100th anniversary of macromolecular science viewpoint : re-engineering cellular interfaces with synthetic macromolecules using metabolic glycan labeling

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

Optimization and stability of cell–polymer hybrids obtained by “clicking” synthetic polymers to metabolically labeled cell surface glycans

Re-engineering of mammalian cell surfaces with polymers enables the introduction of functionality including imaging agents, drug cargoes or antibodies for cell-based therapies, without resorting to genetic techniques. Glycan metabolic labeling has been reported as a tool for engineering cell surface glycans with synthetic polymers through the installation of biorthogonal handles, such as azides. Quantitative assessment of this approach and the robustness of the engineered coatings has yet to be explored. Here, we graft poly(hydroxyethyl acrylamide) onto azido-labeled cell surface glycans using strain-promoted azide–alkyne “click” cycloaddition and, using a combination of flow cytometry and confocal microscopy, evaluate the various parameters controlling the outcome of this “grafting to” process. In all cases, homogeneous cell coatings were formed with >95% of the treated cells being covalently modified, superior to nonspecific “grafting to” approaches. Controllable grafting densities could be achieved through modulation of polymer chain length and/or concentration, with longer polymers having lower densities. Cell surface bound polymers were retained for at least 72 h, persisting through several mitotic divisions during this period. Furthermore, we postulate that glycan/membrane recycling is slowed by the steric bulk of the polymers, demonstrating robustness and stability even during normal biological processes. This cytocompatible, versatile and simple approach shows potential for re-engineering of cell surfaces with new functionality for future use in cell tracking or cell-based therapies

Asymmetric trehalose analogues to probe disaccharide processing pathways in mycobacteria

The uptake and metabolism of the disaccharide trehalose by Mycobacterium tuberculosis is essential for the virulence of this pathogen. Here we describe the chemoenzymatic synthesis of new azido-functionalised asymmetric trehalose probes that resist degradation by mycobacterial enzymes and are used to probe trehalose processing pathways in mycobacteria

Low DMSO cryopreservation of stem cells enabled by macromolecular cryoprotectants

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

Extracellular antifreeze protein significantly enhances the cryopreservation of cell monolayers

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–1, 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

Covalent cell surface recruitment of chemotherapeutic polymers enhances selectivity and activity

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 EC50 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

Photochemical “in-air” combinatorial discovery of antimicrobial co-polymers

There is an urgent need to identify new, non‐traditional antimicrobials. The discovery of new polymeric antimicrobials is limited by current low‐throughput synthetic tools, which means that limited chemical space has been explored. Herein, we employ photochemical “in‐air” reversible addition–fragmentation chain‐transfer (RAFT) polymerization with microwell plates, using liquid‐handling robots to assemble large libraries of cationic polymers, without the need for degassing or purification steps, facilitating transfer to screening. Several lead polymers were identified including a co‐polymer with propylene glycol side chains with significantly enhanced antimicrobial activity and increased therapeutic window. Mechanistic studies showed that this polymer was bacteriostatic, and surprisingly did not lyse the cell membranes, implying an alternative mode of action. This versatile method using simple robotics will help to develop new biomaterials with emergent properties