NanoDrill - A new versatile research tool - high spatial resolution light activated molecular nanomachines

An important need for future bio-medical and fundamental cell biology is the targeted in vivo destruction of selected cells and cell types. Cancer affects every country regardless of financial status, medical infrastructure or the availability of highly-skilled specialists and is responsible for 8.2 million deaths/year worldwide. Our vision for the future is to develop a series of light-activated molecular nanomachines - Nano-drills - to target cancerous cells selectively and safely eradicate them. Currently this can only be facilitated using highly invasive surgical or radiotherapeutic procedures that are often harmful to administer.

We have already demonstrated that our Nano-drills (molecular Nano-Machines, MNMs) can selectively destroy cells using small quantities of ultraviolet activation light. The next phase of our research is four-fold. First, we must enable multi-two-photon (2PE) activation on our existing microscope system using biologically safe near-infra-red (NIR) light for live-cell studies. Second, extend the family of cell type and pathological condition specific nano-drills to allow a wide range of cancer cells to study. Third, design nano-drills that can be activated using plain visible light in order to comply with already established protocols in biomedical research. Fourth, create a new breed of nano-drills that will first be internalised by the targeted cancer cells and subsequently allowing eradication to be triggered them from within. This will promote a more controlled single-cell precision research tool.

Preliminary studies showed that our MNMs can be successfully activated using 2PE. In order to continue this research, we need a fast, high resolution live cell capable Multi-Photon Microscope (MPM) to image live cells. Unfortunately, due to the nature and unique instrumental requirement an off the shelf MPM system costs in the region of £500,000. However, we propose to develop a suitable custom system by adapting existing equipment at fraction of the cost. We will equip our Leica SP5 II confocal system with a tuneable high repetition rate state-of-the-art NIR laser. This experimental modification will allow us to directly compare live-cell molecular nanomachine activation with both UV and 2PE excitation and exceed the capabilities of a standard off-the-shelf microscope. We have already purchased all associated auxiliary optical components needed for attaching the NIR laser. We advanced the instrument build to a stage, where after sourcing the required laser system a simple 'plug and play' approach will yield the multi-photon upgrade on our instrument within a few weeks.

Once fully developed it could form a new extremely high precision biological research tool and opens new horizons toward wound healing and tumour/cancer progression studies. It could pave the way towards a non-invasive treatment of chemotherapeutic agent resistant cancers, such as metastatic breast cancer. This project has a potential game-changing impact on a global scale.

The key innovation is the development of a fundamentally new single-cell precision biological research tool using cell type and metabolic/morphological state specific molecular nanomachines and biologically safe near infra-red activation. In order to achieve this, we have set out to achieve the following work-packages:
1) Enabling 2PE activation on our existing microscope system light for live-cell MNM studies. We have advanced the instrument build to a stage, where after sourcing the required NIR laser system a simple 'plug and play' approach will yield the multi-photon upgrade on our instrument within a few weeks.
2) Extending the family of cell type specific MNMs to target skin, colorectal, breast and prostate cancer cell lines by incorporating short target peptide sequences, using copper-catalyzed cycloaddition. Using PC3 cells, MNM cell membrane anchoring and targeted necrotic cell destruction has already been successfully demonstrated (Nature, 2017, 548, 567-572).
3) Developing MNMs with excitation/activation wavelengths extending into the visible range (above 405 nm) by attaching electron donating functional groups onto the rotor moiety at position 2 or 4 or by substitution of the thioxanthene stator moiety with a 9H-fluorene. These will introduce a bathochromic activation wavelength shift and also enhance multi-photon cross-section.
4) Establishing whether a more biologically favoured apoptotic cell death pathway could be promoted by incorporating hydrophilic pendant side chains in order to facilitate membrane transport, uptake and promote subsequent light activated cell-death form within the cell.
Combining WP2 and 4 will pave the way towards the development and study of MNMs with hybrid substitution. Once fully developed and validated these MNM will open new horizons toward wound healing or tumour/cancer progression and improve and extend molecular toolbox of biological and biomedical researchers wold-wide.

Impact will be delivered by fulfilling the proposed research's key objectives and provide the stakeholders in various sectors with a novel potentially non-invasive photodynamic therapy platform using light activated molecular nanaomachines and biologically safe multi-photon excitation.
The project will ensure and deliver short and long term impact across four key areas:
People:
The training delivered to the PRDA will in part address a skill shortage in a key area affecting national competitiveness in optically active nano-materials and optical imaging research worldwide. The UK has an international standing in the fields of bio-imaging and chemical-biology and the generation of new knowledge in these areas that the project will produce will help to maintain this global advantage via contributing into a novel promising aspect of global healthcare. The PI is committed with an extensive track-record of the University's outreach programmes (i.e. Durham Celebrate Science, 3 days-7000 people/year since 2008). These activities include bringing science to students and teachers and the general public. The public engagement activities will include the use of social media to inform the public of the progress of the project and highlighting the research at public events (e.g. Royal Society Summer Science Fair, invited speaker, 8 public lectures, 2015-2018; Science Museum Lates 2015/2017). Our aim is not only to educate and inspire the next generation of scientists but also raise the public's awareness of our research. We propose to use our combined findings and create a suite of demonstration material, accompanied by adaptable outreach activities that can be delivered to all ages at Science fairs, demonstration lectures or in schools.
Society:
The development and validation of a fundamentally new single-cell precision biological research tool using cell type and metabolic/morphological state specific molecular nanomachines and biologically safe near infra-red activation. Once fully developed and validated it could open new horizons toward wound healing or tumour/cancer progression and improve and extend molecular toolbox of biological and biomedical researchers wold-wide.
Economic :
This directly links with the societal impact detailed above The proposed project has already drawn attention from potential academic, industrial and commercial partners due to the preliminary single photon studies being recently published in Nature. The generation of novel IP will help to develop new markets for commercial exploitation in the UK and worldwide.

Knowledge:
On a fundamental level this project will deliver increasing understanding of the potential that lies within light activated cell type and condition specific molecular nanomachines and will benefit researchers both in academia and industry to work towards extend molecular toolbox of bioscientists and biomedical researchers wold-wide and fill a current void in available resources.
Using the traditional academic channels results will be published via scientific publications submitted to relevant high impact journals and findings will also be disseminated via conference attendance and invited talks. Future outcomes will enable the applicant to base further grant proposals submitted to relevant funding agencies and, since the molecular machines and their associated methodologies are already patent protected by the applicants, private investment or licensing deals to relevant industrial partners. Results generated would not only open new revenues of funding to the applicants but would also enable them to establish vital consultancy links with industrial partners and biomedical/healthcare agencies.