Publications by authors named "Matthew I Gibson"

124 Publications

Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes.

RSC Med Chem 2021 May 18;12(6):982-993. Epub 2021 May 18.

Department of Chemistry, University of Warwick Gibbet Hill Road Coventry CV4 7AL UK

Cell cryopreservation is an essential tool for drug toxicity/function screening and transporting cell-based therapies, and is essential in most areas of biotechnology. There is a challenge, however, associated with the cryopreservation of cells in monolayer format (attached to tissue culture substrates) which gives far lower cell yields (<20% typically) compared to suspension freezing. Here we investigate the mechanisms by which the protective osmolyte l-proline enhances cell-monolayer cryopreservation. Pre-incubating A549 cells with proline, prior to cryopreservation in monolayers, increased post-thaw cell yields two-fold, and the recovered cells grow faster compared to cells cryopreserved using DMSO alone. Further increases in yield were achieved by adding polymeric ice recrystallization inhibitors, which gave limited benefit in the absence of proline. Mechanistic studies demonstrated a biochemical, rather than biophysical ( not affecting ice growth) mode of action. It was observed that incubating cells with proline (before freezing) transiently reduced the growth rate of the cells, which was not seen with other osmolytes (betaine and alanine). Removal of proline led to rapid growth recovery, suggesting that proline pre-conditions the cells for cold stress, but with no impact on downstream cell function. Whole cell proteomics did not reveal a single pathway or protein target but rather cells appeared to be primed for a stress response in multiple directions, which together prepare the cells for freezing. These results support the use of proline alongside standard conditions to improve post-thaw recovery of cell monolayers, which is currently considered impractical. It also demonstrates that a chemical biology approach to discovering small molecule biochemical modulators of cryopreservation may be possible, to be used alongside traditional (solvent) based cryoprotectants.
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http://dx.doi.org/10.1039/d1md00078kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221256PMC
May 2021

Introducing affinity and selectivity into galectin-targeting nanoparticles with fluorinated glycan ligands.

Chem Sci 2020 Nov 16;12(3):905-910. Epub 2020 Nov 16.

Department of Chemistry, University of Warwick CV4 7AL UK

Galectins are potential biomarkers and therapeutic targets. However, galectins display broad affinity towards β-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--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 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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http://dx.doi.org/10.1039/d0sc05360kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179109PMC
November 2020

Covalent cell surface recruitment of chemotherapeutic polymers enhances selectivity and activity.

Chem Sci 2021 Feb 12;12(12):4557-4569. Epub 2021 Feb 12.

Department of Chemistry, University of Warwick Coventry CV4 7AL UK

Synthetic macromolecular chemotherapeutics inspired by host defence peptides can disrupt cell membranes and are emerging as agents for the treatment of cancer and infections. However, their off-target effects remain a major unmet challenge. Here we introduce a covalent recruitment strategy, whereby metabolic oligosaccharide engineering is used to label targeted cells with azido glycans, to subsequently capture chemotherapeutic polymers by a bio-orthogonal click reaction. This results in up to 10-fold reduction in EC and widening of the therapeutic window. Cell death is induced by not only membrane leakage, but also by apoptosis due to the conjugated chemotherapeutic being internalised by glycan recycling. Covalent recruitment also lead to increased penetration and significant cell death in a 3-D tumour model in just 3 hours, whereas doxorubicin required 24 hours. This conceptual approach of 'engineering cells to capture polymers' rather than 'engineering polymers to target cells' will bring new opportunities in non-traditional macromolecular therapeutics.
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http://dx.doi.org/10.1039/d0sc06580cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179505PMC
February 2021

Understanding selectivity of metabolic labelling and click-targeting in multicellular environments as a route to tissue selective drug delivery.

J Mater Chem B 2021 Jul;9(26):5365-5373

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, VIC 3052, Australia. and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, VIC 3052, Australia.

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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http://dx.doi.org/10.1039/d1tb00721aDOI Listing
July 2021

A multi-OMIC characterisation of biodegradation and microbial community succession within the PET plastisphere.

Microbiome 2021 06 21;9(1):141. Epub 2021 Jun 21.

School of Life Sciences, University of Warwick, Coventry, UK.

Background: Plastics now pollute marine environments across the globe. On entering these environments, plastics are rapidly colonised by a diverse community of microorganisms termed the plastisphere. Members of the plastisphere have a myriad of diverse functions typically found in any biofilm but, additionally, a number of marine plastisphere studies have claimed the presence of plastic-biodegrading organisms, although with little mechanistic verification. Here, we obtained a microbial community from marine plastic debris and analysed the community succession across 6 weeks of incubation with different polyethylene terephthalate (PET) products as the sole carbon source, and further characterised the mechanisms involved in PET degradation by two bacterial isolates from the plastisphere.

Results: We found that all communities differed significantly from the inoculum and were dominated by Gammaproteobacteria, i.e. Alteromonadaceae and Thalassospiraceae at early time points, Alcanivoraceae at later time points and Vibrionaceae throughout. The large number of encoded enzymes involved in PET degradation found in predicted metagenomes and the observation of polymer oxidation by FTIR analyses both suggested PET degradation was occurring. However, we were unable to detect intermediates of PET hydrolysis with metabolomic analyses, which may be attributed to their rapid depletion by the complex community. To further confirm the PET biodegrading potential within the plastisphere of marine plastic debris, we used a combined proteogenomic and metabolomic approach to characterise amorphous PET degradation by two novel marine isolates, Thioclava sp. BHET1 and Bacillus sp. BHET2. The identification of PET hydrolytic intermediates by metabolomics confirmed that both isolates were able to degrade PET. High-throughput proteomics revealed that whilst Thioclava sp. BHET1 used the degradation pathway identified in terrestrial environment counterparts, these were absent in Bacillus sp. BHET2, indicating that either the enzymes used by this bacterium share little homology with those characterised previously, or that this bacterium uses a novel pathway for PET degradation.

Conclusions: Overall, the results of our multi-OMIC characterisation of PET degradation provide a significant step forwards in our understanding of marine plastic degradation by bacterial isolates and communities and evidences the biodegrading potential extant in the plastisphere of marine plastic debris. Video abstract.
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http://dx.doi.org/10.1186/s40168-021-01054-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215760PMC
June 2021

The polymeric glyco-linker controls the signal outputs for plasmonic gold nanorod biosensors due to biocorona formation.

Nanoscale 2021 Jun;13(24):10837-10848

Health Unit, Flemish Institute for Technological Research (VITO), Boeretang 200, Mol, BE-2400, Belgium.

Gold nanorods (GNRs) are a promising platform for nanoplasmonic biosensing. The localised surface plasmon resonance (LSPR) peak of GNRs is located in the near-infrared optical window and is sensitive to local binding events, enabling label-free detection of biomarkers in complex biological fluids. A key challenge in the development of such sensors is achieving target affinity and selectivity, while both minimizing non-specific binding and maintaining colloidal stability. Herein, we reveal how GNRs decorated with galactosamine-terminated polymer ligands display significantly different binding responses in buffer compared to serum, due to biocorona formation, and how biocorona displacement due to lectin binding plays a key role in their optical responses. GNRs were coated with either poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) or poly(N-hydroxyethyl acrylamide) (PHEA) prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerisation and end-functionalised with galactosamine (Gal) as the lectin-targeting unit. In buffer Gal-PHEA-coated GNRs aggregated upon soybean agglutinin (SBA) addition, whereas Gal-PHPMA-coated GNRs exhibited a red-shift of the LSPR spectrum without aggregation. In contrast, when incubated in serum Gal-PHPMA-coated nanorods showed no binding response, while Gal-PHEA GNRs exhibited a dose-dependent blue-shift of the LSPR peak, which is the opposite direction (red-shift) to what was observed in buffer. This differential behaviour was attributed to biocorona formation onto both polymer-coated GNRs, shown by differential centrifugal sedimentation and nanoparticle tracking analysis. Upon addition of SBA to the Gal-PHEA coated nanorods, signal was generated due to displacement of weakly-bound biocorona components by lectin binding. However, in the case of Gal-PHPMA which had a thicker corona, attributed to lower polymer grafting densities, addition of SBA did not lead to biocorona displacement and there was no signal output. These results show that plasmonic optical responses in complex biological media can be significantly affected by biocorona formation, and that biocorona formation itself does not prevent sensing so long as its exact nature (e.g. 'hard versus soft') is tuned.
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http://dx.doi.org/10.1039/d1nr01548fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223873PMC
June 2021

Red Blood Cell Cryopreservation with Minimal Post-Thaw Lysis Enabled by a Synergistic Combination of a Cryoprotecting Polyampholyte with DMSO/Trehalose.

Biomacromolecules 2021 Jun 7. Epub 2021 Jun 7.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.

From trauma wards to chemotherapy, red blood cells are essential in modern medicine. Current methods to bank red blood cells typically use glycerol (40 wt %) as a cryoprotective agent. Although highly effective, the deglycerolization process, post-thaw, is time-consuming and results in some loss of red blood cells during the washing procedures. Here, we demonstrate that a polyampholyte, a macromolecular cryoprotectant, synergistically enhances ovine red blood cell cryopreservation in a mixed cryoprotectant system. Screening of DMSO and trehalose mixtures identified optimized conditions, where cytotoxicity was minimized but cryoprotective benefit maximized. Supplementation with polyampholyte allowed 97% post-thaw recovery (3% hemolysis), even under extremely challenging slow-freezing and -thawing conditions. Post-thaw washing of the cryoprotectants was tolerated by the cells, which is crucial for any application, and the optimized mixture could be applied directly to cells, causing no hemolysis after 1 h of exposure. The procedure was also scaled to use blood bags, showing utility on a scale relevant for application. Flow cytometry and adenosine triphosphate assays confirmed the integrity of the blood cells post-thaw. Microscopy confirmed intact red blood cells were recovered but with some shrinkage, suggesting that optimization of post-thaw washing could further improve this method. These results show that macromolecular cryoprotectants can provide synergistic benefit, alongside small molecule cryoprotectants, for the storage of essential cell types, as well as potential practical benefits in terms of processing/handling.
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http://dx.doi.org/10.1021/acs.biomac.1c00599DOI Listing
June 2021

A minimalistic cyclic ice-binding peptide from phage display.

Nat Commun 2021 05 11;12(1):2675. Epub 2021 May 11.

Laboratoire des Polymères, Institut des Matériaux and Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Rather than relying on the (limited) existing structure-property relationships that have been established for IBPs, here we report the use of phage display for the identification of short peptide mimics of IBPs. To this end, an ice-affinity selection protocol is developed, which enables the selection of a cyclic ice-binding peptide containing just 14 amino acids. Mutational analysis identifies three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics simulations reveal that the side chain of Thr10 hydrophobically binds to ice revealing a potential mechanism. To demonstrate the biotechnological potential of this peptide, it is expressed as a fusion ('Ice-Tag') with mCherry and used to purify proteins directly from cell lysate.
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http://dx.doi.org/10.1038/s41467-021-22883-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113477PMC
May 2021

Polymer Self-Assembly Induced Enhancement of Ice Recrystallization Inhibition.

J Am Chem Soc 2021 May 4;143(19):7449-7461. Epub 2021 May 4.

Department of Chemistry, University of Warwick, Gibbet Hill Road, CV4 7AL Coventry, U.K.

Ice binding proteins modulate ice nucleation/growth and have huge (bio)technological potential. There are few synthetic materials that reproduce their function, and rational design is challenging due to the outstanding questions about the mechanisms of ice binding, including whether ice binding is essential to reproduce all their macroscopic properties. Here we report that nanoparticles obtained by polymerization-induced self-assembly (PISA) inhibit ice recrystallization (IRI) despite their constituent polymers having no apparent activity. Poly(ethylene glycol), poly(dimethylacrylamide), and poly(vinylpyrrolidone) coronas were all IRI-active when assembled into nanoparticles. Different core-forming blocks were also screened, revealing the core chemistry had no effect. These observations show ice binding domains are not essential for macroscopic IRI activity and suggest that the size, and crowding, of polymers may increase the IRI activity of "non-active" polymers. It was also discovered that poly(vinylpyrrolidone) particles had ice crystal shaping activity, indicating this polymer can engage ice crystal surfaces, even though on its own it does not show any appreciable ice recrystallization inhibition. Larger (vesicle) nanoparticles are shown to have higher ice recrystallization inhibition activity compared to smaller (sphere) particles, whereas ice nucleation activity was not found for any material. This shows that assembly into larger structures can increase IRI activity and that increasing the "size" of an IRI does not always lead to ice nucleation. This nanoparticle approach offers a platform toward ice-controlling soft materials and insight into how IRI activity scales with molecular size of additives.
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http://dx.doi.org/10.1021/jacs.1c01963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154521PMC
May 2021

Physicochemical Approach to Understanding the Structure, Conformation, and Activity of Mannan Polysaccharides.

Biomacromolecules 2021 04 17;22(4):1445-1457. Epub 2021 Mar 17.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy.

Extracellular polysaccharides are widely produced by bacteria, yeasts, and algae. These polymers are involved in several biological functions, such as bacteria adhesion to surface and biofilm formation, ion sequestering, protection from desiccation, and cryoprotection. The chemical characterization of these polymers is the starting point for obtaining relationships between their structures and their various functions. While this fundamental correlation is well reported and studied for the proteins, for the polysaccharides, this relationship is less intuitive. In this paper, we elucidate the chemical structure and conformational studies of a mannan exopolysaccharide from the permafrost isolated bacterium strain 273-4. The mannan from the cold-adapted bacterium was compared with its dephosphorylated derivative and the commercial product from . Starting from the chemical structure, we explored a new approach to deepen the study of the structure/activity relationship. A pool of physicochemical techniques, ranging from small-angle neutron scattering (SANS) and dynamic and static light scattering (DLS and SLS, respectively) to circular dichroism (CD) and cryo-transmission electron microscopy (cryo-TEM), have been used. Finally, the ice recrystallization inhibition activity of the polysaccharides was explored. The experimental evidence suggests that the mannan exopolysaccharide from bacterium has an efficient interaction with the water molecules, and it is structurally characterized by rigid-rod regions assuming a 14-helix-type conformation.
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http://dx.doi.org/10.1021/acs.biomac.0c01659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045027PMC
April 2021

Ice recrystallisation inhibiting polymer nano-objects saline-tolerant polymerisation-induced self-assembly.

Mater Horiz 2020 Jul 14;8(7):1883-1887. Epub 2020 May 14.

Department of Chemistry, University of Warwick, CV4 7AL, UK.

Chemical tools to modulate ice formation/growth have great (bio)-technological value, with ice binding/antifreeze proteins being exciting targets for biomimetic materials. Here we introduce polymer nanomaterials that are potent inhibitors of ice recrystallisation using polymerisation-induced self-assembly (PISA), employing a poly(vinyl alcohol) graft macromolecular chain transfer agent (macro-CTA). Crucially, engineering the core-forming block with diacetone acrylamide enabled PISA to be conducted in saline, whereas poly(2-hydroxypropyl methacrylate) cores led to coagulation. The most active particles inhibited ice growth as low as 0.5 mg mL, and were more active than the PVA stabiliser block alone, showing that the dense packing of this nanoparticle format enhanced activity. This provides a unique route towards colloids capable of modulating ice growth.
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http://dx.doi.org/10.1039/D0MH00354ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116880PMC
July 2020

The atomistic details of the ice recrystallisation inhibition activity of PVA.

Nat Commun 2021 02 26;12(1):1323. Epub 2021 Feb 26.

Department of Chemistry, University of Warwick, Coventry, UK.

Understanding the ice recrystallisation inhibition (IRI) activity of antifreeze biomimetics is crucial to the development of the next generation of cryoprotectants. In this work, we bring together molecular dynamics simulations and quantitative experimental measurements to unravel the microscopic origins of the IRI activity of poly(vinyl)alcohol (PVA)-the most potent of biomimetic IRI agents. Contrary to the emerging consensus, we find that PVA does not require a "lattice matching" to ice in order to display IRI activity: instead, it is the effective volume of PVA and its contact area with the ice surface which dictates its IRI strength. We also find that entropic contributions may play a role in the ice-PVA interaction and we demonstrate that small block co-polymers (up to now thought to be IRI-inactive) might display significant IRI potential. This work clarifies the atomistic details of the IRI activity of PVA and provides novel guidelines for the rational design of cryoprotectants.
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http://dx.doi.org/10.1038/s41467-021-21717-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910567PMC
February 2021

Ice recrystallisation inhibiting polymers prevent irreversible protein aggregation during solvent-free cryopreservation as additives and as covalent polymer-protein conjugates.

Eur Polym J 2020 Nov;140:110036

Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.

Protein storage and transport is essential to deliver therapies (biologics), enzymes for biotechnological applications, and underpins fundamental structural and molecular biology. To enable proteins to be stored and transported it is often essential to freeze them, requiring cryoprotectants such as glycerol or trehalose. Here we explore the mechanisms by which poly(vinyl alcohol), PVA, a potent ice recrystallisation inhibitor protects proteins during freeze/thaw to enable solvent-free cryopreservation with a focus on comparing mixing, verses polymer-protein conjugation. A panel of poly(vinyl alcohol)s are investigated including commercial, well-defined (from RAFT), and PVA-protein conjugates, to map out PVA's efficacy. Enzymatic activity recovery of lactate dehydrogenase was found to correlate with post-thaw aggregation state (less aggregated protein had greater activity), which was modulated by PVA's ice recrystallisation inhibition activity. This macromolecular cryoprotectant matched the performance of glycerol, but at lower additive concentrations (as low as 1 mg.mL). It was also demonstrated that storage at -20 °C, rather than -80 °C was possible using PVA as a cryoprotectant, which is not possible with glycerol storage. A second protein, green-fluorescent protein (GFP), was used to enable screening of molecular weight effects and to obtain PVA-GFP bioconjugates. It was observed that covalent attachment of RAFT-derived PVA showed superior cryoprotectant activity compared to simple mixing of the polymer and protein. These results show that PVA is a real alternative to solvent-based protein storage with potential in biotechnology, food and therapeutics. PVA is already approved for many biomedical applications, is low cost and available on a large scale, making it an ideal cryoprotectant formulation enhancer.
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http://dx.doi.org/10.1016/j.eurpolymj.2020.110036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709485PMC
November 2020

The SARS-COV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device.

ACS Cent Sci 2020 Nov 23;6(11):2046-2052. Epub 2020 Sep 23.

Department of Chemistry, University of Warwick, Coventry, CV4 7AL, U.K.

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 -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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http://dx.doi.org/10.1021/acscentsci.0c00855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523238PMC
November 2020

Low DMSO Cryopreservation of Stem Cells Enabled by Macromolecular Cryoprotectants.

ACS Appl Bio Mater 2020 Sep 18;3(9):5627-5632. Epub 2020 Aug 18.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom.

Mesenchymal stromal (stem) cells have potential in regenerative medicine and modulating the immune system. To deliver any cell-based therapy to the patient, it must be cryopreserved, most commonly in DMSO, which impacts cell function and causes clinical side effects. Here we report the use of a synthetically scalable polyampholyte to rescue the cryopreservation of mesenchymal stromal cells in low [DMSO] cryopreservation. Flow cytometry showed retention of key markers of multipotency comparable to 10% (v/v) DMSO, and the cells could be differentiated, showing this polymer material can be used to improve, or replace, current cryopreservation strategies.
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http://dx.doi.org/10.1021/acsabm.0c00638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509910PMC
September 2020

Protecting Group Free Synthesis of Glyconanoparticles Using Amino-Oxy-Terminated Polymer Ligands.

Bioconjug Chem 2020 10 21;31(10):2392-2403. Epub 2020 Sep 21.

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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http://dx.doi.org/10.1021/acs.bioconjchem.0c00465DOI Listing
October 2020

100th Anniversary of Macromolecular Science Viewpoint: Re-Engineering Cellular Interfaces with Synthetic Macromolecules Using Metabolic Glycan Labeling.

ACS Macro Lett 2020 Jul 25;9(7):991-1003. Epub 2020 Jun 25.

Department of Chemistry and Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.

Cell-surface functionality is largely programmed by genetically encoded information through modulation of protein expression levels, including glycosylation enzymes. Genetic tools enable control over protein-based functionality, but are not easily adapted to recruit non-native functionality such as synthetic polymers and nanomaterials to tune biological responses and attach therapeutic or imaging payloads. Similar to how polymer-protein conjugation evolved from nonspecific PEGylation to site-selective bioconjugates, the same evolution is now occurring for polymer-cell conjugation. This Viewpoint discusses the potential of using metabolic glycan labeling to install bio-orthogonal reactive cell-surface anchors for the recruitment of synthetic polymers and nanomaterials to cell surfaces, exploring the expanding therapeutic and diagnostic potential. Comparisons to conventional approaches that target endogenous membrane components, such as hydrophobic, protein coupling and electrostatic conjugation, as well as enzymatic and genetic tools, have been made to highlight the huge potential of this approach in the emerging cellular engineering field.
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http://dx.doi.org/10.1021/acsmacrolett.0c00317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377358PMC
July 2020

Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants.

Biomacromolecules 2020 07 22;21(7):2864-2873. Epub 2020 Jun 22.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom.

The storage and transport of cells is a fundamental technology which underpins cell biology, biomaterials research, and emerging cell-based therapies. Inspired by antifreeze and ice-binding proteins in extremophiles, macromolecular (polymer) cryoprotectants are emerging as exciting biomaterials to enable the reduction and/or replacement of conventional cryoprotective agents such as DMSO. Here, we critically study post-thaw cellular outcomes upon addition of macromolecular cryoprotectants to provide unambiguous evidence that post-thaw culturing time and a mixture of assays are essential to claim a positive outcome. In particular, we observe that only measuring the viability of recovered cells gives false positives, even with non-cryoprotective polymers. Several systems gave apparently high viability but very low total cell recovery, which could be reported as a success but in practical applications would not be useful. Post-thaw culture time is also shown to be crucial to enable apoptosis to set in. Using this approach we demonstrate that polyampholytes (a rapidly emerging class of cryoprotectants) improve post-thaw outcomes across both measures, compared to poly(ethylene glycol), which can give false positives when only viability and short post-thaw time scales are considered. This work will help guide the discovery of new macromolecular cryoprotectants and ensure materials which only give positive results under limited outcomes can be quickly identified and removed.
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http://dx.doi.org/10.1021/acs.biomac.0c00591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362331PMC
July 2020

Combinatorial Biomaterials Discovery Strategy to Identify New Macromolecular Cryoprotectants.

ACS Macro Lett 2020 Feb 7;9(2):290-294. Epub 2020 Feb 7.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.

Cryoprotective agents (CPAs) are typically solvents or small molecules, but there is a need for innovative CPAs to reduce toxicity and increase cell yield, for the banking and transport of cells. Here we use a photochemical high-throughput discovery platform to identify macromolecular cryoprotectants, as rational design approaches are currently limited by the lack of structure-property relationships. Using liquid handling systems, 120 unique polyampholytes were synthesized using photopolymerization with RAFT agents. Cryopreservation screening identified "hit" polymers and nonlinear trends between composition and function, highlighting the requirement for screening, with polymer aggregation being a key factor. The most active polymers reduced the volume of dimethyl sulfoxide (DMSO) required to cryopreserve a nucleated cell line, demonstrating the potential of this approach to identify materials for cell storage and transport.
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http://dx.doi.org/10.1021/acsmacrolett.0c00044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175595PMC
February 2020

X-ray diffraction to probe the kinetics of ice recrystallization inhibition.

Analyst 2020 May;145(10):3666-3677

Department of Chemistry, University of Warwick, Gibbet Hill Road, CV4 7AL, UK. and Warwick Medical School, University of Warwick, Gibbet Hill Road, CV4 7AL, UK.

Understanding the nucleation and growth of ice is crucial in fields ranging from infrastructure maintenance, to the environment, and to preserving biologics in the cold chain. Ice binding and antifreeze proteins are potent ice recrystallization inhibitors (IRI), and synthetic materials that mimic this function have emerged, which may find use in biotechnology. To evaluate IRI activity, optical microscopy tools are typically used to monitor ice grain size either by end-point measurements or as a function of time. However, these methods provide 2-dimensional information and image analysis is required to extract the data. Here we explore using wide angle X-ray scattering (WAXS/X-ray powder diffraction (XRD)) to interrogate 100's of ice crystals in 3-dimensions as a function of time. Due to the random organization of the ice crystals in the frozen sample, the number of orientations measured by XRD is proportional to the number of ice crystals, which can be measured as a function of time. This method was used to evaluate the activity for a panel of known IRI active compounds, and shows strong agreement with results obtained from cryo-microscopy, as well as being advantageous in that time-dependent ice growth is easily extracted. Diffraction analysis also confirmed, by comparing the obtained diffraction patterns of both ice binding and non-binding additives, that the observed hexagonal ice diffraction patterns obtained cannot be used to determine which crystal faces are being bound. This method may help in the discovery of new IRI active materials as well as enabling kinetic analysis of ice growth.
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http://dx.doi.org/10.1039/c9an02141hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116874PMC
May 2020

Polymer-Stabilized Sialylated Nanoparticles: Synthesis, Optimization, and Differential Binding to Influenza Hemagglutinins.

Biomacromolecules 2020 04 31;21(4):1604-1612. Epub 2020 Mar 31.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.

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 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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http://dx.doi.org/10.1021/acs.biomac.0c00179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173702PMC
April 2020

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters.

Environ Microbiol 2020 04 27;22(4):1356-1369. Epub 2020 Feb 27.

School of Life Sciences, University of Warwick, Warwick, UK.

Pristine marine environments are highly oligotrophic ecosystems populated by well-established specialized microbial communities. Nevertheless, during oil spills, low-abundant hydrocarbonoclastic bacteria bloom and rapidly prevail over the marine microbiota. The genus Alcanivorax is one of the most abundant and well-studied organisms for oil degradation. While highly successful under polluted conditions due to its specialized oil-degrading metabolism, it is unknown how they persist in these environments during pristine conditions. Here, we show that part of the Alcanivorax genus, as well as oils, has an enormous potential for biodegrading aliphatic polyesters thanks to a unique and abundantly secreted alpha/beta hydrolase. The heterologous overexpression of this esterase proved a remarkable ability to hydrolyse both natural and synthetic polyesters. Our findings contribute to (i) better understand the ecology of Alcanivorax in its natural environment, where natural polyesters such as polyhydroxyalkanoates (PHA) are produced by a large fraction of the community and, hence, an accessible source of carbon and energy used by the organism in order to persist, (ii) highlight the potential of Alcanivorax to clear marine environments from polyester materials of anthropogenic origin as well as oils, and (iii) the discovery of a new versatile esterase with a high biotechnological potential.
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http://dx.doi.org/10.1111/1462-2920.14947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187450PMC
April 2020

Plasticizer Degradation by Marine Bacterial Isolates: A Proteogenomic and Metabolomic Characterization.

Environ Sci Technol 2020 02 14;54(4):2244-2256. Epub 2020 Jan 14.

School of Life Sciences, University of Warwick , Coventry CV4 7AL , U.K.

Many commercial plasticizers are toxic endocrine-disrupting chemicals that are added to plastics during manufacturing and may leach out once they reach the environment. Traditional phthalic acid ester plasticizers (PAEs), such as dibutyl phthalate (DBP) and bis(2-ethyl hexyl) phthalate (DEHP), are now increasingly being replaced with more environmentally friendly alternatives, such as acetyl tributyl citrate (ATBC). While the metabolic pathways for PAE degradation have been established in the terrestrial environment, to our knowledge, the mechanisms for ATBC biodegradation have not been identified previously and plasticizer degradation in the marine environment remains underexplored. From marine plastic debris, we enriched and isolated microbes able to grow using a range of plasticizers and, for the first time, identified the pathways used by two phylogenetically distinct bacteria to degrade three different plasticizers (i.e., DBP, DEHP, and ATBC) via a comprehensive proteogenomic and metabolomic approach. This integrated multi-OMIC study also revealed the different mechanisms used for ester side-chain removal from the different plasticizers (esterases and enzymes involved in the β-oxidation pathway) as well as the molecular response to deal with toxic intermediates, that is, phthalate, and the lower biodegrading potential detected for ATBC than for PAE plasticizers. This study highlights the metabolic potential that exists in the biofilms that colonize plastics-the Plastisphere-to effectively biodegrade plastic additives and flags the inherent importance of microbes in reducing plastic toxicity in the environment.
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http://dx.doi.org/10.1021/acs.est.9b05228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031849PMC
February 2020

"Tuning aggregative versus non-aggregative lectin binding with glycosylated nanoparticles by the nature of the polymer ligand".

J Mater Chem B 2020 01 28;8(1):136-145. Epub 2019 Nov 28.

Department of Chemistry, University of Warwick, Gibbet Hill Road, CV4 7AL, Coventry, UK. and Warwick Medical School, University of Warwick, Gibbet Hill Road, CV4 7AL, Coventry, UK.

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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http://dx.doi.org/10.1039/c9tb02004gDOI Listing
January 2020

Synthesis of Anthracene Conjugates of Truncated Antifreeze Protein Sequences: Effect of the End Group and Photocontrolled Dimerization on Ice Recrystallization Inhibition Activity.

Biomacromolecules 2019 12 26;20(12):4611-4621. Epub 2019 Nov 26.

Biomacromolecular antifreezes distinguish ice from water, function by binding to specific planes of ice, and could have many applications from cryobiology to aerospace where ice is a problem. In biology, antifreeze protein (AFP) activity is regulated by protein expression levels via temperature and light-regulated expression systems, but in the laboratory (or applications), the antifreeze activity is "always on" without any spatial or temporal control, and hence methods to enable this switching represent an exciting synthetic challenge. Introduction of an abiotic functionality into short peptides (e.g., from solid-phase synthesis) to enable switching is also desirable rather than on full-length recombinant proteins. Here, truncated peptide sequences based on the consensus repeat sequence from type-I AFPs (TAANAAAAAAA) were conjugated to an anthracene unit to explore their photocontrolled dimerization. Optimization of the synthesis to ensure solubility of the hydrophobic peptide included the addition of a dilysine solubilizing linker. It was shown that UV-light exposure triggered reversible dimerization of the AFP sequence, leading to an increase in molecular weight. Assessment of the ice recrystallization inhibition activity of the peptides before and after dimerization revealed only small effects on activity. However, it is reported here for the first time that addition of the anthracene unit to a 22-amino-acid truncated peptide significantly enhanced ice recrystallization inhibition compared to the free peptide, suggesting an accessible synthetic route to allow AFP activity using shorter, synthetically accessible peptides with a photoreactive functionality.
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http://dx.doi.org/10.1021/acs.biomac.9b01538DOI Listing
December 2019

Dimeric benzoboroxoles for targeted activity against Mycobacterium tuberculosis.

Org Biomol Chem 2019 11;17(43):9524-9528

School of Life Sciences, University of Warwick, CV4 7AL, UK.

Dimeric benzoboroxoles that are covalently linked by a short scaffold enhance selective anti-tubercular activity. These multimeric benzoboroxole compounds are capable of engaging the specific extracellular Mycobacterium tuberculosis glycans, do not lead to the evolution of resistance and bypass the need to cross the impermeable mycobacterial cell envelope barrier.
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http://dx.doi.org/10.1039/c9ob02222hDOI Listing
November 2019

High-Throughput Tertiary Amine Deoxygenated Photopolymerizations for Synthesizing Polymer Libraries.

Macromolecules 2019 Oct 2;52(20):7603-7612. Epub 2019 Oct 2.

Department of Chemistry and Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, U.K.

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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http://dx.doi.org/10.1021/acs.macromol.9b01714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812069PMC
October 2019

Extracellular Antifreeze Protein Significantly Enhances the Cryopreservation of Cell Monolayers.

Biomacromolecules 2019 10 19;20(10):3864-3872. Epub 2019 Sep 19.

Department of Chemistry , University of Warwick , Gibbet Hill Road , Coventry CV4 7AL , United Kingdom.

The cryopreservation of cells underpins many areas of biotechnology, healthcare, and fundamental science by enabling the banking and distribution of cells. Cryoprotectants are essential to prevent cold-induced damage. Here, we demonstrate that extracellular localization of antifreeze proteins can significantly enhance post-thaw recovery of mammalian cell monolayers cryopreserved using dimethyl sulfoxide, whereas they show less benefit in suspension cryopreservation. A type III antifreeze protein (AFPIII) was used as the macromolecular ice recrystallization inhibitor and its intra/extracellular locations were controlled by using Pep-1, a cell-penetrating peptide. Flow cytometry and confocal microscopy confirmed successful delivery of AFPIII. The presence of extracellular AFPIII dramatically increased post-thaw recovery in a challenging 2-D cell monolayer system using just 0.8 mg·mL, from 25% to over 60%, whereas intracellularly delivered AFPIII showed less benefit. Interestingly, the antifreeze protein was less effective when used in suspension cryopreservation of the same cells, suggesting that the cryopreservation format is also crucial. These observations show that, in the discovery of macromolecular cryoprotectants, intracellular delivery of ice recrystallization inhibitors may not be a significant requirement under "slow freezing" conditions, which will help guide the design of new biomaterials, in particular, for cell storage.
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http://dx.doi.org/10.1021/acs.biomac.9b00951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794639PMC
October 2019

Enhancement of Macromolecular Ice Recrystallization Inhibition Activity by Exploiting Depletion Forces.

ACS Macro Lett 2019 Aug 9;8(8):1063-1067. Epub 2019 Aug 9.

Department of Chemistry, Warwick Medical School, and Centre for Scientific Computing, University of Warwick Coventry, CV4 7AL, United Kingdom.

Antifreeze (glyco) proteins (AF(G)Ps) are potent inhibitors of ice recrystallization and may have biotechnological applications. The most potent AF(G)Ps function at concentrations a thousand times lower than synthetic mimics such as poly(vinyl alcohol), PVA. Here, we demonstrate that PVA's ice recrystallization activity can be rescued at concentrations where it does not normally function, by the addition of noninteracting polymeric depletants, due to PVA forming colloids in the concentrated saline environment present between ice crystals. These depletants shift the equilibrium toward ice binding and, hence, enable PVA to inhibit ice growth at lower concentrations. Using theory and experiments, we show this effect requires polymeric depletants, not small molecules, to enhance activity. These results increase our understanding of how to design new ice growth inhibitors, but also offer opportunities to enhance activity by exploiting depletion forces, without re-engineering ice-binding materials. It also shows that when screening for IRI activity that polymer contaminants in buffers may give rise to false positive results.
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http://dx.doi.org/10.1021/acsmacrolett.9b00386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711362PMC
August 2019
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