Biomimetic microscale platforms for the visualization of biological processes : from GUVs towards artificial cells

First in order to explore the possibilities of building a synthetic artificial cell following a biomimetic approach, we engineered synthetic polymer-based giant unilamellar vesicles (GUVs) with selective membrane permeability. Since the membranes of polymeric GUVs present a high impermeability compared to natural lipid membranes, membranes are selectively permeabilized in a biomimetic approach by the insertion of the small pore-forming peptide gramicidin (gA) as gA biopores are known to allow the transport of protons and monovalent ions. Whilst gA has been inserted in lipid membranes in numerous research studies, the challenge of inserting the bacterial pore into polymer membranes is greater because of the significant difference between the pore length and the thickness of the polymer membrane (more than 3.5 times). Confocal laser scanning microscopy (CLSM) was used to show that neither the size, nor the morphology of the GUVs was affected by successful insertion of gA and further to visualize the pH change inside the cavity of GUVs in real-time by recording videos. In order to demonstrate the successful insertion of gA, a pH-sensitive dye is encapsulated inside the cavity of GUVs and proton gradients between the environment of GUVs and their inner cavity serves to assess the exchange of protons across the membrane upon gA insertion. The results showed that gA was successfully inserted and remained functional in polymer membranes with thickness of 9.2–12.1 nm. Larger membrane thicknesses did not allow gA insertion, and 12.1 nm represents a limit for the mismatch between the pore length and the membrane thickness. Our gA-GUVs are therefore pH-regulating and maintain their integrity in different pH conditions in a cell-like manner. This bio-mimetic approach to use ion channels with specific selectivity for insertion in polymer membranes is an elegant strategy to develop mimics of biomembranes or for supporting the design of bioreactors.
Next, a functional cell mimetic compartment is developed by the insertion of the bacterial membrane protein (OmpF) in thick synthetic polymer membranes of an artificial GUV compartment that encloses the oxidative enzyme horseradish peroxidase. In this manner a simple and robust cell mimic is designed, that supports a rudimental form of metabolism. The biopore serves as a gate, which allows substrates to enter the cavities of the GUVs, where they are converted into the resorufin-like products by the encapsulated enzyme, and then released in the environments of GUVs.
Our bio-equipped GUVs facilitate the control of specific catalytic reactions in confined micro-scale spaces mimicking cell size and architecture and thus provide a straightforward approach serving to obtain deeper insights in the real-time of biological processes inside cells.
This elegant strategy of equipping both GUV membranes and GUV cavities with biomolecules, opens the way towards cell-like compartments as novel materials with bio-functionality is the combination of synthetic micrometer-sized giant unilamellar vesicles (GUVs) with biomolecules because it enables studying the behavior of biomolecules and processes within confined cavities.
Finally a visionary strategy for creating the first bioinspired molecular factory with functionality as a real cell-mimic based on micrometer-sized giant plasma membrane vesicles (GPMVs) is addressed. GPMVs are cell-derived giant vesicles consisting of an outer compartment architecture (membrane) and an inner composition, which both directly mirror the composition of cells from which they originate except the larger organelles like for example nuclei and Golgi apparatus making measurements easier and are the closest cell-mimic available on the market up to now. In a step towards the development of bioinspired molecular factories with functionality as cell mimics, we generate the next generation of cell mimics by the production of sophisticated hybrid molecular factories based on GPMVs, which are equipped with a synthetic molecular machinery inside their cavities that provides functionality. Such a hierarchical approach in compartmentalization allows the lower-level synthetic functional compartments encapsulated within the cavity of the GPMV to act as independent anatomically discreet units that specialize in their own function, making them nanoscale versions of nature own organelles.
Towards the first bioinspired molecular factory enzyme-equipped polymersomes with a reconstituted membrane protein (OmpF) are encapsulated inside the GPMVs as enzymatic nanocompartment spaces, where they retain their structure and functionality. When substrates were added to the outer solution of the GPMVs, it was shown that they could penetrate both the membrane of the GPMVs and the inner compartment membranes of the synthetic nanoreactors equipped with OmpF pores. In this respect the equipment of the catalytic nanocompartment spaces with OmpF was essential as it allowed the enzyme to perform in the inner cavities. Successful substrate conversion was visualized by following the fluorescent product of the enzymatic reaction (resorufin-like product), which could leave the polymersome and diffuse inside the GPMV cavity. Finally we demonstrate that equipped GPMVs can act as artificial cell mimics – retaining their membrane and inner composition if they are injected into multicellular organisms – Zebrafish embryos. To the best of our knowledge, this is the first time that a molecular factory functioning as a cell-like mimic has been be constructed by using a top down-bottom up approach and has been tested in vivo by taking advantage of the fundamental nature of GPMVs.