Cryopreservation and Rapid Recovery of Differentiated Intestinal Epithelial Barrier Cells at Complex Transwell Interfaces Is Enabled by Chemically Induced Ice Nucleation.

Cell-based models, such as organ-on-chips, can replace and inform in vivo (animal) studies for drug discovery, toxicology, and biomedical science, but most cannot be banked "ready to use" as they do not survive conventional cryopreservation with DMSO alone. Here, we demonstrate how macromolecular ice nucleators enable the successful cryopreservation of epithelial intestinal models supported upon the interface of transwells, allowing recovery of function in just 7 days post-thaw directly from the freezer, compared to 21 days from conventional suspension cryopreservation. Caco-2 cells and Caco-2/HT29-MTX cocultures are cryopreserved on transwell inserts, with chemically induced ice nucleation at warmer temperatures resulting in increased cell viability but crucially retaining the complex cellular adhesion on the transwell insert interfaces, which other cryoprotectants do not. Trans-epithelial electrical resistance measurements, confocal microscopy, histology, and whole-cell proteomics demonstrated the rapid recovery of differentiated cell function, including the formation of tight junctions. Lucifer yellow permeability assays confirmed that the barrier functions of the cells were intact. This work will help solve the long-standing problem of transwell tissue barrier model storage, facilitating access to advanced predictive cellular models. This is underpinned by precise control of the nucleation temperature, addressing a crucial biophysical mode of damage.

Sialic acids in infection and their potential use in detection and protection against pathogens.

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.

Cirrhotic hepatocellular carcinoma-based decellularized liver cancer model for local chemoembolization evaluation.

Transarterial chemoembolization (TACE) is a common treatment for unresectable intermediate stage hepatocellular carcinoma (HCC) and involves the combination of chemotherapy agents and embolic materials to target and block the blood supply to the tumor, leading to localized treatment. However, the selection of clinical chemoembolization agents remains limited, and the effectiveness of various agents is still under investigation. Meanwhile, replicating the complex vasculature and extracellular matrix (ECM) circumstances of HCC in in vitro models for evaluating embolic agents proves to be challenging. Herein, we developed a decellularized cancerous liver model with translucent appearance, a complicated hepatic vascular system and tissue-specific ECM for the evaluation of embolic agents. Inkpad oil and microparticles were used to illustrate different systems of vascular structures between healthy and HCC rats' livers. Quantitative analysis with AngioTool revealed significant differences in vessel density and lacunarity between the two groups. Proteomics showed higher secretion of collagens in the HCC rat liver models than in healthy livers. Utilizing this in vitro model, we investigated the impact of tumor-specific vascular structure and ECM composition on chemoembolization performance, the two key factors inaccessible by currently available drug release testing platforms. Our findings revealed that the presence of an aberrant vascular system and the distorted ECM within the model led to drug retention. This preclinical model holds great promise as a valuable tool for evaluating embolic agents and studying their performance in the tumor microenvironment. STATEMENT OF SIGNIFICANCE: Transarterial chemoembolization (TACE), which employs drug-eluting embolic agents to obstruct the tumor-feeding vessels while locally releasing chemotherapeutic drugs into the tumor, has become the first-line treatment of unresectable liver cancer over past two decades. Nevertheless, the advancement of effective drug-eluting embolic agents has been retarded due to the lack of appropriate in vitro models for assessing the local embolization and chemotherapy performances in TACE. Here we developed a cirrhotic hepatocellular carcinoma-based decellularized liver cancer model, which preserves the aberrant vasculatures and tumor-specific extracellular matrix of liver cancer, for TACE evaluation. This model incorporates a blood flow simulation component to assess the dynamics of drug release behaviors of chemoembolic agents within tumor-mimicking conditions, more accurately replicating the in vivo environment for the locoregional assessments as compared to conventional in vitro models.

Screening of Hydrophilic Polymers Reveals Broad Activity in Protecting Phages during Cryopreservation.

Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. Exceptions were poly(methacrylic acid) and poly(acrylic acid), which can inhibit phage-infection with bacteria, making post-thaw recovery challenging to assess. A particular benefit of a polymeric cryopreservation formulation is that the polymers do not function as carbon sources for the phage hosts (bacteria) and hence do not interfere with post-thaw measurements. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.

Post-thaw application of ROCK-inhibitors increases cryopreserved T-cell yield.

Emerging cell-based therapies such as CAR-T (Chimeric Antigen Receptor T) cells require cryopreservation to store and deliver intact and viable cells. Conventional cryopreservation formulations use DMSO to mitigate cold-induced damage, but do not address all the biochemical damage mechanisms induced by cold stress, such as programmed cell death (apoptosis). Rho-associated protein kinases (ROCK) are a key component of apoptosis, and their activation contributes to apoptotic blebbing. Here we demonstrate that the ROCK inhibitor fasudil hydrochloride, when supplemented into the thawing medium of T-cells increases the overall yield of healthy cells. Cell yield was highest using 5 or 10% DMSO cryopreservation solutions, with lower DMSO concentrations (2.5%) leading to significant physical damage to the cells. After optimisation, the post-thaw yield of T-cells increased by approximately 20% using this inhibitor, a significant increase in the context of a therapy. Flow cytometry analysis did not show a significant reduction in the relative percentage of cell populations undergoing apoptosis, but there was a small reduction in the 8 hours following thawing. Fasudil also led to a reduction in reactive oxygen species. Addition of fasudil into the cryopreservation solution, followed by dilution (rather than washing) upon thaw also gave a 20% increase in cell yield, demonstrating how this could be deployed in a cell-therapy context, without needing to change clinical thawing routines. Overall, this shows that modulation of post-thaw biochemical pathways which lead to apoptosis (or other degradative pathways) can be effectively targeted as a strategy to increase T-cell yield and function post-thaw.

Cryopreservation of assay-ready hepatocyte monolayers by chemically-induced ice nucleation: preservation of hepatic function and hepatotoxicity screening capabilities.

Cell culture plays a critical role in biomedical discovery and drug development. Primary hepatocytes and hepatocyte-derived cell lines are especially important cellular models for drug discovery and development. To enable high-throughput screening and ensure consistent cell phenotypes, there is a need for practical and efficient cryopreservation methods for hepatocyte-derived cell lines and primary hepatocytes in an assay-ready format. Cryopreservation of cells as adherent monolayers in 96-well plates presents unique challenges due to low volumes being susceptible to supercooling, leading to low recovery and well-to-well variation. Primary cell cryopreservation is also particularly challenging due to the loss of cell viability and function. In this study, we demonstrate the use of soluble ice nucleator materials (IN) to cryopreserve a hepatic-derived cell line (HepG2) and primary mouse hepatocytes, as adherent monolayers. HepG2 cell recovery was near 100% and ∼75% of primary hepatocytes were recovered 24 hours post-thaw compared to just 10% and 50% with standard 10% DMSO, respectively. Post-thaw assessment showed that cryopreserved HepG2 cells retain membrane integrity, metabolic activity, proliferative capacity and differentiated hepatic functions including urea secretion, cytochrome P450 levels and lipid droplet accumulation. Cryopreserved primary hepatocytes exhibited reduced hepatic functions compared to fresh hepatocytes, but functional levels were similar to commercial suspension-cryopreserved hepatocytes, with the added benefit of being stored in an assay-ready format. In addition, normal cuboidal morphology and minimal membrane damage were observed 24 hours post-thaw. Cryopreserved HepG2 and mouse hepatocytes treated with a panel of pharmaceutically active compounds produced near-identical dose-response curves and EC50 values compared to fresh hepatocytes, confirming the utility of cryopreserved bankable cells in drug metabolism and hepatotoxicity studies. Cryopreserved adherent HepG2 cells and primary hepatocytes in 96 well plates can significantly reduce the time and resource burden associated with routine cell culture and increases the efficiency and productivity of high-throughput drug screening assays.

Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation.

Cell therapies such as allogenic CAR T-cell therapy, natural killer cell therapy and stem cell transplants must be cryopreserved for transport and storage. This is typically achieved by addition of dimethyl sulfoxide (DMSO) but the cryoprotectant does not result in 100% cell recovery. New additives or technologies to improve their cryopreservation could have major impact for these emerging therapies. l-Proline is an amino acid osmolyte produced as a cryoprotectant by several organisms such as the codling moth Cydia pomonella and the larvae of the fly Chymomyza costata, and has been found to modulate post-thaw outcomes for several cell lines but has not been studied with Jurkat cells, a T lymphocyte cell line. Here we investigate the effectiveness of l-proline compared to d-proline and l-alanine for the cryopreservation of Jurkat cells. It is shown that 24-hour pre-freezing incubation of Jurkat cells with 200 mM l-proline resulted in a modest increase in cell recovery post-thaw at high cell density, but a larger increase in recovery was observed at the lower cell densities. l-Alanine was as effective as l-proline at lower cell densities, and addition of l-proline to the cryopreservation media (without incubation) had no benefit. The pre-freeze incubation with l-proline led to significant reductions in cell proliferation supporting an intracellular, biochemical, mechanism of action which was shown to be cell-density dependent. Controls with d-proline were found to reduce post-thaw recovery attributed to osmotic stress as d-proline cannot enter the cells. Preliminary analysis of apoptosis/necrosis profiles by flow cytometry indicated that inhibition of apoptosis is not the primary mode of action. Overall, this supports the use of l-proline pre-conditioning to improve T-cell post-thaw recovery without needing any changes to cryopreservation solutions nor methods and hence is simple to implement.

Programmable Monodisperse Glyco-Multivalency Using Self-Assembled Coordination Cages as Scaffolds.

The multivalent presentation of glycans leads to enhanced binding avidity to lectins due to the cluster glycoside effect. Most materials used as scaffolds for multivalent glycan arrays, such as polymers or nanoparticles, have intrinsic dispersity: meaning that in any sample, a range of valencies are presented and it is not possible to determine which fraction(s) are responsible for binding. The intrinsic dispersity of many multivalent glycan scaffolds also limits their reproducibility and predictability. Here we make use of the structurally programmable nature of self-assembled metal coordination cages, with polyhedral metal-ion cores supporting ligand arrays of predictable sizes, to assemble a 16-membered library of perfectly monodisperse glycoclusters displaying valencies from 2 to 24 through a careful choice of ligand/metal combinations. Mono- and trisaccharides are introduced into these clusters, showing that the synthetic route is tolerant of biologically relevant glycans, including sialic acids. The cluster series demonstrates increased binding to a range of lectins as the number of glycans increases. This strategy offers an alternative to current glycomaterials for control of the valency of three-dimensional (3-D) glycan arrays, and may find application across sensing, imaging, and basic biology.

Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation.

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.

High Molecular Weight Polyproline as a Potential Biosourced Ice Growth Inhibitor: Synthesis, Ice Recrystallization Inhibition, and Specific Ice Face Binding.

Ice-binding proteins (IBPs) from extremophile organisms can modulate ice formation and growth. There are many (bio)technological applications of IBPs, from cryopreservation to mitigating freeze-thaw damage in concrete to frozen food texture modifiers. Extraction or expression of IBPs can be challenging to scale up, and hence polymeric biomimetics have emerged. It is, however, desirable to use biosourced monomers and heteroatom-containing backbones in polymers for in vivo or environmental applications to allow degradation. Here we investigate high molecular weight polyproline as an ice recrystallization inhibitor (IRI). Low molecular weight polyproline is known to be a weak IRI. Its activity is hypothesized to be due to the unique PPI helix it adopts, but it has not been thoroughly investigated. Here an open-to-air aqueous N-carboxyanhydride polymerization is employed to obtain polyproline with molecular weights of up to 50000 g mol-1. These polymers were found to have IRI activity down to 5 mg mL-1, unlike a control peptide of polysarcosine, which did not inhibit all ice growth at up to 40 mg mL-1. The polyprolines exhibited lower critical solution temperature behavior and assembly/aggregation observed at room temperature, which may contribute to its activity. Single ice crystal assays with polyproline led to faceting, consistent with specific ice-face binding. This work shows that non-vinyl-based polymers can be designed to inhibit ice recrystallization and may offer a more sustainable or environmentally acceptable, while synthetically scalable, route to large-scale applications.

Chemically Induced Extracellular Ice Nucleation Reduces Intracellular Ice Formation Enabling 2D and 3D Cellular Cryopreservation.

3D cell assemblies such as spheroids reproduce the in vivo state more accurately than traditional 2D cell monolayers and are emerging as tools to reduce or replace animal testing. Current cryopreservation methods are not optimized for complex cell models, hence they are not easily banked and not as widely used as 2D models. Here we use soluble ice nucleating polysaccharides to nucleate extracellular ice and dramatically improve spheroid cryopreservation outcomes. This protects the cells beyond using DMSO alone, and with the major advantage that the nucleators function extracellularly and hence do not need to permeate the 3D cell models. Critical comparison of suspension, 2D and 3D cryopreservation outcomes demonstrated that warm-temperature ice nucleation reduces the formation of (fatal) intracellular ice, and in the case of 2/3D models this reduces propagation of ice between adjacent cells. This demonstrates that extracellular chemical nucleators could revolutionize the banking and deployment of advanced cell models.

Ice nucleation in aqueous solutions of short- and long-chain poly(vinyl alcohol) studied with a droplet microfluidics setup.

Poly(vinyl alcohol) (PVA) has ice binding and ice nucleating properties. Here, we explore the dependence of the molecular size of PVA on its ice nucleation activity. For this purpose, we studied ice nucleation in aqueous solutions of PVA samples with molar masses ranging from 370 to 145 000 g mol-1, with a particular focus on oligomer samples with low molar mass. The experiments employed a novel microfluidic setup that is a follow-up on the previous WeIzmann Supercooled Droplets Observation on a Microarray (WISDOM) design by Reicher et al. The modified setup introduced and characterized here, termed nanoliter Bielefeld Ice Nucleation ARraY (nanoBINARY), uses droplet microfluidics with droplets (96 ± 4) µm in diameter and a fluorinated continuous oil phase and surfactant. A comparison of homogeneous and heterogeneous ice nucleation data obtained with nanoBINARY to those obtained with WISDOM shows very good agreement, underpinning its ability to study low-temperature ice nucleators as well as homogeneous ice nucleation due to the low background of impurities. The experiments on aqueous PVA solutions revealed that the ice nucleation activity of shorter PVA chains strongly decreases with a decrease in molar mass. While the cumulative number of ice nucleating sites per mass nm of polymers with different molar masses is the same, it becomes smaller for oligomers and completely vanishes for dimer and monomer representatives such as 1,3-butanediol, propan-2-ol, and ethanol, most likely because these molecules become too small to effectively stabilize the critical ice embryo. Overall, our results are consistent with PVA polymers and oligomers acting as heterogeneous ice nucleators.

Anionic Synthetic Polymers Prevent Bacteriophage Infection.

Bioprocessing and biotechnology exploit microorganisms (such as bacteria) for the production of chemicals, biologics, therapies, and food. A major unmet challenge is that bacteriophage (phage) contamination compromises products and necessitates shut-downs and extensive decontamination using nonspecific disinfectants. Here we demonstrate that poly(acrylic acid) prevents phage-induced killing of bacterial hosts, prevents phage replication, and that induction of recombinant protein expression is not affected by the presence of the polymer. Poly(acrylic acid) was more active than poly(methacrylic acid), and poly(styrenesulfonate) had no activity showing the importance of the carboxylic acids. Initial evidence supported a virustatic, not virucidal, mechanism of action. This simple, low-cost, mass-produced additive offers a practical, scalable, and easy to implement solution to reduce phage contamination.

Cryopreservation of Liver-Cell Spheroids with Macromolecular Cryoprotectants.

Spheroids are a powerful tool for basic research and to reduce or replace in vivo (animal) studies but are not routinely banked nor shared. Here, we report the successful cryopreservation of hepatocyte spheroids using macromolecular (polyampholyte) cryoprotectants supplemented into dimethyl sulfoxide (DMSO) solutions. We demonstrate that a polyampholyte significantly increases post-thaw recovery, minimizes membrane damage related to cryo-injury, and remains in the extracellular space making it simple to remove post-thaw. In a model toxicology challenge, the thawed spheroids matched the performance of fresh spheroids. F-actin staining showed that DMSO-only cryopreserved samples had reduced actin polymerization, which the polyampholyte rescued, potentially linked to intracellular ice formation. This work may facilitate access to off-the-shelf and ready-to-use frozen spheroids, without the need for in-house culturing. Readily accessible 3-D cell models may also reduce the number of in vivo experiments.

Discrimination between protein glycoforms using lectin-functionalised gold nanoparticles as signal enhancers.

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.

Synthesis of Poly(2-(methylsulfinyl)ethyl methacrylate) via Oxidation of Poly(2-(methylthio)ethyl methacrylate): Evaluation of the Sulfoxide Side Chain on Cryopreservation.

Conventional cryopreservation solutions rely on the addition of organic solvents such as DMSO or glycerol, but these do not give full recovery for all cell types, and innovative cryoprotectants may address damage pathways which these solvents do not protect against. Macromolecular cryoprotectants are emerging, but there is a need to understand their structure-property relationships and mechanisms of action. Here we synthesized and investigated the cryoprotective behavior of sulfoxide (i.e., "DMSO-like") side-chain polymers, which have been reported to be cryoprotective using poly(ethylene glycol)-based polymers. We also wanted to determine if the polarized sulfoxide bond (S+O- character) introduces cryoprotective effects, as this has been seen for mixed-charge cryoprotective polyampholytes, whose mechanism of action is not yet understood. Poly(2-(methylsulfinyl)ethyl methacrylate) was synthesized by RAFT polymerization of 2-(methylthio)ethyl methacrylate and subsequent oxidation to sulfoxide. A corresponding N-oxide polymer was also prepared and characterized: (poly(2-(dimethylamineoxide)ethyl methacrylate). Ice recrystallization inhibition assays and differential scanning calorimetry analysis show that the sulfoxide side chains do not modulate the frozen components during cryopreservation. In cytotoxicity assays, it was found that long-term (24 h) exposure of the polymers was not tolerated by cells, but shorter (30 min) incubation times, which are relevant for cryopreservation, were tolerated. It was also observed that overoxidation to the sulfone significantly increased the cytotoxicity, and hence, these materials require a precision oxidation step to be deployed. In suspension cell cryopreservation investigations, the polysulfoxides did not increase cell recovery 24 h post-thaw. These results show that unlike hydrophilic backboned polysulfides, which can aid cryopreservation, the installation of the sulfoxide group onto a polymer does not necessarily bring cryoprotective properties, highlighting the challenges of developing and discovering macromolecular cryoprotectants.

Polymer-tethered glyconanoparticle colourimetric biosensors for lectin binding: structural and experimental parameters to ensure a robust output.

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.

Poly(vinyl alcohol) Molecular Bottlebrushes Nucleate Ice.

Ice binding proteins (IBP) have evolved to limit the growth of ice but also to promote ice formation by ice-nucleating proteins (INPs). IBPs, which modulate these seemingly distinct processes, often have high sequence similarities, and molecular size/assembly is hypothesized to be a crucial determinant. There are only a few synthetic materials that reproduce INP function, and rational design of ice nucleators has not been achieved due to outstanding questions about the mechanisms of ice binding. Poly(vinyl alcohol) (PVA) is a water-soluble synthetic polymer well known to effectively block ice recrystallization, by binding to ice. Here, we report the synthesis of a polymeric ice nucleator, which mimics the dense assembly of IBPs, using confined ice-binding polymers in a high-molar-mass molecular bottlebrush. Poly(vinyl alcohol)-based molecular bottlebrushes with different side-chain densities were synthesized via a combination of ring-opening metathesis polymerization (ROMP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization, using "grafting-to" and "grafting-through" approaches. The facile preparation of the PVA bottlebrushes was performed via selective hydrolysis of the acetate of the poly(vinyl acetate) (PVAc) side chains of the PVAc bottlebrush precursors. Ice-binding polymer side-chain density was shown to be crucial for nucleation activity, with less dense brushes resulting in colder nucleation than denser brushes. This bio-inspired approach provides a synthetic framework for probing heterogeneous ice nucleation and a route toward defined synthetic nucleators for biotechnological applications.

Polymer-tethered glycosylated gold nanoparticles recruit sialylated glycoproteins into their protein corona, leading to off-target lectin binding.

Upon exposure to biological fluids, the fouling of nanomaterial surfaces results in non-specific capture of proteins, which is particularly important when in contact with blood for in vivo and ex vivo applications. It is crucial to evaluate not just the protein components but also the glycans attached to those proteins. Polymer-tethered glycosylated gold nanoparticles have shown promise for use in biosensing/diagnostics, but the impact of the glycoprotein corona has not been established. Here we investigate how polymer-tethered glycosylated gold nanoparticles interact with serum proteins and demonstrate that the protein corona introduces new glycans and hence off-specific targeting capability. Using a panel of RAFT-derived polymers grafted to the gold surface, we show that the extent of corona formation is not dependent on the type of polymer. In lectin-binding assays, a glycan (galactose) installed on the chain-end of the polymer was available for binding even after protein corona formation. However, using sialic-acid binding lectins, it was found that there was significant off-target binding due to the large density of sialic acids introduced in the corona, confirmed by western blotting. To demonstrate the importance, we show that the nanoparticles can bind Siglec-2, an immune-relevant lectin post-corona formation. Pre-coating with (non-glycosylated) bovine serum albumin led to a significant reduction in the total glycoprotein corona. However, sufficient sialic acids were still present in the residual corona to lead to off-target binding. These results demonstrate the importance of the glycans when considering the protein corona and how 'retention of the desired function' does not rule out 'installation of undesired function' when considering the performance of glyco-nanomaterials.

Synthesis of poly(vinyl alcohol) by blue light bismuth oxide photocatalysed RAFT. Evaluation of the impact of freeze/thaw cycling on ice recrystallisation inhibition.

Poly(vinyl alcohol), PVA, is the most potent polymeric ice recrystallisation inhibitor (IRI), mimicking a complex function of ice binding proteins. The IRI activity of PVA scales with its molecular weight and hence broad molecular weight distributions in free radical-derived PVAs lead to activity measurements dominated by small amounts of heavier fractions. Well-defined PVA can be prepared by thermally initiated RAFT/MADIX polymerization using xanthates by the polymerization of the less activated monomer vinyl acetate. The low conversions and molecular weights obtained during this approach, often requires feeding of additional initiator and bulk polymerization. Here we employ bismuth oxide photo-RAFT in solution, using blue light (450 nm), rather than previously reported white light, to obtain a library of PVA's. The use of blue light enabled quantitative conversion and acceptable dispersities. Purple light (380 nm) was also used, but asymmetric molecular weight distributions were obtained in some cases. High concentrations of high molecular weight PVA is known to form cryogels during freeze/thaw which has led to speculation this might limit the use of PVA in environments where the temperature cycles e.g. the construction industry. After 4 freeze/thaw cycles there was only small changes in observable IRI for all synthesised PVAs and two commercial standards. In an extended test, activity was retained after 100 freeze/thaw cycles, mitigating concerns that PVA could not be used in situations where freeze/thaw cycles occur. This work presents a convenient method to obtain well-defined PVAs for cryoscience studies compared to conventional thermal-RAFT and indicates that cryogelation concerns may not prevent their use.