Correction: SARS-CoV-2 and approaches for a testing and diagnostic strategy.

Correction for 'SARS-CoV-2 and approaches for a testing and diagnostic strategy' by Delyan R. Hristov et al., J. Mater. Chem. B, 2021, 9, 8157-8173, DOI: https://doi.org/10.1039/D1TB00674F.

Add Sugar to Chitosan: Mucoadhesion and In Vitro Intestinal Permeability of Mannosylated Chitosan Nanocarriers.

Crosslinked chitosan nanocarriers (140-160 nm) entrapping coumarin-6 (λex/em = 455/508 nm) with or without surface mannosylation were synthesized and assessed for cytotoxicity, adherence and cellular uptake in Caco-2 cells, flux across Caco-2 monolayers, and mucoadhesion to porcine mucin. Mannosylated and non-mannosylated nanocarriers demonstrated biocompatibility with slow release of coumarin-6 at pH 6.8 and 7.4 over 24 h. Adherence of the non-mannosylated nanocarriers (50 and 150 µg/mL) to Caco-2 cells was ~10% over 24 h, whereas cellular uptake of 25-30% was noted at 4 h. The mannosylated nanocarriers showed a similar adherence to non-mannosylated nanocarriers after 24 h, but a lower cellular uptake (~20%) at 1 h, comparable uptake at 4 h, and a higher uptake (~25-30%) at 24 h. Overall, the nanocarriers did not affect the integrity of Caco-2 monolayers. Mannosylated nanocarriers elicited higher Papp of 1.6 × 10-6 cm/s (50 µg/mL) and 1.2 × 10-6 (150 µg/mL) than the non-mannosylated ones: 9.8 × 10-7 cm/s (50 µg/mL) and 1.0 × 10-6 (150 µg/mL) after 2 h. Non-mannosylated chitosan nanocarriers elicited enhanced adhesion to porcine gut mucin via mucin-filled microchannels due to higher cationic charge density. These results underpin the importance of surface chemistry in the biological interactions of nanocarriers, while highlighting the role of surface hydrophilicity in mucopermeation due to mannosylation.

SARS-CoV-2 and approaches for a testing and diagnostic strategy.

The COVID-19 pandemic has led to an unprecedented global health challenge, creating sudden, massive demands for diagnostic testing, treatment, therapies, and vaccines. In particular, the development of diagnostic assays for SARS-CoV-2 has been pursued as they are needed for quarantine, disease surveillance, and patient treatment. One of the major lessons the pandemic highlighted was the need for fast, cheap, scalable and reliable diagnostic methods, such as paper-based assays. Furthermore, it has previously been suggested that paper-based tests may be more suitable for settings with lower resource availability and may help alleviate some supply chain challenges which arose during the COVID-19 pandemic. Therefore, we explore how such devices may fit in a comprehensive diagnostic strategy and how some of the challenges to the technology, e.g. low sensitivity, may be addressed. We discuss the properties of the SARS-CoV-2 virus itself, the COVID-19 disease pathway, and the immune response. We then describe the different diagnostic strategies that have been pursued, focusing on molecular strategies for viral genetic material, antigen tests, and serological assays, and innovations for improving the diagnostic sensitivity and capabilities. Finally, we discuss pressing issues for the future, and what needs to be addressed for the ongoing pandemic and future outbreaks.

Impact of dynamic sub-populations within grafted chains on the protein binding and colloidal stability of PEGylated nanoparticles.

Polyethylene glycol grafting has played a central role in preparing the surfaces of nano-probes for biological interaction, to extend blood circulation times and to modulate protein recognition and cellular uptake. However, the role of PEG graft dynamics and conformation in determining surface recognition processes is poorly understood primarily due to the absence of a microscopic picture of the surface presentation of the polymer. Here a detailed NMR analysis reveals three types of dynamic ethylene glycol units on PEG-grafted SiO2 nanoparticles (NPs) of the type commonly evaluated as long-circulating theranostic nano-probes; a narrow fraction with fast dynamics associated with the chain ends; a broadened fraction spectrally overlapped with the former arising from those parts of the chain experiencing some dynamic restriction; and a fraction too broad to be observed in the spectrum arising from units closer to the surface/graft which undergo slow motion on the NMR timescale. We demonstrate that ethylene glycol units transition between fractions as a function of temperature, core size, PEG chain length and surface coverage and demonstrate how this distribution affects colloidal stability and protein uptake. The implications of the findings for biological application of grafted nanoparticles are discussed in the context of accepted models for surface ligand conformation.

The Immunoprobe Aggregation State is Central to Dipstick Immunoassay Performance.

As new infectious disease outbreaks become more likely, it is important to be able to develop and deploy appropriate testing in time. Paper-based immunoassays are rapid, cheap, and easy to produce at scale and relatively user friendly but often suffer from low selectivity and sensitivity. Understanding the molecular mechanisms of paper immunoassays may help improve and hasten development and therefore production and market availability. Here, we study how the behavior of nanoparticle-antibody immunoprobes in paper dipstick immunoassays is impacted by synthesis strategy and surface chemistry architecture. We conjugate gold nanoparticles to polyclonal anti-immunoglobulin G (IgG) and anti-zika NS1 antibodies by electrostatic adsorption and N-hydroxysuccinimide (NHS) and hydrazide (Hz) chemistries. The immunoprobes were used in paper immunoassays and the effective affinity for the antigen was quantified from the test line intensities, as well as the distribution of the immunoprobes throughout the strips. The results show that nanoparticle colloidal stability, both post synthesis and during antigen binding, is a key factor and affects immunoassay results and performance, often through reduction or loss of signal.

Labrasol® is an efficacious intestinal permeation enhancer across rat intestine: Ex vivo and in vivo rat studies.

Labrasol® ALF (Labrasol®), is a non-ionic surfactant excipient primarily used as a solubilising agent. It was investigated here as an intestinal permeation enhancer in isolated rat colonic mucosae in Ussing chamber and in rat in situ intestinal instillations. Labrasol® comprises mono-, di- and triglycerides and mono- and di- fatty acid esters of polyethylene glycol (PEG)-8 and free PEG-8, with caprylic (C8)- and capric acid (C10) as the main fatty acids. Source components of Labrasol® as well as Labrasol® modified with either C8 or C10 as the sole fatty acid components were also tested to determine which element of Labrasol® was responsible for its permeability-enhancing properties. Labrasol® (4, 8 mg/mL) enhanced the transport of the paracellular markers, [14C] mannitol, FITC-dextran 4000, and FITC-insulin across colonic mucosae. The enhancement was non-damaging, transient, and molecular weight-dependent. The PEG ester fraction of Labrasol® also had enhancing properties. When insulin was administered with Labrasol® in instillations, it had a relative bioavailability of 7% in jejunum and 12% in colon. C8- and C10 versions of Labrasol® and the PEG ester fraction also induced similar bioavailability values in jejunal instillations: 6, 5 and 7% respectively. Inhibition of lipases in instillations did not reduce the efficacy of Labrasol®, suggesting that its mechanism as a PE is not simply due to liberated medium chain fatty acids. Labrasol® acts as an efficacious intestinal permeation enhancer and has potential for use in oral formulations of macromolecules and BCS Class III molecules.

Designing Paper-Based Immunoassays for Biomedical Applications.

Paper-based sensors and assays have been highly attractive for numerous biological applications, including rapid diagnostics and assays for disease detection, food safety, and clinical care. In particular, the paper immunoassay has helped drive many applications in global health due to its low cost and simplicity of operation. This review is aimed at examining the fundamentals of the technology, as well as different implementations of paper-based assays and discuss novel strategies for improving their sensitivity, performance, or enabling new capabilities. These innovations can be categorized into using unique nanoparticle materials and structures for detection via different techniques, novel biological species for recognizing biomarkers, or innovative device design and/or architecture.

Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers.

Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In this work, Caco-2 intestinal epithelial cells were used as a well-established model for the intestinal barrier, and the uptake, trafficking and translocation of model silica nanoparticles of different sizes were investigated using a combination of imaging, flow cytometry and transport studies. Compared to typical observations in standard cell lines commonly used for in vitro studies, silica nanoparticle uptake into well-developed Caco-2 cellular barriers was found to be very low. Instead, nanoparticle association to the apical outer membrane was substantial and these particles could easily be misinterpreted as internalised in the absence of imaging. Passage of nanoparticles through the barrier was very limited, suggesting that the low amount of internalised nanoparticles was due to reduced uptake into cells, rather than a considerable transport through them.

Efficacy assessment of self-assembled PLGA-PEG-PLGA nanoparticles: Correlation of nano-bio interface interactions, biodistribution, internalization and gene expression studies.

The aim of our study was to develop and compare the biological performance of two types of biodegradable SN-38 loaded nanoparticles (NPs) with various surface properties, composed of low and high Mw triblock PLGA-PEG-PLGA copolymers, applying rational quality and safety by design approach. Therefore, along with the optimization of crucial physico-chemical properties and in order to evaluate the therapeutical potential and biocompatibility of prepared polymeric nanoparticles, analysis of nano-bio interactions, cell internalization, gene expression and biodistribution studies were performed. The optimized formulations, one of low Mw and one composed of high Mw PLGA-PEG-PLGA copolymer, exhibited different characteristics in terms of surface properties, particle size, zeta potential, drug loading, protein adsorption and biodistribution, which may be attributed to the variations in nano-bio interface interactions due to different NP building blocks length and Mw. On the contrary to protein adsorption and biodistribution studies, both types of NPs exhibited similar results during cell internalization and gene expression studies performed in cell culture medium containing serum proteins. This pool of useful data for internalization and efficacy as well as the notable advance in the circulation time of low Mw NPs may be further employed for shaping the potential of the designed nanocarriers.

Using single nanoparticle tracking obtained by nanophotonic force microscopy to simultaneously characterize nanoparticle size distribution and nanoparticle-surface interactions.

Comprehensive characterization of nanomaterials for medical applications is a challenging and complex task due to the multitude of parameters which need to be taken into consideration in a broad range of conditions. Routine methods such as dynamic light scattering or nanoparticle tracking analysis provide some insight into the physicochemical properties of particle dispersions. For nanomedicine applications the information they supply can be of limited use. For this reason, there is a need for new methodologies and instruments that can provide additional data on nanoparticle properties such as their interactions with surfaces. Nanophotonic force microscopy has been shown as a viable method for measuring the force between surfaces and individual particles in the nano-size range. Here we outline a further application of this technique to measure the size of single particles and based on these measurement build the distribution of a sample. We demonstrate its efficacy by comparing the size distribution obtained with nanophotonic force microscopy to established instruments, such as dynamic light scattering and differential centrifugal sedimentation. Our results were in good agreement to those observed with all other instruments. Furthermore, we demonstrate that the methodology developed in this work can be used to study complex particle mixtures and the surface alteration of materials. For all cases studied, we were able to obtain both the size and the interaction potential of the particles with a surface in a single measurement.

Identification of Receptor Binding to the Biomolecular Corona of Nanoparticles.

Biomolecules adsorbed on nanoparticles are known to confer a biological identity to nanoparticles, mediating the interactions with cells and biological barriers. However, how these molecules are presented on the particle surface in biological milieu remains unclear. The central aim of this study is to identify key protein recognition motifs and link them to specific cell-receptor interactions. Here, we employed an immuno-mapping technique to quantify epitope presentations of two major proteins in the serum corona, low-density lipoprotein and immunoglobulin G. Combining with a purpose-built receptor expression system, we show that both proteins present functional motifs to allow simultaneous recognition by low-density lipoprotein receptor and Fc-gamma receptor I of the corona. Our results suggest that the "labeling" of nanoparticles by biomolecular adsorption processes allows for multiple pathways in biological processes in which they may be "mistaken" for endogenous objects, such as lipoproteins, and exogenous ones, such as viral infections.

Mapping of Molecular Structure of the Nanoscale Surface in Bionanoparticles.

Characterizing the orientation of covalently conjugated proteins on nanoparticles, produced for in vitro and in vivo targeting, though an important feature of such a system, has proved challenging. Although extensive physicochemical characterization of targeting nanoparticles can be addressed in detail, relevant biological characterization of the nanointerface is crucial in order to select suitable nanomaterials for further in vitro or in vivo experiments. In this work, we adopt a methodology using antibody fragments (Fab) conjugated to gold nanoparticles (immunogold) to map the available epitopes on a transferrin grafted silica particle (SiO2-PEG8-Tf) as a proxy methodology to predict nanoparticle biological function, and therefore cellular receptor engagement. Data from the adopted method suggest that, on average, only ∼3.5% of proteins grafted on the SiO2-PEG8-Tf nanoparticle surface have a favorable orientation for recognition by the cellular receptor.

PEO-PPO-PEO/Poly(DL-lactide-co-caprolactone) Nanoparticles as Carriers for SN-38: Design, Optimization and Nano-Bio Interface Interactions.

Encapsulation of extremely hydrophobic substances such as SN-38 into nanoparticles, is a promising approach to solve the solubility issue and enable drug administration. Moreover, nanocarriers' tumor homing behavior, targeted and controlled release at the site of action will optimize therapeutic potency and decrease toxicity of the incorporated drug substance. However, the enormous drug hydrophobicity might limit the capacity for encapsulation as the premature drug precipitation will contribute to fast free drug crystal growth, low drug incorporation and huge waste of the active material. In this article we defined the optimal region for manufacturing of SN-38 loaded PEO-PPO-PEO/P(DL)LCL nanoparticles (NPs) with high efficacy of encapsulation, suitable particle size and different surface properties, using D-optimal design and nanoprecipitation as production method. Further we made an approach to investigate the interactions with macromolecules at the nano-bio interface which are predetermined by the physico-chemical and surface properties of the NPs, and are important determinants for the biological identity of the nanoparticles, the potential for evasion of the physiological barriers and the efficacy of localization at the site of action. Here we present in depth analysis of the behavior of two types of nanoparticles with different surface properties through structured protein interaction and bioreactivity experiments in order to presuppose NP performance and toxicological profile in biological environment.

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.

Controlling aqueous silica nanoparticle synthesis in the 10-100 nm range.

We describe the control of size and homogeneity in silica nanoparticle dispersions, prepared by a two-phase arginine catalysed aqueous method, through varying the upper organic solvent phase. The final particle dispersion characteristics can be controlled by varying features including solvent type and interfacial area, related to the rate of monomer transfer at the TEOS/water interface.

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.

Stabilising fluorescent silica nanoparticles against dissolution effects for biological studies.

Here we show that commonly employed Stöber type fluorescently labelled silica nanoprobes degrade by hydrolytic dissolution, accelerated under biological media conditions as compared to water alone. We have thus developed a method to greatly improve their stability under such conditions.