key: cord-0995307-1rzmzn6a authors: Gregoriadis, Gregory title: Liposomes and mRNA: two technologies together create a COVID-19 vaccine date: 2021-08-06 journal: Med Drug Discov DOI: 10.1016/j.medidd.2021.100104 sha: 543530cf710261c5d094f1c7bab2adde6e7cb6c2 doc_id: 995307 cord_uid: 1rzmzn6a The urgency to understand and modify immune responses has never been as great universally as during the present Coronavirus time. It has been suggested that using established techniques, a small piece of the so-called spike protein of the Coronavirus injected into humans in the form of mRNA could raise an immune response against the expressed protein, in turn killing or inactivating the invading Coronavirus. Unfortunately, however, the mRNA was found to be too vulnerable to survive in the body long enough on injection to produce the spike protein and an immune response to it. But as it happens, a solution was to hand, one waiting to be discovered. In the mid-1960's, the biophysicist Alec Bangham and colleagues at the Babraham Institute in Cambridge observed bimolecular leaflet membrane structures formed on addition of water to dry phospholipid [1, 2] . The membrane structures were initially named 'Banghasomes', then 'Phospholipid vesicles', 'Liposomes' and more recently 'Lipid nanoparticles', a name that does not seem to add significantly to its meaning as liposomes can also be of nano size. Regardless, 'Liposomes' have dominated as a name. And because of their similarity to cell membranes, liposomes were adopted as a model for the study of cell membrane biophysics. A few years later, however, the future of liposomes was to change direction from serving as a model of cell membranes to that of a drug delivery system. The author, together with Brenda Ryman at the Royal Free Hospital School of Medicine, London, in search of a system that could deliver drugs to specific areas in the body, used liposomes to that effect. Initial work in animals confirmed liposomes as a promising drug delivery system [3] [4] [5] . It was eventually adopted by a myriad of workers, with dozens of drugs and other agents entrapped in liposomes of varying size (e.g., 20 nm to several microns in diameter), lipid composition, surface charge, ability to accommodate water soluble or lipid soluble materials and, if needed, to provide a pegylated vesicle surface, thus rendering liposomes a delivery system of multiple uses. These include the licencing of a plethora of therapeutics, for instance for cancer and antimicrobial therapy [6] [7] [8] [9] . injected subcutaneously or intravenously with tetanus toxoid or with the toxoid entrapped in negatively charged liposomes [10, 11] . The antibody response to the liposomal toxoid was far greater than that of the toxoid as such, thus confirming liposomes as an immunological adjuvant for vaccines. Positively charged liposomes were superior as adjuvants to those that were negatively charged. Moreover, whereas re-injection of pre-immunised animals with tetanus toxoid (as when booster injections are required) led to serum sickness and death, animals re-injected with the liposomal toxoid remained healthy. The immunological adjuvant property of liposomes was further demonstrated using the hepatitis B surface antigen [12] , and by the production of two additional liposome-based vaccines by Berna, namely Epaxal for hepatitis A, and Inflexal V for influenza, both approved for use in humans. It was only to be expected that sooner or later, liposomes would be used in genomic vaccines as well as other liposome-based applications of nucleic acids. This was in fact predicted at the very beginning of liposome research on the drug delivery potential of liposomes [5] . Because of the vulnerability of liposomes in the circulating blood and the potential leakage of entrapped labile solutes (for example, nucleic acids), it was essential that liposomal membranes were rendered stable in the presence of blood. This was achieved by the judicious choice of liposomal lipids. We were able to show that liposomal membrane stability in blood is achieved by using a long chain phospholipid [13] , for instance dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, or dioleoyl phosphatidyl ethanolamine, each supplemented with equimolar cholesterol [14] . Such liposomes were also supplemented with a cationic lipid, for instance 1,2-dioleoyl-3 (trimethylammonium) propane (DOTAP) which binds to the nucleic acid thus leading to high values of nucleic acid (e.g., mRNA) association with, or entrapment into, liposomes. Alternatively, one could use an equally effective ionizable aminolipid [15] . Our work with plasmid DNA (pRc/CMV HBS coding for the hepatitis B surface antigen, S region) entrapped in liposomes of a lipid composition identical to that just described, has shown that the injected liposomal plasmid not only expresses itself to produce the protein antigen, both humoral and cell mediated immune responses to the antigen are far greater than when the free plasmid is injected [16] . In view of the above, the following scenario for the creation of an anti-COVID-19 vaccine was made possible: mRNA coding for the protein spike of the Coronavirus, would be entrapped into liposomes that are designed to remain stable in the circulating blood [17] until they are taken up by phagocytic cells in the body by endocytosis [5] . It has been suggested that within the cytosol, there will be destabilisation of the endosomal membrane whereupon, through lateral diffusion of anionic lipids from the cytoplasm-facing endosomal monolayer, mRNA will be displaced from the complex and released into the cytosol [18] . The mRNA will then be expressed as the spike protein in turn promoting an immune response to it that will kill or inactivate the invading virus. The mechanism by which liposomes act as an immunological adjuvant to augment the immune response to the spike protein, is not clear at present. It is a fact, however, that a liposome-based mRNA anti-COVID-19 vaccine has been created by Pfizer/BioNTech and Moderna, and already administered worldwide into millions. Both vaccines [19] , developed within the last two or three years, are made of components [10, 11, 13, 14, 18] previously shown to maintain the stability of liposomes in blood and to promote immune responses. The components include distearoyl phosphatidyl choline (the lipid that provides the liposomal bilayer and hence the basis of liposomal adjuvanticity), cholesterol (which contributes to the stability of the liposomal membrane in the presence of blood), and a cationic or an ionizable lipid that contributes to improving liposomal adjuvanticity [19] . The presence of the pegylated lipid in both vaccines ensures the production of liposomes free of vesicle surface to surface interactions. It follows that without the liposomal bilayers and the components within, there would not be a liposome-based anti-COVID-19 vaccine. Table 1 . Advantages of using liposomes to deliver mRNA into cells. Liposomes are biodegradable, easy to prepare and can entrap mRNA quantitatively. 2 Liposome-entrapped mRNA fully protected from nuclease attack in the blood circulation. 3 Liposomal mRNA enters the cytoplasm of cells by endocytosis. Cationic liposomal mRNA escapes the lysosomotropic pathway to end up intact in the cytoplasm. Within the cytoplasm, mRNA is expressed as the spike protein whereupon, by an as yet unclear mechanism, liposomes or their remnants exert their immunological adjuvant action. Regardless of the minutiae of vesicle onomatopoeia, it is remarkable that it took fifty or so years for the two technologies, mRNA and lipid vesicles, to come together at more or less the same time of their need. Diffusion of univalent ions across the lamellae of swollen phospholipids Enzyme entrapment in liposomes Fate of protein-containing liposomes injected into rats. An approach to the treatment of storage diseases Lysosomal localization of -fructofuranosidase-containing liposomes injected into rats The carrier potential of liposomes in biology and medicine Targeting of drugs: implications in medicine Liposomal drug delivery systems: from concept to clinical applications Liposomes Came First: The Early History of Liposomology Liposomes as immunological adjuvants Entrapment of proteins in liposomes prevents allergic reactions in pre-immunised mice Hepatitis B surface antigen-containing liposomes enhance humoral and cell-mediated immunity to the antigen Cholesterol content of small unilamellar liposomes controls phospholipid loss to high density lipoproteins in the presence of serum The phospholipid component of small unilamellar liposomes controls the rate of clearance of entrapped solutes from the circulation Delivery of selfamplifying mRNA vaccines by cationic lipid nanoparticles: The impact of cationic lipid selection Genetic vaccines: strategies for optimization Liposome-mediated DNA vaccination: the effect of vesicle composition How are nucleic acids released in cells from cationic lipid-nucleic acid complexes? Lipid Nanoparticles-From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement The author is grateful to Sir Brian Heap at the University of Cambridge for insightful discussions during the preparation of the manuscript. In the interest of transparency, we ask you to disclose all relationships/activities/interests listed below that are related to the content of your manuscript. "Related" means any relation with forprofit or not-for-profit third parties whose interests may be affected by the content of the manuscript. Disclosure represents a commitment to transparency and does not necessarily indicate a bias. If you are in doubt about whether to list a relationship/activity/interest, it is preferable that you do so. The following questions apply to the author's relationships/activities/interests as they relate to the current manuscript only. The author's relationships/activities/interests should be defined broadly. 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