In depth characterisation of the biomolecular coronas of polymer coated inorganic nanoparticles with differential centrifugal sedimentation.

Advances in nanofabrication methods have enabled the tailoring of new strategies towards the controlled production of nanoparticles with attractive applications in healthcare. In many cases, their characterisation remains a big challenge, particularly for small-sized functional nanoparticles of 5 nm diameter or smaller, where current particle sizing techniques struggle to provide the required sensitivity and accuracy. There is a clear need for the development of new reliable characterisation approaches for the physico-chemical characterisation of nanoparticles with significant accuracy, particularly for the analysis of the particles in the presence of complex biological fluids. Herein, we show that the Differential Centrifugal Sedimentation can be utilised as a high-precision tool for the reliable characterisation of functional nanoparticles of different materials. We report a method to correlate the sedimentation shift with the polymer and biomolecule adsorption on the nanoparticle surface, validating the developed core-shell model. We also highlight its limit when measuring nanoparticles of smaller size and the need to use several complementary methods when characterising nanoparticle corona complexes.

Bovine intra-mammary challenge with Streptococcus dysgalactiae spp. Dysgalactiae to explore the effect on the response of Complement activity.

Recently published work as described by the authors highlighted the extent of Complement activity in bovine milk. Localised mastitis infection occurring in the mammary glands of dairy cows is readily detectable by the levels of somatic cells in milk. Thus, it is opportune to monitor Complement activity in milks in association with the animal's innate immune response to mammary infection. Preliminary screening of milk samples taken randomly showed that milk with a high somatic cell count (SCC) reduced growth of the Complement-sensitive strain E. coli O111 to a greater extent (P < 0·05) than when the marker microorganism was grown in milk heated for the purpose of inactivating Complement. A follow-up study set out to determine the effect on Complement activity when a sub-clinical mastitis infection was induced in the mammary gland of four lactating dairy cows. The effect of Str. dysgalactiae spp. dysgalactiae inoculation into selected individual udder quarters of the mammary glands of each animal was followed by monitoring of SCC levels in the milks from the segregated udder samples during subsequent milking. At 72 and 96 h post inoculation (PI), the SCCs for the challenged quarter were increased compared to normal values. At the same time, the bactericidal sequestration assay identified increased E. coli O111 inhibition that can be directly linked to greater Complement activity in those quarter milks affected by induced inflammation. Thus, it can be identified that the high SCC milks were more effective in limiting E. coli O111 growth. Milks from the unchallenged quarters in all four cows were significantly less effective at reducing growth of the assay strain (P < 0·05). An ELISA assay targeting specific activation components of the Complement pathways confirmed that greater bacterial inhibition observed during the bactericidal sequestration assay was attributable to higher Complement activity in the milk samples from the affected quarters, i.e., with higher SCC. The induced infection was confirmed as self-limiting in three of the affected animals and their SCC returned to normal levels within 14 d PI, while the fourth cow required brief antibiotic intervention.

Understanding the Kinetics of Protein-Nanoparticle Corona Formation.

When a pristine nanoparticle (NP) encounters a biological fluid, biomolecules spontaneously form adsorption layers around the NP, called "protein corona". The corona composition depends on the time-dependent environmental conditions and determines the NP's fate within living organisms. Understanding how the corona evolves is fundamental in nanotoxicology as well as medical applications. However, the process of corona formation is challenging due to the large number of molecules involved and to the large span of relevant time scales ranging from 100 µs, hard to probe in experiments, to hours, out of reach of all-atoms simulations. Here we combine experiments, simulations, and theory to study (i) the corona kinetics (over 10-3-103 s) and (ii) its final composition for silica NPs in a model plasma made of three blood proteins (human serum albumin, transferrin, and fibrinogen). When computer simulations are calibrated by experimental protein-NP binding affinities measured in single-protein solutions, the theoretical model correctly reproduces competitive protein replacement as proven by independent experiments. When we change the order of administration of the three proteins, we observe a memory effect in the final corona composition that we can explain within our model. Our combined experimental and computational approach is a step toward the development of systematic prediction and control of protein-NP corona composition based on a hierarchy of equilibrium protein binding constants.

A casein hydrolysate protects mice against high fat diet induced hyperglycemia by attenuating NLRP3 inflammasome-mediated inflammation and improving insulin signaling.

SCOPE: Activation of the nod-like receptor protein 3 (NLRP3) inflammasome is required for IL-1ß release and is a key component of obesity-induced inflammation and insulin resistance. This study hypothesized that supplementation with a casein hydrolysate (CH) would attenuate NLRP3 inflammasome mediated IL-1ß secretion in adipose tissue (AT) and improve obesity-induced insulin resistance. METHODS AND RESULTS: J774.2 macrophages were LPS primed (10 ng/mL) and stimulated with adenosine triphosphate (5 mM) to assess NLRP3 inflammasome activity. Pretreatment with CH (1 mg/mL; 48 h) reduced caspase-1 activity and decreased IL-1ß secretion from J774.2 macrophages in vitro. 3T3-L1 adipocytes cultured with conditioned media from CH-pretreated J774.2 macrophages demonstrated increased phosphorylated (p)AKT expression and improved insulin sensitivity. C57BL/6JOLaHsd mice were fed chow or high fat diet (HFD) for 12 wk ± CH resuspended in water (0.5% w/v). CH supplementation improved glucose tolerance in HFD-fed mice as determined by glucose tolerance test. CH supplementation increased insulin-stimulated pAKT protein levels in AT, liver, and muscle after HFD. Cytokine secretion was measured from AT and isolated bone marrow macrophages cultured ex vivo. CH supplementation attenuated IL-1ß, tumor necrosis factor alpha (TNF-α) and IL-6 secretion from AT and IL-1ß, IL-18, and TNF-α from bone marrow macrophages following adenosine triphosphate stimulation ex vivo. CONCLUSION: This novel CH partially protects mice against obesity-induced hyperglycemia coincident with attenuated IL-1ß secretion and improved insulin signaling.

Emulsification properties of pea protein isolate using homogenization, microfluidization and ultrasonication.

Pea protein isolate (PPI) is used in many food formulations, due to its low cost, commercial availability and excellent amino acid profile. The objective of this study was to determine the emulsification properties of PPI. Particle size of PPI powders showed neither temperature (25-65°C) nor time (up to 24h) increased solubilisation of powder particles during mixing. Heating PPI dispersions (10%, w/w, protein) from 45 to 90°C led to an increase in storage modulus (G'; Pa) at 71°C, indicating the onset of protein aggregation. Gel formation occurred at 79°C (G'>1Pa). Pea protein-stabilised emulsions made using homogenization (15MPa; 1 pass) or microfluidization (50MPa; 1 pass) resulted in the formation of cold-set gels, with gel strength increasing with increasing oil concentration and fluidic pressure. Droplet size and viscosity of pea protein-stabilised emulsions decreased and increased, respectively, with increasing ultrasonication time. Overall, ultrasonication (<50°C) can create a uniform droplet size emulsion, while, homogenization and microfluidization can produce cold-set gels for use in a wide-range of food applications.

Tuning of nanoparticle biological functionality through controlled surface chemistry and characterisation at the bioconjugated nanoparticle surface.

We have used a silica - PEG based bionanoconjugate synthetic scheme to study the subtle connection between cell receptor specific recognition and architecture of surface functionalization chemistry. Extensive physicochemical characterization of the grafted architecture is capable of capturing significant levels of detail of both the linker and grafted organization, allowing for improved reproducibility and ultimately insight into biological functionality. Our data suggest that scaffold details, propagating PEG layer architecture effects, determine not only the rate of uptake of conjugated nanoparticles into cells but also, more significantly, the specificity of pathways via which uptake occurs.

Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

Mapping protein binding sites on the biomolecular corona of nanoparticles.

Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized 'corona' of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.

The "sweet" side of the protein corona: effects of glycosylation on nanoparticle-cell interactions.

The significance of a protein corona on nanoparticles in modulating particle properties and their biological interactions has been widely acknowledged. The protein corona is derived from proteins in biological fluids, many of which are glycosylated. To date, the glycans on the proteins have been largely overlooked in studies of nanoparticle-cell interactions. In this study, we demonstrate that glycosylation of the protein corona plays an important role in maintaining the colloidal stability of nanoparticles and influences nanoparticle-cell interactions. The removal of glycans from the protein corona enhances cell membrane adhesion and cell uptake of nanoparticles in comparison with the fully glycosylated form, resulting in the generation of a pro-inflammatory milieu by macrophages. This study highlights that the post-translational modification of proteins can significantly impact nanoparticle-cell interactions by modulating the protein corona properties.

Nanomaterials: impact on cells and cell organelles.

Colloidal nanoparticles designed for the interactions with cells are very small, nanoscale objects usually consisting of inorganic cores and organic shells that are dispersed in a buffer or biological medium. By tuning the material properties of the nanoparticles a number of different biological applications of nanomaterials are enabled i.e. targeting, labelling, drug delivery, use as diagnostic tools or therapy. For all biological applications of nanoparticles, it is important to understand their interactions with the surrounding biological environment in order to predict their biological impact, in particular when designing the nanoparticles for diagnostic and therapeutic purpose. Due to the high surface-to-volume ratio, the surface of nanomaterials is very reactive. When exposed to biological fluids, the proteins and biomolecules present therein tend to associate with the nanoparticles' surface. This phenomenon is defined as biomolecular corona formation. The biomolecular corona plays a key role in the interaction between nanoparticles and biological systems, impacting on how these particles interact with biological systems on a cellular and molecular level. This book chapter describes the nature of the interactions at the bio-nano interface, shows the design strategy of nanoparticles for nanomedicine, and defines the concepts of biomolecular corona and biological identity of nanoparticles. Moreover, it describes the interaction of functionalised nanomaterials with cell organelles and intracellular fate of nanoparticles and it shows therapeutic application of gold nanoparticles as dose enhancers in radiotherapy.

Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface.

Nanoparticles have been proposed as carriers for drugs, genes and therapies to treat various diseases. Many strategies have been developed to target nanomaterials to specific or over-expressed receptors in diseased cells, and these typically involve functionalizing the surface of nanoparticles with proteins, antibodies or other biomolecules. Here, we show that the targeting ability of such functionalized nanoparticles may disappear when they are placed in a biological environment. Using transferrin-conjugated nanoparticles, we found that proteins in the media can shield transferrin from binding to both its targeted receptors on cells and soluble transferrin receptors. Although nanoparticles continue to enter cells, the targeting specificity of transferrin is lost. Our results suggest that when nanoparticles are placed in a complex biological environment, interaction with other proteins in the medium and the formation of a protein corona can 'screen' the targeting molecules on the surface of nanoparticles and cause loss of specificity in targeting.