Usurping the scalpel: non-invasive oxygen nanosensors to refine data acquisition

Measuring oxygen within cells and tissue is fundamentally important for our understanding of the development, progression, and therapy of many diseases, including heart disease, lung disease, stroke, multiple sclerosis, diabetic retinopathy, vessel occlusion, and cancer. However, methods to measure oxygen in humans are limited in their applicability. Therefore, biomedical researchers rely upon animal research to investigate oxygen levels within cells and tissue. However, it is still common for biomedical researchers to use techniques that necessitate animal sacrifice. Consequently, large numbers of animals are sacrificed in biomedical studies of disease development, with each animal providing a relatively sparse amount of data.

The aim of this research is to develop an effective non-invasive technology to study oxygen within blood, cells and tissue of laboratory animals in a repeatable manner over extended time-periods. By "usurping the scalpel", this technology will have many benefits, including reducing animal sacrifice in scientific research, reducing the cost of research, and providing better data to biomedical researchers.

This fellowship will develop nanosensors to non-invasively report oxygen levels deep within tissue using cell-friendly near-infrared light. The nanosensors will be formed around a nanoscale crystal known as an upconversion nanoparticle, which emits red light when stimulated by near infrared light. These optical properties allow light from a just a few dozen of these nanosensors to be measured through several centimetres of overly tissue. Such tissue would normally have to be surgically removed for imaging with conventional optical probes, with the animal likely being sacrificed after the procedure. The nanosensors will be constructed so that the intensity of red-light emission changes in the presence of oxygen: in effect they will act as an optical dimmer switch controlled by localised oxygen levels. The nanosensors will be guided and anchored to cells and tissues of interest by modifying their surface with appropriate biorecognition molecules. Further, the nanosensors can be modified to ensure that they are not harmful to cells.

The nanosensors will be tested in blood, within cells, within bacteria, and within artificial constructs called phantoms, which will simulate the tissue of living animals. Testing will be conducted to ensure that the nanosensors are robust against variables such as pH, temperature, dissolved gases, and tissue pigmentation. This testing will demonstrate that the nanosensors are fit for deployment within living animals.

This work will pave the way for the oxygen nanosensors to be developed in collaboration with commercial and academic partners. Dissemination of the nanosensor technology to biomedical end-users will reduce the number of animals sacrificed in biomedical research, providing additional benefits in reduced costs for research, reduced time-burdens, and improved data quality. This will add value to the UK economy and help meet the UK government's strategic aim of reducing animal sacrifice in scientific research.

The aim of this research is to develop nanosensors to provide repeated non-invasive measurement of oxygen within the blood, cells, and tissue of laboratory animals over the extended time-periods required to study disease progression and therapy. This would reduce current reliance on highly-invasive methods which necessitate immediate animal sacrifice, thereby significantly reducing animal usage in biomedical research. This proposal meets 2 BBSRC responsive mode remits: (A) 'Technology Development for the biosciences', and (B) 'The replacement, refinement, and reduction (3Rs) in research using animal models'.

The key innovation is the development of novel nanosensor technology to non-invasively report the concentration of oxygen (O2) within specifically targeted cells/tissue and blood even through several centimetres of overlying tissue. The nanosensors will be constructed around upconversion nanoparticles, which are excited by near infrared photons (~980 nm) and emit visible photons (~660 nm) via multi-photon upconversion. These properties enable nanosensor imaging through deep-tissue tissue with no photobleaching, phototoxicity, or visible autofluorescence. The novel sensing mechanism behind these nanosensors will be patented.

To pave the way for eventual in vivo application of the O2 nanosensors, the core objectives are:

(a) Achieving repeatable synthesis of nanoconstructs and demonstrating wavelength selective excitation of sensor vs reference nanoconstructs for in situ ratiometric readout.
(b) Establishing calibration curves of emission ratio vs variables such as pH, temperature, CO2, and tissue parameters.
(c) Demonstrating repeatability and reversibility of nanosensor O2 measurements.
(d) Demonstrating applicability to oximetry of whole blood within deep-tissue phantoms.
(e) Demonstrating biocompatibility and targeted O2 measurement within mammalian cells.
(f) Demonstrating sensing of O2 and nitric oxide consumed/emitted by bacteria.

(A) IMPACT/BENEFITS ARISING FROM REDUCING ANIMAL USE IN STUDIES OF OXYGEN TRANSPORT

The UK government and BBSRC have committed to reduce the use of animals in scientific research, with a delivery plan in place since 2014.[1]. This is done by supporting the '3Rs' principle: replace, reduce, and refine. This plan acknowledges the need for new technologies and the need to train future research leaders.

By funding this fellowship proposal, BBSRC will help develop a technology with the potential to drastically reduce the number of animals used in studies of oxygen transport (e.g. a 7x reduction in worked example below) with proportional cost-savings to funders.

WORKED EXAMPLE:
Currently, monitoring progression of spinal cord tissue hypoxia in a rat model of multiple sclerosis over one week requires using the lethal method of immunohistochemical tissue standing/sectioning.[2] Each day, 5 disease model animals and 5 control animals are sacrificed. Over 7 days, this totals 70 animals sacrificed. Each animal has the following costs: purchase (£30) and feeding/housing (£10 per day for 12 weeks). For 70 animals, this is a total cost of £10,500. By instead using non-invasive nanosensors for repeated targeted measurement of oxygen within tissue over consecutive days, only 10 animals have to be sacrificed to glean equivalent information (5 model animals and 5 controls), resulting in a 7x reduction in animal usage and associated cost i.e. from £10,500 to £1500.

EXAMPLAR OUTCOMES:
1. 7x decrease in animals sacrificed in a week-long sub-study.
2. Proportional decrease in cost to funders (i.e. feeding/housing animals).
3. Increase in research efficiency (time + cost savings), boosting value for money.

BENIFICARIES:
* Biomedical Researchers.
* UK Government & UKRI funding agencies, particularly BBSRC and MRC.
* 3rd-sector (charity) funders: e.g. British Heart Foundation and Cancer Research UK.

[1] HM Government. (2014) 'Working to reduce the use of animals in research'. Ref: ISBN 978-1-78246-264-4, BIS/14/589
[2] Davies et al. (2013) 'Neurological deficits caused by tissue hypoxia in neuroinflammatory disease.' Annals of Neurology. 74 (6). p815-825.

(B) IMPACT/BENEFITS TO THE UK KNOWLEDGE ECONOMY AND TO THE NORTH OF ENGLAND

The UK government's industrial strategy is centred around a world-leading knowledge-led economy, underpinned by university research and partnership with business.[3] Additionally, the UK Government has identified that the North of England requires investment as a matter of strategic priority.[4]

Funding this fellowship proposal will help grow the UK's knowledge economy by enabling intellectual property to be secured and disseminated to commercial partners for commercial exploitation. This will benefit economy of the north of England by developing and transferring new biotechnology knowledge and developing commercial partnerships. The North East will gain world-leading knowledge in the development and application of these biosensors. This will help secure economic investment, e.g. by attracting consultancy opportunities via Durham University, which will help stimulate the northern regional economy.

BENIFICARIES:
*Northern regional economy
*UK government.
*Future commercial partners (TBC)

[3] HM Government, (2018). 'Industrial strategy: building a Britain fit for the future'.
[4] HM Government (2016). 'Northern Powerhouse Strategy'. Ref: ISBN 978-1-911375-53-1, PU1992

(C) IMPACT/BENEFITS ARISING FROM PUBLIC ENGAGEMENT
The fellow will participate in public engagement activities, with emphasis on communicating the importance and benefits of non-invasive optical biosensing technologies. The fellow will present appropriate themed demonstration at the annual Durham University Celebrate Science Festival (~4000 public visitors over 3 days), and Durham University Schools Science Festival, reaching ~150 school pupils over 3 days.

BENEFICIARIES: the public and school pupils.