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Advances in lipid nanoparticle (LNP) design have contributed notably to the emergence of the current clinically approved mRNA-based vaccines and are of high relevance for delivering mRNA to combat diseases where therapeutic alternatives are sparse. LNP-assisted mRNA delivery utilizes ionizable lipid-mediated cargo translocation across the endosomal membrane driven by the acidification of the endosomal environment. However, this process occurs at a low efficiency, a few percent at the best. Utilizing surface-sensitive fluorescence microscopy with a single LNP and mRNA resolution, we have investigated pH-controlled interactions between individual LNPs and a planar anionic supported lipid bilayer (SLB) formed on nanoporous silica, mimicking the electrostatic conditions of the early endosomal membrane. For LNPs with an average diameter of 140 nm, fusion with the anionic SLB preferentially occurred when the pH was reduced from 6.6 to 6.0. Furthermore, there was a delay in the onset of LNP fusion after the pH drop, and upon fusion, a significant fraction (>70%) of mRNA was released into the acidic solution representing the endosomal lumen, while a fraction of mRNA remained bound to the SLB even after reversing the pH to neutral cytosolic conditions. Finally, a comparison of the fusion efficiency of two LNP formulations with different surface concentrations of gel-forming lipids correlated with differences in the protein translation efficiency previously observed in human primary cell transfection studies. Together, these findings emphasize the relevance of biophysical investigations of ionizable lipid-containing LNP-assisted mRNA delivery mechanisms while potentially also offering means to optimize the design of LNPs with enhanced endosomal escape capabilities.

Polycatecholamine coatings have attracted significant attention in the past 10 years owing to their ability to functionalize a wide range of materials. Here we apply the use of such coatings to drug nanocrystals, made from a poorly soluble drug compound, to postfunctionalize the nanocrystal surface with the aim of providing steric stabilization and extending their circulation time after intravenous injection. We show that both polydopamine and polynorepinephrine can be used to successfully modify drug nanocrystals and subsequently incorporate end-functionalized PEG to the surface. Even though high grafting densities of PEG were achieved, we observed rapid clearance and increased liver uptake for polycatecholamine functionalized drug nanocrystals. Using both surface sensitive model systems and protein corona profiling, we determine that the rapid clearance was correlated with an increase in adsorption of proteins involved in coagulation to the polycatecholamine surface, with fibrinogen being the most abundant. Further analysis of the most abundant proteins revealed a significant increase in thiol-rich proteins on polycatecholamine coated surfaces. The observed interaction with coagulation proteins highlights one of the current challenges using polycatecholamines for drug delivery but might also provide insights to the growing use of these materials in hemostatic applications.

Hydrophobic ion pairing (HIP) can successfully increase the drug loading and control the release kinetics of ionizable hydrophilic drugs, addressing challenges that prevent these molecules from reaching the clinic. Nevertheless, polymeric nanoparticle (PNP) formulation development requires trial-and-error experimentation to meet the target product profile, which is laborious and costly. Herein, we design a preformulation framework (solid-state screening, computational approach, and solubility in PNP-forming emulsion) to understand counterion–drug–polymer interactions and accelerate the PNP formulation development for HIP systems. The HIP interactions between a small hydrophilic molecule, AZD2811, and counterions with different molecular structures were investigated. Cyclic counterions formed amorphous ion pairs with AZD2811; the 0.7 pamoic acid/1.0 AZD2811 complex had the highest glass transition temperature (Tg; 162 °C) and the greatest drug loading (22%) and remained as phase-separated amorphous nanosized domains inside the polymer matrix. Palmitic acid (linear counterion) showed negligible interactions with AZD2811 (crystalline-free drug/counterion forms), leading to a significantly lower drug loading despite having similar log Pand pKawith pamoic acid. Computational calculations illustrated that cyclic counterions interact more strongly with AZD2811 than linear counterions through dispersive interactions (offset π–π interactions). Solubility data indicated that the pamoic acid/AZD2811 complex has a lower organic phase solubility than AZD2811-free base; hence, it may be expected to precipitate more rapidly in the nanodroplets, thus increasing drug loading. Our work provides a generalizable preformulation framework, complementing traditional performance-indicating parameters, to identify optimal counterions rapidly and accelerate the development of hydrophilic drug PNP formulations while achieving high drug loading without laborious trial-and-error experimentation.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

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.

The purpose of this study was to investigate in vivointestinal precipitation of a model drug mebendazole, a basic BCS class II drug, using dogs with intestinal stomas for administration or sampling. After oral administration of a solution with an expected intestinal supersaturation of approximately 20 times the solubility, the measured supersaturation in dog intestinal fluid (DIF) was up to 10 times and, on average, only 11% of the given dose was retrieved as solid drug in the collected fluid from the stoma. The drug was rapidly absorbed with >90% of the total systemic exposure reached within three hours after duodenal administration of a solution. In silicoabsorption modeling showed that in vivodata were reasonably well described by a nonprecipitating solution. An in vitromodel of precipitation in DIF predicted that the intestinal concentration of dissolved mebendazole would be less than 1/5 of the initial concentration within 10 min at concentrations comparable to in vivo. It was concluded that intestinal precipitation did not have any major influence on mebendazole absorption. The extent of precipitation was overpredicted in vitrogiven the in vivoabsorption rate, and further work is needed to identify in vitrofactors that could enable more accurate in vivopredictions of intestinal precipitation from solutions.

Dissolution models require, at their core, an accurate diffusion model. The accuracy of the model for diffusion-dominated dissolution is particularly important with the trend toward micro- and nanoscale drug particles. Often such models are based on the concept of a “diffusion layer.” Here a framework is developed for diffusion-dominated dissolution models, and we discuss the inadequacy of classical models that are based on an unphysical constant diffusion layer thickness assumption, or do not correctly modify dissolution rate due to “confinement effects”: (1) the increase in bulk concentration from confinement of the dissolution process, (2) the modification of the flux model (the Sherwood number) by confinement. We derive the exact mathematical solution for a spherical particle in a confined fluid with impermeable boundaries. Using this solution, we analyze the accuracy of a time-dependent “infinite domain model” (IDM) and “quasi steady-state model” (QSM), both formally derived for infinite domains but which can be applied in approximate fashion to confined dissolution with proper adjustment of a concentration parameter. We show that dissolution rate is sensitive to the degree of confinement or, equivalently, to the total concentration Ctot. The most practical model, the QSM, is shown to be very accurate for most applications and, consequently, can be used with confidence in design-level dissolution models so long as confinement is accurately treated. The QSM predicts the ratio of diffusion layer thickness to particle radius (the Sherwood number) as a constant plus a correction that depends on the degree of confinement. The QSM also predicts that the time required for complete saturation or dissolution in diffusion-controlled dissolution experiments is singular (i.e., infinite) when total concentration equals the solubility. Using the QSM, we show that measured differences in dissolution rate in a diffusion-controlled dissolution experiment are a result of differences in the degree of confinement on the increase in bulk concentration independent of container geometry and polydisperse vs single particle dissolution. We conclude that the constant diffusion-layer thickness assumption is incorrect in principle and should be replaced by the QSM with accurate treatment of confinement in models of diffusion-controlled dissolution.

A simple turbidimetric method was developed to measure the bulk concentration of drug in nanosuspensions. The bulk concentrations measured were in the range from 1 μM to 1 mM. The accuracy of the method was checked by determination of the bulk concentration of crystalline nanosuspensions, i.e., the crystalline solubility, which compared favorably to solubilities measured by a conventional method. Results obtained for amorphous nanosuspensions agreed with predictions using a theory describing the relative solubility between a supercooled liquid and a crystal. Further, it was found that the bulk concentration in Ostwald ripening inhibited amorphous nanosuspensions and could be lowered by incorporation of higher amounts of the inhibitor, in agreement with predictions using the Bragg−Williams theory of nonideal solutions.

Amorphous drug nanosuspensions are prone to particle growth due to Ostwald ripening. By incorporating a second component of extremely low aqueous solubility, Ostwald ripening can be inhibited. These studies indicate that to inhibit ripening, the drug/inhibitor mixture (in the particles) must form a single phase. The drug/inhibitor mixture can be characterized by the interaction parameter χ using the Bragg-Williams theory, in which single phase mixtures are obtained for χ < 2. The χ parameter can be calculated from the (crystalline) solubility of the drug in the inhibitor, provided the inhibitor is a liquid, and the melting entropy and temperature of the drug.

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.