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A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.
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Almacenaje de Medicamentos , Hidrogeles , Desnaturalización Proteica , Estabilidad Proteica , Proteínas , Jeringas , Humanos , Excipientes , Liofilización , Hidrogeles/química , Proteínas/administración & dosificación , Proteínas/química , Proteínas/economía , Trehalosa , Congelación , Refrigeración , Vacunas contra Papillomavirus/química , Almacenaje de Medicamentos/economía , Almacenaje de Medicamentos/métodosRESUMEN
In structural terms, the sialic acids are a large family of nine carbon sugars based around an alpha-keto acid core. They are widely spread in nature, where they are often found to be involved in molecular recognition processes, including in development, immunology, health and disease. The prominence of sialic acids in infection is a result of their exposure at the non-reducing terminus of glycans in diverse glycolipids and glycoproteins. Herein, we survey representative aspects of sialic acid structure, recognition and exploitation in relation to infectious diseases, their diagnosis and prevention or treatment. Examples covered span influenza virus and Covid-19, Leishmania and Trypanosoma, algal viruses, Campylobacter, Streptococci and Helicobacter, and commensal Ruminococci.
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Glycoforms (and other post-translational modifications) of otherwise identical proteins can indicate pathogenesis/disease state and hence new tools to detect and sense a protein's glycosylation status are essential. Antibody-based assays against specific protein sequences do not typically discriminate between glycoforms. Here we demonstrate a 'sandwich' bio-assay approach, whereby antibodies immobilised onto biolayer interferometry sensors first select proteins, and then the specific glycoform is identified using gold nanoparticles functionalised with lectins which provide signal enhancement. The nanoparticles significantly enhance the signal relative to lectins alone, allowing glycoform specific detection as low as 0.04 µg mL-1 (1.4 nM) in buffer, and crucially there is no need for an enrichment step and all steps can be automated. Proof of concept is demonstrated using prostate specific antigen: a biomarker for prostate cancer, where glycoform analysis could distinguish between cancerous and non-cancerous status, rather than only detecting overall protein concentration.
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Lectinas , Nanopartículas del Metal , Humanos , Masculino , Anticuerpos , Glicosilación , Oro , Lectinas/metabolismo , Neoplasias de la Próstata/diagnóstico , Antígeno Prostático Específico/análisisRESUMEN
Glycan-lectin interactions play essential roles in biology; as the site of attachment for pathogens, cell-cell communication, and as crucial players in the immune system. Identifying if a new glycan (natural or unnatural) binds a protein partner, or if a new protein (or mutant) binds a glycan remains a non-trivial problem, with few accessible or low-cost tools available. Micro-arrays allow for the interrogation of 100's of glycans but are not widely available in individual laboratories. Biophysical techniques such as isothermal titration calorimetry, surface plasmon resonance spectrometry, biolayer interferometry and nuclear magnetic resonance spectroscopy all provide detailed understanding of glycan binding but are relatively expensive. Glycosylated plasmonic nanoparticles based on gold cores with polymeric tethers have emerged as biosensors to detect glycan-protein binding, based on colourimetric (red to blue) outputs which can be easily interpreted by a simple UV-visible spectrometer or by eye. Despite the large number of reports there are no standard protocols for each system or recommended start points, to allow a new user to deploy this technology. Here we explore the key parameters of nanoparticle size, polymeric tether length and gold concentration to provide some guidelines for how polymer-tethered glycosylated gold nanoparticles can be used to probe a new glycan/protein interactions, with minimal optimisation barriers. This work aimed to remove the need to explore chemical and nanoparticle space and hence remove a barrier for other users when deploying this system. We show that the concentration of the gold core is crucial to balance strong responses versus false positives and recommend a gold core size and polymer tether length which balances sufficient colloidal stability and output. Whilst subtle differences between glycans/lectins will impact the outcomes, these parameters should enable a lab user to quickly evaluate binding using minimal quantities of the glycan and lectin, to select candidates for further study.
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The COVID-19 pandemic has highlighted the need for innovative biosensing, diagnostic, and surveillance platforms. Here we report that glycosylated, polymer-stabilized, gold nanorods can bind the SARS-CoV-2 spike protein and show correlation to the presence of SARS-CoV-2 in primary COVID-19 clinical samples. Telechelic polymers were prepared by reversible addition-fragmentation chain-transfer polymerization, enabling the capture of 2,3-sialyllactose and immobilization onto gold nanorods. Control experiments with a panel of lectins and a galactosamine-terminated polymer confirmed the selective binding. The glycosylated rods were shown to give dose-dependent responses against recombinant truncated SARS-CoV-2 spike protein, and the responses were further correlated using primary patient swab samples. The essentiality of the anisotropic particles for reducing the background interference is demonstrated. This highlights the utility of polymer tethering of glycans for plasmonic biosensors of infection.
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COVID-19 , Nanotubos , COVID-19/diagnóstico , Oro , Humanos , Pandemias , Polímeros , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Lateral flow devices are rapid (and often low cost) point-of-care diagnostics-the classic example being the home pregnancy test. A test line (the stationary phase) is typically prepared by the physisorption of an antibody, which binds to analytes/antigens such as viruses, toxins, or hormones. However, there is no intrinsic requirement for the detection unit to be an antibody, and incorporating other ligand classes may bring new functionalities or detection capabilities. To enable other (nonprotein) ligands to be deployed in lateral flow devices, they must be physiosorbed to the stationary phase as a conjugate, which currently would be a high-molecular-weight carrier protein, which requires (challenging) chemoselective modifications and purification. Here, we demonstrate that poly(vinylpyrrolidone), PVP, is a candidate for a polymeric, protein-free test line, owing to its unique balance of water solubility (for printing) and adhesion to the nitrocellulose stationary phase. End-functionalized PVPs were prepared by RAFT polymerization, and the model capture ligands of biotin and galactosamine were installed on PVP and subsequently immobilized on nitrocellulose. This polymeric test line was validated in both flow-through and full lateral flow formats using streptavidin and soybean agglutinin and is the first demonstration of an "all-polymer" approach for installation of capture units. This work illustrates the potential of polymeric scaffolds as anchoring agents for small-molecule capture agents in the next generation of robust and modular lateral flow devices and that macromolecular engineering may provide real benefit.
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Lateral flow immuno-assays, such as the home pregnancy test, are rapid point-of-care diagnostics that use antibody-coated nanoparticles to bind antigens/analytes (e.g., viruses, toxins or hormones). Ease of use, no need for centralized infrastructure and low-cost, makes these devices appealing for rapid disease identification, especially in low-resource environments. Here glycosylated polymer-coated nanoparticles are demonstrated for the sensitive, label-free detection of lectins in lateral flow and flow-through. The systems introduced here use glycans, not antibodies, to provide recognition: a "lateral flow glyco-assay," providing unique biosensing opportunities. Glycans are installed onto polymer termini and immobilized onto gold nanoparticles, providing colloidal stability but crucially also introducing assay tunability and selectivity. Using soybean agglutinin and Ricinus communis agglutinin I (RCA120 ) as model analytes, the impact of polymer chain length and nanoparticle core size are evaluated, with chain length found to have a significant effect on signal generation-highlighting the need to control the macromolecular architecture to tune response. With optimized systems, lectins are detectable at subnanomolar concentrations, comparable to antibody-based systems. Complete lateral flow devices are also assembled to show how these devices can be deployed in the "real world." This work shows that glycan-binding can be a valuable tool in rapid diagnostics.
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Oro , Nanopartículas del Metal , Bioensayo , Oro/química , Lectinas/metabolismo , PolímerosRESUMEN
The COVID-19 pandemic, and future pandemics, require diagnostic tools to track disease spread and guide the isolation of (a)symptomatic individuals. Lateral-flow diagnostics (LFDs) are rapid and of lower cost than molecular (genetic) tests, with current LFDs using antibodies as their recognition units. Herein, we develop a prototype flow-through device (related, but distinct to LFDs), utilizing N-acetyl neuraminic acid-functionalized, polymer-coated, gold nanoparticles as the detection/capture unit for SARS-COV-2, by targeting the sialic acid-binding site of the spike protein. The prototype device can give rapid results, with higher viral loads being faster than lower viral loads. The prototype's effectiveness is demonstrated using spike protein, lentiviral models, and a panel of heat-inactivated primary patient nasal swabs. The device was also shown to retain detection capability toward recombinant spike proteins from several variants (mutants) of concern. This study provides the proof of principle that glyco-lateral-flow devices could be developed to be used in the tracking monitoring of infectious agents, to complement, or as alternatives to antibody-based systems.
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COVID-19 , Nanopartículas del Metal , Oro , Humanos , Pandemias , Polisacáridos , SARS-CoV-2RESUMEN
Cancer cells generally exhibit higher metabolic demands relative to that of normal tissue cells. This offers great possibilities to exploit metabolic glycoengineering in combination with bio-orthogonal chemistry reactions to achieve tumour site-targeted therapeutic delivery. This work addresses the selectivity of metabolic glycan labelling in diseased (i.e., cancer) versus normal cells grown in a multicellular environment. Dibenzocylooctyne (DBCO)-bearing acetylated-d-mannosamine (Ac4ManNDBCO) was synthesised to metabolically label three different types of cell lines originating from the human lung tissues: A549 adenocarcinomic alveolar basal epithelial cells, MeT5A non-cancerous mesothelial cells, and MRC5 non-cancerous fibroblasts. These cell lines displayed different labelling sensitivity, which trended with their doubling time in the following order: A549 ≈ MeT5A > MRC5. The higher metabolic labelling efficiency inherently led to a higher extent of specific binding and accumulation of the clickable N3-conjugated gold nanoparticles (N3-AuNps, core diameter = 30 nm) in the DBCO-glycan modified A549 and MeT5A cells, but to a less prominent effect in MRC5 cells. These findings demonstrate that relative rates of cell metabolism can be exploited using metabolic labelling to recruit nanotherapeutics whilst minimising non-specific targeting of surrounding tissues.
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Ciclooctanos/metabolismo , Sistemas de Liberación de Medicamentos , Oro/metabolismo , Hexosaminas/metabolismo , Nanopartículas del Metal/química , Polisacáridos/metabolismo , Línea Celular , Química Clic , Ciclooctanos/química , Células Epiteliales/química , Células Epiteliales/metabolismo , Fibroblastos/química , Fibroblastos/metabolismo , Oro/química , Hexosaminas/química , Humanos , Estructura Molecular , Tamaño de la Partícula , Polisacáridos/química , Propiedades de SuperficieRESUMEN
[This corrects the article DOI: 10.1021/acscentsci.0c00855.].
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Multivalent glycosylated materials (polymers, surfaces, and particles) often show high affinity toward carbohydrate binding proteins (e.g., lectins) due to the nonlinear enhancement from the cluster glycoside effect. This affinity gain has potential in applications from diagnostics, biosensors, and targeted delivery to anti-infectives and in an understanding of basic glycobiology. This perspective highlights the question of selectivity, which is less often addressed due to the reductionist nature of glycomaterials and the promiscuity of many lectins. The use of macromolecular features, including architecture, heterogeneous ligand display, and the installation of non-natural glycans, to address this challenge is discussed, and examples of selectivity gains are given.
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There is an urgent need to understand the behavior of the novel coronavirus (SARS-COV-2), which is the causative agent of COVID-19, and to develop point-of-care diagnostics. Here, a glyconanoparticle platform is used to discover that N-acetyl neuraminic acid has affinity toward the SARS-COV-2 spike glycoprotein, demonstrating its glycan-binding function. Optimization of the particle size and coating enabled detection of the spike glycoprotein in lateral flow and showed selectivity over the SARS-COV-1 spike protein. Using a virus-like particle and a pseudotyped lentivirus model, paper-based lateral flow detection was demonstrated in under 30 min, showing the potential of this system as a low-cost detection platform.
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Glycomaterials display enhanced binding affinity to carbohydrate-binding proteins due to the nonlinear enhancement associated with the cluster glycoside effect. Gold nanoparticles bearing glycans have attracted significant interest in particular. This is due to their versatility, their highly tunable gold cores (size and shape), and their application in biosensors and diagnostic tools. However, conjugating glycans onto these materials can be challenging, necessitating either multiple protecting group manipulations or the use of only simple glycans. This results in limited structural diversity compared to glycoarrays which can include hundreds of glycans. Here we report a method to generate glyconanoparticles from unprotected glycans by conjugation to polymer tethers bearing terminal amino-oxy groups, which are then immobilized onto gold nanoparticles. Using an isotope-labeled glycan, the efficiency of this reaction was probed in detail to confirm conjugation, with 25% of end-groups being functionalized, predominantly in the ring-closed form. Facile post-glycosylation purification is achieved by simple centrifugation/washing cycles to remove excess glycan and polymer. This streamlined synthetic approach may be particularly useful for the preparation of glyconanoparticle libraries using automation, to identify hits to be taken forward using more conventional synthetic methods. Exemplar lectin-binding studies were undertaken to confirm the availability of the glycans for binding and show this is a powerful tool for rapid assessment of multivalent glycan binding.
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Oro/química , Nanopartículas del Metal/química , Polímeros/química , Polisacáridos/química , Aminación , Ligandos , Nanotecnología , Polímeros/síntesis química , Polisacáridos/síntesis químicaRESUMEN
During influenza infection, hemagglutinins (HAs) on the viral surface bind to sialic acids on the host cell's surface. While all HAs bind sialic acids, human influenza targets terminal α2,6 sialic acids and avian influenza targets α2,3 sialic acids. For interspecies transmission (zoonosis), HA must mutate to adapt to these differences. Here, multivalent gold nanoparticles bearing either α2,6- or α2,3-sialyllactosamine have been developed to interrogate a panel of HAs from pathogenic human, low pathogenic avian, and other species' influenza. This method exploits the benefits of multivalent glycan presentation compared to monovalent presentation to increase affinity and investigate how multivalency affects selectivity. Using a library-orientated approach, parameters including polymer coating and core diameter were optimized for maximal binding and specificity were probed using galactosylated particles and a panel of biophysical techniques [ultraviolet-visible spectroscopy, dynamic light scattering, and biolayer interferometry]. The optimized particles were then functionalized with sialyllactosamine and their binding analyzed against a panel of HAs derived from pathogenic influenza strains including low pathogenic avian strains. This showed significant specificity crossover, which is not observed in monovalent formats, with binding of avian HAs to human sialic acids and vice versa in agreement with alternate assay formats. These results demonstrate that precise multivalent presentation is essential to dissect the interactions of HAs and may aid the discovery of tools for disease and zoonosis transmission.
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Gripe Humana , Nanopartículas del Metal , Animales , Oro , Hemaglutininas , Humanos , PolímerosRESUMEN
Galectins are potential biomarkers and therapeutic targets. However, galectins display broad affinity towards ß-galactosides meaning glycan-based (nano)biosensors lack the required selectivity and affinity. Using a polymer-stabilized nanoparticle biosensing platform, we herein demonstrate that the specificity of immobilised lacto-N-biose towards galectins can be 'turned on/off' by using site-specific glycan fluorination and in some cases reversal of specificity can be achieved. The panel of fluoro-glycans were obtained by a chemoenzymatic approach, exploiting BiGalK and BiGalHexNAcP enzymes from Bifidobacterium infantis which are shown to tolerate fluorinated glycans, introducing structural diversity which would be very laborious by chemical methods alone. These results demonstrate that integrating non-natural, fluorinated glycans into nanomaterials can encode unprecedented selectivity with potential applications in biosensing.
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Glycan-lectin interactions drive a diverse range of biological signaling and recognition processes. The display of glycans in multivalent format enables their intrinsically weak binding affinity to lectins to be overcome by the cluster glycoside effect, which results in a non-linear increase in binding affinity. As many lectins have multiple binding sites, upon interaction with glycosylated nanomaterials either aggregation or surface binding without aggregation can occur. Depending on the application area, either one of these responses are desirable (or undesirable) but methods to tune the aggregation state, independently from the overall extent/affinity of binding are currently missing. Herein, we use gold nanoparticles decorated with galactose-terminated polymer ligands, obtained by photo-initiated RAFT polymerization to ensure high end-group fidelity, to show the dramatic impact on agglutination behaviour due to the chemistry of the polymer linker. Poly(N-hydroxyethyl acrylamide) (PHEA)-coated gold nanoparticles, a polymer widely used as a non-ionic stabilizer, showed preference for aggregation with lectins compared to poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA)-coated nanoparticles which retained colloidal stability, across a wide range of polymer lengths and particle core sizes. Using biolayer interferometry, it was observed that both coatings gave rise to similar binding affinity and hence provided conclusive evidence that aggregation rate alone cannot be used to measure affinity between nanoparticle systems with different stabilizing linkers. This is significant, as turbidimetry is widely used to demonstrate glycomaterial activity, although this work shows the most aggregating may not be the most avid, when comparing different polymer backbones/coating. Overall, our findings underline the potential of PHPMA as the coating of choice for applications where aggregation upon lectin binding would be problematic, such as in vivo imaging or drug delivery.
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Resinas Acrílicas/metabolismo , Lectinas de Plantas/metabolismo , Ácidos Polimetacrílicos/metabolismo , Proteínas de Soja/metabolismo , Sitios de Unión , Glicosilación , Nanopartículas del Metal , Unión ProteicaRESUMEN
The huge chemical space potential of synthetic polymers means that in many studies only a small parameter range can be realistically synthesized in a short time and hence the "best" formulations may not be optimum. Throughput is traditionally limited by the need for deoxygenation in radical polymerizations, but advances in photopolymerization now provide opportunities for "in-air" polymerizations. Here, we have developed a protocol using liquid handling robots (or multichannel pipettes) with blue light photolysis of reversible addition fragmentation chain transfer agents and tertiary amine deoxygenation to enable the synthesis of polymer libraries in industry-standard 96-well plates with no specialized infrastructure and no degassing step. The roles of solvents and amine deoxygenators are explored to optimize the polymerization, particularly to look at alternatives to dimethyl sulfoxide (DMSO) for hydrophobic monomer (co)polymerization. DMSO is shown to aid the degassing process but is not easy to remove and hence prevents isolation of pure polymers. In contrast, using dioxane in-plate evaporation or precipitation of the tertiary amine allowed isolation of polymers in-plate. This removed all reaction components yielding pure polymers, which is not easily achieved with systems using metal catalysts and secondary reductants, such as ascorbic acid. As an example of the throughput, in just under 40 h, 392 polymers were synthesized and subsequently analyzed direct from plates by a 96-well plate sampling size exclusion chromatography system to demonstrate reproducibility. Due to less efficient degassing in dioxane (compared to DMSO), the molecular weight and dispersity control were limited in some cases (with acrylates giving the lowest dispersities), but the key aim of this system is to access hundreds of polymers quickly and in a format needed to enable testing. This method enables easy exploration of chemical space and development of screening libraries to identify hits for further study using precision polymerization methods.
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There is an urgent need to identify new, non-traditional antimicrobials. The discovery of new polymeric antimicrobials is limited by current low-throughput synthetic tools, which means that limited chemical space has been explored. Herein, we employ photochemical "in-air" reversible addition-fragmentation chain-transfer (RAFT) polymerization with microwell plates, using liquid-handling robots to assemble large libraries of cationic polymers, without the need for degassing or purification steps, facilitating transfer to screening. Several lead polymers were identified including a co-polymer with propylene glycol side chains with significantly enhanced antimicrobial activity and increased therapeutic window. Mechanistic studies showed that this polymer was bacteriostatic, and surprisingly did not lyse the cell membranes, implying an alternative mode of action. This versatile method using simple robotics will help to develop new biomaterials with emergent properties.
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Antibacterianos/síntesis química , Polímeros/síntesis química , Cationes , Evaluación Preclínica de Medicamentos , Escherichia coli/efectos de los fármacos , Humanos , Luz , Metacrilatos/química , Mycobacterium/efectos de los fármacos , Procesos Fotoquímicos , Polimerizacion , Bibliotecas de Moléculas Pequeñas/síntesis químicaRESUMEN
Because of the emergence of antimicrobial resistance to traditional small-molecule drugs, cationic antimicrobial polymers are appealing targets. Mycobacterium tuberculosis is a particular problem, with multi- and total drug resistance spreading and more than a billion latent infections globally. This study reports nanoparticles bearing variable densities of poly(dimethylaminoethyl methacrylate) and the unexpected and distinct mechanisms of action this multivalent presentation imparts against Escherichia coli versus Mycobacterium smegmatis (model of M. tuberculosis), leading to killing or growth inhibition, respectively. A convergent "grafting to" synthetic strategy was used to assemble a 50-member nanoparticle library, and using a high-throughput screen identified that only the smallest (2 nm) particles were stable in both saline and complex cell media. Compared with the linear polymers, the nanoparticles displayed two- and eight-fold enhancements in antimicrobial activity against M. smegmatis and E. coli, respectively. Mechanistic studies demonstrated that the antimicrobial particles were bactericidal against E. coli due to rapid disruption of the cell membranes. Conversely, against M. smegmatis the particles did not lyse the cell membrane but rather had a bacteriostatic effect. These results demonstrate that to develop new polymeric antituberculars the widely assumed, broad spectrum, membrane-disrupting mechanism of polycations must be re-evaluated. It is clear that synthetic nanomaterials can engage in more complex interactions with mycobacteria, which we hypothesize is due to the unique cell envelope at the surface of these bacteria.
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Antibacterianos/farmacología , Antituberculosos/farmacología , Escherichia coli/efectos de los fármacos , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Nanopartículas , Polímeros/farmacología , Antibacterianos/química , Antituberculosos/química , Pruebas de Sensibilidad Microbiana , Nanopartículas/químicaRESUMEN
Nature dynamically controls carbohydrate expression on cells rather than static presentation. Here we report synthetic glycosylated nanoparticles that contain polymeric 'gates' to enable external control (via temperature changes) of glycan surface expression, as an alternative to enzymatic control in nature. This approach offers a new dynamic multivalent scaffold for glycan recognition.