Liposome interactions with biological systems: a journey into cells

Nanomedicine has been rapidly developed in the last decades. However, the clinical translation of nano-formulations remain challenging and relatively few nanomedicines have been approved for clinical use. One of the main obstacles in the development of nanomedicines is the still limited understanding of nanomaterial interactions with biological systems. In this context, using liposomes, one of the most clinically established nano-formulations, as a nanomedicine model, this Thesis studied nanocarrier behavior in complex biological systems, in order to explore new strategies to guide the design of more effective nano-formulations. Liposome composition was changed systematically in order to obtain formulations with different biological outcomes. This allowed to gain a better understanding on how nanomaterial design can be tuned to control the protein corona forming on their surface once introduced in serum, modulate cell uptake efficiency and kinetics, affect the mechanisms cells used for their internalization and the kinetics of drug release following uptake by cells. Additionally, biomimetic nanotechnology, an alternative strategy to fabricate nanoparticles with defined interactions with biological systems, was exploited to dope liposomes with cell membranes from stromal and leukemia cells in the context of acute myeloid leukemia (AML). Our results suggested that stromal cell membrane-doped liposomes had the potential to be developed as a tool to characterize the interactions between stromal and leukemia cells and to identify novel targets for AML treatment. The results presented in this Thesis have helped to deepen our knowledge on how complex biological systems affect nanomaterial behavior

Investigation of CO2 storage capacity in open saline aquifers with numerical models

AbstractAccurate calculation of carbon dioxide (CO2) storage capacity in deep saline aquifers is a challenging task. However an assessment must be performed to determine whether there is sufficient capacity in a storage site for any CO2 sequestration project. We evaluated the CO2 storage capacity for a simplified reservoir system, which the layered potential storage formations are overlaid by sealing cap rock. This study aims at determining CO2 storage capacity for the injection of CO2 in opened saline formations using numerical simulation method. In this study, a 3D numerical model was developed for the investigation. Detailed processes for the storage capacity estimation are derived. The impact of injection strategy and the residual gas saturation on storage capacity was investigated. It is shown that both of the injection strategy and the residual gas saturation have a great impact on the spatial distribution of CO2 plume and the effective storage of CO2 in the reservoir. Simulation results also indicate that the injection well distribution may significantly influence the use of the formation porous space for CO2 storage. From this study, we may conclude that the most accurate way to estimate storage capacity is through construction of a basin scale three-dimensional numerical model for specific storage site by incorporating detailed geological information of the site and injection scheme used

Effects of Protein Source on Liposome Uptake by Cells:Corona Composition and Impact of the Excess Free Proteins

Corona formation in biological fluids strongly affects nanomedicine interactions with cells. However, relatively less is known on additional effects from the free proteins in solution. Within this context, this study aims to gain a better understanding of nanomaterial-cell interactions in different biological fluids and, more specifically, to disentangle effects due to corona composition and those from the free proteins in solution. To this aim, the uptake of liposomes in medium with bovine and human serum are compared. Uptake efficiency in the two media differs strongly, as also corona composition. However, in contrast with similar studies on other nanomaterials, despite the very different corona, when the two corona-coated liposomes are exposed to cells in serum free medium, their uptake is comparable. Thus, in this case, the observed differences in uptake depend primarily on the presence and source of the free proteins. Similar results are obtained when testing the liposomes on different human cells, as well as in murine cells and in the presence of murine serum. Overall, these results show that the protein source affects nanomedicine uptake not only due to effects on corona composition, but also due to the presence and composition of the free proteins in solution

Corona Composition Can Affect the Mechanisms Cells Use to Internalize Nanoparticles

Nano-sized objects, such as nanoparticles and other drug carriers used in nanomedicine, once in contact with biological environments are modified by adsorption of biomolecules on their surface. The presence of this corona strongly affects the following interactions at cell and organism levels. It has been shown that corona proteins can be recognized by cell receptors. However, it is not known whether the composition of this acquired layer can also affect the mechanisms nanoparticles use to enter cells. This is of particular importance when considering that the same nanoparticles can form different coronas for instance In Vitro when exposed to cells in different serum amounts, or In Vivo depending on the exposure or administration route. Thus, in this work, different coronas were formed on 50 nm silica by exposing them to different serum concentrations. The uptake efficiency in HeLa cells was compared, and the uptake mechanisms were characterized using transport inhibitors and RNA interference. The results showed that the nanoparticles were internalized by cells via different mechanisms when different coronas were formed, and only for one corona condition uptake was mediated by the LDL receptor. This suggested that corona of different composition can be recognized differently by cell receptors, and this in turn leads to internalization via different mechanisms. Similar studies were performed using other cells, including A549 cells and primary HUVEC, and different nanoparticles, namely 100 nm liposomes and 200 nm silica. Overall, the results confirmed that the corona composition can affect the mechanism of nanoparticle uptake by cells

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.</p

Accelerating O-redox kinetics with carbon nanotubes for stable lithium-rich cathodes

Lithium-rich cathodes (LRCs) show great potential to improve the energy density of commercial lithium-ion batteries owing to their cationic and anionic redox characteristics. Herein, a complete conductive network using carbon nanotubes (CNTs) additives to improve the poor kinetics of LRCs is fabricated. Ex situ X-ray photoelectron spectroscopy first demonstrates that the slope at a low potential and the following long platform can be assigned to the transition metal and oxygen redox, respectively. The combination of galvanostatic intermittent titration technique and electrochemical impedance spectroscopy further reveal that a battery with CNTs exhibited accelerated kinetics, especially for the O-redox process. Consequently, LRCs with CNTs exhibit a much better rate and cycling performance (approximate to 89% capacity retention at 2 C for over 200 cycles) than the Super P case. Eventually, TEM results imply that the improved electrochemical performance of the CNTs case also benefits from its more stable bulk and surface structures. Such a facile conductive additive modification strategy also provides a universal approach for the enhancement of the electron diffusion properties of other electrode materials.Web of Science67art. no. 220044

Tuning Liposome Stability in Biological Environments and Intracellular Drug Release Kinetics

Ideal drug carriers should be stable in biological environments but eventually release their drug load once inside the targeted cells. These two aspects can be in contrast with each other, thus they need to be carefully tuned in order to achieve the desired properties for specific applications. Quantifying drug release profiles in biological environments or inside cells can be highly challenging, and standard methods to determine drug release kinetics in many cases cannot be applied to complex biological environments or cells. Within this context, the present work combined kinetic studies by flow cytometry with aging experiments in biological fluids and size-exclusion chromatography to determine drug release profiles in biological environments and inside cells. To this purpose, anionic and zwitterionic liposomes were used as model nanomedicines. By changing lipid composition, liposome stability in serum and intracellular release kinetics could be tuned and formulations with very different properties could be obtained. The methods presented can be used to characterize liposome release profiles in complex biological media, as well as inside cells. In this way, liposome composition can be tuned in order to achieve formulations with optimal balance between stability and release kinetics for specific applications

Tuning Liposome Composition to Modulate the Corona Forming in Human Serum and Uptake by Cells

Nano-sized objects such as liposomes are modified by adsorption of biomolecules in biological fluids. The resulting corona critically changes nanoparticle behavior at cellular level. A better control of corona composition could allow to modulate uptake by cells. Within this context, in this work, liposomes of different charge were prepared by mixing negatively charged and zwitterionic lipids to different ratios. The series obtained was used as a model system with tailored surface properties to modulate corona composition and determine the effects on liposome interactions with cells. Uptake efficiency and uptake kinetics of the different liposomes were determined by flow cytometry and fluorescence imaging. Particular care was taken in optimizing the methods to isolate the corona forming in human serum to prevent liposome agglomeration and to exclude residual free proteins which could confuse the results. Thanks to the optimized methods, mass spectrometry of replicate corona isolations showed excellent reproducibility and this allowed semi-quantitative analysis to determine for each formulation the most abundant proteins in the corona. The results showed that by changing the fraction of zwitterionic and charged lipids in the bilayer, the amount and identity of the most abundant proteins adsorbed from serum differed. Interestingly, the formulations also showed very different uptake kinetics. Similar approaches can be used to tune lipid composition in a systematic way in order to obtain formulations with the desired corona and cell uptake behavior. Statement of Significance Liposomes and other nano-sized objects when introduced in biological fluids are known to adsorb biomolecules forming the so-called nanoparticle corona. This layer strongly affects the subsequent interactions of liposomes with cells. Here, by tuning lipid composition in a systematic way, a series of liposomes with tailored surface properties has been prepared to modulate the corona forming in human serum. Liposomes with very different cellular uptake kinetics have been obtained and their corona was identified in order to determine the most enriched proteins on the different formulations. By combining corona composition and uptake kinetics candidate corona proteins associated with reduced or increased uptake by cells can be identified and the liposome formulation can be tuned to obtain the desired uptake behavior

Comparison of the uptake mechanisms of zwitterionic and negatively charged liposomes by HeLa cells

Zwitterionic molecules are used as an alternative to PEGylation to reduce protein adsorption on nanocarriers. Nonetheless, little is known on the effect of zwitterionic modifications on the mechanisms cells use for nanocarrier uptake. In this study, the uptake mechanism of liposomes containing zwitterionic or negatively charged lipids was characterized using pharmacological inhibitors and RNA interference on HeLa cells to block endocytosis. As expected, introducing zwitterionic lipids reduced protein adsorption in serum, as well as uptake efficiency. Blocking clathrin-mediated endocytosis strongly decreased the uptake of the negatively charged liposomes, but not the zwitterionic ones. Additionally, inhibition of macropinocytosis reduced uptake of both liposomes, but blocking actin polymerization had effects only on the negatively charged ones. Overall, the results clearly indicated that the two liposomes were internalized by HeLa cells using different pathways. Thus, introducing zwitterionic lipids affects not only protein adsorption and uptake efficiency, but also the mechanisms of liposome uptake by cells