GBMs.
Glioblastoma multiforme (GBMs) is the most invasive tumor from central nervous system (CNS) and its invasiveness is related to interaction between glycoproteins of extracellular matrix and integrins present in tumor cells. Integrins α5β1, αvβ5, e αvβ3 are overexpressed in GBMs and they could be used as target molecules in the treatment and diagnostic imaging of the tumors from CNS. The disintegrins cRGDfV, cRGDfK and vicrostatin have been studied for the treatment of GBMs because they inhibit the integrins αvβ3, αvβ5, and α5β1, and, therefore, they show potent anti˗tumor properties. However, the administration of therapeutic proteins is limited by their low stability in physiological fluids and low penetration in biological membranes. To overcome these problems, drug delivery systems have been developed and alternative route of administration, such as nasal administration, has been used. Thus, the aim of this study was the development of chitosan nanoparticles, employing factorial design, by the means of ionotropic gelification and the encapsulation of disintegrins cRGDfV e cRGDfK. The chitosan˗based drug delivery system increased the mucoadhesion, which could promote the blood˗brain barrier permeation of disintegrins cRGDfV and cRGDfK. Another strategy aimed in this work was the use of chitosan nanoparticles selected by factorial design to be modified with PEG by supramolecular interactions and also by covalent interactions with methoxy˗polyethylene glycol and maleimide˗polyethylene glycol˗hydroxisuccinimide. The modified nanoparticles by covalent interactions were functionalized through linkage of disintegrin cRGDfK on nanoparticles’ surface in order to improve the targeting of nanoparticles to tumor environment by specific interactions with the integrins overexpressed in the GBMs. Theranostic carboxymethylchitosan nanoparticles containing the contrast agent gadolinium˗diethylenetriamine˗pentaacetate˗meglumine were also developed; these nanoparticles was developed by eletrostatic interactions, and then they were modified by covalent interactions to attach the desintegrin vicrostatin on the surface of these nanoparticles. All formulations obtained were characterized by size distribution, zeta potential, scanning eletron microscopy, atomic force microscopy, nuclear magnetic resonance, differential scanning calorimetry, circular dichroism and Fourier transform infrared spectrocopy. In in vitro cytotoxicity assays using GBMS cells, it was possible to observe the differences in the cell viability when the desintegrins cRGDfV and cRGDfK were encapsuled into nanoparticles and when the desintegrin cRGDfK is on the surface of nanoparticles. The nanoparticles with cRGDfK on the surface presented a significative decrease in cell viability as compared to nanoparticles which the disintegrins are encapsulated. In the uptake and internalization assays by flow citometry and confocal microscopy, the cRGDfK attached to chitosan nanoparticles and vicrostatin attached to the theranostic carboxymethylchitosan nanoparticles showed a specific interaction with the integrins, leading to higher uptake of nanoparticles by GBMs cells, and, thus, promoting an anti˗tumor effect due to integrins inhibition. In the case of theranostic nanoparticles, an increase in uptake of gadolinium could happen and it could facilitate the diagnosis by image. The nanoparticles developed can be exploited as a platform in order to assist in the treatment and diagnosis by image of the GBMs.
Descrição
Palavras-chave
Desintegrinas, Diagnóstico por imagem, Gadolínio, Integrinas, Nanopartículas, Quitosana, Disintegrins, Diagnostic imaging, Gadolinium, Integrins, Nanoparticles, Chitosan