Periodic Reporting for period 1 - EXTREME (Engineering Extracellular Matrices for Controlling Structure and Dynamics of Lipid Bilayer Membranes)
Reporting period: 2020-06-01 to 2022-05-31
Cell membrane is primarily composed of about five-nanometers-thick lipid bilayer and defines the physical boundaries of all living cells. It is through this thin layer the cells interact with their physical environment and perform many critical activities like endocytosis, exocytosis, ion-transport, cell-cell communication and cell division. Therefore, understanding the complex hierarchical structure and dynamics of lipid membrane is an essential ingredient to precisely control these vital processes. Biological membranes are in contact with extracellular matrices (ECM)- complex viscoelastic media primarily made of flexible and semi-flexible polymers. Relaxation of the polymers occurs at time scales comparable to those of membrane undulations; it is expected that the lipid motion as well as membrane fluctuations are coupled dynamically. As a result, the membrane dynamics can be altered externally by modulating the viscoelastic properties of the ECM. However, most of the studies so far have been performed on membranes that are isolated from their native environment and embedded in simple solvent media. The overall objective of this EU-funded project entitled ‘Engineering Extracellular Matrices for Controlling Structure and Dynamics of Lipid Bilayer Membranes’ (EXTREME) is to understand, at the fundamental level, how the elasticity of ECM modifies the structure and dynamics of lipid membranes. For this purpose, we used unilamellar lipid vesicles (liposomes) as model artificial cells, and aqueous poly(ethylene glycol) solutions and hydrogels of varying stiffnesses to mimic the viscoelastic properties of the embedded media. By using various state-of-the art characterization techniques, we were able to experimentally correlate, for the first time, the viscoelastic properties of the embedded media and multiscale dynamics of vesicles, from microviscosity in bilayer to bulk diffusion. The results show that, unlike the conventional cell specific chemistry-based approaches, the membrane fluidity can be controlled by engineering the ECM media and opens up exciting new possibilities for developing efficient drug-delivery mechanism and membrane based lipid therapies.
The project achieved its primary objectives and milestones, with relatively minor deviations due to Covid-19 pandemic. With this Action, the experienced researcher gained experience in new techniques, become more skilled in multidisciplinary research areas, dissemination of scientific results, supervising a graduate student, knowledge transfer and management of a research group.
The project achieved its primary objectives and milestones, with relatively minor deviations due to Covid-19 pandemic. With this Action, the experienced researcher gained experience in new techniques, become more skilled in multidisciplinary research areas, dissemination of scientific results, supervising a graduate student, knowledge transfer and management of a research group.
The project was organized to have three main work packages. In the first part, we investigated the structure of unilamellar vesicles in the polymeric microenvironment. Using aqueous solutions of poly (ethylene glycol) (PEG) with a wide range of molecular weights (from 1.5 kDa to 400 kDa) and nearly monodisperse 100 nm unilamellar vesicles of DMPC/DMPG liposomes, we studied the effect of polymeric media on the structure and dynamics of lipid membranes. The addition of PEG chains did not cause fusion or unilamellar to multilamellar transition. However, the chains formed adsorbed polymer layer on the vesicles due to the physical interactions between the membrane and the polymer. This layer was found to have a profound effect on particle mobility and microviscosity. The phase transition of the liposome bilayer, from gel to fluid state, was affected by the polymers in a way that a remarkable thermal-thickening depending on the chain length, unilamellarity and size, was observed at the macroscopic level. Such a thermally activated relative viscosity increase can be also useful for biolubricants and stimuli responsive materials.
The vesicles were then embedded in the hydrogel matrix. PEG hydrogels containing liposomes were produced by photocrosslinking, and the elasticity of the hydrogels was tuned from 1 Pa to 200 Pa to mimic the polymeric nature of the different tissues. Liposomes remained unilamellar in the crosslinked polymer environment, and their stability was assessed. Liposomes acted as reinforcing nanoparticles and resulted in nearly 2-fold increased elasticity values compared to neat PEG hydrogels. In this part, we successfully produced hydrogel-liposome composite for dynamical studies.
Upon completing the structural and bulk characterization of the liposomes in ECM mimetic media, how the network strength affected the membrane at the nanoscale was investigated. Within composite hydrogel-liposome, the crosslinked polymer environment caused a slowdown in lipid motion. Liposome release from gels with varying stiffnesses as well as small molecule release from the composite ‘lipo-hydrogels’ were evaluated.
The project started two months after the first Covid-case was reported in my host country and official pandemic announcement. The project was almost entirely experimental, thus, required physical presence in the lab. This was not always possible due to special pandemic regulations. However, the situation was managed quite well by my host institution; we were able to access our research labs and campus facilities albeit for limited days and time. We were also able to remotely access to the labs. All these helped greatly to stay close to the original research plan. As a result, a full article related to PEG-liposome solutions was published in an open-access peer-reviewed journal. A review article on deformation effect on liposome properties is now under peer assessment and another full-article about the liposome-hydrogel system will be shortly submitted to a journal. The results were also shared with the scientific community in two major international conferences, and with the public using social media platforms. A scattering workshop was organized to meet researchers with beamline scientists from the large facilities around the world.
The vesicles were then embedded in the hydrogel matrix. PEG hydrogels containing liposomes were produced by photocrosslinking, and the elasticity of the hydrogels was tuned from 1 Pa to 200 Pa to mimic the polymeric nature of the different tissues. Liposomes remained unilamellar in the crosslinked polymer environment, and their stability was assessed. Liposomes acted as reinforcing nanoparticles and resulted in nearly 2-fold increased elasticity values compared to neat PEG hydrogels. In this part, we successfully produced hydrogel-liposome composite for dynamical studies.
Upon completing the structural and bulk characterization of the liposomes in ECM mimetic media, how the network strength affected the membrane at the nanoscale was investigated. Within composite hydrogel-liposome, the crosslinked polymer environment caused a slowdown in lipid motion. Liposome release from gels with varying stiffnesses as well as small molecule release from the composite ‘lipo-hydrogels’ were evaluated.
The project started two months after the first Covid-case was reported in my host country and official pandemic announcement. The project was almost entirely experimental, thus, required physical presence in the lab. This was not always possible due to special pandemic regulations. However, the situation was managed quite well by my host institution; we were able to access our research labs and campus facilities albeit for limited days and time. We were also able to remotely access to the labs. All these helped greatly to stay close to the original research plan. As a result, a full article related to PEG-liposome solutions was published in an open-access peer-reviewed journal. A review article on deformation effect on liposome properties is now under peer assessment and another full-article about the liposome-hydrogel system will be shortly submitted to a journal. The results were also shared with the scientific community in two major international conferences, and with the public using social media platforms. A scattering workshop was organized to meet researchers with beamline scientists from the large facilities around the world.
The aim of this project was to unravel the effect of the polymeric extracellular matrix (ECM) on the multiscale dynamics of the lipid bilayers using liposomes as artificial cell membranes and polymeric matrix as models for the external environment. The previous studies approached the problem from the chemistry point of view by altering lipid bilayer composition. However, this is mostly cell specific. This project uses biophysical approaches and proposes a more generic method to modulate cell membrane dynamics via engineering the complex extracellular microenvironment. There are strong evidences that the membrane stiffness is significantly altered in tumor tissue, and membrane permeability is strongly altered in some diseases, such as type-2 diabetes and Alzheimer's; the results from this action are expected to offer novel strategies for therapies against these membrane-associated diseases,
Moreover, like our model PEG-liposome system, the mRNA-based Covid-19 vaccines utilize PEGylated lipids to create lipid nanoparticle solutions. The role of PEG is to enhance the colloidal stability, and biodistribution. Since the local viscoelasticity depends strongly on the PEG chain length, the lipid nanoparticles may experience a different different microenvironment compared to their polymer-free state. This can significantly alter the delivery efficiency of vaccines and other drug molecules. However, how PEGlylation affects the membrane permability is not well know. Our results shed-light on some of these fundamental questions related to public health.
Moreover, like our model PEG-liposome system, the mRNA-based Covid-19 vaccines utilize PEGylated lipids to create lipid nanoparticle solutions. The role of PEG is to enhance the colloidal stability, and biodistribution. Since the local viscoelasticity depends strongly on the PEG chain length, the lipid nanoparticles may experience a different different microenvironment compared to their polymer-free state. This can significantly alter the delivery efficiency of vaccines and other drug molecules. However, how PEGlylation affects the membrane permability is not well know. Our results shed-light on some of these fundamental questions related to public health.