Silvestri, Simona (2024) Modulating Lipid Bilayer Fluidity for enhanced delivery of active and biological compounds. [Tesi di dottorato]

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Abstract

Synthetic and Natural lipid-based systems, such as Lipid Nanoparticles or small Extracellular vesicles (sEVs), are attracting particular interest due to their distinctive pharmacokinetic profiles and physico-chemical properties. Among them, it is worth mentioning the low toxicity, the ability to house both hydrophilic and hydrophobic compounds and the reduced off-target effects. Moreover, one key peculiarity lies in their biomimeticity, which combines with low immunogenicity and improves both the transport and the interaction in biological environments, also favoured by the high flexibility and permeability. The lipid membrane forming the surface of the systems dictates the achievement of these characteristics and a unique mechanical profile, that is tightly regulated by entropic demands and strongly dependent on external conditions. Consequently, a characteristic dynamic Fluidity appears owing to remodelling pathways that are continuously triggered. Moreover, lipid bilayers under stimulation are characterized by a solid-fluid duality that is linked to the thermodynamic state, the molecular composition, the organization, and the type of stresses they are subjected to. Commonly, they behave fluid-like when laterally stressed, while they resist deformations when transversally stressed. Fluidity emerges as a key regulator of these shape- and compositional- changes, being also responsible for relevant processes like vesicle fusion and fission, membrane remodelling, biological and synthetic identities. Spanning from technological needs to biological demands at the nano-bio-interactions level, this intrinsic mechanical property can be exploited to improve the clinical translation of both synthetic and natural lipidic systems. Indeed, the thermodynamical knowledge of the processes and the properties of materials can be matched to control the interaction happening at the material-environment interface. In this way, it is possible to handle synthetic or natural components by identifying physico-chemical parameters to play with, in order to obtain improved outcomes. The goal of this PhD project is to investigate the role, organization, thermodynamics, and transformation of lipid bilayers under different deformative pathways. Microfluidic platforms were designed and developed to apply various pressure-based hydrodynamic stimuli on both natural and synthetic lipid bilayers. These technologies offer a novel approach to addressing challenges related to the clinical application of small Extracellular Vesicles (sEVs), which are naturally produced by cells and released into biological fluids via exocytosis. While they have currently extensively studied since being recognized as relevant molecules for intercellular communications, biological investigations, diagnostic and therapeutic applications, their clinical use is still hindered due to technical challenges. Indeed, samples preparation remains time-consuming and costly due to huge amount of cells as source of sEVs, complex purification steps required to remove contaminants and lack of standardized characterization techniques. In this work, the dynamic fluidity of LBs has been exploited to induce controlled, temporary and non-destructive membrane destabilizations, in a dual-mode setup for the high-throughput isolation and loading of sEVs. Starting from a high-pressure configuration, a complex force field was firstly employed to stimulate cells, by tuning surface destabilizations that accelerated the secretion of sEVs. This allowed for a significant concentration of usable vesicles to be obtained from a smaller quantity of cells, demonstrating the system's potentiality to reduce the high costs associated with sEVs manufacturing. The effectiveness of this stimulation was verified by comparing data from the stimulated samples to those processed using standard techniques. In a second application, the same high-pressure derived deformative field was used on sEVs that had already been isolated, purified and co-incubated with active biological molecules. The goal was to enhance the sEVs loading capacity by temporarily modifying their surfaces. The next step was the transition from high- to low- pressure microfluidics, enabling the selection of relevant forces to be investigated. While the high-pressure setup did not permit differentiation of dominant contributions, the design and development of low-pressure microfluidic devices enabled the isolation of single or simple stress types. Moreover, the introduction of lab-scaling devices enabled the extension of the applicability of our approach to more cells sources to be processed for sEVs secretion and more molecules to be encapsulated. Therefore, various geometries were selected and simulated with the aid of a CFD tool to design the experimental parameters properly, and they were fabricated and exploited for cells and sEVs stimulations. Overall, this research work has demonstrated the potential to exploit dynamic Fluidity to reversibly overcome the semi-permeability of lipidic membranes, without changing the physicochemical properties of the stimulated entities. This finding paved the way for a new approach aimed at deepening the thermodynamic understanding of fluidic systems and developing microfluidic platforms to tune controlled surface destabilizations through fluid-dynamics.

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