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CAM-RIG ConfocAl Microscopy and real-time Rheology of dynamIc hyroGels Project Objectives

In pursuit of deeper understanding of dynamic hydrogels Prof. Oren A. Scherman, Professor of Supramolecular and Polymer Chemistry and Director of the Melville Laboratory for Polymer Synthesis reveals how the ERC funded CAM-RIG (Confocal Microscopy and real-time Rheology of dynamic hydrogels) project is providing state of the art innovation to understand hydrogel behaviour at a molecular level in real time. Hydrogels, in the simplest of descriptions, are water-based jellies. They have a range of potential real-world uses depending on their composition. For example, hydrogels can be used for applications including soaps, cosmetics, soft contact lenses or even just for filling empty spaces. Use of hydrogels as 3D support structures for cells, as a means of delivering bioactive molecules in the form of targeted medicines, for wound-healing applications, and to study biological processes, are all known. The versatility of their properties means they have huge potential in myriad areas. In particular, their tunable material properties make them ideal candidates for new biomaterials, for example, to replace human parts like ligaments. In short, they are a ‘wonder material’ but by the very nature of their versatility they can be ‘slippery’ when it comes to studying them.

The power of dynamic behaviour Before comprehending CAM-RIG’s achievements, we need to first differentiate between types of hydrogels. A hydrogel is a network of polymer chains that is held together by crosslinks, which hold the (polymer) strands together. These can be classed as either physical (dynamic, break and reform) or chemical (permanent, do not break) crosslinks.

“There are two different types of hydrogels - dynamic and covalent (static). We’re interested in studying dynamic hydrogels,” explains Professor Scherman. “With dynamic hydrogels you have materials held together by interactions that can readily break and reform. For example, if you have a dynamic hydrogel in a syringe and you push the plunger, the sheer force of pushing the mass of material out through a tiny orifice means all

crosslinks means that the hydrogel can be readily formulated and, for instance, a drug can be added to the gel, which will then diffuse out of the gel to the surrounding area as the crosslinks break and reform.” The hydrogels can also be used to deliver or grow cells and recently we have even begun to grow organoids in the gels. In these cases, the dynamic crosslinks allow the cells to move and grow without restrictions.”

By solving the intricate, microscopic mysteries of dynamic hydrogels a whole world of exciting and sometimes life-changing applications will be possible for future generations to benefit from. of those dynamic interactions that are holding together the big chains, they can be broken in an instant and then immediately they fall back into place when the material exits the needle, so the gel goes from being a network to a flowing fluid, and then immediately goes back to being a network. You can completely change the structure of the gel. In contrast, if you loaded a static hydrogel in a syringe you couldn’t push it through a needle.” This property is important, especially as we want to use these gels for biomedical applications. The reversible nature of the

Seeing hydrogels up close The CAM-RIG project has pioneered a new state-of-the-art experimental set up. It couples together two pieces of characterisation methodologies - super resolution microscopy and rheology - to allow tracking of changes in the hydrogels at a molecular level in real time. The crosslinks within the gels used by Scherman are specially designed to be fluorescent when the crosslink is together (‘on state’) and optically dark when the crosslink is broken (‘off state’). The super resolution microscope images the crosslinks within the hydrogels while the

Image showing CAM-RIG setup, which combines super resolution fluorescence microscopy with piezo axial vibratory rheology.

rheometer simultaneously probes the material properties of the gel. The combined information allows for insight into how to fine-tune the hydrogel design towards a specific function or application, key when developing materials for use in the biomedical realm. “We are trying to get both spatial and temporal information of what’s actually happening with the crosslinks in the hydrogel rather than an ensemble average of what’s happening, so basically it’s about seeing a higher level of detail, as without the detail you’re just left with an average and it doesn’t give you any real information,” says Scherman, adding an analogy for clarity. “It’s like looking at a telescopic image of the Milky Way without any type of temporal or spatial resolution where you just see a smudge of light but if you’re able to get a fixed point in time – a snapshot – you would be able to see all of the stars at specific locations and how bright they are, so it’s like taking a smudge off of a telescope lens and giving you crystal clear information at a point in time.”

Pioneering new methods To achieve the aims of the project the team of researchers needed a piece of equipment that did not yet exist, so they were tasked to build a device that could reveal spatial and temporal resolution at extremely high resolution for the type of time scales and distance scales that are relevant. “At the start we knew what we wanted to be able to see but had to think about how to put together a set up that would allow us to do this. We decided to couple a super resolution microscope together to a rheometer to allow us to visualise the crosslinks whilst interrogating the material properties. The challenge came when we had to make these two pieces of equipment ‘speak’ to each other; in equipment terms you could say we have melded together a magnifying glass with a stopwatch and a hammer. These are tools that don’t normally work together in unison so making those connections was key.” To further complicate the development of this hybrid set up, there were nuances that

the equipment needed. Rheometers typically have a lag time before they output information about strain rates, this missing window was where most of the meaningful data that we needed was located. The other problem was that typical microscopy is not super resolution and limited to distance scales that are not below the diffraction limit of light. “We needed to find a way to put together super resolution microscopy with rheological measurements whose time scales could be immediate because the timescale of the ‘onoff’ transient crosslinks can be as short as sub-second. In collaboration with Dr Steven Lee, an expert in super-resolution microscopy, we found a way to do this and have spent the last two years building and testing our set up, which we call CAM-RIG. CAM-RIG includes spatial resolution that has a sub diffraction limit on the order of 10 nanometers or less and in addition it gives us time resolution on the order of thousands per second.” The science that will come out of the CAMRIG project will have direct implications in the advancement of biomaterials that the Scherman group are developing. They are world leaders in the development of biocompatible hydrogels for localised drug delivery and the insight from CAM-RIG will feed directly into how to design and develop new hydrogels for specific applications. The knowledge and insight gained by visualising these crosslinks within dynamic hydrogels can lead directly to advanced biomaterial applications in a wider sense by opening up the ability to precisely tune hydrogel physical properties. Halfway through the project, there are already many interested parties seeing great potential for industrial, healthcare and commercial applications. “We work with Cancer Research UK in Cambridge and the Brain Tumour Charity and of course we have a large amount of interaction with industry.” By solving the intricate, microscopic mysteries of dynamic hydrogels a whole world of exciting and sometimes lifechanging applications will be possible for future generations to benefit from.

CAM-RIG pioneers the combination of stateof-the-art characterisation techniques into a unique experimental setup, namely superresolution microscopy imaging modalities with simultaneous rheological measurements to investigate fundamental structure-property relationships of polymer networks and dynamic hydrogels. Using CAM-RIG makes it possible, for the first time, to deconvolute the molecularlevel dynamics of supramolecular physical crosslinks from chain entanglement of the polymeric networks and understand their relative contributions on the resultant properties of the hydrogels. We are using the knowledge gained through CAM-RIG to design and develop new supramolecular (bio)materials for a range of realworld applications.

Project Funding

Funded under the European Union’s Horizon 2020 research and innovation program under grant agreement 726470. (EU contribution: €2,038,120).

Contact Details

Project Coordinator, Oren A. Scherman Professor of Supramolecular and Polymer Chemistry Director of the Melville Laboratory University of Cambridge T: +44 1223 748850 E: oas23@cam.ac.uk W: https://www.ch.cam.ac.uk/group/scherman/

Professor Oren A. Scherman

Prof. Oren A. Scherman is a supramolecular and polymer chemist with a PhD in Chemistry from the California Institute of Technology (Caltech). He is the Director of the Melville Laboratory for Polymer Synthesis in the Chemistry Department in Cambridge. His research focuses on dynamic supramolecular self-assembly at interfaces through the application of macrocyclic host-guest chemistry, using cucurbiturils in the development of novel supramolecular systems. The Scherman group exploits control over these molecular level interactions to design and fabricate soft materials with integrated function, exploring topics that include biocompatible hydrogels for drug-delivery applications, sensing and catalysis, tough supramolecular polymer networks and bioinspired supramolecular fibres.

(a) Schematic to highlight covalent vs. dynamic hydrogel networks. (b) Supramolecular ‘on’-’off’ reporting model hydrogel used within CAM-RIG.

EU Research