The storage and distribution of vital protein therapeutics presents several complex challenges. Many medicines and vaccines need stable, temperature-controlled environments and chemical additives (excipients) such as preservatives to keep them effective and safe for use.
This requires cold storage infrastructure and reliable energy sources which not only puts the responsibility on the user but causes accessibility and affordability challenges, especially in developing countries where resources are limited.
Now researchers from the UK Universities of Manchester, Glasgow and Warwick have designed the world’s first hydrogel technology for the storage and distribution of crucial medicines and other biopharmaceuticals without the need for refrigeration or chemical additives. The aim is to provide more robust and equitable storage and delivery systems, benefitting everyone worldwide.
The novel hydrogel cargo-system paves the way for affordable, energy efficient and user-friendly ways of supplying patients and clinics with vital protein-based drugs for health conditions like diabetes and cancer. The hydrogel also offers exciting potential for diagnostics and biotechnology.
Fig. 1: Beamline I22 in a typical experimental configuration. The SAXS detector is positioned behind the large tank on the blue frame toward the centre of the image. The tank contains beam stops, that prevent the intense X-ray beam from damaging the sensitive detector. The sample position is just out of shot, to the right of the image. To access very small angles, and thus interrogate large distances in the sample, the distance between sample and detector is quite large. If this was all air, that would scatter the X-rays further and blur the data. Instead, the large tank and horizontal tubes are evacuated to a few millionths of an atmosphere with powerful vacuum pumps. Credit: Sean Dillow. Copyright: Diamond Light Source Ltd.
Published in the journal Nature on 24 July 2024, the research involved a series of tests to show how the hydrogel works on two proteins: insulin to treat diabetes, and beta-galactosidase, an enzyme with life science and biotechnology applications. Synchrotron science played an important role in the early development and testing of the hydrogel using Diamond’s I22 (Fig.1) and B21 (Fig.2) beamlines.
The research was supported with funding from the European Union’s Horizon 2020 programme, the European Research Council, the Royal Society, the Engineering and Physical Sciences Research Council (EPSRC), the University of Glasgow, and UK Research and Innovation (UKRI).
The new hydrogel system – made mostly of water – has been designed to stabilise proteins, protecting their properties and functionality under stresses such as vibration and temperatures as high as 50 degrees centigrade.
Key to this breakthrough is the hydrogel’s mechanical structure. The unique gel was developed using a Low Molecular Weight Gelator (LMWG) and a chemical trigger that prompts the gelator molecules to self-assemble into very long, three dimensional fibres which tangle together to create a spaghetti-like network.
When the proteins are added, they become lodged in the spaces between the fibres, where they are unable to mix and aggregate (mixing and aggregation can prevent or limit effectiveness). The fibres are both stiff and brittle which ensures ease of protein release, while the protein remains unchanged throughout the process (Fig.3).
Andy Smith, Senior Beamline Scientist at Diamond’s I22 beamline, said:
Although there has been previous research at Diamond on alternatives to the cold chain problem using gels [2], these latest hydrogel results are an exciting breakthrough. In this study, the research team got the idea of using the fragility of the gel as an attribute, exploiting the brittleness of the gel’s fibres to enable ease of protein entry and release. It was a kind of eureka moment when it became clear that the hydrogel could be used to encapsulate proteins and other things and get the proteins back out intact and useable.
The hydrogels used for experiments in this study were made at Diamond by Dave Adams’ group from the University of Glasgow. Dave Adams is one of the co-authors of the Nature paper and has many years of experience making gel-type materials and employing neutron and synchrotron sources to investigate how the chemistry of the gelator affects its structure.
During the initial stages of the research (October 2020), the gels were explored with time-resolved experiments on Diamond’s beamline I22 using small-angle X-ray scattering (SAXS) techniques. The resulting scattering patterns revealed several structural parameters in the gels, which were then analysed using mathematical models.
Fig.4 - Initial gel studies. SAXS patterns of gels made in presence of calcium chloride (black) and without CaCl2 (red). The fits obtained through model fitting are overlayed on each spectrum (details in Supplementary Information). The plotted data show the averaged scattering pattern obtained from five measurements across the sample. The error bars are generated during data processing by calculating scattering signal uncertainty in the detected data according to previously published methods.
Andy explains:
Our I22 beamline played a foundational role in investigating the structure of the gel material, how the gel’s molecular fibres fit together and how they performed in situ under various conditions. For example, a process called ‘in situ rheology’ was employed where the gel material is loaded onto a rheometer and sheared to investigate the gel’s stiffness. The rheology equipment was placed on the I22 beamline, and the gel structure examined in real time as it was being poked and pushed, providing insights into the mechanical properties of the gel under conditions of stress.
Changing the chemistry of the gelator or the speed of gelation, and then analysing the effects on the gel’s structure, enabled different types of fibres to be tested, including varying lengths and flexibility to create hydrogel with optimal conditions.
Once the team had a viable set of gels that could be used to start the encapsulation process, various tests were done at other research centres to provide the full picture [1]. Stress tests included testing the effectiveness of the hydrogel suspension for insulin and the enzyme beta-galactosidase under high temperature conditions and under the strain of rotating it at 600 revolutions per minute, in addition to transporting samples of proteins suspended in hydrogel in the post for several days. The team also conducted experiments to test protein release using an ordinary syringe, fitted with a special filter, filled with the protein-storing gel (Fig. 5).
Matthew Gibson, from the University of Manchester and co-author of the Nature paper, adds:
Protein therapies normally require excipients, such as polymers and sugars, which stabilise them against temperature and aggregation stressors, during freezing, freeze drying or liquid phase storage. Other gel-based systems have been reported for stabilising proteins at room temperature, but they typically required addition of a chemical trigger (so use needs to add a new compound) and following break-down of the gel, all those components are mixed in with the protein. In our system, we release the protein just by pressing down on the plunger in a syringe, so it is very easy. Furthermore, as it is released, our technology separates the gel from the protein so only protein in pure buffer is released. Thus, we have solved the question of how to release the protein in a user-friendly method, and to remove all the excipients in the same action.
After the results were peer-reviewed, further experiments were conducted at Diamond’s B21 beamline to confirm that the protein which entered the gel was the same after it was expressed. This involved a day of BioSAXS experiments (in December 2023) - simple protein structure measurements before and after expression. Further biochemical tests were conducted elsewhere, with equally pleasing results [1].
Dave reports:
The beamlines at Diamond Light Source do exactly what we need. Andy Smith [from beamline I22] is an amazing person to work with and Nathan Cowieson was kind enough to let us run the last necessary experiment for referee comments on beamline B21.
The full scope in terms of longevity and breath of protein cargoes continue to be investigated by the research team while pursuing commercial opportunities, follow-on funding, and investment to take the findings further. With a patent-pending, different market segments are being explored where the novel hydrogels can make impact, including vaccines for low-resource environments, therapeutic proteins and enzymes, and antibody therapies.
“The gel is easy to make and to scale and could be made easily on site, such as a clinical setting,” says Dave. “I see no issue with it being made in any location; the ingredients are all pretty simple.”
At Diamond, the current length scale on beamline I22 ranges from a few nanometers up to a few hundred nanometers but this is not yet sufficient to reveal the full length of the hydrogel’s fibres. Forthcoming plans to upgrade beamline I22 [3] will enable further insights and tests with the gel materials, covering the entire length of the hydrogel’s fibres.
To find out more about the I22 beamline, or to discuss potential applications, please contact Principal Beamline Scientist, Nick Terrill: nick.terrill@diamond.ac.uk or Senior Beamline Scientist, Andy Smith: andy.smith@diamond.ac.uk
To find out more about the B21 beamline, or to discuss potential applications, please contact Principal Beamline Scientist, Nathan Cowieson: nathan.cowieson@diamond.ac.uk
[1] Bianco, S., Hasan, M., Ahmad, A. et al. Mechanical release of homogenous proteins from supramolecular gels. Nature 631, 544–548 (2024). https://doi.org/10.1038/s41586-024-07580-0
[2] The approach of Doekhie et al. was to wrap the protein in a silica jacket to protect it, and then chemically release it later. Doekhie, A., Dattani, R., Chen, YC. Et al. Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization. Sci Rep 10, 9243 (2020). https://doi.org/10.1038/s41598-020-65876-3
[3] More details about upgrades to beamline I22 can be found here: https://www.diamond.ac.uk/Instruments/Soft-Condensed-Matter/small-angle/I22/Projects/Current-projects.html, accessed on the 10.8.2024
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