RESUMEN
Fluorescent probes are used in drug nanocarrier pre-clinical studies or as active compounds in theranostics and photodynamic therapy. In the biological medium, nanoparticles interact with proteins, which can result in the off-target release of their cargo. The present study used asymmetric flow field-flow fractionation with online multi-angle laser light scattering and fluorescence detection (AF4-MALLS-FLD) to study the release, transfer, and partition of fluorescent dyes from polymeric nanoparticles (NP). NP formulations containing the dyes Rose Bengal, Rhodamine B, DiI, 3-(α-azidoacetyl)coumarin and its polymer conjugate, Nile Red, and IR780 and its polymer conjugate were prepared. NP suspensions were incubated in a medium with serum proteins and then analyzed by AF4. AF4 allowed efficient separation of proteins (< 10 nm) from fluorescently labeled NP (range of 54 - 180 nm in diameters). The AF4 analyses showed that some dyes, such as Rose Bengal, IR780, and Coumarin were transferred to a high extent (68-77%) from NP to proteins. By contrast, for DiI and dye-polymer conjugates, transfer occured to a lower extent. The studies of dye release kinetics showed that the transfer of IR780 from NP to proteins occurs at a high extent (~50%) and rate, while Nile Red was slowly released from the NP over time with reduced association with proteins (~20%). This experiment assesses the stability of fluorescence labeling of nanocarriers and probes the transfer of fluorescent dyes from NP to proteins, which is otherwise not accessible with commonly used techniques of separation, such as dialysis and ultrafiltration/centrifugation employed in drug encapsulation and release studies of nanocarriers. Determining the interaction and transfer of dyes to proteins is of utmost importance in the pre-clinical evaluation of drug nanocarriers for improved correlation between in vitro and in vivo studies.
Asunto(s)
Proteínas Sanguíneas/análisis , Portadores de Fármacos/química , Colorantes Fluorescentes/química , Fraccionamiento de Campo-Flujo/métodos , Nanopartículas/química , Polímeros/química , Adsorción , Fluorescencia , Humanos , Hidrodinámica , Cinética , Oxazinas/química , Teoría Cuántica , Rodaminas/química , Dispersión de RadiaciónRESUMEN
Antimicrobial and antitumor activities of resveratrol, a compound found mainly in grapes, have already been demonstrated. However, its low bioavailability is a limiting factor for therapeutic application. Polymeric micelles can be an approach to solve this problem since they can encapsulate hydrophobic substances. We developed and characterized micellar formulations containing resveratrol and evaluated their cytotoxic and antimicrobial effects. The formulations were prepared by the cold dispersion method with different concentrations of F127 (5 or 10% w/w) and resveratrol (500 or 5000 µM). The formulations were characterized according to size, polydispersity index, pH, encapsulation rate and in vitro release. Cytotoxic effect was evaluated on a bladder cancer cell line and antimicrobial effect was evaluated on E. coli, S. aureus and C. albicans. One of the formulations (10% w/w of F127 and 5000 µM of resveratrol) was a monodispersed solution with high encapsulation rate, thus it was chosen for the cytotoxicity and antimicrobial assays. MS- 10+RES-3 was able to preserve the antimicrobial and cytotoxic activity of resveratrol. This is the first study that evaluated antimicrobial potential and cytotoxicity of micelles containing resveratrol on bladder cancer cells and the results showed that micellar nanostructures could ensure the maintenance of the biological activity of resveratrol.
Asunto(s)
Neoplasias de la Vejiga Urinaria , Células , Resveratrol/análisis , Neoplasias/patología , Soluciones/administración & dosificación , Técnicas In Vitro/instrumentación , Línea Celular/clasificación , Vitis/clasificación , Concentración de Iones de Hidrógeno , MicelasRESUMEN
The interaction of polymer nanocapsules (NC) prepared from four biodegradable polyesters with variable polymer hydrophobicity (PCL, PLA, PLGA and PLA-PEG) was investigated in the non-phagocytic Vero, Caco-2 and HepG2 cell lines. The NC, labeled with the highly lipophilic fluorescent indocarbocyanine dye DIL, had very similar sizes (approx. 140â¯nm) and negative zeta-potentials. Asymmetric flow field-flow fractionation evidenced NC colloidal stability and negligible transfer of the dye to serum proteins in the incubation medium. The cytotoxicity of the NC was evaluated via MTT assay over a large polymer concentration range (1-1000⯵g/mL) and time of exposure (2, 24 and 48â¯h). The NC were safe in vitro up to a concentration of approx. 100⯵g/mL or higher, depending on the cell line and nature of the polymer. Vero cells were more sensitive to the NC, in particular NC of the more hydrophobic polymer. The cells were exposed to endocytosis inhibitors, incubated with NC, and the cell-associated fluorescence was quantified by spectrofluorometry. HepG2 cells presented a 1.5-2-fold higher endocytic capacity than Caco-2 and Vero cells. The main mechanism of NC uptake was caveolin-mediated endocytosis in HepG2 and Vero cells, and macropinocytosis in Caco-2 cells. Polymer hydrophobicity had an effect on the level of NC associated to HepG2 cells and to a lesser extent on the endocytosis mechanisms in Vero and Caco-2 cells. The NC uptake levels and endocytosis mechanisms differed significantly between cell lines tested.