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. The characterization strategy and the analytical measurements described here will have an important role during formulation development and in determining robust quality attributes ultimately supporting the quality assessment of these innovative RNA therapeutics.

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. The good agreement between DLS values and those obtained from gel filtration chromatography is a consequence of the high similarity in structure, shape, and density within the globin superfamily. Moreover, globins and heme proteins in general share similar spectroscopic properties, thereby reducing possible systematic errors associated with the absorption of the probe radiation by the chromophore.

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. We demonstrate the statistically identical characterisation of the new scheme and dipping against the reference measurements, but in sample volumes reduced by 1 and 3 orders of magnitude, respectively, key for high value applications such as pharmaceutical development where sample costs of $100 k per mg are common and in the environmental & medical sciences where samples may be difficult or unethical to collect in bulk. •Use of the capillary method to characterize high value samples in the lowest, reproducible volume.•Pitfalls and subsequent development of the best reproducible method.

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. This strategy enabled a time-resolved analysis of the formation of ER-enriched protein complexes in distinct nucleoplasmic compartments. Such complexes represent important but hitherto uncharacterized macromolecular structures involved in ER function. Different, long-lasting effects were observed depending on the type of ligand. For example, 4 h after pulsed application of the partial antagonist 4-hydroxytamoxifen, the second receptor population exhibited a speckled pattern in the cell nucleus that overlapped with the first receptor population pattern. This novel finding suggests that the intranuclear positioning of receptor aggregates is not random but influenced in a ligand-dependent manner. The antagonist ICI 182,780 (7-alpha-[9-(4.4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10)-triene-3,17-beta-diol), a potent drug used in cancer treatment, led to down-regulation of the first receptor population and newly expressed receptors accumulated in the cytoplasm. In contrast, the natural agonist 17beta-estradiol resulted in significantly shorter effects. Four hours after ligand application, newly expressed receptors were homogeneously distributed in the nucleus as in untreated control cells. We present the pulse labeling of AGT-ER fusion proteins with different fluorophores as a novel tool for investigating the functional regulation of nuclear receptors in individual cells.

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. We found that ER forms a limited number of distinct complexes with a varying population by dynamic interaction with other nuclear components. Dose-response curves of different ligands could be obtained for each receptor interaction. The potential to identify interacting proteins was demonstrated by comparing DDA of the ER cofactor SRC-3 attached to yellow fluorescent protein (YFP) with those of YFP-ER. Our findings open up new routes to elucidating transcription regulation and to detecting and distinguishing pharmacologically and toxicologically active compounds in vivo. Moreover, DDA provides a general approach to monitoring biochemical networks in individual living cells.