Resumen de Study of rbc shape transitions induced by nanoparticles
This thesis describes the study of the properties of extracellular medium on the cryopreservation of red blood cells and the potential application of silica nanoparticles as co-agents for the intracellular delivery of trehalose, a natural cryoprotectant.
Cryopreservation of red blood cells is an important process to ensure a stock of blood for transfusion, in particular, in areas where the supply is limited, such as war areas or third world countries. Current methodologies use glycerol as a cryoprotectant, and storage at -80ᵒC. In order to avoid osmotic shock, excess glycerol must be removed upon thawing, requiring further processing and specialized equipment. As such, new methodologies which can avoid the extensive processing time and specialized storage equipment need to be developped.
Trehalose, a natural-found sugar, has been shown to possess promising properties as a cryoprotectant and has been proposed as alternative for the use of glycerol for the cryopreservation of red blood cells. However, its use has been limited by its inability to permeate through the cell membrane of cells. To surpass this problem, different co-agents have been proposed, most notably cell-penetrating peptides and nanoparticles. While cell-penetrating peptides allow control of their action upon a pre-established trigger (e.g. lower pH) they may remain attached to the cell membrane, and possibly lead to long term problems. Apatite nanoparticles have been shown to create a transient pore allowing trehalose to cross the lipid bilayer, while nanoparticles remain on the outer leaflet of the membrane. Nanoparticle-cell interaction has also been shown to be a reversible interaction, allowing nanoparticles to detach from the cells. This work opens the door for different biocompatible nanoparticles which may produce similar results, namely silica nanoparticles, which are known to interact with the cell membrane without inducing high toxicity.
On the first phase of the work described in this thesis I study different freeze-thawing condition and different properties of the extracellular medium on the cryopreservation of red blood cells, namely, osmolarity, sugar molecules. I describe an improvement to the analysis of the freezing and thawing points of blood by applying an image analysis technique to data of freeze-thawing curves obtained by freeze drying microscopy. Different freeze-thawing strategies were compared via hemolysis assay. Results suggest two main freeze-thawing strategies: immersion in liquid Nitrogen, followed by fast thawing at 37 ᵒC; and by placing the sample in a pre-cooled to -40 ᵒC media, followed by slow thawing methods. Study of solvent properties suggests osmolarity plays an important role in cryopreservation. By forcing intracellular water to leave the cell via osmosis, it is possible to achieve a cryoprotective effect (however less effective) in the absence of cryoprotectant. When comparing different sugar molecules, trehalose shows a stronger protective effect than glucose or sucrose. While this effect is not fully understood, it was suggested that trehalose acts by stabilizing the lipid bilayer, or by promoting vitrification as opposed to crystallization upon freezing. To understand possible alterations to red blood cells, samples were imaged using brightfield microscopy and their morphology of the population was analyzed using a single-cell analysis method. Results suggest that cell size is larger after freezing, however this requires further investigation.
The second phase of the work investigates the interaction of differently charged silica nanoparticles with red blood cells. Silica nanoparticles’ toxicity was evaluated via hemolysis assay, showing no effect up to 1mg/mL, after which cell survivability is compromised, especially for the least negatively charged nanoparticles. After interaction with the nanoparticles, freely floating red blood cells were imaged using laser scanning confocal microscopy. Due to the movement of the cells, resulting images showed a defect where cells are distorted along XZ and XY planes. A new post-processing high-throughput method was developed to correct these distortions. This method uses single-cell analysis to identify the cells along the z-stack followed by a registration technique to correct the drift. Upon correction cells were analyzed for the number of clusters, showing inverse correlation to the amount of negative charge on the silica nanoparticles. The results were confirmed with an experimental model system, GUVs. In this case however nanoparticles attached homogeneously and not as clusters, suggesting the glycocalyx may play a more important role than initially considered.
Overall, as long as the concentration of nanoparticles is below the 1 mg/mL threshold, SiNPs are a stable component, not showing clear signs of toxicity to RBCs. This enables future applications to be developed as co-agents for intracellular delivery.
