The unparalleled wonder of silk

22 April 2020

Dr Asma Khalid enjoys wearing silk dresses, and appreciates diamonds for their beauty —but she never expected both silk and diamonds to end up being the cornerstone of her work as a physicist. Yet they have opened up a whole new way to see deep in the body, sense infections on the skin and even deliver drugs in controlled amounts.

As a PhD student at the University of Melbourne, she had been working with nanodiamonds, particles of solid carbon arranged in a crystal structure that are less than one thousandth of a millimetre in size. Because they’re inert to biological structures and have excellent light-emitting properties, nanodiamonds are being widely explored in biology as sensitive tools for diagnostic imaging and sensing.

‘Nanodiamonds are fluorescent, they glow brightly when we excite them with a laser. However they have a rough surface and tend to clump together a lot,’ said Dr Khalid, now a Vice-Chancellor’s Postdoctoral Fellow at RMIT University and an Associate Investigator at the CNBP. ‘We wanted to improve their surface and optical properties by coating them with a material that was still biocompatible in the body. That’s when we tried silk.’

One of the advantages of silk is that it has great optical properties, such as being optically transparent. ‘Silk actually enhances the brightness of the nanodiamonds significantly. And when we implanted a silk-coated hybrid inside mice, we found the silk dissolved in the body without causing any inflammation.’

Her resulting paper in Biomedical Optics Express generated a lot of interest, leading Dr Khalid to a scholarship to visit Prof Fiorenzo Omenetto’s Silk Lab at Tufts University in Boston, which has pioneered the use of silk in photonics and biotechnology. There, she found the inspiration to work in the multidisciplinary field of silk optics.

‘I learned how to extract silk from cocoons and transform that liquid silk into a range of different structures, like implantable films, injectable nanoparticles, 2D and 3D printed silk, and several other structures and devices for biophotonics and biomedical applications,’ she recounted.

‘I also produced silk-coated nanodiamond spheres, which worked really well as super-bright cell imaging tools, and drug-loaded nanodiamond silk spheres that could be used as vehicles for controlled release of drugs in anticancer treatment,’ she said.  ‘The hybrid spheres can release small amounts of drug over the period of weeks as the silk dissolves, and because the nanodiamonds fluoresce, we can track the release of the drug.’

The work led to 2 new scientific papers, and also piqued the interest of Tufts University in the optical properties of nanodiamonds as imaging and biosensing tools. When Dr Khalid returned to Australia and joined RMIT, she brought the Tufts and University of Melbourne collaboration with her. ‘So, we combined our work and got very interesting applications in imaging, sensing, drug release cell growth and tissue regenerating.’

A nanodiamond-silk membrane developed by Asma and team. Credit: RMIT University

Dr Khalid started her own lab at CNBP’s RMIT node to produce liquid silk nanoparticles from cocoons, creating fibre coatings, and electro-spun membranes — sheets of interconnected, uniformly sized fibres that can be combined with a host of useful particles for health monitoring applications.

At the time, Dr Jiawen Li, a CNBP biomedical engineer collaborating with Prof Heike Ebendorff-Heidepriem’s CNBP team at the University of Adelaide, was experimenting with a new class of ultra-thin fibre endoscopes that could take measurements deep in the body of a living organism, rather than having to use samples taken from the body.

‘It might be a tumour deep in the lung and we want to know whether it’s malignant or not by measuring the pH level or some target molecules without having to remove a piece of tissue for biopsy,’ she said. ‘Or instead of drawing blood and then getting the chemical composition of something like calcium or glucose, we want to insert these really tiny optical fibres, similar to that of a human hair, which are minimally invasive, to take real-time localised measurements at its original position without the need to wait for the lab result.’

‘The problem is that to enable these fibres to take measurements, you usually need to use harsh chemicals, which is not good for putting into the body,’ Dr Li added. ‘Or those chemicals might interfere with the information we’re trying to collect, so that you’re changing what you’re trying to observe.’

Dr Khalid suggested using silk, and the duo tried coating Dr Li’s optical fibres with it and testing them for biosensing and bioimaging in mice.

‘I couldn’t believe how simple it was,’ Dr Li said. ‘The silk is totally biocompatible; it’s not treated as a foreign intruder. And the whole process of adding sensors, like fluorophores, on top of the fibre is done at room temperature by just dipping the fibre into solution for 30 seconds.’

The next challenge was ensuring that when incorporating biomarkers into silk, the sensors stayed attached and worked as expected. That’s when organic chemists Aimee Horsfall and Patrick Capon, PhD students working under Prof Andrew Abell, CNBP’s chief investigator for biosensors research, joined the effort.

‘We needed to test the robustness of the system,’ said Horsfall. ‘If we poke it into somebody to reach an organ or tumour, will the biomarkers stay attached and function properly, and what are the limitations of the system?’

They started with a sensor for pH. But making the silk cladding work as a reliable sensor housing turned out to be a challenge: no matter how many ways they tweaked the chemistry of the silk, not all of the biomarkers stuck fast. After months of dead ends, they changed tack, and looked for a bonding peptide that might help anchor the sensors directly to the silk.

After bathing the fibre in a mixture of the silk and sensor attached to the silk-binding peptide for 30 seconds, the critical step turned out to be dipping the silk-coated fibre immediately into methanol for 10 seconds.

‘That changes the structure of the silk and makes it much more crystalline, which really attaches it to the fibre strongly,’ said Capon. ‘We’ve tested the fibre and verified we can hook up a laser and get a fluorescent signal from the sensor that’s anchored there. And we’ve found the only way we’re able to make the bonding fail is to literally break the fibre.’

Now that CNBP researchers have proven sensors can be dependably attached, they’re expanding from pH to hydrogen peroxide sensors for sperm cell health; sensors for metal ions like calcium and zinc for use in fertility and IVF; in fact, a whole range of applications to target specific ailments and conditions. Clearly, silk is going to be the workhorse for sensor work in fibre optics.

‘There’s a lot of work to do, but we’re pretty excited,’ said Horsfall. Capon agreed: ‘Every time we talk, we seem to come up with another 3 new ideas. How many sensors can we pack onto a fibre? What kind of applications can we find that biologists are interested in, or that clinical staff need?’

Meanwhile, Dr Khalid has expanded her work into silk-coated fluorescent nanoparticles for use in anticancer studies in a collaboration with CNBP’s Macquarie University node; silk coated magnetic particles for brain imaging along with engineering group at RMIT; and metal oxide-silk nanoparticles as biodegradable cell imaging tools with the University of Melbourne. She is also developing other exciting hybrid silk materials to address the current limitations in wound and burn care technologies.

‘I always used to think of silk as a fabric,’ Dr Khalid mused. ‘But when I did the literature review, I realised that it’s been used in medicine for centuries as a suture. Silk and diamond may be fancy materials, but they also have got great optical properties and a lot of benefits in health and medicine.’