Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications.

Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.

Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS).

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

Quantitative size-resolved characterization of mRNA nanoparticles by in-line coupling of asymmetrical-flow field-flow fractionation with small angle X-ray scattering.

We present a generically applicable approach to determine an extensive set of size-dependent critical quality attributes inside nanoparticulate pharmaceutical products. By coupling asymmetrical-flow field-flow fractionation (AF4) measurements directly in-line with solution small angle X-ray scattering (SAXS), vital information such as (i) quantitative, absolute size distribution profiles, (ii) drug loading, (iii) size-dependent internal structures, and (iv) quantitative information on free drug is obtained. Here the validity of the method was demonstrated by characterizing complex mRNA-based lipid nanoparticle products. The approach is particularly applicable to particles in the size range of 100 nm and below, which is highly relevant for pharmaceutical products-both biologics and nanoparticles. The method can be applied as well in other fields, including structural biology and environmental sciences.

Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy.

Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions.

Self-amplifying RNA vaccine protects mice against lethal Ebola virus infection.

Emerging and re-emerging viruses, such as Zaire Ebola virus (EBOV), pose a global threat and require immediate countermeasures, including the rapid development of effective vaccines that are easy to manufacture. Synthetic self-amplifying RNAs (saRNAs) attend to these needs, being safe and strong immune stimulators that can be inexpensively produced in large quantities, using cell-free systems and good manufacturing practice. Here, the first goal was to develop and optimize an anti-EBOV saRNA-based vaccine in terms of its antigen composition and route of administration. Vaccinating mice with saRNAs expressing the EBOV glycoprotein (GP) alone or in combination with the nucleoprotein (NP) elicited antigen-specific immune responses. GP-specific antibodies showed neutralizing activity against EBOV. Strong CD4+ T cell response against NP and GP and CD8+ T cell response against NP were detected by ELISpot assays. Intramuscular vaccination with saRNAs conferred better immune response than intradermal. Finally, mice vaccinated in a prime-boost regimen with saRNAs encoding both GP and NP or with GP alone survived an EBOV infection. In addition, a single dose of GP and NP saRNAs was also protective against fatal EBOV infection. Overall, saRNAs expressing viral antigens represent a promising vaccine platform.

Hybrid core-shell particles for mRNA systemic delivery.

mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines.

3D Melanoma Cocultures as Improved Models for Nanoparticle-Mediated Delivery of RNA to Tumors.

Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention. This study investigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cell culture models of melanoma. Insights into cell-type specific targeting, non-cell-autonomous effects, and penetration capacity in tumor and stroma cells of the mRNA lipoplex nanoparticles were obtained. It was shown that both coculture of different cell types as well as three-dimensional cell growth characteristics can modulate distribution and transfection efficiency of mRNA lipoplex formulations. The results demonstrate that three-dimensional coculture spheroids can provide a valuable surplus of information in comparison to adherent cells. Thus, they may represent in vitro models with enhanced predictivity for the in vivo activity of cancer nanotherapeutics.

pH-Dependent Behavior of Ionizable Cationic Lipids in mRNA-Carrying Lipoplexes Investigated by Molecular Dynamics Simulations.

Lipid-based nanoparticles and lipoplexes containing ionizable lipids are among the most successful nanocarriers for mRNA-based therapies. The molecular structure of these assemblies is still not fully understood, as well as the role played by the ionizable lipids. SAXS experiments have shown that lipoplexes including the ionizable lipid 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), under specific conditions, have a lamellar structure, where lipid bilayers are separated by mRNA-rich layers, with an overall spacing between 6.5 and 8.0 nm and a complex pH-dependence. Here, the structure and dynamics of these lipoplexes are investigated at varying pH and mRNA concentration using multiscale molecular dynamics simulations. It is observed that the interaction between DODMA and RNA is slightly attractive only at low pH levels, while it becomes effectively repulsive at high and intermediate pH. This results into a pH-dependent relocation of the RNA inside the multilayers, from the lipid head groups at low pH to a more uniform distribution inside the hydrophilic slabs of the multilayers at high pH. It is also observed that at high pH, DODMA lipids shift toward the hydrophobic part of the bilayer, consequently increasing their leaflet-flipping rate, a phenomenon which may ultimately affect the fusion process of the lipoplex with the endosomal membrane.

Immunogenicity of stabilized HIV-1 Env trimers delivered by self-amplifying mRNA.

Self-amplifying mRNA (saRNA) represents a promising platform for nucleic acid delivery of vaccine immunogens. Unlike plasmid DNA, saRNA does not require entry into the nucleus of target cells for expression, having the capacity to drive higher protein expression compared to mRNA as it replicates within the cytoplasm. In this study, we examined the potential of stabilized native-like HIV-1 Envelope glycoprotein (Env) trimers to elicit immune responses when delivered by saRNA polyplexes (PLXs), assembled with linear polyethylenimine. We showed that Venezuelan equine encephalitis virus (VEEV) saRNA induces a stronger humoral immune response to the encoded transgene compared to Semliki Forest virus saRNA. Moreover, we characterized the immunogenicity of the soluble and membrane-bound ConSOSL.UFO Env design in mice and showed a faster humoral kinetic and an immunoglobulin G (IgG)2a skew using a membrane-bound design. The immune response generated by PLX VEEV saRNA encoding the membrane-bound Env was then evaluated in larger animal models including macaques, in which low doses induced high IgG responses. Our data demonstrated that the VEEV saRNA PLX nanoparticle formulation represents a suitable platform for the delivery of stabilized HIV-1 Env and has the potential to be used in a variety of vaccine regimens.

Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA.

BACKGROUND: RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. Here, we have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was in vitro transcribed in cell lines and evaluated for expression. RNA-lipoplex nanoparticles were then assembled by complexing RNA with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice. RESULTS: NIS expression was detected on the membrane of two cell lines as early as 6 h after transfection and gradually decreased over 48 h. In vivo and ex vivo PET/MRI of anionic spleen-targeting or cationic lung-targeting NIS-RNA lipoplexes revealed a visually detectable rapid increase of Iodine-124 uptake in the spleen or lung compared to control-RNA-lipoplexes, respectively, with minimal background in other organs except from thyroid, stomach and salivary gland. CONCLUSIONS: The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.

Heparin modulates the cellular uptake of nanomedicines.

Liposomal formulations are used to improve the safety and cellular absorption of conventional drugs by limiting their interaction with phagocytes. The uptake behaviour of these nanocarriers is affected by the blood composition, and accordingly the presence of an anticoagulant in the blood could have a critical impact on the efficiency of nanomedicines. For the negatively charged liposomes, such as AmBisome®, no significant change in the uptake could be observed when co-incubated with heparin and primary phagocytes. Yet, we observed that a peak of the uptake extent of cationic liposomes was reached at a clinically relevant concentration of heparin for phagocytes and cancer cells. Hence, we recommend avoiding treatment of a heparinized patient with cationic nanomedicines because unexpectedly high uptake can occur in phagocytes.

Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering.

Messenger ribonucleic acid (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clinical practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clinical efficacy. In this study, pH-induced changes in internal molecular organization and overall physicochemical characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were determined for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate determination of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ determination of the structural response of the lipoplex systems to changes of the environmental pH similar to that observed for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA.

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.

An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma.

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)-an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma-in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice.

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses.

New vaccine platforms are needed to address the time gap between pathogen emergence and vaccine licensure. RNA-based vaccines are an attractive candidate for this role: they are safe, are produced cell free, and can be rapidly generated in response to pathogen emergence. Two RNA vaccine platforms are available: synthetic mRNA molecules encoding only the antigen of interest and self-amplifying RNA (sa-RNA). sa-RNA is virally derived and encodes both the antigen of interest and proteins enabling RNA vaccine replication. Both platforms have been shown to induce an immune response, but it is not clear which approach is optimal. In the current studies, we compared synthetic mRNA and sa-RNA expressing influenza virus hemagglutinin. Both platforms were protective, but equivalent levels of protection were achieved using 1.25 µg sa-RNA compared to 80 µg mRNA (64-fold less material). Having determined that sa-RNA was more effective than mRNA, we tested hemagglutinin from three strains of influenza H1N1, H3N2 (X31), and B (Massachusetts) as sa-RNA vaccines, and all protected against challenge infection. When sa-RNA was combined in a trivalent formulation, it protected against sequential H1N1 and H3N2 challenges. From this we conclude that sa-RNA is a promising platform for vaccines against viral diseases.

Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration.

Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.

Translating nanoparticulate-personalized cancer vaccines into clinical applications: case study with RNA-lipoplexes for the treatment of melanoma.

The development of nucleic acid based vaccines against cancer has gained considerable momentum through the advancement of modern sequencing technologies and on novel RNA-based synthetic drug formats, which can be readily adapted following identification of every patient's tumor-specific mutations. Furthermore, affordable and individual 'on demand' production of molecularly optimized vaccines should allow their application in large groups of patients. This has resulted in the therapeutic concept of an active personalized cancer vaccine, which has been brought into clinical testing. Successful trials have been performed by intranodal administration of sterile isotonic solutions of synthetic RNA vaccines. The second generation of RNA vaccines which is currently being developed encompasses intravenously injectable RNA nanoparticle formulations (lipoplexes), made up from lipid excipients, denoted RNA(LIP). A first product that has made its way from bench to bedside is a therapeutic vaccine for intravenous administration based on a fixed set of four RNA lipoplex drug products, each encoding for one shared tumor antigen (Lipoplex Melanoma RNA Immunotherapy, 'Lipo-MERIT'). This article describes the steps for translating these novel RNA nanomedicines into clinical trials.

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy.

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid.

Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150mmolL(-1) sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.