Implementation of mRNA-Lipid Nanoparticle Technology in Atlantic Salmon ().

Background: This study was conducted to investigate whether mRNA vaccine technology could be adapted for the ectothermic vertebrate Atlantic salmon (). Lipid nanoparticle (LNP) technology has been developed and optimized for mRNA vaccines in mammals, stabilizing mRNA and facilitating its delivery into cells. However, its utility at the temperatures and specific biological environments present in ectotherms remains unclear.

Toward an international standardisation roadmap for nanomedicine.

The French National Metrology Institute (LNE) initiated a series of events to identify priorities for test methods and their harmonisation that directly address regulatory needs in Nanomedicine. One of these workshops entitled "The International Standardisation Roadmap for Nanomedicine" held in October 2023 (Paris, France) brought together key experts in the characterisation of nanomedicines and medical products containing nanomaterials, including the Joint Research Centre of the European Commission, SINTEF Industry and the metrology institutes of France, the UK, the USA and Canada, two flagship initiatives of the European Commission (PHOENIX and SAFE-n-MEDTECH Open Innovation Test Beds), representatives of a working party on mRNA vaccines at the European Directorate for the Quality of Medicines (EDQM) and members of international standardisation and pre-normative organisations (including CEN, ISO, ASTM, VAMAS). Two take-home message came out from the discussion.

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts.

The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts.

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.

Cryo-XPS for Surface Characterization of Nanomedicines.

Nanoparticles used for medical applications commonly possess coatings or surface functionalities intended to provide specific behavior , for example, the use of PEG to provide stealth properties. Direct, quantitative measurement of the surface chemistry and composition of such systems in a hydrated environment has thus far not been demonstrated, yet such measurements are of great importance for the development of nanomedicine systems. Here we demonstrate the first use of cryo-XPS for the measurement of two PEG-functionalized nanomedicines: a polymeric drug delivery system and a lipid nanoparticle mRNA carrier.

Physicochemical characterization and quantification of nanoplastics: applicability, limitations and complementarity of batch and fractionation methods.

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)-multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC-MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies.

A Decision Support System for preclinical assessment of nanomaterials in medical products: the REFINE DSS.

The application of nanomaterials in medicine has led to novel pharmaceuticals and medical devices that have demonstrated a strong potential for increasing the efficacy/performance and safety of therapeutic and diagnostic procedures to address a wide range of diseases. However, the successful translation of these technologies from their inception (proof-of-concept) to clinical practice has been challenged by substantial gaps in the scientific and technical capacity of R&D companies, especially SMEs, to keep up with the ever-evolving regulatory expectations in the emerging area of nanomedicine. To address these challenges, the EU Horizon 2020 project REFINE has developed a Decision Support System (DSS) to support developers of nanotechnology-enabled health products in bringing their products to the clinic.

Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques.

Novel vaccine platforms for delivery of nucleic acids based on viral and non-viral vectors, such as recombinant adeno associated viruses (rAAV) and lipid-based nanoparticles (LNPs), hold great promise. However, they pose significant manufacturing and analytical challenges due to their intrinsic structural complexity. During product development and process control, their design, characterization, and quality control require the combination of fit-for-purpose complementary analytical tools.

Quantitative Proteomic Analysis of Biogenesis-Based Classification for Extracellular Vesicles.

Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results.

Quantifying protein aggregation kinetics using electrospray differential mobility analysis.

Protein aggregation is a critical concern in bioprocessing, where its presence can result in serious adverse interactions in clinical end-use applications. In this study, an aerosol-based technique, electrospray differential mobility analysis (ES-DMA), was used to quantify thermally-induced protein aggregation kinetics for bovine serum albumin (BSA) and α-chymotrypsinogen A (α-chymo), employing a new methodology to modify the solution for compatibility with the electrospray process. Results are compared orthogonally with asymmetrical-flow field-flow fractionation (AF4), a hydrodynamic separation technique with UV detection.

Characterization of Size and Aggregation for Cellulose Nanocrystal Dispersions Separated by Asymmetrical-Flow Field-Flow Fractionation.

Cellulose nanocrystals (CNCs) derived from various types of cellulose biomass have significant potential for applications that take advantage of their availability from renewable natural resources and their high mechanical strength, biocompatibility and ease of modification. However, their high polydispersity and irregular rod-like shape present challenges for the quantitative dimensional determinations that are required for quality control of CNC production processes. Here we have fractionated a CNC certified reference material using a previously reported asymmetrical-flow field-flow fractionation (AF4) method and characterized selected fractions by atomic force microscopy (AFM) and transmission electron microscopy.

Asymmetrical flow field-flow fractionation of white wine chromophoric colloidal matter.

Two analytical separation methods-size-exclusion chromatography and asymmetrical flow field-flow fractionation-were implemented to evaluate the integrity of the colloidal composition of Chardonnay white wine and the impact of pressing and fermentations on the final macromolecular composition. Wine chromophoric colloidal matter, representing UV-visible-absorbing wine macromolecules, was evaluated by optical and structural measurements combined with the description of elution profiles obtained by both separative techniques. The objective of this study was to apply these two types of fractionation on a typical Chardonnay white wine produced in Burgundy and to evaluate how each of them impacted the determination of the macromolecular chromophoric content of wine.