Covalent recruitment of polymers and nanoparticles onto glycan-engineered cells enhances gene delivery during short exposure.

Non-viral gene delivery with cationic polymers/nanoparticles relies on iterative optimization of the carrier to achieve delivery. Here we demonstrate, instead, that precision engineering of cell surfaces to covalently capture a polyplex accelerates gene delivery within just 10 min of exposure. Azides were installed into cell-surface sialic acids, which enabled the rapid and selective recruitment of cyclooctyne-functional polyplexes, leading to increased delivery of fluorescent cargo, and also increased plasmid expression and siRNA knockdown. Covalent delivery enhancement was also shown for a polymer-coated nanoparticle delivery system. This validates using cellular metabolic engineering (or other synthetic biology) tools to overcome payload delivery challenges.

Trehalose in cryopreservation. Applications, mechanisms and intracellular delivery opportunities.

Cryopreservation is crucial to fields including immune and stem cell therapies, reproductive technology, blood banking, regenerative medicine and across all biotechnology. During cryopreservation, cryoprotectants are essential to protect cells from the damage caused by exposure to freezing temperatures. The most common penetrating cryoprotectants, such as DMSO and glycerol do not give full recovery and have a cytotoxicity limit on the concentration which can be applied. The non-reducing disaccharide trehalose has been widely explored and used to supplement these, inspired by its use in nature to aid survival at extreme temperatures and/or desiccation. However, trehalose has challenges to its use, particular its low membrane permeability, and how its protective role compares to other sugars. Here we review the application of trehalose and its reported benefit and seek to show where chemical tools can improve its function. In particular, we highlight emerging chemical methods to deliver (as cargo, or via selective permeation) into the intracellular space. This includes encapsulation, cell penetrating peptides or (selective) modification of hydroxyls on trehalose.

Induced Extracellular Ice Nucleation Protects Cocultured Spheroid Interior and Exterior during Cryopreservation.

Spheroids and other 3D cellular models more accurately recapitulate physiological responses when compared to 2D models and represent potential alternatives to animal testing. The cryopreservation of spheroids remains challenging, limiting their wider use. Standard DMSO-only cryopreservation results in supercooling to low subzero temperatures, reducing viability, shedding surface cells, and perforating spheroid interiors. Here, cocultured spheroids with differentially labeled outer cell layers allow spatial evaluation of the protective effect of macromolecular ice nucleators by microscopy and histology. Extracellular nucleation is shown to reduce damage to both interior and exterior regions of the spheroids, which will support the development of "off-the-shelf" 3D models.

Data-driven discovery of potent small molecule ice recrystallisation inhibitors.

Controlling the formation and growth of ice is essential to successfully cryopreserve cells, tissues and biologics. Current efforts to identify materials capable of modulating ice growth are guided by iterative changes and human intuition, with a major focus on proteins and polymers. With limited data, the discovery pipeline is constrained by a poor understanding of the mechanisms and the underlying structure-activity relationships. In this work, this barrier is overcome by constructing machine learning models capable of predicting the ice recrystallisation inhibition activity of small molecules. We generate a new dataset via experimental measurements of ice growth, then harness predictive models combining state-of-the-art descriptors with domain-specific features derived from molecular simulations. The models accurately identify potent small molecule ice recrystallisation inhibitors within a commercial compound library. Identified hits can also mitigate cellular damage during transient warming events in cryopreserved red blood cells, demonstrating how data-driven approaches can be used to discover innovative cryoprotectants and enable next-generation cryopreservation solutions for the cold chain.

Synthesis and screening of a library of Lewisx deoxyfluoro-analogues reveals differential recognition by glycan-binding partners.

Glycan-mediated interactions play a crucial role in biology and medicine, influencing signalling, immune responses, and disease pathogenesis. However, the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognised by multiple biologically distinct protein receptors. To address this specificity challenge, we report the enzymatic synthesis of a 150-member library of site-specifically fluorinated Lewisx analogues ('glycofluoroforms') using naturally occurring enzymes and fluorinated monosaccharides. Subsequent incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognise Lewisx. Notably, we show that for two proteins with unique binding sites for Lewisx, glycofluoroforms exhibited enhanced binding to one protein, whilst reduced binding to the other, with selectivity governed by fluorination patterns. We finally showcase the potential diagnostic utility of this approach in glycofluoroform-mediated bacterial toxin detection by lateral flow.

Harnessing glycofluoroforms for impedimetric biosensing.

Glycans play a major role in biological cell-cell recognition and signal transduction but have found limited application in biosensors due to glycan/lectin promiscuity; multiple proteins are capable of binding to the same native glycan. Here, site-specific fluorination is used to introduce protein-glycan selectivity, and this is coupled with an electrochemical detection method to generate a novel biosensor platform. 3F-lacto-N-biose glycofluoroform is installed onto polymer tethers, which are subsequently immobilised onto gold screen printed electrodes, providing a non-fouling surface. The impedance biosensing platform is shown to selectively bind cancer-associated galectin-3 compared to control glycans and proteins. To improve the analytical capability, Bayesian statistical analysis was deployed in the equivalent circuit fitting of electrochemical impedance spectroscopy data. It is shown that Markov Chain Monte Carlo (MCMC) analysis is a helpful method for visualising experimental irreproducibility, and we apply this as a quality control step.

Acidic polymers reversibly deactivate phages due to pH changes.

Bacteriophages are promising as therapeutics and biotechnological tools, but they also present a problem for routine and commercial bacterial cultures, where contamination must be avoided. Poly(carboxylic acids) have been reported to inhibit phages' ability to infect their bacterial hosts and hence offer an exciting route to discover additives to prevent infection. Their mechanism and limitations have not been explored. Here, we report the role of pH in inactivating phages to determine if the polymers are unique or simply acidic. It is shown that lower pH (=3) triggered by either acidic polymers or similar changes in pH using HCl lead to inhibition. There is no inhibitory activity at higher pHs (in growth media). This was shown across a panel of phages and different molecular weights of commercial and controlled-radical polymerization-derived poly(acrylic acid)s. It is shown that poly(acrylic acid) leads to reversible deactivation of phage, but when the pH is adjusted using HCl alone the phage is irreversibly deactivated. Further experiments using metal binders ruled out ion depletion as the mode of action. These results show that polymeric phage inhibitors may work by unique mechanisms of action and that pH alone cannot explain the observed effects whilst also placing constraints on the practical utility of poly(acrylic acid).

Cryopreserved Kidney Epithelial (Vero) Cell Monolayers for Rapid Viral Quantification, Enabled by a Combination of Macromolecular Cryoprotectants.

Plaque assays quantify the amount of active, replicating virus to study and detect infectious diseases by application of samples to monolayers of cultured cells. Due to the time taken in thawing, propagating, plating, counting, and then conducting the assay, the process can take over a week to gather data. Here, we introduce assay-ready cryopreserved Vero monolayers in multiwell plates, which can be used directly from the freezer with no cell culture to accelerate the process of plaque determination. Standard dimethyl sulfoxide cryopreservation resulted in just 25% recovery, but addition of polyampholytes (macromolecular cryoprotectants) increased post-thaw recovery and viability in 12- and 24-well plate formats. Variability between individual wells was reduced by chemically induced ice nucleation to prevent supercooling. Cryopreserved cells were used to determine influenza viral plaques in just 24 h, matching results from nonfrozen controls. This innovation may accelerate viral detection and quantification and facilitate automation by eliminating extensive cell culturing.

Mechanical release of homogenous proteins from supramolecular gels.

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.

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.

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.

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.

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.