Lipid shape and packing are key for optimal design of pH-sensitive mRNA lipid nanoparticles.

The ionizable-lipid component of RNA-containing nanoparticles controls the pH-dependent behavior necessary for an efficient delivery of the cargo-the so-called endosomal escape. However, it is still an empirical exercise to identify optimally performing lipids. Here, we study two well-known ionizable lipids, DLin-MC3-DMA and DLin-DMA using a combination of experiments, multiscale computer simulations, and electrostatic theory. All-atom molecular dynamics simulations, and experimentally measured polar headgroup pKa values, are used to develop a coarse-grained representation of the lipids, which enables the investigation of the pH-dependent behavior of lipid nanoparticles (LNPs) through Monte Carlo simulations, in the absence and presence of RNA molecules. Our results show that the charge state of the lipids is determined by the interplay between lipid shape and headgroup chemistry, providing an explanation for the similar pH-dependent ionization state observed for lipids with headgroup pKa values about one-pH-unit apart. The pH dependence of lipid ionization is significantly influenced by the presence of RNA, whereby charge neutrality is achieved by imparting a finite and constant charge per lipid at intermediate pH values. The simulation results are experimentally supported by measurements of α-carbon 13C-NMR chemical shifts for eGFP mRNA LNPs of both DLin-MC3-DMA and DLin-DMA at various pH conditions. Further, we evaluate the applicability of a mean-field Poisson-Boltzmann theory to capture these phenomena.

Screening of the binding affinity of serum proteins to lipid nanoparticles in a cell free environment.

Lipid nanoparticles (LNPs) are promising drug and gene carriers. Upon intravenous administration, LNPs' experience different degree of cellular uptake depending on their formulation. Currently, in vitro and in vivo studies are the gold standard for assessing the fate of nano carriers once administered, but they are time consuming and expensive. In this work, we propose a time and cost-effective method to screen a wide range of LNP formulations and select the most promising candidates for in vitro and in vivo studies. Two different approaches were explored to investigate the binding affinity between LNPs and serum proteins using sensor functionalisation with either protein specific antibody or PEG specific antibody. The first approach allowed to identify the presence of a specific protein in the protein corona of lipid particles (reconstituted and native high-density lipoproteins (rHDL and HDL), and low-density lipoproteins LDL); while the second one provided a versatile platform for the immobilisation of pegylated-particles in order to follow the interaction with serum proteins and hence predict the composition of LNP protein corona. Sensing was done using Quartz Crystal Microbalance with Dissipation (QCM-D) but the approach is extendable to other surface sensing techniques such as Surface Plasmon Resonance (SPR) or ellipsometry.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles.

The development of safe and efficacious gene vectors has limited greatly the potential for therapeutic treatments based on messenger RNA (mRNA). Lipid nanoparticles (LNPs) formed by an ionizable cationic lipid (here DLin-MC3-DMA), helper lipids (distearoylphosphatidylcholine, DSPC, and cholesterol), and a poly(ethylene glycol) (PEG) lipid have been identified as very promising delivery vectors of short interfering RNA (siRNA) in different clinical phases; however, delivery of high-molecular weight RNA has been proven much more demanding. Herein we elucidate the structure of hEPO modified mRNA-containing LNPs of different sizes and show how structural differences affect transfection of human adipocytes and hepatocytes, two clinically relevant cell types. Employing small-angle scattering, we demonstrate that LNPs have a disordered inverse hexagonal internal structure with a characteristic distance around 6 nm in presence of mRNA, whereas LNPs containing no mRNA do not display this structure. Furthermore, using contrast variation small-angle neutron scattering, we show that one of the lipid components, DSPC, is localized mainly at the surface of mRNA-containing LNPs. By varying LNP size and surface composition we demonstrate that both size and structure have significant influence on intracellular protein production. As an example, in both human adipocytes and hepatocytes, protein expression levels for 130 nm LNPs can differ as much as 50-fold depending on their surface characteristics, likely due to a difference in the ability of LNP fusion with the early endosome membrane. We consider these discoveries to be fundamental and opening up new possibilities for rational design of synthetic nanoscopic vehicles for mRNA delivery.

Probing adsorption of DSPE-PEG2000 and DSPE-PEG5000 to the surface of felodipine and griseofulvin nanocrystals.

Nanosized formulations of poorly water-soluble drugs show great potential due to improved bioavailability. In order to retain colloidal stability, the nanocrystals need to be stabilized. Here we explore the use of the poly(ethylene glycol) (PEG) conjugated phospholipids DSPE-PEG2000 and DSPE-PEG5000 as stabilizers of felodipine and griseofulvin nanocrystals. Nanocrystal stability and physicochemical properties were examined and the interaction between the PEGylated lipids and the nanocrystal surface as well as a macroscopic model surface was investigated. Using quartz crystal microbalance with dissipation monitoring both mass adsorption and the thickness of the adsorbed layer were estimated. The results indicate that the PEGylated lipids are adsorbed as flat layers of around 1-3nm, and that DSPE-PEG5000 forms a thicker layer compared with DSPE-PEG2000. In addition, the mass adsorption to the drug crystals and the model surface are seemingly comparable. Furthermore, both DSPE-PEG2000 and DSPE-PEG5000 rendered stable drug nanocrystals, with a somewhat higher surface binding and stability seen for DSPE-PEG2000. These results suggest DSPE-PEG2000 and DSPE-PEG5000 as efficient nanocrystal stabilizers, with DSPE-PEG2000 giving a somewhat higher surface coverage and superior colloidal stability, whereas DSPE-PEG5000 shows a more extended structure that may have advantages for prolongation of circulation time in vivo and facilitation for targeting modifications.

Interactions of PAMAM dendrimers with negatively charged model biomembranes.

We have investigated the interactions between cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-L-serine (PS) phospholipids. Two types of model membranes were used: solid-supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free-standing bilayers, formed at the interface between two aqueous droplets in oil (droplet interface bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The charge density of the bilayer affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimer-bilayer interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells.

Interactions of small dendrimers with sodium dodecyl sulfate at the air-water interface.

We have determined how the bulk behavior of mixtures of small cationic poly(amidoamine) dendrimers (generation 2, PAMAM-G2) and sodium dodecyl sulfate (SDS) affects the structure and composition of the adsorbed layers at the air-water interface. The aim is to reveal how the size of a well-defined hyperbranched polyelectrolyte affects the interfacial and bulk solution behavior of mixtures with oppositely charged surfactants, when the size of the polyelectrolyte approaches that of the surfactant. A combination of electrophoretic mobility, UV-vis spectroscopy, dynamic light scattering, and small-angle X-ray scattering measurements have been employed to characterize the interactions in the bulk solution. PAMAM-G2 associates strongly with SDS in the bulk, forming large aggregates where the size and the charge depend on the bulk composition. We show that kinetically trapped aggregates can be formed at compositions outside the equilibrium two-phase region, and the positively charged aggregates are larger than the negative ones. Surface tensiometry, neutron reflectometry, and ellipsometry have been used to reveal the properties of the interfacial layers. The interfacial structures formed depend strongly on the bulk composition: structured layers are present for samples inside the two-phase region, whereas intact nanostructured aggregates adsorb for samples just outside the two-phase region. The interfacial behavior of PAMAM-G2/SDS mixtures is compared with that of small amines or multivalent ions and oppositely charged surfactants. The implications of aggregate adsorption, dissociation, and spreading processes are discussed as well as the potential of small dendrimers for applications involving the delivery of functional molecules to interfaces.

Direct impact of nonequilibrium aggregates on the structure and morphology of Pdadmac/SDS layers at the air/water interface.

We discuss different nonequilibrium mechanisms by which bulk aggregates directly modify, and can even control, the interfacial structure and morphology of an oppositely charged polyelectrolyte/surfactant (P/S) mixture. Samples are categorized at the air/water interface with respect to the dynamic changes in the bulk phase behavior, the bulk composition, and the sample history using complementary surface-sensitive techniques. First, we show that bulk aggregates can spontaneously interact with the adsorption layer and are retained in it and that this process occurs most readily for positively charged aggregates with an expanded structure. In this case, key nonequilibrium issues of aggregate dissociation and spreading of surface-active material at the interface have a marked influence on the macroscopic interfacial properties. In a second distinct mechanism, aggregates inherently become trapped at the interface during its creation and lateral flocculation occurs. This irreversible process is most pronounced for aggregates with the lowest charge. A third mechanism involves the deposition of aggregates at interfaces due to their transport under gravity. The specificity of this process at an interface depends on its location and is mediated by density effects in the bulk. The prevalence of each mechanism critically depends on a number of different factors, which are outlined systematically here for the first time. This study highlights the sheer complexity by which aggregates can directly impact the interfacial properties of a P/S mixture. Our findings offer scope for understanding seemingly mysterious irreproducible effects which can compromise the performance of formulations in wide-ranging applications from foams to emulsions and lubricants.

Multilayers at interfaces of an oppositely charged polyelectrolyte/surfactant system resulting from the transport of bulk aggregates under gravity.

We show conclusively that multilayers at interfaces of an oppositely charged polyelectrolyte/surfactant system can result from the transport under gravity of bulk aggregates with internal molecular structure. This process was demonstrated by measurements of poly(diallyldimethylammonium chloride)/sodium dodecyl sulfate solutions at the air/liquid and solid/liquid interfaces using neutron reflectometry. In the latter case a novel approach involving the comparison of reflection up versus down measurements provided key evidence. Interfacial multilayers indicated by a strong Bragg peak and clear off-specular scattering are exhibited under three conditions: (1) only for samples in the phase separation region, (2) only for fresh samples where a suspension of bulk aggregates remains in solution, and (3) only when the creaming or sedimentation process occurs in the direction of the interface under examination. This bulk transport mechanism is an alternative route of formation of interfacial multilayers to surface induced self-assembly. The two processes evidently give rise to interfaces with very different structural and rheological properties. Such directionality effects in the formation of nanostructured liquid interfaces may have implications for a broad range of soft matter and biophysical systems containing macromolecules such as synthetic polymers, proteins, or DNA.

Effects of bulk colloidal stability on adsorption layers of poly(diallyldimethylammonium chloride)/sodium dodecyl sulfate at the air-water interface studied by neutron reflectometry.

We show for the oppositely charged system poly(diallyldimethylammonium chloride)/sodium dodecyl sulfate that the cliff edge peak in its surface tension isotherm results from the comprehensive precipitation of bulk complexes into sediment, leaving a supernatant that is virtually transparent and a depleted adsorption layer at the air/water interface. The aggregation and settling processes take about 3 days to reach completion and occur at bulk compositions around charge neutrality of the complexes which lack long-term colloidal stability. We demonstrate excellent quantitative agreement between the measured surface tension values and a peak calculated from the surface excess of surfactant in the precipitation region measured by neutron reflectometry, using the approximation that there is no polymer left in the liquid phase. The nonequilibrium nature of the system is emphasized by the production of very different interfacial properties from equivalent aged samples that are handled differently. We go on to outline our perspective on the "true equilibrium" state of this intriguing system and conclude with a comment on its practical relevance given that the interfacial properties can be so readily influenced by the handling of kinetically trapped bulk aggregates.