Point of care nanotechnology for early blood clot detection and characterisation in disease screening, theranostic and self monitoring applications

Lead Research Organisation: School of Pharmacy
Department Name: Pharmaceutics

Abstract

Thromboembolic disease and associated blood coagulation abnormalities cause significant morbidity and mortality in Western society, with stroke being the third leading cause of death in the UK. The incidence of stroke increases markedly with age and is often higher in socially deprived areas. In stroke, the processes of endothelial and vascular damage, activation of the coagulation cascade and decreased fibrinolysis result in abnormal clots, often with excessively cross-linked fibrin networks. An unsatisfactory aspect of work in this area is that the microstructures of such clots are usually reported in only adjectival terms (e.g., dense or tight ) - usually on the basis of a visual inspection of fragments of dessicated clots in SEM micrographs. Early detection of clots is vital. Early clotting events may contribute to a pro-thrombotic state which exacerbates the disease state and thrombotic states can be followed rapidly by haemorrhagic states due to adverse changes in clot structure. The available therapeutic options informed by early detection and characterisation are greatly enhanced.New technology is essential to address shortcomings in this area. This project will exploit our recent advances in blood clot detection and ultra-sensitive nanomaterials development for device applications to overcome these shortcomings. Under a Royal Society Brian Mercer Award and an EPSRC Portfolio Partnership Award, in collaboration with the NHS, we have developed a new haemorheological technique for the early detection and characterisation of blood clots. This has led to the discovery that the incipient clot's fractal microstructure is a biomarker for the conditions of clot formation, including therapeutic intervention. The significance of this discovery stems from the incipient clot's role as the microstructural template for ensuing clot development. In parallel work we have demonstrated the controlled reproducible growth of vertical arrays of ZnO nanowires and have confirmed their electrical current generation capabilities. Our Grand Challenge proposal involves combining this nanotechnology with our haemorheological work to develop the first point of care (POC) device capable of the early detection and characterisation of abnormal clots. By a point of care device we refer to technology suitable for widespread use outside hospitals (i.e., within pharmacies and surgeries) and which will ultimately be developed for use by patients at home. This will exploit the piezoelectric properties of ZnO nanowire arrays as a transducer to detect shear wave propagation within coagulating blood. Our aim is to drastically improve the sensitivity of early clot detection for more accurate assessments of (i) coagulation abnormalities and (ii) therapeutic targeting of abnormal clots at the earliest stage of development. The project involves in vivo and in vitro disease model (Stroke) work at University of London, and work intended to enable our device to perform a therapeutic function. In this way we propose to lay the foundations for a POC system for Patient Self Assessment and Patient Self Management in anticoagulant applications, in addition to a new technological basis for thromboembolic disease screening. The project also includes anticoagulated Stroke patient volunteers at Morriston NHS Hospital.We have a highly multidisciplinary Team with internationally leading expertise in rheometry and haemorheology; nanotechnology, nanomaterials and nanofabrication; nanomedicine and drug delivery; and human-device interaction aspects of medical instrument design. We have two partners. The first is the NHS who will provide clinical facilities and governance of clinical research. Our second partner is Boots Centre for Innovation (BCI) whose involvement anticipates healthcare provision involving POC applications in next-generation pharmacies. BCI's role is to inform design relating to customer needs/experience, the POC market and environment.

Publications

10 25 50
 
Description Electro-responsive polymer matrices were developed as the therapeutic component of the first point of care (POC) device capable of the early detection and characterisation of abnormal clots. Stimuli-responsive hydrogel hybrids were engineered to allow the device to perform a therapeutic function, and to deliver drug in a pulsatile manner, remotely controlled by the ON/OFF application of an electric voltage generated by the device when the detection of a clot occurs. The drug delivery system is composed of multi-walled carbon nanotubes (MWNTs) incorporated in an electro-responsive hydrogel matrix such as poly (methyl methacrylate) (PMAA). The use of MWNTs considerably improved the sensitivity and response of the hydrogel hybrids to the applied electric field compared to the hydrogels without MWNTs. The pulsatile release of a model drug in the blood upon the application of the electric field was also demonstrated in vivo when the hybrid gels were subcutaneously implanted in mice. The MWNT hybrid gels demonstrated higher drug release and sharper responses to the electric field, significantly outperforming the blank gel during the same time period and under the same conditions of stimulation. An optimisation of the system was explored by using graphene sheets as nanofillers of the PMAA hydrogel matrix. These novel graphene gel hybrids demonstrated higher drug release capabilities than the MWNT hybrid gels.



A systemic model of thrombosis was selected to enable comparison with clinical blood samples and lay the groundwork for point-of-care device testing. Intravenous injection of thrombin was used to induce a widespread prothrombotic state without causing animal death and reduced occurrence of stroke compared to other methods to induce systemic thrombosis. The thrombin-induced systemic model was characterised and compared to healthy animals using a wide range of techniques. Traditional methods were used in conjunction with our haemorheological technique for the early detection and characterisation of blood clots. The prothrombotic state of the disease model was confirmed by: platelet aggregate formation (full blood profile); a 4-fold increase in the number of activated platelets (flow cytometry); a ten-fold increase in thrombin-antithrombin levels (ELISA); observation of more compact fibrin networks in the clot structure (scanning electron microscopy) and thromboembolisms (histological analysis). Together these data confirm that this in vivo model is prothrombotic. Employing our haemorheological technique, clotting time was reduced by half and the mature clot was 2.5 times more rigid in structure in blood from the disease model. This is the first time that haemorheological measurements have been made in mouse. It is possible to measure blood samples from both healthy and diseased mice. This in vivo model of thrombosis is suitable for validation of the point-of-care device.
Exploitation Route Work under the grant involving samples of blood was conducted principally in collaboration with our partner, ABMU NHS Trust at the NHS Morriston Hospital EPSRC-NHS Clinical Haemorheology Labaoratory (opened in 2005). The routes for exploiting such sensitive and stimuli-responsive delivery systems for industrial applications rely on the capacity of these gels to be easily attached on, and form a part of the device developed for early clot detection. The pulsatile delivery of anticoagulant agents such as heparin or warfarin could be generated subsequently to the early detection of a clot combining diagnosis and therapy in a single device.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.nanomedicinelab.com/cooperation-and-support/collaborations/
 
Description The significant clinical (screening and monitoring) potential of the new haemorheological biomarkers exploited in the work under report led to the award of £1.5M in 2011 to establish a new Haemostasis Biomarker Research Unit at Morriston NHS Hospital. Within this new Unit the prototype/proof of concept point of care rheometrical devices developed during the work under report are being assessed and validated alongside conventional haemorheological and clot assay techniques, with 9 new staff appointments (Clinical Fellows, Clinical Haemorheologists and Research Nurses) taking the technology from the laboratory to use at the point of care in (i) operating theatres; (ii) treatment rooms and (iii) on patient wards, in collaboration with partner NHS Consultants in seven carefully chosen Care Pathways (including stroke, sepsis, diabetes, cancer and trauma). In addition, these point of care assessment/validation studies include the effects of fluid dilution, temperature change and aniplatelt therapy including aspirin).
Sector Cultural,Policy & Public Services
Impact Types Cultural

 
Description Engineering Responsive Nanomaterials for Pulsatile Neural Regeneration
Amount £117,753 (GBP)
Funding ID EP/K001558/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 11/2012 
End 12/2013
 
Title Subcutaneous gel release model 
Description Subcutaneous implantation of hybrid gel, stimulation of drug release by electrical stimulation and subsequent blood sampling and analysis. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Subsequent studies from other laboratories have repeated the experimental method described with success 
 
Title Systemic thrombosis mouse model 
Description Intravenous injection of 5U/ml thrombin into mouse tail vein to induce a procoagulant state. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact N/A 
 
Description Development of new device. 
Organisation Hemair Systems
Country India 
Sector Private 
PI Contribution 1 day visit to Swansea University for discussions with Biotech company, Hemair, and Rhodri Williams regarding the development of a new device.
Start Year 2010
 
Description Development of wireless drug delivery system with magnetic field actuation Description * 
Organisation ETH Zurich
Department Institute of Robotics and Intelligent Systems
Country Switzerland 
Sector Public 
PI Contribution A navigatable and wirelessly actuated by a magnetic field polymer matrix was engineered and developed for controlled and targeted drug delivery.
Start Year 2011
 
Description Functionalisation of carbon based nanomaterials with contrast agents for diagnosis using the MRI technique 
Organisation Eindhoven University of Technology
Country Netherlands 
Sector Academic/University 
PI Contribution Collaboration with Department of Biomedical Engineering in Eindhoven University of Technology for the functionalisation of carbon based nanomaterials with contrast agents for diagnosis using the MRI technique.
Start Year 2010
 
Description The effect of carbon nanotubes on rheological properties 
Organisation Swansea University
Country United Kingdom 
Sector Academic/University 
PI Contribution 2 day visit to Swansea University to perform haemorheological experimentation in human blood with Karl Hawkins. The effect of carbon nanotubes on rheological properties was examined.
Start Year 2011
 
Description Track nanoparticles in vivo 
Organisation Helmholtz Zentrum München
Department Institute for Biological and Medical Imaging
Country Germany 
Sector Public 
PI Contribution 5 day visit to the Institute for Biological and Medical Imaging (IBMI), Helmholtz Centre, Munich to track nanoparticles in vivo using multi-spectral optoacoustic technology.
Start Year 2012
 
Title Development of wireless drug delivery system with magnetic field actuation 
Description A navigatable and wirelessly actuated by a magnetic field polymer matrix was engineered and developed for controlled and targeted drug delivery. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2012 
Impact Further studies on magnetic actuation of micro- and nano-particles 
 
Title Haemorheological technique in mouse blood 
Description Haemorheological measurements have not been performed previously. The technique used for human blood was adapted for use small volumes of mouse blood sample (approx 600ul). This has made it possible to compare haemorheological data from mouse to clinical samples. Dr Methven did the adaptation of human technique and development of protocol for heamorheological technique in mouse blood experiments. Dr Karl Hawkins did the rheological training, development of protocol for heamorheological technique in mouse blood experiments. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2012 
Impact Preclinical evidence to educate clinical trial design 
 
Title Injectable hydrogel hybrids for controlled drug delivery 
Description An injectable electro-responsive hybrid polymeric implant for the pulsatile and targeted delivery in the brain of Retinoic acid (RA), a growth factor that plays a crucial role in neuron patterning, differentiation and survival in the brain was developed. 
Type Of Technology Systems, Materials & Instrumental Engineering 
Year Produced 2012 
Impact Prototype for future studies 
 
Title MWNT/Graphene Hydrogel hybrids 
Description PMAA-MWNT hydrogel hybrids were fabricated by in-situ radical polymerisation; the concentration of multi walled carbon nanotubes (MWNTs) and of the cross-linker was optimised in order to obtain hydrogels with enhanced responses to the electric field. The incorporation of MWNTs within the polymeric network, confirmed by SEM, improved the physical properties of the hybrids by decreasing significantly the bulk resistivity of the polymeric matrix and improving the thermal properties of the polymeric matrix. 
Type Of Technology New Material/Compound 
Year Produced 2012 
Impact N/A 
 
Title Mimicking clinical experiments with mouse blood 
Description High-specification rheometer was transported to and used at School of Pharmacy for mouse blood samples to mimic the clinical data obtained at Swansea University and CHL Morriston Hospital. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2012 
Impact Prototype for clinical trial design 
 
Title Novel MEMS druge delivery device 
Description A novel MEMS based drug delivery device has been developed, consisting of an array of metallic contacts on silicon and Pyrex glass wafers. The meander structured device creates a uniform electric field which stimulates drug release. An electro-active hydrogel based polymer matrix has also been developed, which responds to an electrical stimulus and shrinks or de-swells on application of an electric field from the fabricated device. Different drug candidates can be encapsulated within the polymer matrix. The de-swelling of the polymer enables the encapsulated drug to be released from the matrix. 
Type Of Technology Physical Model/Kit 
Year Produced 2011 
Impact Prototype for further industrial development 
 
Description Nanomedicine Lab website 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The Nanomedicine Lab describes the in its website the project and the acknowledges its contribution to advance the research conducted.
Year(s) Of Engagement Activity 2009
URL http://www.nanomedicinelab.com/cooperation-and-support/collaborations/
 
Description Nanonews Net on the PNAS article 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact http://www.nanonewsnet.ru/news/2011/dlya-lecheniya-ishemicheskogo-insulta-vpervye-oprobovany-uglerodnye-nanotrubki-dostavlyayu.
Year(s) Of Engagement Activity 2011
URL http://www.nanonewsnet.ru/news/2011/dlya-lecheniya-ishemicheskogo-insulta-vpervye-oprobovany-uglerod...
 
Description Nanotubes inject stroke therapy into rats' brains 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact http://www.rsc.org/chemistryworld/News/2011/June/21061102.asp.
Year(s) Of Engagement Activity 2011
URL http://www.rsc.org/chemistryworld/News/2011/June/21061102.asp
 
Description Talk softly but carry a tiny stick: Stroke prevention and recovery with nanotube-delivered siRNA 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact http://medicalxpress.com/news/2011-07-softly-tiny-recovery-nanotube-delivered-sirna.html
.
Year(s) Of Engagement Activity 2011
URL http://medicalxpress.com/news/2011-07-softly-tiny-recovery-nanotube-delivered-sirna.html