Fabrication, surface engineering, cellular uptake and cytotoxicity of metallic and polymeric nanoparticles
- Autores: Yuan Qiu
- Directores de la Tesis: Sergio Moya (dir. tes.), David Mecerreyes Molero (tut. tes.)
- Lectura: En la Universidad del País Vasco - Euskal Herriko Unibertsitatea ( España ) en 2014
- Idioma: inglés
- Tribunal Calificador de la Tesis: Edwin Donath (presid.), Galder Kortaberria Altzerreka (secret.), Ángel Millán Escolano (voc.), Jacqueline Forcada Garcia (voc.), Mihaela Delcea (voc.)
- Materias:
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- Resumen
This PhD dissertation is focused on the fabrication and surface engineering of metallic, metal oxide and polymeric nanoparticles (NPs), and the study of their cytotoxicity, intracellular behavior, trafficking, localization and biological fate in vitro.
The role of the surface coating of NPs and its charge to control the interaction with cells was studied with CeO2 NPs (CNPs) coated with either positively or negatively charged polyelectrolyte brushes via in situ Atomic Transfer Radical Polymerization (ATRP).
Polymer brush provides higher surface charge density and better colloidal stability in aqueous media to CNPs. CNPs coated with polyelectrolyte brushes were fluorescently labeled. The fluorescent dye was attached directly to the surface of the CNPs remaining inside the polymer shell. In this way the dye will remain entrapped in the brush and will not impact on the surface properties of polymer brush coated CNPs. Polymer brush coated CNPs showed slower cellular uptake than unmodified CNPs and the uptake mechanisms were also altered. Moreover, polymer brush coated CNPs showed higher colocalization with acidic cell compartments in the cell, meaning that these CNPs were mostly internalized through endosomal and lysosomal involved endocytosis. The different surface charge resulted in different levels of cytotoxicity. The effect of surface modified CNPs on the intracellular generation of reactive oxygen species (ROS) was also examined. Cells with internalized polymer brush coated CNPs showed a much lower ROS level than control cells after exposure to ROS inducer. This indicates that the antioxidant properties of the CNPs were also presented in the polymer coated CNPs.
The intracellular dynamics of NPs inside cells was studied by means of Fluorescence Correlation Spectroscopy (FCS). Glucose derivative conjugated gold NPs (Glc-Au NPs) were chosen as model NPs for these studies since they have little non specific interactions with proteins due to the glucose derivative coating, and should therefore display a lesser degree of aggregation. Glc-Au NPs formed aggregates when incubated in DMEM with 10% FBS, as hinted by the change in their FCS correlation value reflecting a change in the relative concentration of single NPs compared to those in distilled water. The diffusion time increased from 113.2 ms in distilled water to 243.3 ms in DMEM with 10% FBS. The corresponding concentration decreased from 77.4 nM to 37.0 nM. The hydrodynamic size increased from 2 nm to 4.4 nm.
Intracellularly, the formation of large aggregates of NPs that behave as an immobilized fraction limits the applicability of FCS. To apply FCS intracellularly to measure NP diffusion, a prebleaching strategy was employed. The prebleaching eliminates the fluorescence of large aggregates and makes it possible to obtain meaningful correlation curves by FCS. Then, intracellular dynamics, concentration, hydrodynamic radius, etc. of Glc-Au-Hi NPs were studied. Measured diffusion times range from 238 ms to 780 ms. The narrow range of diffusion times allowed us to conclude that only single NPs and small aggregates of NPs contribute to the mobile fraction. Approximately 88 % of the aggregates are between 3.5 nm and 7.0 nm. Occasionally aggregates over 780 ms (~12%) can be measured but most of these aggregates can be considered as immobile. In addition, FCS data hinted that NPs are localized in aqueous environments within the cell.
Biodegradable poly (lactide-co-glycolide) (PLGA) NPs with BSA as a stabilizer were fabricated through O/W emulsion method. The NPs were prepared with PLGA polymers with 15 % or 35 % glycolide. The degradation of PLGA NPs has been studied under physiological and intracellular conditions by means of flow cytometry (FACS). In a physiological solution, FACS can trace the variations in the fluorescence of rhodamine B labeled PLGA NPs with 15 % glycolide (PLGA 15) due to the release of labeled polymer as degradation proceeds over 20 days while no change in fluorescence is observed for the PLGA NPs with 35 % (PLGA 35) after 70 days. Degradation was confirmed by Transmission Electron Microscopy, Dynamic Light Scattering and zeta potential measurements. Intracellular degradation of PLGA NPs was followed by FACS measuring the changes in fluorescence intensity per cell over time. Rhodamine B displays a higher quantum yield in a polar environment like that of the cells than in a non-polar media like inside the PLGA NPs. When labeled PLGA molecules are released from the PLGA NPs into the cell environment, their fluorescence will increase. An increase in fluorescence intensity was observed during the first 24h for cells with PLGA15 NPs while no changes in fluorescence were observed of PLGA35, meaning that PLGA15 degraded during the first 24 hours while PLGA35 did not. Additionally, Confocal Raman Microscopy (CRM) was used to trace degradation intracellularly at single cell level by recording the relative changes of the bands at 1768 cm-1 and at 872 cm-1. The band at 1768 cm-1 corresponds to C=O vibration of the ester group of PLGA. As degradation takes place, the hydrolysis of the ester should decrease the intensity of the band at 1768 cm-1. The band at 872 cm-1 is assigned to the C-COO vibration of lactic acid, which will not be affected by the hydrolysis process during the degradation. Therefore, the change of ratio between the band at 1768cm-1 and that of 872 cm-1 reveals the change of the amount of ester group during the degradation. The results from CRM showed that intracellular PLGA15 NPs had a decrease in the intensity of the ester group after incubated with cells for 120h.Whereas PLGA35 NPs did not show a significant change after 120h incubation with cells. The decrease intensity of the ester group indicates the break of ester bond and the degradation of NPs. This corroborated the degradation of the PLGA15 and the stability of the PLGA35 NPs intracellularly.
Finally, PLGA NPs incorporating quantum dots (QDs), superparamagnetic iron oxide nanoparticles (SPIONs) and gold NPs (Au NPs) were fabricated via the W/O/W double emulsion method. QDs and SPIONs were entrapped inside PLGA NPs during emulsification while Au NPs were assembled on top of the PLGA NPs via electrostatic interactions. The uptake of the hybrid PLGA NPs by human neutrophils was studied by FACS and Confocal Laser Scanning Microscopy (CLSM). In addition, the induction of reactive oxygen species (ROS) in neutrophils after incubation with the hybrid PLGA NPs was assessed. Magnetophoresis experiments showed that neutrophils with internalized hybrid PLGA NPs can be effectively laterally displaced towards the magnetic field.
Magnetic Resonance Imaging (MRI) of the hybrid PLGA NPs resulted in images with a contrast enhancement linearly dependent on the concentration of the hybrid PLGA NPs.
The hybrid NPs have potential for in vivo applications, e.g., tumor visualization and localized photothermal treatment.
