Subtle pH variation around pH 4.0 affects aggregation kinetics and aggregate characteristics of recombinant human insulin

Insulin is a biotherapeutic protein, which, depending on environmental conditions such as pH, has been shown to form a large variety of aggregates with different structures and morphologies. This work focuses on the formation and characteristics of insulin particulates, dense spherical aggregates having diameters spanning from nanometre to low-micron size. An in-depth investigation of the system is obtained by applying a broad range of techniques for particle sizing and characterisation. An interesting observation was achieved regarding the formation kinetics and aggregate characteristics of the particulates; a subtle change in the pH from pH 4.1 to pH 4.3 markedly affected the kinetics of the particulate formation and led to different particulate sizes, either nanosized or micronsized particles. Also, a clear difference between the secondary structure of the protein particulates formed at the two pH values was observed, where the nanosized particulates had an increased content of aggregated β-structure compared to the micronsized particles. The remaining characteristics of the particles were identical for the two particulate populations. These observations highlight the importance of carefully studying the formulation design space and of knowing the impact of parameters such as pH on the aggregation to secure a drug product in control. Furthermore, the identification of particles only varying in few parameters, such as size, are considered highly valuable for studying the effect of particle features on the immunogenicity potential.Drug Delivery Technolog

Waterborne Electrospinning of α-Lactalbumin Generates Tunable and Biocompatible Nanofibers for Drug Delivery

This is the author accepted manuscript. The final version is available from the American Chemical Society via the DOI in this recordProtein-based drug carriers are an interesting alternative to traditional polymeric drug delivery systems due to their intrinsic biocompatibility and biodegradability. Electrospinning of neat proteins holds advantages over electrospinning of protein mixtures, e.g., whey isolates, such as better control of the physicochemical and biological function of the resulting nanofiber-based system. In this study, we explore electrospinning of the isolated milk protein α-lactalbumin (ALA), which is a whey protein with important nutritional and pharmacological properties. Via waterborne electrospinning of ALA with a minimum amount of poly(ethylene oxide) (PEO) as a cospininng polymer, nanofibers of high protein content were successfully produced (up to 84% (w/w)). We demonstrate the ability to produce ALA-based nanofibers with a high degree of tunability in terms of size, stability in water, and mechanical properties. The nanofibers displayed excellent biocompatibility in vitro as the viability of cultured TR146 human buccal epithelium and NIH 3T3 murine fibroblast cells was not influenced by exposure to ALA-based nanofibers. ALA-based nanofibers were loaded with up to 6% (w/w) ampicilin, and the nanofibers were capable of maintaining the activity of the antibiotic after electrospinning and cross-linking. Using such a property of the material, we demonstrate that ampicillin-loaded nanofibers successfully inhibit the growth of Gram-negative bacteria in vitro. Importantly, after treatment with ampicillin-loaded nanofibers, no bacterial regrowth was observed, which indicates that this treatment may clear eventual persisters to ampicillin. Finally, the structural properties of the native functional protein were maintained after release of ALA from the nanofibers. This promotes our platform, not only as a sustainable protein-based drug delivery system, but also as an innovative solid form of ALA for food and pharmaceutical applications.Villum FoundationDanish Council for Independent Research; Technology and ProductionRoyal SocietyMedical Research Council (MRC)Wellcome Trus

Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.

Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.This is the author's accepted manuscript and will be under embargo until the 3rd of September 2015. The final version is published by ACS in The Journal of Physical Chemistry Letters here: http://pubs.acs.org/doi/abs/10.1021/jz501614e

The route to protein aggregate superstructures: particulates and amyloid-like spherulites

Depending on external conditions, native proteins may change their structure and undergo different association routes leading to a large scale polymorphism of the aggregates. This feature has been widely observed but is not fully understood yet. This review focuses on morphologies, physico-chemical properties and mechanisms of formation of amyloid structures and protein superstructures. In particular, the main focus will be on protein particulates and amyloid-like spherulites, briefly summarizing possible experimental methods of analysis. Moreover, we will highlight the role of protein conformational changes and dominant forces in driving association together with their connection with the final aggregate structure. Eventually, we will discuss future perspectives in this field and we will comment what is, in our opinion, urgently needed

Tracking the heterogeneous distribution of amyloid spherulites and their population balance with free fibrils

The analysis of amyloidogenic systems reveals the appearance of distinct states of aggregation for amyloid fibrils. For different proteins and under specific experimental conditions, amyloid spherulites are recognized as a significant component occurring in several protein model systems used for in vitro fibrillation studies. In this work we have developed an approach to characterize solutions containing a mixture of amyloid spherulites and individual fibrils. Using bovine insulin as the model system, sedimentation kinetics for the amyloid aggregates were followed using a combination of UV-Vis spectroscopy and cross-polarized optical microscopy. Spherulites were identified as the species undergoing sedimentation. A simple mathematical approach allows the description of the kinetics in terms of decay time/rate distribution. Moreover, based on the sedimentation kinetics, a rough estimate of the balance between amyloid spherulites and individual fibrils can be provided. Fitting the experimental data with the proposed physico-chemical approach shows self-consistent results in reasonable agreement with quantitative imaging analysis previously reported. Our results provide new physical insights into the analysis of amyloidogenic systems, providing a method to characterize the heterogeneous distribution of amyloid spherulites and simultaneously distinguish spherulites and free fibril populations. Importantly, the method can be generally applied to the characterization of polydisperse solutions containing optically traceable spherical particles in the micrometric range

Imprese che esportano e imprese che vorrebbero esportare: un confronto

Sulla base di un'indagine diretta presso un campione rappresentativo di imprese esportatrici e non esportatrici, si procede a una stima dei differenziali di produttività e a un confronto relativo a variabili economiche e relazioni fra imprese esportatrici, imprese non esportatrici e imprese potenzialmente esportatric