Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nano-delivery system for therapeutic nucleic acids. The great effort put in the development of ionizable lipids with increased in vivo potency brought LNPs from the laboratory benches to the FDA approval of patisiran in 2018 and the ongoing clinical trials for mRNA-based vaccines against SARS-CoV-2. Despite these success stories, several challenges remain in RNA delivery, including what is known as “endosomal escape.” Reaching the cytosol is mandatory for unleashing the therapeutic activity of RNA molecules, as their accumulation in other intracellular compartments would simply result in efficacy loss. In LNPs, the ability of ionizable lipids to form destabilizing non-bilayer structures at acidic pH is recognized as the key for endosomal escape and RNA cytosolic delivery. This is motivating a surge in studies aiming at designing novel ionizable lipids with improved biodegradation and safety profiles. In this work, we describe the journey of RNA-loaded LNPs across multiple intracellular barriers, from the extracellular space to the cytosol. In silico molecular dynamics modeling, in vitro high-resolution microscopy analyses, and in vivo imaging data are systematically reviewed to distill out the regulating mechanisms underlying the endosomal escape of RNA. Finally, a comparison with strategies employed by enveloped viruses to deliver their genetic material into cells is also presented. The combination of a multidisciplinary analytical toolkit for endosomal escape quantification and a nature-inspired design could foster the development of future LNPs with improved cytosolic delivery of nucleic acids

Heat Transfer Through a Rarefied Polyatomic Gas Confined Between Parallel Plates

The conductive heat transfer through rarefied polyatomic gases confined between parallel plates maintained at different temperatures is investigated. The approach is based on the Holway kinetic model as well as on the Boltzmann equation via the DSMC scheme supplemented by the Borgnakke-Larsen collision model. The hard sphere model is applied. Results are presented for the total as well as for the translational and rotational parts of the heat flux in the whole range of the Knudsen number and for various temperature differences. The effect of the thermal accommodation coefficient is also examined. The results obtained by the Holway model and the DSMC method are in very good agreement and they compare well with experimental data available in the literature. Qualitatively the behavior of the dimensionless total macroscopic quantities is similar to that of the monatomic ones but the heat fluxes of polyatomic gases are significantly higher than the corresponding monatomic ones. It is clearly demonstrated that heat transfer simulations through rarefied polyatomic gases in MEMS cannot rely on typical monatomic modeling and on the contrary, reliable kinetic modeling for polyatomic gases must be implemented

Helical Inclusions in Phospholipid Membranes: Lipid Adaptation and Chiral Order

The lipid bilayer is a flexible matrix that is able to adapt in response to the perturbation induced by inclusions, such as peptides and proteins. Here we use molecular dynamics simulations with a coarse-grained model to investigate the effect of a helical inclusion on a lipid bilayer in the liquid disordered phase. We show that the helical inclusion induces a collective tilt of acyl chains, with a small, yet unambiguous difference between a right- and a left-handed inclusion. This behavior is rationalized using the elastic continuum theory: The magnitude of the chiral (twist) deformation of the bilayer is determined by the interaction at the lipid/inclusion interface, and the decay length is controlled by the elastic properties of the bilayer. The lipid reorganization can thus be identified as a generic mechanism that, together with specific interactions, contributes to chiral recognition in phospholipid bilayers. An enhanced response is expected in highly ordered environments, such as rafts in biomembranes, with a potential impact on membrane-mediated interactions between inclusions

α-Helical structures drive early stages of self-assembly of amyloidogenic amyloid polypeptide aggregate formation in membranes

The human islet amyloid polypeptide (hIAPP) is the primary component in the toxic islet amyloid deposits in type-2 diabetes. hIAPP self-assembles to aggregates that permeabilize membranes and constitutes amyloid plaques. Uncovering the mechanisms of amyloid self-assembly is the key to understanding amyloid toxicity and treatment. Although structurally similar, hIAPP's rat counterpart, the rat islet amyloid polypeptide (rIAPP), is non-toxic. It has been a puzzle why these peptides behave so differently. We combined multiscale modelling and theory to explain the drastically different dynamics of hIAPP and rIAPP: The differences stem from electrostatic dipolar interactions. hIAPP forms pentameric aggregates with the hydrophobic residues facing the membrane core and stabilizing water-conducting pores. We give predictions for pore sizes, the number of hIAPP peptides, and aggregate morphology. We show the importance of curvature-induced stress at the early stages of hIAPP assembly and the a-helical structures over ß-sheets. This agrees with recent fluorescence spectroscopy experiments

Conductive heat transfer in a rarefied polyatomic gas confined between coaxial cylinders

The conductive heat transfer through rarefied gases composed by rigid rotators and confined between coaxially placed cylinders maintained at different temperatures is investigated on the basis of the Holway and Rykov kinetic models as well as on the Boltzmann equation via the DSMC scheme supplemented by the Borgnakke-Larsen collision model. The translational and rotational parts as well as the total temperature and heat flux distributions are computed and their behavior in terms of the gas rarefaction, the temperature difference between the cylinders and the ratio of the radii is investigated. The two kinetic models and the DSMC method provide results which are in good agreement for HS and VHS molecules. Furthermore, very good agreement with available experimental data for polyatomic gases has been observed at small and large temperature differences validating the implemented modeling. Qualitatively the behavior of the dimensionless total macroscopic quantities is similar to that of the monatomic ones. Quantitatively however, the heat fluxes of polyatomic gases are significantly higher than the corresponding monatomic ones. Also, as the amount of the elastic compared to the inelastic collisions is increased, the translational heat fluxes are increased and they tend to the monatomic ones, while always the rotational heat fluxes are about 50% and 75% of the translational ones for linear and non-linear rigid rotators, respectively. It is clearly demonstrated that heat transfer simulations through rarefied polyatomic gases in MEMS and other devices cannot rely on typical monatomic modeling. On the contrary, reliable kinetic modeling for polyatomic gases must be implemented. (C) 2014 Elsevier Ltd. All rights reserved

A statistical assessment of persistent micropollutants occurrence, fate and health risk using censored water quality data

In the recent years, the presence of micropollutants in drinking water has become an issue of growing global concern. Great attention is paid to persistent toxic micropollutants that belong to several families (e.g. pesticides, perfluorinated compounds, pharmaceuticals, endocrine disrupting compounds) and are present at trace concentrations (ranging from ng/l to μg/l) in aquatic environments [1]. Due to their low concentration, monitoring databases are usually rich in censored data (e.g. samples with concentrations reported below the limit of quantification (LOQ)) that are typically eliminated or replaced with a value between 0 and LOQ [2]. These traditional methods present some limitations and can lead to erroneous conclusions on the presence of persistent micropollutants in the source water, treatment efficiencies, quality of the produced water and associated human health risk. Alternative methods, based on the principles of survival analysis, allow to estimate the statistical distribution of the whole dataset, combining the values above the LOQ with the information contained in the proportion of censored data [3]. The methods applied in this work are Maximum Likelihood Estimation or non-parametric techniques (e.g. Kaplan-Meier). Monitoring data of 5,362 groundwater (GW) and 12,344 drinking water samples collected from 2012 to 2017 in the city of Milan, Italy were analysed. Several persistent micropollutants, including pesticides and perfluorinated compounds, were selected for this study. This study demonstrated the benefits of the innovative methods in the assessment of data statistical distribution, highlighting the more accurate estimation of the distribution median, 95° and 98° quantiles, especially for high percentages of censored data. The resulting statistical distributions were used for several applications: time trend evaluation in GW micropollutant concentrations, optimization of well management, treatment efficiency evaluation. Moreover, they have been applied to assess the residual health risk associated with low concentration micropollutants and the risk reduction resulting by treatment and/or management intervention in the drinking water treatment plants. This study highlighted high discrepancy in the results obtained with traditional and innovative techniques related to the evaluation of the presence, fate and health risk associated to persistent and toxic micropollutants

Protein Adsorption at the Air-Water Interface by a Charge Sensing Interferometric Technique

Protein uptake at the interface of a millimeter-sized air bubble in water is investigated by a recently developed differential interferometric technique. The technique allows the study of capillary waves with amplitudes around 10-9 m, excited at the surface of the bubble by an electric field of intensity on the order of 10 V/cm. When one studies the resonant modes of the bubble (radial and shape modes), it is possible to assess variations of interfacial properties and, in particular, of the net surface charge as a function of bulk protein concentration. Sensing the interfacial charge, the technique enables us to follow the absorption process in conditions of low concentrations, not easily assessable by other methods. We focus on bovine serum albumin (BSA) and lysozyme as representatives of typical globular proteins. To provide comprehensive insight into the novelty of the technique, we also investigated the equilibrium adsorption of sodium dodecyl sulfate (SDS) ionic surfactant for bulk concentrations at hundreds of times lower than the Critical Micelle Concentration (CMC). Results unveil how the absorption of charged molecules affects the amplitudes of the bubble resonant modes even before affecting the frequencies in a transition-like fashion. Different adsorption models are proposed and developed. They are validated against the experimental findings by comparing frequency and amplitude data. By measuring the charging rate of the bubble interface, we have followed the absorption kinetics of BSA and lysozyme recognizing a slow, energy barrier limited phenomena with characteristic times in agreement with data in the literature. The evaluation of the surface excess concentration (\u393) of BSA and SDS at equilibrium is obtained by monitoring charge uptake. At the investigated low bulk concentrations, reliable comparisons with literature data from equilibrium surface tension isotherm models are reported

Transient step-like kinetics of enzyme reaction on fragmented-condensed micellar substrates

We followed the process of enzymatic digestion of ganglioside GD1a, operated by sialidase on aggregated micelles. The product is the ganglioside GM1, lacking the external sialic acid. The structural aspects and the kinetics connected to the process occurring on a fragmented-condensed substrate, the ganglioside micelles, are investigated by small angle X-ray scattering (SAXS). Observed at short times, the kinetics of the reaction shows a transient step-like decay, while it tends to a smooth Michaelis-Menten kinetics in the late stages. We propose a model, based on the fragmented-condensed nature of the substrate, that well reproduces the experimental observation without invoking any feedback mechanism in the reaction, usually required for an oscillatory behavior. The model predicts an initial regime dominated by the strict enzyme-substrate interaction, with a step-like appearance

Embedding Hydrogels into Microfluidic Chips: Vascular Transport Analyses and Drug Delivery Optimization

Current two-dimensional in vitro culture conditions do not accurately replicate the hierarchical complexity of the in vivo tissue microenvironment. Microfluidic chips have emerged as alternative tools that address interdisciplinary aspects of tissue/organ and disease modeling and aid the investigation of nanomedicine delivery and efficacy. In this chapter, we discuss the design considerations for hydrogel-embedded microfluidic chips developed in our laboratory. We also describe different nanoconstructs and nanomedicines, which have been analyzed using hydrogel-embedded microfluidic chips. Finally, we highlight computational methods used to study particles under flow conditions and briefly mention the potential applications of the approaches that bring together all these aspects of biomedical engineering to advance drug development and nanomedicine toward the clinical setting.</p