Development of Heterodyne Coherent Anti-Stokes Raman Scattering Microscopy for Monitoring Nanoparticle Drug Delivery

Lead Research Organisation: University of Exeter
Department Name: Physics

Abstract

At present, 95% of all potential new drug compounds cannot be directly administered as a pharmaceutical due to poor biocompatibility, i.e. they have poor solubility, unacceptable levels of toxicity, or become metabolised by the body before reaching the site of interest. Nanoparticle drug delivery can overcome these problems by encapsulating the compounds in particles less than 1 thousandth of a millimetre in size. Moreover, nanoparticles can act as depositories for controlled drug release and can be tailored to actively target specific sites within the body.Nanoparticle drug delivery is known to greatly improve the effectiveness of a pharmaceutical and has found a wide range of applications, with different routes of administration including oral, intravenous, transcutaneous and ocular. However, the mechanisms by which these nanoparticles travel through, interact with, and modify tissues and how this relates to the improved drug performance are still unclear. These are critical questions that need to be answered in order to develop future pharmaceuticals, with lower dosing rates and reduced side effects. Our ability to answers these questions is greatly hindered by that fact that there is currently no imaging modality available to directly visualise such small particles and the structure and function of the surrounding tissue, without the aid of contrast agents. Current imaging modalities derive image contrast of the nanoparticles by means of external labels. Moreover, with these techniques it not possible to detect when and how the nanoparticles release the drug without replacing the drug with an active contrast agent. We propose to test the feasibility of a novel type of optical microscopy for performing label-free measurements.Coherent Anti-Stokes Raman Scattering, or CARS, microscopy is an optical technique in which image contrast is derived from the intrinsic chemical makeup of a sample. Preliminary work has shown that CARS can be used to image nanoparticle drug carriers against a background of biological tissues. However, modifications are required to the instrument before the technique can be fully exploited. In this proposal we plan to make such modifications and test the effectiveness of the new system for monitoring nanoparticle drug delivery to tissues and cells.A successful outcome of this project will produce a tool that can provide new information of the fundamental mechanisms underlying nanoparticle drug delivery. It will allow pharmacologists to rationally design more efficient, safer, and less invasive drug delivery systems.

Publications

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Belsey NA (2014) Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies. in Journal of controlled release : official journal of the Controlled Release Society

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Watts AJ (2016) Effect of Microplastic on the Gills of the Shore Crab Carcinus maenas. in Environmental science & technology

 
Description At present, 95% of all potential new drug compounds cannot be directly administered as a pharmaceutical due to poor biocompatibility, i.e. they have poor solubility, unacceptable levels of toxicity, or become metabolised by the body before reaching the site of interest. Nanoparticle drug delivery can overcome these problems by encapsulating the compounds in particles less than 1 thousandth of a millimetre in size. Moreover, nanoparticles can act as depositories for controlled drug release and can be tailored to actively target specific sites within the body.

Nanoparticle drug delivery is known to greatly improve the effectiveness of a pharmaceutical and has found a wide range of applications, with different routes of administration including oral, intravenous, transcutaneous and ocular. However, the mechanisms by which these nanoparticles travel through, interact with, and modify tissues and how this relates to the improved drug performance are still unclear. These are critical questions that need to be answered in order to develop future pharmaceuticals, with lower dosing rates and reduced side effects. Our ability to answers these questions is greatly hindered by that fact that there is currently no imaging modality available to directly visualise such small particles and the structure and function of the surrounding tissue, without the aid of contrast agents.

Current imaging modalities derive image contrast of the nanoparticles by means of external labels. Moreover, with these techniques it not possible to detect when and how the nanoparticles release the drug without replacing the drug with an active contrast agent. We showed that with a novel type of optical microscopy (Heterodyne detected Coherent Raman Scattering) it is possible to perform label-free imaging of polymer nanoparticles in tissues at the cellular scale.



The Key findings from or work are:

1) Heterodyne CRS microscopy provides additional sensitivity over standard CRS microscopy.

2) That heterodyne detection can be used to enhance the detection of other Coherent Raman processes (e.g. Stimulated Raman Scattering (SRS)).

3) SRS has specific advantages over other Heterodyne CRS for imaging nanomedicines.

4) Heterodyne CRS suffers significant non-resonant background from strongly absorbing compounds. Specific examples investigated were hemoglobin and chlorophyll.

5) The background can be separated from the SRS signal using phase-sensitive detection allow (for the first time) chemically specific imaging based on vibrational spectroscopy in highly absorbing media (such as blood and plant tissues).

6) A combination of H-CRS and SRS can be used with great effect to image the location of nanomedicines at the cellular scale in ex-vivo tissues and in in-vitro cell cultures.
Exploitation Route The novel analytical tools developed in this project have applications across a wide range of commercial sectors, including the pharmaceutical, agrochemical, and consumer goods industries. The findings of this research are being followed up through a TSB award with commercial partners (Nanomerics). In this project SRS is being used as a research tool to aid the development a novel nanomedicine.



The discovery that H-CRS allows vibrational spectroscopy to be performed in plant tissues is of great interest to the agrochemical industry. We have established a long-term collaboration with Syngenta (the worlds largest agrochemical company) to explore several potential applications of SRS for agrochemical R&D.



The additional detection sensitivity afforded by SRS provides sufficient to monitor the diffusion of small molecules through the skin. We have undertaken contract research projects (funded by both GSK and Unilever) to apply this technique to proved novel information regarding the rate of depth-penetration of skin care products into the skin at the cellular level. In both cases SRS provided novel information that has been used to support company claims on the performance of new skin care products.



A TSB project has been funded in partnership with a UK based laser manufacturer to develop a compact laser and detection module aimed at non-expert users to that heterodyne detection techniques can be exploited by the wider biological community.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The project developed a novel label-free microscopy technique based on heterodyne detection of coherent Raman scattering microscopy (CARS) specifically for application in the development of nanoparticle drug delivery systems. This initial project carried out in collaboration with the London School of Pharmacy (UCL), demonstrated that heterodyne CARS is a extremely powerful tool that provides new insight into the mechanisms of uptake of nanoparticles into biological tissues. Having established this capabilities Exeter and the London School of Pharmacy (UCL) have continued and strengthened their collaboration through two further EPSRC awards and a TSB award lead by Nanomerics (a UCL spin-out company) to develop a novel nanoparticle drug delivery system and take it to clinical trials. The label-free imaging capability developed in this initial project has play a vital role in providing the mechanisms of enhanced uptake required by the regulatory bodies. The heterodyne CARS detection technique developed in this project has also unexpected impact in the agrochemical industry. Early on in the project it was found that the novel detection scheme had potential to allow Raman imaging to be performed in heavily pigmented samples and preliminary data suggested that our techniques could be used to image agrochemical compounds in living plants. This data lead to a research project fully funded for 3 years by Syngenta, to fully investigate the exploitation of CARS microscopy for agrochemical R&D. The ability to visualise the environmental fate of microplastics at the cellular level in marine animals played a key role in both the elucidating mechanisms by which they harm organisms and in convincing policy makers to ban their use in cosmetic products. The unique strength of the techniques developed and implemented by the Moger Group was the ability to, for the first time, unequivocally identify microplastics in marine organisms based on the intrinsic chemical signatures of the polymers from which the particles were composed. The research underpinning these imaging techniques originally aimed to develop label-free microscopy for monitoring the uptake of nanomedicines in mammalian tissues. Nanoparticle drug delivery was known to greatly improve the efficacy of pharmaceuticals however; the mechanisms by which nanoparticles improved drug performance was unclear. Research in the Moger Group between 2009-2012 developed and applied techniques based on coherent Raman scattering (CRS) to image polymeric nanoparticles in animals dosed with nanomedicines based on their intrinsic vibrational signature. The ability to track nanomedicines at the cellular level without using fluorescent labels represented a major advance in drug developmental capability and allowed, for the first time, clarification of the delivery mechanisms which aided the rational engineering of particles for the appropriate clinical condition. This capability, serendipitously, turned out to be extremely important for visualising microplastics since particles in organisms collected from the marine environment are by their nature unlabelled. Between 2013-2017 research was undertaken in collaboration with Tamara Galloway (Exeter) to further develop and apply CRS techniques to visualise the uptake and accumulation of microplastics in various marine organisms.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Chemicals,Environment,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description EPSRC Programme Grant
Amount £5,752,646 (GBP)
Funding ID EP/R020965/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 02/2018 
End 01/2023
 
Description Enabling Technologies for Advanced Multiphoton Microscopy
Amount £142,275 (GBP)
Funding ID 101787 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 04/2014 
End 04/2016
 
Description Exploring agrochemical applications of coherent Raman scattering
Amount £172,000 (GBP)
Organisation Syngenta International AG 
Sector Public
Country Global
Start 01/2011 
End 03/2014
 
Description Frequency-Modulated Stimulated Raman Scattering Microscopy for Label-Free Functional Imaging In-Planta
Amount £149,939 (GBP)
Funding ID BB/K013602/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2013 
End 02/2015
 
Description SRS Imaging of anti-dandruff actives in pig/human skin
Amount £24,000 (GBP)
Organisation Unilever 
Sector Private
Country United Kingdom
Start 03/2012 
End 07/2012
 
Description Technologies for the Treatment of Brain Diseases
Amount £235,691 (GBP)
Funding ID EP/G061564/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 04/2009 
End 04/2012
 
Description In Planta Label-Free Imaging of Agrochemical AIs and their Metabolites using Coherent Raman Scattering Microscopy 
Organisation Syngenta International AG
Country Global 
Sector Public 
PI Contribution Funded collaboration with Syngenta (leading agrochemical company) to explore the potential of applying the heterodyne detected coherent Raman schemes developed in EP/G028362/1 to overcome the issues associated with performing Raman analysis in plant tissues. This initial interaction with Syngenta was extremely successful. The application of heterodyne detection for Raman imaging in living plant tissues was shown to have great potential to overcome some the limitations of technologies currently used in agrochemcial R&D and have lead to two further (fully funded) projects with Syngenta and the submission of an collaborative EPSRC application.
Start Year 2011