Preparation of asymmetric liposomes by chemically fueled reversible reactions

Objectives of the proposal:

The existence of bilayer asymmetry (i.e. having different lipid composition in the inner and outer leaflet of the lipid bilayer) is a key aspect of biological organisms. Such property gives cells the ability to distinguish between their inside and their environment, enabling one-way transport of both necessary nutrients and unnecessary waste. Moreover, asymmetric liposomes are being explored as drug delivery systems because of the possibility to tailor their inner layer to the necessities of the payload and their outer layer to the needs of the receiving organism. However, the obtention of these asymmetric liposomes is far from straightforward, thus limiting their possible applications. The way that living cells achieve membrane asymmetry is by the action of transporter proteins known as flippases (which move lipids from the inner to the outer leaflet) and floppases (which move lipids from the outer to the inner leaflet). Developing a synthetic mimic of these proteins would provide an easy method to build asymmetric vesicles. As shown by the PI this year (Fracassi, Seoane et al. Nat. Chem. 2025), it is possible to use chemically fueled reversible reactions between n-hydroxysuccinimide (NHS) and fatty acids to mimic the cellular process of lipid remodeling. This is done by synthesizing NHS-bearing single-chain phospholipids which can react with fatty acids in the presence of a stoichiometric activator to form labile NHS-ester- containing dual-chain phospholipids that can self-assemble into lipid nanoparticles (LNPs) which, overtime, disassemble after the hydrolysis of the NHS-ester bond by the water present in the medium.

With this precedent in mind, the goal of this project is to develop a synthetic system to imitate flippase activity that would allow for a straightforward construction of asymmetric liposomes. For this project, we take inspiration form the communication to design a different system where reversible NHS-ester linkages are used to mimic cellular processes. In this case, the chemically fueled reversible NHS-ester between free NHS and fatty acids formation will be used to imitate floppase activity (moving lipids from the outer layer to the inner layer).

Description and work plan:

To successfully carry out this project, it will be divided into smaller work packages, which will serve as milestones to monitor the progress of the project:

WP1: Synthesis and characterization of the reporting fluorophore (1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH): The research will start by synthesizing the fluorophore which is described in the literature, crucial to validate the generation of the asymmetric liposomes. This fluorophore has been chosen due to its properties: On one hand, it is positively charged, which means that it associates better with negatively charged liposomes than neutral or positive ones. On the other hand, it is solvatochromic, meaning that its fluorescence intensity increases when it is associated with the liposomes.

WP2: Generation of symmetric oleate-containing LNPs and evaluation of the responsiveness of the fluorophore: Once the behavior of the fluorophore has been determined, we will proceed to evaluate the possibility of synthesizing the asymmetric liposomes. To do so, we will prepare liposomes with different compositions adding negatively charged oleate in their composition. Once this has been done, the change of brightness of the fluorophore depending on the charge of the liposomes by monitoring the total fluorescence intensity of the fluorophore when added to liposomes with different compositions. This will help to qualitatively construct a model correlating total fluorescence with total charge of the outer leaflet of the liposome.

WP3: Generation of asymmetric LNPs: This goal will be achieved by the addition of NHS and chemical fuel to oleate-containing liposomes. These reagents will react specifically with the oleate, generating a neutral labile NHS-ester which can migrate to the inner layer of the liposome and hydrolyze in its lumen, leading to the membrane asymmetry. The depletion of negative charge from the outer layer of the liposome will be monitored by the addition of the solvatochromic positively charged fluorophore to the external solution, which will change total fluorescence intensity in the sample (due to less association of the fluorophore with the bilayer). Experimentally, unillamelar liposomes, bearing different mixtures of phospholipids and oleate will be constructed by extrusion. Then, the vesicles will be treated with the fluorophore and with or without the different reagents needed for the system (fuel and/or NHS). Fluorescence measurements will be carried out and the difference of intensity of the emission will be used to assess the extent of the disappearance of the negative charge from the outer leaflet of the LNPs.

The funding received will be to hire a technician to aid with the project (18% of the budget) used to buy the necessary reagents (lipids, fuels, NHS, reagents for the preparation of the fluorophore) and the extruder to prepare the unilamellar vesicles (43%) of the budget and publication costs of the results in a high-impact journal (25% of the budget). The remaining 14% of the budget will be cofinanced by the PI

Expected results:

The straightforward obtention of asymmetric LNPs is a longstanding challenge within the field and will provide a method to synthetically imitate natural behavior as well as to construct novel pharmaceutically interesting drug delivery systems. Consequently, the following results are expected from this project:

I-A publication on a high-ranked journal (JACS, ACIE, Chem Sci...) that will position the name of URL researchers in the field of dissipative systems to mimic cellular processes and build drug delivery platforms and will help to achieve further collaboration with other academic institutions and/or industry.
II-This proposal is included in the area of dissipative chemistry which is not the focus of other groups at URL. By working in this area, we will expand the number of research topics covered by URL, which will also increase the number of possible collaborations.
III-Since the PI of the project is a young researcher in the beginning of his career (having applied to the "Proyectos de Generación de concimiento" from the ministry of science in 2024 and 2025 and the BBVA fellowship in 2024) , the funding provided by this grant will help him kickstart his career by providing him with corresponding-author publications which will help him secure further funding for related projects (for instance by producing data that will be used in the application of the "Proyectos de Generación de concimiento" in 2026), increasing the amount of funding received by the URL.