Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.

The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques.

An optimized filling method for capillary DLS.

Capillary dynamic light scattering (DLS) is a new, simple and enabling technique, that increases the size range of DLS by over an order of magnitude in a cheap, disposable, but high optical quality, glass capillary. Sample loading for other capillary-based modalities, such as blood analysis, is typically achieved by dipping the capillary into the bulk sample, however, DLS is exquisitely sensitive to static scattering such as from a fluid meniscus or sample dried on the outside of the capillary and is sometimes used for extended measurement times where evaporation must be avoided. In this work, we carefully validate capillary dipping and sealing with a clay plug for DLS against reference measurements in a high quality 1 cm cuvette and then introduce a simple capillary loading scheme that reproducibly places a >3 μl sample in the correct location for a DLS measurement.

Polymerized and polyethylene glycol-conjugated hemoglobins: a globin-based calibration curve for dynamic light scattering analysis.

Dynamic light scattering (DLS) is a technique capable of determining the hydrodynamic radius of proteins. From this parameter, a molecular weight can be assessed provided that an appropriate calibration curve is available. To this goal, a globin-based calibration curve was used to determine the polymerization state of a recombinant hemoglobin-based oxygen carrier and to assess the equivalent molecular weight of hemoglobins conjugated with polyethylene glycol molecules.

Distribution plasticity of the human estrogen receptor alpha in live cells: distinct imaging of consecutively expressed receptors.

An amino-terminal fusion of the human estrogen receptor alpha (ER) with human O6-alkylguanine-DNA alkyltransferase (AGT) enabled the observation and distinction of consecutively expressed ER populations by sequential pulse labeling of the AGT tag with different fluorescent O(6)-alkylguanine derivatives in live cells. The application of agonists and antagonists led to the characteristic speckled redistribution of fluorescent receptors in the nucleus as visualized by confocal microscopy. To investigate where newly synthesized receptors were localized in individual cells with respect to their older relatives in response to extracellular chemical signals, receptor expression was continued for 4 h and newly synthesized receptors were labeled with a new fluorophore spectrally distinct from the first probe.

Diffusion-time distribution analysis reveals characteristic ligand-dependent interaction patterns of nuclear receptors in living cells.

Nuclear receptors initiate transcription, interact with regulatory proteins, and are influenced by hormones, drugs, and pollutants. Herein, we discover ligand-specific mobility patterns of human estrogen receptor-alpha (ER) in living cells using diffusion-time distribution analysis (DDA). This novel method, based on fluorescence correlation spectroscopy (FCS), is especially suited to unraveling multiple protein interactions in vivo at native expression levels.