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ABSTRACTThe 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. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

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. First, developing standard test methods and Reference Materials (RMs) for nanomedicines is a key priority for the European Commission and various stakeholders. Furthermore, there was a unanimous recognition of the need for a unified approach between standardisation committees, regulators and the nanomedicine community. At the USA, Canadian and European level, examples of success stories and of future initiative have been discussed. Future perspectives include the creation of a dedicated Working Group under CEN/TC 352 to consolidate efforts and develop a nanomedicine standardisation roadmap., (© 2024. The Author(s).)

Background: This study was conducted to investigate whether mRNA vaccine technology could be adapted for the ectothermic vertebrate Atlantic salmon ( Salmo salar ). 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. In addition, it is unknown if modified mRNA containing non-canonical nucleotides can correctly translate in salmonid cells., Methods: We used an mRNA transcript coding for enhanced green fluorescence protein, flanked by the untranslated regions of the hemagglutinin-esterase gene of the infectious salmon anemia virus, and a 120-base-long poly(A) tail. The mRNA was generated via in vitro transcription where uridine residues were replaced with N1-methyl-pseudouridines, and then encapsulated in LNPs., Results: When transfected into the salmonid cell line CHH-1, the mRNA-LNP construct induced expression of EGFP. Furthermore, when mRNA-LNPs were injected intramuscularly into salmon, in vivo protein expression was demonstrated via immunohistochemistry. EGFP was observed in cells infiltrating the spaces between muscle cells in a focal inflammatory response., Conclusion: The results indicate that N1-methyl-pseudouridine-modified mRNA encapsulated in LNPs can be used to express antigens of interest in salmonid fish.

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., Competing Interests: Declaration of competing interest Roland Drexel and Florian Meier are employees of Postnova Analytics GmbH; Hanna Jankevics, Natalia Markova, Michele Carboni are employees of Malvern Panalytical Ltd. The other authors do not have any conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nanoparticles used for medical applications commonly possess coatings or surface functionalities intended to provide specific behavior in vivo , 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. The observed differences between cryo-XPS and standard XPS measurements indicate the potential of cryo-XPS for providing quantitative measurements of such nanoparticle systems in hydrated conditions.

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. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)-based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC-MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation., (© 2023. The Author(s).)

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. The REFINE DSS has been developed to support experts, innovators, and regulators in the implementation of intelligent testing strategies (ITS) for efficient preclinical assessment of nanotechnology-enabled health products. The DSS applies logical rules provided by REFINE experts which generate prioritized lists of assays to be performed (i.e. ITSs) for physicochemical characterisation and for immunotoxicological endpoints. The DSS has been tested against several case studies and was validated by internal project experts as well as external ones., (© 2022. The Author(s).)

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. Moreover, an in-depth methodological expertise and holistic approach to data analysis are required for robust measurements and to enable an adequate interpretation of experimental findings. Here the combination of complementary label-free biophysical techniques, including dynamic light scattering (DLS), multiangle-DLS (MADLS), Electrophoretic Light Scattering (ELS), nanoparticle tracking analysis (NTA), multiple detection SEC and differential scanning calorimetry (DSC), have been successfully used for the characterization of physical and chemical attributes of rAAV and LNPs encapsulating mRNA. Methods' performance, applicability, dynamic range of detection and method optimization are discussed for the measurements of multiple critical physical-chemical quality attributes, including particle size distribution, aggregation propensity, polydispersity, particle concentration, particle structural properties and nucleic acid payload.

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. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.

Introduction: The main objective of the present prospective study was to assess mechanical complications associated with an original intramedullary nail with limited and controlled blade back-out in the treatment of trochanteric fracture., Material and Method: All patients treated for trochanteric fracture in a single orthopedic/traumatologic surgery department over a 2-year period were included. Minimum follow-up was 6 months. Fracture stability was assessed on the AO criteria. The TFP® intramedullary nail has a monobloc helicoid blade. Its main feature is the controlled and limited blade back-out, optimizing fracture site compression in weight-bearing, without the drawback of excessive back-out. The main endpoint was onset of mechanical complications: cut-out, intra-articular protrusion, non-union, and pain. Baumgaertner's Tip-Apex Distance (TAD), blade centering within the femoral head and fracture reduction were also assessed., Results: One hundred thirty-eight patients (mean age, 83 years) were operated on, and 118 followed up. There were 9 mechanical complications (7.6%): 4 cut-outs (3.4%), 3 intra-articular protrusions (2.5%), 1 non-union (0.8%) and 1 case of pain (0.8%). TAD length was not associated with complications rate. Poor reduction was significantly associated with more complications (p=0.02), as was blade malpositioning. Mean back-out was 3.3mm, affecting 22 nails (19%). There were no complications in case of back-out, versus a 9.4% rate in absence of back-out, although this difference was not significant (p=0.21). There were no postoperative infections., Conclusion: The TFP® nail is useful for fixation of trochanteric fracture, whether stable or unstable, due to its low rate of mechanical complications compared to the literature., (Copyright © 2020. Published by Elsevier Masson SAS.)

Introduction: Periprosthetic tibial fracture after total knee arthroplasty (TKA) is rare, but jeopardizes implant survival. The main objective of the present study was to assess treatment efficacy, on the hypothesis that surgery provides good long-term results., Material and Methods: A two-center retrospective study included 15 patients (6 male, 9 female: mean age, 71.8±10.2 years), managed between 1997 and 2017 for isolated tibial fracture after TKA. Patients were assessed clinically (IKS, inverted Oxford, Parker and SF-12 scores) and radiologically. Complications and revision surgeries were collated. Fractures were classified on the SoFCOT classification: 9 stable implants (4 type B1, 5 type C1), 4 periprosthetic osteolyses (1 type A3, 2 type B3, 1 type C3), and 2 loosenings (type A2). Treatments comprised: non-operative treatment (1 bed-ridden patient), 11 osteosyntheses for fracture on stable implant (2 standard plates, 7 locking plates, 2 intramedullary nailings), and 3 implant replacements by cemented long stem models for loosening., Results: Mean follow-up was 28 months (range, 12-120 months). Consolidation was achieved in 13 cases, at a mean 15 weeks. Complications comprised: 4 infections, 2 cases of secondary displacement, and 2 of non-union. Surgical revision was required in 8 cases, including 2 secondary implant revision procedures. Functional results were good in 10 cases. At last follow-up, mean Parker score was 7 (range, 4-8.5), Oxford score 32 (range, 16-39), and IKS score 150 (range, 85-167) with knee and function scores respectively 78 (range, 55-86) and 75 (range, 30-85)., Conclusion: Radiologic and clinical results were encouraging, but with impaired quality of life and a high rate of complications., Level of Evidence: IV, retrospective cohort study., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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. Measurements were conducted over a range of protein concentrations and temperatures. Both techniques successfully resolved the protein monomer and dimer populations, allowing quantification of monomer loss. BSA and α-chymo exhibited second and first order kinetics, respectively, confirming different limiting steps for the two species. The Arrhenius equation yielded activation energies for BSA of (240 ± 20) kJ mol -1 and (190 ± 10) kJ mol -1 by ES-DMA and AF4, respectively. The rates determined by ES-DMA were equal to or slightly faster than those measured by AF4, so instrumental differences were analyzed to identify potential sources of bias. An important factor may be the applicable concentration range for each method; notably, AF4 operates at the mg mL -1 level, while ES-DMA is sensitive at μg mL -1 and therefore requires much smaller samples for analysis (typically several μL are injected). The limitations of each method are detailed in the discussion and demonstrate the importance of orthogonal measurement strategies for the analysis of protein kinetics. ES-DMA provides a potentially useful alternative to size exclusion chromatography to screen the stability of formulation conditions for protein therapeutics; neither ES-DMA nor AF4 rely on column interactions for separation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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. This work was aimed at addressing discrepancies in length between fractionated and unfractionated CNC and obtaining less polydisperse samples with fewer aggregates to facilitate microscopy dimensional measurements. The results demonstrate that early fractions obtained from an analytical scale AF4 separation contain predominantly individual CNCs. The number of laterally aggregated "dimers" and clusters containing 3 or more particles increases with increasing fraction number. Size analysis of individual particles by AFM for the early fractions demonstrates that the measured CNC length increases with increasing fraction number, in good agreement with the rod length calculated from the AF4 multi-angle light scattering data. The ability to minimize aggregation and polydispersity for CNC samples has important implications for correlating data from different sizing methods.

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. UV-visible and fluorescence measurements of collected fractions were successfully applied. An additional proteomic study revealed that grape and microorganism proteins largely impacted the composition of chromophoric colloidal matter of Chardonnay wines. Asymmetrical flow field-flow fractionation appeared to be more reliable and less invasive with respect to the native chemical environment of chromophoric wine macromolecules, and hence is recommended as a tool to fractionate chromophoric colloidal matter in white wines. Graphical Abstract An innovative macromolecular separation method based on Asymmetrical Flow Field-Flow Fractionation was developed to better control colloidal dynamics across Chardonnay white winemaking.