Determination of the structure and morphology of gold nanoparticle–HSA protein complexes

A method to measure the number of proteins bound to each nanoparticle and changes in the protein structure is reported

Evaluation of the critical quality attributes of lipid nanoparticles stored under different conditions

Lipid nanoparticles (LNPs) are emerging new modalities for mRNA therapeutics which have been in the spotlight for the past decade. Since these are relatively new drug delivery systems compared to conventional medicines, new analytical techniques for the robust characterization of their critical quality attributes (CQAs) are needed [1]. It has been reported that several stimuli can affect the stability of the LNPs such as leakage of the nucleic acid cargo from the nanoparticle and LNP aggregation, resulting in low translation efficiency. Hence, understanding the duration of stability is key during formulation development. The aim of the present study is to evaluate the stability of PolyA-LNPs: 1) Stored at different temperatures (4oC and 25oC); 2) Dialysed in the absence and presence of cryoprotectant sucrose. We measured the impact of the above storage conditions on LNP physicochemical parameters

Rational design of multi-functional gold nanoparticles with controlled biomolecule adsorption: a multi-method approach for in-depth characterization

Multi-functionalized nanoparticles are of great interest for diagnostic and therapeutic applications

Physicochemical characterisation of poly(A) lipid nanoparticles : effect of cryoprotectant and temperature storage conditions

Lipid nanoparticles (LNPs) are emerging new modalities for mRNA therapeutics which have been in the spotlight for the past decade. Since these are relatively new drug delivery systems compared to conventional medicines, new analytical techniques for the robust characterization of their critical quality attributes (CQAs) are needed [1]. It has been reported that several stimuli can affect the stability of the LNPs such as leakage of the nucleic acid cargo from the nanoparticle and LNP aggregation, resulting in low translation efficiency [2]. Hence, understanding the duration of stability is key during formulation development. The aim of the present study is to evaluate the stability of PolyA-LNPs: 1) Stored at different temperatures (4oC and 25oC); 2) Dialysed in the absence and presence of cryoprotectant sucrose. We measured the impact of the above storage conditions on LNP physicochemical parameters

Assessing lipid nanoparticle protein corona formation and cytocompatibility

Lipid nanoparticles (LNPs) represent an emerging new modality for mRNA delivery. Following administration and interaction with blood constituents, LNPs form a corona complex consisting of proteins adsorbed on the surface altering their stability, biological identity, and fate. Cytocompatibility of the LNPs is an important factor when considering their safety efficacy in delivering the encapsulated drug dose, lipid choice and the specific target cells. The aim of this study was to investigate the changes in LNP physical parameters in physiologically-relevant media. Key attributes such as particle size, polydispersity index and zeta-potential were measured using Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA). Cytocompatibility was assessed via CellTiter-Glo® assay. Following 24-hour incubation of LNPs with Bovine Serum Albumin (BSA), the LNP z-average increased from 92.4 (± 49.0) nm to 131.4 (± 64.9) nm indicating interaction between LNPs and BSA. A decrease in percentage cell viability was demonstrated with increased lipid concentration for MCF-7 and A549 cell lines. This work shows changes in LNP physicochemical properties in the presence of protein and biologically relevant conditions consistent with protein surface adsorption. The cytocompatibility of LNPs can be associated with the type of lipids used in the synthesis of LNPs

The use of orthogonal analytical approaches to profile lipid nanoparticle physicochemical attributes

Lipid nanoparticles (LNPs) have become a major disruptor within the drug delivery field of complex RNA molecules. The wide applicability of prototype nanomedicines has the potential to fill clinical requirements for use against current untreatable diseases. The uptake and implementation of analytical technologies to evaluate these prototype nanomedicines have not experienced similar growth rates, thus hindering the translation of LNPs. Here, we evaluate a model RNA-LNP formulation with a selection of routine and high-resolution orthogonal analytical techniques across studies on the manufacturing process parameter impact and formulation stability evaluation under refrigerated and ultra-low temperatures. We analysed a model cationic RNA-complexed LNP formulation via the process impact on formulation critical quality attributes, short-term refrigerated stability evaluation and frozen-storage stability using zetasizer dynamic light scattering and nanoparticle tracking analysis. We also evaluated freeze-/thaw-induced stress on LNP formulation using high-resolution field-flow fractionation. Statistical analysis and correlations between techniques were conducted to further enhance our understanding of LNP formulation design and its physicochemical attributes to facilitate LNP formulation clinical translation

Interactions nanoparticles-proteins : characterization of the protein corona

De nos jours, les nanomatériaux sont utilisés dans de très nombreux domaines allant de l’agroalimentaire à la santé en passant par l’industrie automobile et le textile. Dans ce contexte, de nombreuses disciplines comme la métrologie et la nanotoxicologie se sont fortement développées. Dans les milieux biologiques, les protéines interagissent avec les nanoparticules pour former une couronne de protéines. Cette couronne de protéines joue un rôle important dans les interactions des nanoparticules avec leur environnement. Comprendre et caractériser les interactions des nanoparticules avec les protéines permettraient d’améliorer l’utilisation des nanoparticules dans différents domaines, notamment dans celui de la santé. Nous avons d’abord mis au point une méthode de purification des nanoparticules permettant de préserver la structure et la composition de la couronne protéique (Asymmetric flow field-flow fractionation, AF4). Nous avons ensuite caractérisé la couronne protéique associée à ces nanoparticules par plusieurs méthodes telles que la sédimentation centrifuge différentielle, la microscopie électronique, des méthodes basées sur la diffusion de la lumière (diffusion dynamique de la lumière (DLS) et la résonance plasmonique de surface localisée ou non), le dichroïsme circulaire classique et le Synchrotron Radiation Circular Dichroism (SRCD, Diamond, UK). Nous avons montré qu’une couronne d’albumine sérique humaine provoque une augmentation du diamètre hydrodynamique des nanoparticules d’or de 14 à 25,3 nm et une diminution de la densité d’un facteur 2. Ceci nous a permis de calculer que 19 molécules d’albumine en moyenne interagissent avec une nanoparticule. Les spectres de dichroïsme circulaire ont permis d’estimer que l’albumine conserve environ 70% de ses structures hélicoïdales lorsqu’elle est complexée avec les nanoparticules. Nous avons estimé l’affinité avec laquelle les nanoparticules d’or interagissent qui est d’environ 351 nM pour l’albumine et 513 nM pour la transthyrétine qui est riche en brins béta. Nous avons également optimisé une méthode de couplage de l’AF4 à un appareil de mesure de la diffusion dynamique de la lumière (DLS) pour améliorer la précision de la mesure du diamètre hydrodynamique des nanoparticules. Cette méthode précise et flexible permettra de caractériser de nombreuses modifications de surface des nanoparticules comme l’ajout de polyéthylène glycol, utilisées pour la conception de nano-médicamentsNowadays, nanomaterials are used in numerous areas ranging from food to health through cars and textile engineering. In this context, many disciplines such as metrology and nanotoxicology, have been developed. In biological fluids, proteins interact with nanoparticles to form the protein corona, which plays an important role in mediating the interactions of the nanoparticles with their environment. Understanding and characterizing the interactions of nanoparticles with proteins and the corona structure would improve their use in various fields and particularly in the health sector. We have first developed a method based on Asymmetric Flow Field Flow Fractionation (AF4) for purifying gold nanoparticles preserving the structure and composition of the protein corona. Then we have characterized the protein corona associated to these nanoparticles by differential centrifugal sedimentation, electron microscopy, light scattering (dynamic light scattering, DLS), surface plasmon resonance (SPR) and localized SPR and by circular dichroism (classical CD and Synchrotron Radiation Circular Dichroism, SRCD, Diamond, UK). We have shown that human serum albumin corona increased the hydrodynamic diameter of the gold nanoparticles from 14 to 25.3 nm and decreases their density by a factor of 2. This enabled us to calculate that 19 albumin molecules on average interact with a nanoparticle. We have estimated by circular dichroism that albumin maintains about 70% of its helical structures when complexed with nanoparticles. The affinity between gold nanoparticles and proteins, is about 351 nM for albumin and 513 nM for transthyretin, which are enriched in helices and beta strands respectively. We have also optimized a coupling method between the AF4 system and the dynamic light scattering apparatus to improve the measurement accuracy of the hydrodynamic diameter of the nanoparticles. This accurate and flexible method will be helpful to characterize many surface modifications of the nanoparticles such as the addition of polyethylene glycol used for the design of nanodrug