WO2012160364A1 - Contrast agents for biophotonic imaging - Google Patents

Abstract

The present invention relates to the use of sterically-stabilised organic conducting polymeric nanoparticles as a contrast agent for biophotonic imaging, especially in optical coherence tomography (OCT) imaging. Exposing biological tissue to said contrast agent (whether in vivo or in vitro) prior to OCT imaging significantly enhances the contrast of the resulting OCT images and allows for enhanced detection and diagnosis of clinical conditions, such as cancerous tumours.

Description

Contrast Agents for Biophotonic Imaging

INTRODUCTION

[0001] This invention relates to contrast agents for use in biophotonic imaging, and especially optical coherence tomography (OCT) imaging. More specifically, the present invention relates the use of organic conducting polymeric nanoparticles as contrast agents for biophotonic imaging. The invention also relates to methods of imaging biological samples using these polymeric nanoparticles as contrast agents.

BACKGROUND

[0002] The ability to clearly view and assess biological tissue for abnormalities can be extremely valuable in the diagnosis of certain clinical conditions, such as cancer. There is therefore significant interest in biophotonic imaging techniques.

[0003] Optical coherence tomography (OCT) is one particularly promising biophotonic imaging technique. OCT is an emerging medical imaging technique discovered in the early 1990s that provides near real-time high resolution cross-sectional images of the epithelium [1 ,2,3]. The basic principle of OCT is similar to that of traditional ultrasound tomography: it allows

construction of diagnostic images by detecting the depth-dependent reflection of near-infrared light (NIR) from tissues using a low coherence interferometer [1 ]. Compared to x-ray computer tomography (CT) and magnetic resonance imaging (MRI), OCT provides near real-time high resolution diagnostic images in cases where excisional biopsy is dangerous. In the past decade, OCT has been widely applied in ophthalmology for the diagnosis of retinal diseases [4]. The development of swept-source optical coherence tomography (SS-OCT) significantly improves sensitivity and hence reduces the image acquisition time required for OCT imaging [5]. There is increasing interest in using OCT for the detection of early-stage epithelial cancers, such as breast [6] and bladder cancers [7], which account for over 85 % of all cancer deaths [8].

[0004] As with many medical imaging techniques, poor image contrast often adversely affects OCT sensitivity in the context of cancer diagnosis. In the past few years, two main classes of OCT contrast agents have been evaluated to improve the sensitivity and specificity of cancer detection [9,10]. Gold nanoshells [1 1 ], gold nanoparticles [12] and iron oxide nanoparticles [13] improve OCT image contrast by increasing the backscattered signal intensity. Alternatively, absorbing contrast agents, such as indocyanine green [14,15], gold nanorods [16,17,18], gold nanocages [19,20], gold-coated liposomes [21 ] and gold nanorings [22,23], have NIR

absorption spectra that overlap with the OCT light source spectra, thus improving contrast by increasing the spectral signature of abnormal tissues. In highly scattering human tissues, absorbing contrast agents should provide stronger contrast effects than scattering contrast agents. In dilute aqueous solution (~ 6.5 μΜ), indocyanine green, which is a Food and Drug Administration (FDA)-approved molecular dye, has a strong near-infrared (NIR) absorption band at around 800 nm [14,15]. However, this absorption diminishes to almost zero beyond 900 nm and its extinction cross-section (σ,_,οοτ) is relatively small. Gold nanorods [16,17,18] and nanocages [19,20] are a class of noble metal-based, OCT-absorbing contrast agents with strong NIR absorption at 800 to 900 nm due to the surface plasmon resonance effect. However, a longer wavelength OCT light source centred at around 1300 nm is preferred for tumour imaging due to lower background scattering. Gold nanorings were the first OCT contrast agent to offer enhanced contrast beyond 1000 nm. Although preliminary results were promising, later studies suggest that this contrast enhancement is actually dominated by light scattering, rather than absorption [22,23].

[0005] It is therefore an object of the present invention to provide improved contrast agents for use in biophotonic imaging, and especially OCT.

SUMMARY OF THE INVENTION

[0006] In a first aspect, the present invention provides the use of sterically-stabilised organic conducting polymeric nanoparticles as defined herein as contrast agents in biophotonic imaging.

[0007] In another aspect, the present invention provides a contrast agent composition, comprising sterically-stabilised organic conducting polymeric nanoparticles as defined herein in admixture with a diluent or carrier. The contrast agent composition is administered to the substrate that is to be imaged prior to the acquisition of the images.

[0008] In another aspect, the present invention provides sterically-stabilised organic conducting polymeric nanoparticles as defined herein, or a contrast agent composition as defined herein, for use in biophotonic imaging.

[0009] In another aspect, the present invention provides a method of imaging a substrate, comprising contacting the substrate with a contrast agent as defined herein, or a contrast agent composition as defined herein; and acquiring images of the substrate.

[0010] In another aspect, the present invention provides a method of diagnosing a clinical condition, said method comprising administering a contrast agent as defined herein, or a contrast agent composition as defined herein, to a substrate; acquiring images of the substrate; and analysing the image to identify any abnormalities in the substrate. In a particular embodiment, the substrate is a human or animal tissue. The clinical condition may be, for example, cancer and the abnormality present in the substrate may be the presence of one or more tumours.

[0011] Any or all of the above methods and uses may be applied in vivo and/or in vitro.

[0012] The sterically-stabilised organic conducting polymeric nanoparticles of the present invention are particularly suited for biophotonic imaging applications, such as OCT imaging, where absorption of near infrared radiation is desirable.

[0013] Advantageously, the sterically-stabilised organic conducting polymeric nanoparticles of the present invention exhibit strong absorption in the near-infrared (NIR) spectrum, i.e. at wavelengths within the range of 700 to 1400 nm. Particularly suitable organic conducting nanoparticles for OCT imaging, especially the OCT imaging of tumours, exhibit absorption at wavelengths within the range of 1200 to 1350 nm and more particularly at 1300 nm.

[0014] In addition, when formulated as a contrast agent composition which comprises the sterically-stabilised organic conducting polymeric nanoparticles dispersed in a diluent, the nanoparticles can be easily administered to the substrate that is to be imaged. Furthermore, the nanoparticles readily diffuse into the substrate to provide the required enhancement in the contrast within the images acquired. In particular, organic conducting nanoparticles are ideal for cancer imaging because they can diffuse rapidly into tumours, often selectively so. When the sterically stabilised organic conducting polymeric nanoparticle contrast agent is applied to a substrate comprising a tumour, the presence of the tumour can be detected via a reduction in back-scattered light intensity at a fixed depth, or an increase in the rate of decay of back- scattered intensity with increasing depth. This technique effectively highlights any tumours present in the image. The contrast agent may also be detected indirectly by localised heating, which causes a detectable change in the refractive index of the surrounding material and/or increased infrared emission. Alternatively, the contrast agent can be heated transiently using pulses of light of duration in the pico-second to milli-second range, in which case acoustic emission can be detected.

BRIEF DESCRIPTION OF THE DRA WINGS

[0015] For a better understanding of the present invention, and to show how particular embodiments may be put into effect, reference is made, by way of example, to the following figures, in which:

[0016] Figure 1 is a TEM image of polyvinyl alcohol)-stabilised polypyrrole nanoparticles, with an inset cartoon schematically representing the location of the adsorbed stabiliser chains;

[0017] Figure 2 is a particle size distribution of the polypyrrole nanoparticles shown in Figure 1 ;

[0018] Figure 3 is a UV-Vis-NIR extinction spectrum (solid line) of polyvinyl alcohol)-stabilised polypyrrole nanoparticles and also the emission spectrum for the standard SS-OCT NIR laser light source (dotted line) centred at around 1300 nm;

[0019] Figure 4 is a UV-Vis-NIR extinction spectrum recorded for an aqueous dispersion of polyvinyl alcohol)-stabilised polypyrrole nanoparticles;

[0020] Figure 5 is a digital image of an intralipid (IL) tissue phantom containing different concentrations of polypyrrole nanoparticles;

[0021] Figure 6 is an OCT image of (a) 2 % intralipid tissue phantom and (b) the same tissue phantom containing 200 μg/mL of 53 ± 7 nm diameter polypyrrole nanoparticles.;

[0022] Figure 7 is selected OCT images obtained for (a) 2 % intralipid tissue phantom and (b) to (e) the same tissue phantom containing (b) 40, (c) 80, (d) 120 and (e) 200 μg mL of polypyrrole nanoparticles;

[0023] Figure 8 is (a) the normalised calibration function (K(z - z )) of the SS-OCT system calibrated using Fresnel reflection of 2 % intralipid tissue phantom at different optical depths; (b) A-scan of 2% intralipid tissue phantom contained 200 μg mL of polypyrrole nanoparticles before (green data points) and after (blue data points) correcting the confocal properties of the SS- OCT system and their best-fits (red solid lines) according to equation (2);

[0024] Figure 9 is a series of demodulated OCT A-scans for 2 % intralipid (IL) tissue phantoms containing various concentrations of polypyrrole nanoparticles;

[0025] Figure 10 shows the variation in attenuation coefficient (μ_(,οοτ) and normalised backscattering albedo (a') for tissue phantoms containing varying concentrations of polypyrrole nanoparticles;

[0026] Figure 1 1 is a polar plot of the scattering pattern of polypyrrole nanoparticles calculated by Mie scattering; and

[0027] Figure 12 is a schematic arrangement of the swept-source OCT system operating at a wavelength centred on 1300 nm.

[0028] Figure 13 is shows the MTT assay results for human dermal fibroblasts incubated with various concentrations of P(2TMOI-MPC) PPy nanoparticles for various times. A strong loss of viability relative to control is indicated for a 1 :10 dilution of the base PPy liquid (which is 1 .44% solids content, undiluted) whereas at lower concentrations there is no evidence of reduced cell viability. Strong sedimentation of the 1 :10 diluted PPy latex was observed in the cell culture well-plate, which may have caused cell death simply by passive nutrient transport blockade.

[0029] Figure 14 shows ex-vivo rabbit corneas incubated with P(2TMOI-MPC)PPy for 5 and 10 minutes, compared to controls. After incubation, free PPy was removed by washing (left column) and by a combination of washing and physical wiping with tissue paper (right column). A marginal darkening relative to control is apparent for the washed samples but is not very evident for washed and wiped samples (right column). This suggests that image darkening most likely results from a thin film of adhered PPy on the corneal surface, rather than to PPy that has diffused throughout a significant thickness of epithelium.

[0030] Figure 15 shows tissue engineered skin constructs containing melenoma cancer cells, incubated with PPy for 1 hour at 37 °C with 5% C0₂. Top image: control; middle image: after washing away free PPy; bottom image: after washing and wiping away adhered PPy. A reduction in image brightness is visible (clearer on the 1 -D profiles in figure 4) which persists after wiping. This suggests that the absorbing agent has penetrated into the surface layers of the composite to a greater extent than for the cornea samples.

[0031] Figure 16 shows profiles taken through the 2-D images shown in Figure 15. The washed and washed/wiped composites produce consistently lower signal brightness than the control.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0032] The term "biophotonic imaging" is used herein to refer to generally to the imaging of biological substrates (or substrates similar in nature thereto) by detecting the interaction of non- ionising photons with the biological substrates. On the micro-scale, biophotonic imaging may include techniques such as microscopy, optical coherence tomography (OCT), and

photoacoustic imaging, whereas on the macro-scale it may include diffuse optical imaging (DOI) and diffuse optical tomography (DOT).

[0033] The term "contrast agent" is used herein to refer to sterically-stabilised polymeric nanoparticles which, when administered to a substrate to be imaged, enhance the contrast of the biophotonic image of the substrate.

[0034] The term "nanoparticles" is used herein to refer to particles, whether spherical or otherwise, having a particle size of less than 300 nm and, more typically, of less than or equal to 100 nm.

[0035] The term "organic conducting polymeric nanoparticles" is used herein to refer to nanoparticles formed from any suitable organic conductive polymer material. Suitably, the organic conducting polymer is selected from polypyrrole, poly(3,4-ethylenedioxythiophene) (PEDOT), or polyaniline.

[0036] The term "sterically stabilised" is used herein to refer to a hydrophilic/aqueously soluble polymer coating provided on the surface of the nanoparticles, which provides a steric barrier to prevent adjacent particles aggregating in aqueous environments.

[0037] The term "particle size" is a term of art which is used herein to refer to either the particle diameter (for spherical particles) or the largest dimension of the particle (for irregular shaped particles).

The Contrast Agent

[0038] As previously stated, the present invention provides the use of sterically-stabilised organic conducting polymeric nanoparticles as defined herein as contrast agents in biophotonic imaging.

[0039] In another aspect of the present invention, there is provided sterically-stabilised organic conducting polymeric nanoparticles as defined herein, or a contrast agent composition as defined herein, for use in biophotonic imaging.

[0040] In an embodiment, the organic conducting polymeric nanoparticles absorb in the visible (i.e. typically 390 to 700 nm range) and/or near infra-red spectrum (i.e. typically 700 to 1400 nm).

[0041 ] In a particular embodiment, the organic conducting polymeric nanoparticles absorb photons in the near infra-red spectrum (i.e. typically 700 to 1400 nm).

[0042] For OCT applications, it is preferable that the particles absorb photons in the range of 1200 to 1350 nm and especially at or around 1300 nm.

[0043] The sterically-stabilised organic conducting polymeric nanoparticles remain dispersed in aqueous environments, such as water. As such, the polymeric nanoparticles do not aggregate to any substantial extent.

[0044] The sterically-stabilised organic conducting polymeric nanoparticles suitably have an average particle size within the range of 5 to 200 nm. In an embodiment, the particle size of the nanoparticles is within the range of 20 and 100 nm. In a particular embodiment, the particle size is within the range of 50 to 100 nm.

[0045] In an embodiment, the nanoparticles are sterically stabilised polypyrrole, poly(3,4- ethylenedioxythiophene) (PEDOT) or polyaniline nanoparticles. In another embodiment, the nanoparticles are sterically stabilised polypyrrole or poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles.

[0046] In a particular embodiment, the nanoparticles are sterically stabilised polypyrrole nanoparticles. A particular advantage of the sterically-stabilised polypyrrole nanoparticles of the present invention is their biocompatibility. This renders them suitable for in vivo as well as in vitro delivery. In particular, recent animal studies have indicated that low concentrations of polypyrrole nanoparticles (<200 nm) have very low long-term cytotoxicity [32]. Polypyrrole nanoparticles exhibit strong absorption in the near infra-red spectrum and particularly at 1300 nm, which makes them particularly suitable for the OCT imaging of tumours.

[0047] Suitably, the structure of polypyrrole can be generally defined by formula I shown below:

Formula I

wherein n is at least 2 and where X^" is a dopant anion, such as chloride (CI ) or tosylate (OTs ).

[0048] The sterically-stabilised polymeric nanoparticles comprise a polymeric core of, for example, polypyrrole, poly(3,4-ethylenedioxythiophene) (PEDOT) or polyaniline, which is coated with a suitable stabiliser. The stabiliser is suitably a water-soluble, biocompatible polymer which is either physically adsorbed or covalently bound to the surface of the

nanoparticle. Preferably, the stabiliser polymer is covalently bound to the nanoparticle surface. In aqueous media, the hydrophilic polymer chains of the stabiliser extend into the surrounding medium to provide a steric barrier which prevents particle aggregation.

[0049] The stabiliser may comprise any suitable hydrophilic polymer chains or polymers comprising sufficient hydrophilic segments or repeat units. Examples of suitable hydrophilic polymers include poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC), poly(glycerol monomethacrylate) (PGMA), poly(N-vinylpyrrolidone), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), or poly(vinylalcohol) (PVA). In a particular embodiment, the stabiliser is PVA.

[0050] The average molecular weight of the polymer stabiliser may be between 1000 and 300000. Suitably, the average molecular weight of the stabiliser polymer is between 2000 and 100000, and more suitably between 5000 and 50000.

[0051 ] In some embodiments, the stabiliser polymer may comprise an ionic species, such as a polyelectrolyte, in which case a suitable counterion is present. Any suitable pharmaceutically acceptable/biocompatible counterion may be used.

[0052] In an embodiment, the stabiliser is adsorbed onto the surface of the nanoparticles. To facilitate adsorption to the surface of the nanoparticles (the nanoparticle or polymeric core), the stabiliser polymer may suitably comprise one or more blocks or segments of hydrophobic polymer which exhibit a higher affinity for the nanoparticle surface and serves to bind the stabiliser polymer to the particle surface. [0053] In an alternative embodiment, the stabiliser is grafted directly onto the polymeric core or grafted to the polymeric core via a suitable grafting moiety. For example, the stabiliser polymer may comprise a co-monomer component that functions as the grafting moiety. The co- monomer will suitably comprise a reactive pendent group that is capable of reacting with the nanoparticle surface to covalently bind the stabiliser polymer to the nanoparticle core. Suitable techniques to achieve this are known in the art. In the case of polypyrrole nanoparticles, the reactive co-monomer may be, for example, a 2- or 3-substituted thiophene which is present as a co-monomer in the stabiliser polymer at an amount of between 1 and 20 mol %, more suitably between 5 and 15 mol %. The pendent thiophene groups can be reacted with the polypyrrole chains to covalently attach the stabiliser polymer to the nanoparticle surface.

[0054] In a particular embodiment, the stabiliser comprises PVA chains physically absorbed on the surface of the nanoparticles.

[0055] The stabiliser may constitute between 5 and 30 % w/w of the sterically-stabilised polymeric nanoparticles. Suitably, the stabiliser comprises between 10 and 20 % w/w of the sterically-stabilised polypyrrole nanoparticles.

[0056] The sterically-stabilised polymeric nanoparticles may be neutral or they may be charged particles. In a particular embodiment, the particle charge is due to cationic and/or anionic moieties present within the stabiliser polymer.

[0057] The nanoparticles defined herein can all be prepared by well established techniques known in the art.

Contrast Agent Composition

[0058] The present invention further provides a contrast agent composition which comprises sterically-stabilised highly absorbing organic conducting polymeric nanoparticles as defined herein in admixture with a diluent or carrier.

[0059] The present invention further provides the use of a contrast agent composition as defined herein in biophotonic applications.

[0060] The present invention also provides a contrast agent composition as defined herein for use in biophotonic imaging.

[0061 ] The composition is suitably a biocompatible or pharmaceutically acceptable

composition, especially when the composition is intended for in vivo delivery. The diluent or carrier is suitably an aqueous diluent, such as water or a physiological buffer, e.g. phosphate buffered saline (PBS).

[0062] Suitably the composition is a dispersion of sterically-stabilised organic conducting polymeric nanoparticles. In a particular embodiment the composition is a highly absorbing aqueous dispersion.

[0063] The composition may suitably comprise 0.05 and 0.20 mg/mL of the sterically-stabilised organic conducting polymeric nanoparticles

[0064] The composition may be in an injectable form. Alternatively, the composition may be formulated as a spray or in any other suitable form for application to the substrate that is to be imaged.

Imaging

[0065] Biological substrates will tend to reflect, absorb and/or scatter incident photons to varying degrees depending, inter alia, on the nature, properties and orientation of the substrate and the wavelength of the photons directed at the sample. As such, images of substrates can be obtained by detecting how photons (typically with characteristic wavelengths in the visible, ultraviolet (UV), or infrared (IR) spectral regions) are reflected, absorbed or scattered within the substrate. The photons are emitted from a suitable source (i.e. a "source light"), such as a laser, superluminescent diode, superfluorescent light source, supercontinuum light source, light emitting diode or incandescent light bulb, and directed at the substrate. Reflected light is suitably detected and analysed. Well-known computer-implemented techniques may be used to construct a biophotonic image of the substrate based on an analysis of any deflected light, typically through making comparisons between the deflected light and the source light

[0066] The source light may emit photons with a wavelength within the range of 200 to 3500 nm. More suitably, for biophotonic applications such as OCT, at least a proportion of the photons need to be within the near infra-red range. For imaging tumours by OCT, the photons ideally have wavelengths within the range of 1200 to 1350 nm, and especially at or around 1300 nm.

[0067] Biophotonic imaging may suitably include micro-scale (including nano-scale) imaging such as microscopy, optical coherence tomography (OCT), and also photoacoustic techniques (which detect ultrasound following nano-scale heating of a substrate, suitably by non-ionising lasers). Biophotonic imaging may also include macro-scale imaging such as diffuse optical imaging (DOI) and diffuse optical tomography (DOT).

[0068] In a particular embodiment, the biophotonic imaging is OCT or photoacoustic imaging. Most suitably, the imaging technique is OCT.

[0069] The substrate is the biological substrate that is to be imaged. The contrast within biophotonic images is enhanced when the substrate is exposed to the sterically stabilised organic conducting polymeric nanoparticles defined herein before imaging takes place.

[0070] The substrate may be any suitable biological material. In a particular embodiment, the substrate is a biological tissue, such as animal or human tissue. Tissue samples may be examined ex vivo by the analysis of, for example, biopsy samples. In a particular embodiment, the tissue is a tissue that is suspected to contain at least one tumour.

[0071 ] In another embodiment, the substrate is imaged in vivo. As such, imaging according to the invention takes place upon the human or animal body, suitably in real time.

[0072] As previously stated, the present invention provides a method of imaging a substrate, comprising contact of at least part of the substrate with a contrast agent as defined herein; and imaging the substrate while the contrast agent is in contact with the substrate.

[0073] Contacting the substrate with sterically-stabilised organic conducting polymeric nanoparticles may suitably comprise applying a contrast agent composition as defined herein to the substrate. Applying the contrast agent composition may suitably involve injecting the composition into or onto the substrate.

[0074] In a particular embodiment, contacting the substrate with a contrast agent takes place in vivo within a human or animal body. The substrate may be, for example, a particular biological tissue. Where the substrate is in vivo, imaging of the substrate also suitably takes place in vivo, suitably in real time.

[0075] In an alternative embodiment, contacting the substrate with a contrast agent takes place in vitro, wherein the substrate is suitably a biological tissue sample. The biological tissue sample may have been suitably abstracted from a patient, for instance as a biopsy. The biological tissue sample may have been suitably extracted from a patient suspected of having a medical condition, such as a proliferative condition (e.g. cancer). For example, the substrate may be suspected to contain a tumour. In such cases, the contrast agent is suitably applied to a part of the substrate suspected of possessing the tumour. Where the substrate is in vitro, imaging of the substrate also takes place in vitro, suitably in real time. Most suitably the imaging is OCT imaging.

[0076] Imaging the substrate may suitably comprise:

i) providing a light source;

ii) irradiating the substrate with source light from the light source;

iii) detecting and analysing light deflected by the substrate; and iv) producing an image of the substrate based on the analysis of the

deflected light. Diagnosis of Medical Conditions

[0077] The contrast agents of the present invention provide enhanced biophotonic images of biological samples. The images generated can be used to enable the diagnosis of certain clinical conditions by enabling abnormalities to be visualised.

[0078] Thus, the present invention further provides a method of diagnosing a medical condition, comprising adding a contrast agent as defined hereinbefore to a biological substrate; imaging the substrate as defined hereinbefore to obtain one or more images; and determining assessing the image for presence of one or more abnormalities indicative of a particular medical condition.

[0079] Computer software can process the images and assist a clinician in the identification and assessment of any tissue abnormalities.

[0080] The medical condition may be, for example, a proliferative disorder, such as cancer.

[0081] Determining the existence or otherwise of the medical condition from the image may suitably comprise examining the substrate image for areas of high source light absorption. For instance, a cancerous tumour may be apparent in the image as a dark area (or light area if the image is inverted) following preferential absorption of the contrast agent in the tumour.

EXAMPLES

Materials

[0082] Pyrrole (> 98 %) was purchased from Aldrich and purified by passing through a column of activated basic alumina before being stored in the dark at - 20 °C prior to use. Iron(lll) trichloride hexahydrate (ACS reagent, 98.0 - 102 %) and polyvinyl alcohol) (PVA, Mw ^« 9,000, 80 % hydrolysed) were purchased from Aldrich and used without further purification. Doubly- deionised water was used for both the polymerisation and also subsequent purification.

Intralipid (10 %) was purchased from Fresenius Kabi Ltd. (UK). Each bag of 500 ml_ intralipid contains 50 g soybean oil, 6 g purified egg phospholipids and 1 1 g glycerol. Intralipid was diluted to 2.0 wt.% using doubly-deionised water prior to the OCT imaging experiments.

EXAMPLE 1

Synthesis of polypyrrole nanoparticles

[0083] Sterically-stabilised polypyrrole (PPy) nanoparticles with a number-average diameter of around 53 ± 7 nm were prepared using a well-established method [25,26]. Briefly, PVA (5.00 g) was dissolved in deionised water (100 ml_) in a 125 ml_ sealed container by stirring at 400 rpm at 20 °C for 15 h. Iron(lll) chloride hexahydrate (6.30 g, 23.33 mmol) was added to the stirred PVA solution. After 30 min., pyrrole (0.69 ml_, 10.00 mmol) was added to the reaction mixture via syringe. Polymerisation was allowed to proceed for 24 h. The resulting black dispersion was first filtered through pre-washed glass wool (Aldrich) under gravity before being centrifuged at 20,000 rpm for 3 h and the resulting black sediment was redispersed in deionised water by ultrasonication for 1 hour. This centrifugation-redispersion cycle was repeated nine times to remove excess PVA stabiliser and soluble Fe(ll) salts. Purified polypyrrole nanoparticles were filtered through pre-washed glass wool to remove any aggregates before further studies.

Synthesis of polypyrrole bulk powder

[0084] Iron(lll) chloride hexahydrate (6.30 g, 23.33 mmol) was dissolved in deionised water (100 ml_) in a 125 ml_ sealed container by stirring at 400 rpm. After 30 min., pyrrole (0.69 ml_, 10.00 mmol) was added to the iron chloride solution via syringe. Polymerisation was allowed to proceed for 24 h at 20 °C. The resulting PPy bulk powder was purified by washing with copious amounts of deionised water in a filter funnel and dried at 40 °C for 24 h before further studies.

Characterisation

Transmission electron microscopy (TEM)

[0085] TEM sample was prepared by drying a drop (10 μΙ_) of highly diluted polypyrrole dispersion onto a carbon-coated TEM grid (from Agar Scientific) and air-dried overnight before analysis using a Philips CM100 electron microscope operating at 100 kV.

Helium pycnometry

[0086] PPy nanoparticles were freeze-dried from aqueous solution prior to solid-state density measurements using a Micrometrics AccuPyc 1330 helium pycnometer conducted at 20 °C. The particle densities of PPy bulk powder and PPy nanoparticles were found to be 1 .440 and 1 .397 g cm^"3 respectively.

Elemental microanalysis

[0087] The PPy samples were freeze-dried before elemental microanalysis. Elemental microanalyses were conducted by the Elemental Analysis Laboratory in the Department of Chemistry at the University of Sheffield. Carbon, hydrogen and nitrogen contents were determined using a Perkin Elmer 2400 Elemental Analyser. Chlorine contents were determined by the standard Schoninger flask combustion technique.

Solids content determination

[0088] The solids content of aqueous PPy dispersions was determined using an Ohaus MB45 Moisture Analyser Balance. All measurements were performed in triplicate.

UV-visible-NIR spectroscopy

[0089] UV-Visible-NIR spectra of intralipid alone and various concentrations of the polypyrrole nanoparticles dispersed in pure water were recorded using a Perkin Elmer Lambda 900 UV-Vis- NIR Spectrometer at 25 °C using 10 mm pathlength poly(methyl methacrylate) disposable micro-cuvettes (Plastibrand^®). All samples were diluted to 1 mg/mL and their solids contents were re-checked using the moisture analyser before serial dilution for the UV-Vis-NIR measurements.

OCT imaging

[0090] OCT imaging was carried out using a swept-source optical coherence tomography system centred at 1300 nm. The system comprises a commercially available Michelson

Diagnostics EX1301 Swept-Source Fourier-Domain Optical Coherence Tomography

Microscope with an in-house fibre-based Michelson interferometer. This set-up consists of a HSL-2000-1 1 MDL swept-source light source operating at 10 kHz centred at a wavelength of 1300 nm (FWHM = 1 10 nm) and a Thorlabs LSM03 OCT scanning lens. To simplify the quantitative analysis, only one of the four imaging channels in the microscope was used in this study. This swept-source OCT system has an axial resolution of 10 μηι in air and a lateral resolution of 25 μηι. A schematic representation of the OCT system is shown in FIG. 12 (set-up of the swept-source OCT system operating at a wavelength of 1300 nm). Five consecutive image frames (1 mm width and 2.4 mm depth in air) were recorded and averaged to give a two- dimensional OCT image comprising 250 axial-scans (A-scans). Axial scans were extracted from the OCT image using a Matlab source code provided by Michelson Diagnostic Ltd. (UK). Curve fitting was carried out using OriginPro version 8.1 . A series of OCT images of 2 % intralipid at different optical depths were recorded and the Fresnel reflections were used to obtain a normalised calibration curve (K(z - z )) for the OCT system (see Figure 8) that corrects the confocal properties of the system. To prevent undesired interactions between intralipid and PPy nanoparticles from affecting the studies, a calculated amount of PPy nanoparticles was added to the intralipid tissue phantoms and shaken gently (by hand, 10 s) prior to performing the OCT imaging experiments within 2 minutes.

Mie scattering calculation

[0091 ] The Mie scattering calculation was performed using a Mie Scattering Theory Calculator program written by Dr. Scott Prahl at the Oregon Health and Science University

(http://omlc.ogi.edu/calc/mie_calc.html/). The complex refractive index for PPy, 1 .35 + 1 .01 /^', used in this Mie calculation was computed from its complex dielectric function, ε = 0.80 + 2.74/^', [43,46] as determined from its reflectance spectrum using the Kramers-Kronig transformation [46,47].

Results and Discussion

[0092] Table 1 summarises the optical properties of polypyrrole nanoparticles and other common OCT-absorbing contrast agents.

Table 1

green

Gold 15 x 45 800 43.4 15

1.30x10 3.50 [b] 16,17,18 nanorods

Gold 37 825 16.85 8.07x10^" 8.97 19,20 nanocages

Gold-coated 890 unknown Unknown Unknown 21 plasmon

resonant

nanoshells

Gold 174 1300 6.21 4.86x10^" 0.47 22,23 nanorings

Polypyrrole 55 1300 29.75 9.47xl0^"lb 213

nanoparticles

[a] Determined by uv-visible absorption spectroscopy in aqueous solution.

[b] Determined by uv-visible absorption spectroscopy using the double-integrating sphere method.

[c] Determined by OCT.

[d] Calculated using Mie theory. [0093] Nanoparticles of less than 100 nm diameter are ideal for cancer imaging since they can diffuse rapidly into tumours [33]. Near-monodisperse PPy nanoparticles were synthesised using a polyvinyl alcohol) (PVA) stabiliser via aqueous dispersion polymerisation.

[0094] Fig. 1 shows a transmission electron microscopy (TEM) image of polyvinyl alcohol- stabilised polypyrrole nanoparticles. The inset cartoon schematically represents the location of the adsorbed stabiliser chains, which dry down to negligible thickness in the absence of any solvent. [0095] Fig. 2 shows particle size distribution of the PPy nanoparticles shown in the TEM image in Fig. 1 . The number of analysed nanoparticles was 94 and the number-average particle diameter is 53 ± 7 nm.

[0096] Comparing the microanalytical nitrogen content of the PPy nanoparticles (N = 13.4 %) with that of the corresponding PPy bulk powder prepared in the absence of any stabiliser (N = 16.6 %), it is estimated that the former system contains approximately 19 wt % PVA stabiliser.

[0097] Fig. 3 shows a UV-Vis-NIR extinction spectrum (solid line) recorded for 50 μg mL polyvinyl alcohol)-stabilised polypyrrole nanoparticles of 53 ± 7 nm diameter dispersed in 9:1 D₂0/H₂0 and also the emission spectrum for the standard SS-OCT NIR laser light source (dotted line) centred at around 1300 nm. In order to remove the NIR absorption band due to H₂0 at 1350 nm, the nanoparticle spectrum was recorded in 90% D₂0 [34]. The inset digital photograph shows a sample of 50 μg mL PPy nanoparticles, illustrating their intense intrinsic black colouration.

[0098] The absorption band with a peak maximum at 415 nm has been previously attributed to the π - π^* transition for the conjugated polypyrrole backbone [35]. The broad absorption band extending from the visible to the NIR region is characteristic of the bipolaronic metallic state of chloride-doped polypyrrole [36]. The NIR absorption peak due to PPy clearly overlaps with the 1300 nm OCT light source, ensuring strong absorption within human tissue.

[0099] Fig. 4 shows a UV-Vis-NIR extinction spectrum of PPy nanoparticles recorded in H₂0. The inset shows the extinction for different concentrations of PPy nanoparticles at 1300 nm. This indicates an extinction coefficient of 31 .4 O.D. cm^"1 mg^"1 ml_ (7.23 mm^"1mg^"1 ml_). [Unit conversion: 1 .00 O.D. cm^"1 mg^"1 ml_= 1 .00 χ 0.10 χ In10 mm^"1mg^"1 ml_ « 0.23mm^"1mg^"1 ml_]

[00100] To demonstrate that PPy nanoparticles can serve as a useful OCT contrast agent for tumour imaging, the dose-dependent effect of tissue phantoms containing different concentrations of PPy nanoparticles was investigated (see Fig. 5 - digital image of the intralipid (IL) tissue phantom containing different concentrations of PPy nanoparticles) using a SS-OCT system operating at a wavelength of 1300 nm. 2 % intralipid was used as a tissue phantom to mimic the scattering properties of abnormal human tissue. Five consecutive image frames (1 mm width and 2.4 mm depth in air) were recorded and averaged to give a two-dimensional OCT image that was composed of 250 axial scans (A-scans). In our SS-OCT system, the

photodetector converts back-scattered light into an OCT signal (SOCT(Z), in dB), which is equivalent to the square root of the depth-dependent reflectivity (R(z)).

[00101 ] Fig. 6 shows an OCT image of (a) 2 % intralipid tissue phantom and (b) the same tissue phantom containing 200 μg/mL of 53 ± 7 nm diameter PPy nanoparticles.

[00102] Fig. 7 shows selected OCT images obtained for (a) 2 % intralipid tissue phantom and (b) to (e) the same tissue phantom containing (b) 40, (c) 80, (d) 120 and (e) 200 μg mL of PPy nanoparticles. The optical depth is indicated by a false colour image and decreases monotonically with increasing concentration of PPy nanoparticles.

[00103] Fig.8 shows: (a) normalised calibration function (K(z - zf) of the SS-OCT system calibrated using Fresnel reflection of 2 % intralipid tissue phantom at different optical depths; (b) A-scan of 2% intralipid tissue phantom containing 200 μg mL of PPy nanoparticles before (lower dotted line) and after (upper dotted line) correcting the confocal properties of the SS-OCT system and their best-fits (solid lines) according to equation (2). This example demonstrates that the calibration function {K{z - zf)) corrects the confocal loss due to loss of focus of the scanning lens at deeper regions of tissue phantom by increasing the magnitude of the gradient of the decay curve.

[00104] Fig. 6 shows the OCT image obtained for the intralipid tissue phantom and the same phantom containing 200 μg/mL PPy nanoparticles. Clearly, the penetration depth of the tissue phantomis gradually reduced as the concentration of PPy nanoparticles is increased (see Fig. 7). The addition of PPy nanoparticles significantly broadens the colour distribution, and thus the contrast, of the uppermost 1 .5 mm of the tissue phantom.

[00105] Quantitative OCT studies were conducted to investigate the origin of the contrast effect. A-scans were extracted from OCT images. The 150 neighbouring A-scans on the far left of each image in FIG.6 were averaged to reduce background noise. If there is no multiple scattering, the reflectivity of the depth-dependent backscattered light follows the Beer-Lambert law [37,38,39].

R(z) = R₀ ^■ μυ^■ exp(-2 · μ_ί ^■ z) (1 )

whe^_fc is the backscattering coefficient and μ^ε the attenuation coefficient of the medium.

[00106] Since our SS-OCT system measures a photocurrent that is equivalent to V^ff(^z)_> the demodulated A-scans follows the relationship [17,18,20,37,38,39]:

¾crO) = So^■ K{z - z_f)^■ /½ · exp (-μ_ί · z) (2) where S_0CT(z) is the depth-dependent OCT signal, 5₀ is the background OCT signal, and K{z - zf) is a normalised calibration function that corrects the confocal properties of the OCT system (see Fig. 8).

[00107] To simplify these studies, the corrected OCT signal-to-noise ratio (S_SNRl>dB (z), in dB) was plotted as a function of depth (z) by defining S_{SNRl dB} (z) as:

10 · log₁₀ (S_OCT(z)/[S₀ ^■ K{z - z_f)]).

[00108] Fig.9 shows demodulated OCT A-scans (dotted lines; N. B. each dot represents one data point) recorded for 2 % intralipid (IL) tissue phantoms containing 0, 40, 80, 120 and 200 μg/ml PPy nanoparticles and their lines of best fit calculated according to equation (2).

[00109] Fig.9 shows the corrected A-scans recorded for tissue phantoms contained 0, 40, 80, 120 and 200 μg/mL PPy nanoparticles of 53 ± 7 nm diameter. The uppermost 0.7 mm of the decay curves fit equation (2) very well. The gradients of these decay curves decrease linearly from -1 .20 mm^"1 to -2.68 mm^"1 as the PPy nanoparticle concentration is increased from 0 to 200 μg/mL, which gives an attenuation coefficient (μ_(,οοτ) of 6.85 mm^"1 mg^"1 ml_ (29.75 O.D. cm^"1 mg^{" 1} ml_) and an extinction cross-section (σ,_,οοτ) of 9.47 ^χ 10^"16 m² [41 ]. Conversely, the intercepts of the decay curves

remain constant, which indicates that PPy nanoparticles do not enhance the backscattering from the intralipid tissue phantom. However, accurately determining the back-scattering coefficient (ji_biocr) and the absorbing coefficient (ji_a,ocr) from demodulated A-scans is not straightforward due to strong Fresnel reflection (reflection at the interface between two homogeneous media with differing refractive indices). Instead, we use the back- scattering albedo (a =μ_1}/μ_ί) Π 7, 18,37], which is sensitive to back-scattering; this parameter is introduced to determine the contrast contribution made by the PPy nanoparticles. The intercepts obtained from curve-fitting

^{were use}d ^t0 estimate the back-scattering albedo of each tissue phantom.

[001 10] Fig. 10 shows the variation in attenuation coefficient (μ_(,οοτ) (solid line (a)) and normalized backscattering albedo (a') (dotted line (b)) for tissue phantoms containing varying concentrations of 53 ± 7 nm diameter PPy nanoparticles. Here the backscattering albedo is normalized relative to the pure intralipid tissue phantom (i.e. in the absence of any PPy nanoparticles).

[001 1 1 ] Fig. 10 shows the plot of backscattering albedo relative to tissue phantom (a') as a function of PPy nanoparticle concentration [42]: a' is gradually attenuated as the PPy nanoparticle concentration in the phantom is increased, while the μ^_οσΓ gradually increases (see dotted line (b)). For example, the a' value of a tissue phantom containing 200 g mL PPy nanoparticles is reduced by 55 % compared to the phantom containing no PPy nanoparticles. This substantial reduction in a' demonstrates the potential for PPy nanoparticles to act as an absorbing contrast agent for OCT imaging.

[001 12] Fig. 1 1 shows a polar plot of the scattering pattern of PPy nanoparticles calculated by Mie scattering. The overall scattering efficiency of the PPy nanoparticles is 0.6 %.

[001 13] Mie scattering calculations [40] were also performed to quantify the OCT results. The extinction coefficient (μ₍,Μί_Θ) , extinction (a_t,_Mie) , backscattering (a_b,Mie) and absorbing (a_a,Mie) cross-section at 1300 nm were estimated to be 26.75 O.D. cm^"1 mg^"1 ml_, 8.5 ^χ 10^"16 m², 2.3 ^χ 10^"18 m² and 8.5 ^χ 10^"16 m², respectively [43]. This calculation also indicates that the scattering cross-section of the PPy nanoparticles is both symmetrical and relatively small compared with the absorbing cross-section (see Fig. 1 1 ). The calculated extinction coefficient (μ₍,Μί_θ) is in good agreement with the extinction coefficient determined by spectroscopy ( }_x,_spea = 31 .4 O.D. cm^"1 mg^"1 ml_) and OCT (μ, = 29.8 O.D. cm^"1 mg^"1 ml_). The extinction coefficient for PPy is comparable to that of gold nanorods and gold nanocages (see Table 1 ). However, the extinction cross-section is about eight times smaller for PPy than for gold nanocages due to the relatively low density of PPy compared to gold [44,45]. Thus the Mie scattering calculation provides evidence to support our quantitative OCT studies.

[00114] In summary, a relatively low concentration of PPy nanoparticles can improve the OCT image contrast of tissue phantoms. Quantitative OCT studies and Mie scattering calculations suggest that the observed contrast is due to strong NIR absorption. Although the extinction cross-section of PPy nanoparticles is somewhat lower than that of gold nanorods and nanocages, PPy is the first absorbing OCT contrast agent that generates a strong contrast effect at around 1300 nm. Contrast enhancement in tissue phantoms suggest that these PPy nanoparticles can improve OCT sensitivity significantly, hence facilitating the early-stage detection of cancers.

Example 2 - MTT Cytotoxicity of PPy Nanoparticles

[00115] Human dermal fibroblasts were harvested from split thickness skin grafts (STSGs) obtained from specimens following routine breast reduction and abdominoplasties. The STSGs were cut into 0.5cm² pieces using a scalpel blade and incubated overnight (12- 24hrs) at 4°C in 0.1 % w/v trypsin. Fetal calf serum (FCS) was added to neutralise the trypsin and the epidermal and dermal layers were carefully separated using a pair of forceps with fine points. The dermal part of the specimen were collected these dermal samples were washed several times in sterile PBS and then finely minced with a scalpel blade. The dermal mince was incubated at 37°C overnight in 10ml of 0.5% collagenase A solution. The following day, the collagenase digest was spun down in a centrifuge at 200 g for l Ominutes. The supernatant was discarded and the pellet was re-suspended in fibroblast medium. Dermal fibroblasts were cultured in fibroblast growth medium consisting of Dulbecco's modified eagles medium (DMEM) supplemented with 10% (v/v) FCS, 100IU/ml penicillin, 2mM L-glutamine, 100μg/ml

streptomycin and 0.625μg/ml amphotericin B. The isolated cells were cultured at 37°C in a 5% C02/95% air humidified incubator. The cells were split 1 :3 every 6-7 days. For sub-culturing cells .the media was aspirated, cells washed twice with PBS and 1 -2 ml of a 1 :1 mixture of 0.1 %w/v trypsin and 0.02% EDTA was added per flask. The flasks containing the cells were placed in the incubator for 5-10 min. After approximately 10 min the cells were visualised under the microscope to check if the cells were rounding up and detaching from the tissue culture plastic. The underside of the flask was tapped repeatedly to dislodge the cells. Media containing FCS was added to inactivate the trypsin/EDTA used to detach the cells. The cells suspension was pipetted into a universal tube and the cells were centrifuged at 200 g for 5mins. The supernatant was discarded and the cells resuspended in 3-5 ml of media. The cells were pipetted up and down 10-15 times to ensure a single cell re-suspension was generated.

Approximately 1 ml of the cell suspension (1 :3-1 :5 split) was added to a 75 cm² flask containing 8-10 ml of pre-warmed media. The flasks were checked daily and re-fed with complete media every two days. Cells were passaged at 80% confluency. The cells were routinely split every 6- 7days. Passage 4-9 fibroblasts were used in all experiments. Human dermal fibroblasts were cryo-preserved in 10%DMSO in FCS for future use and stored in liquid nitrogen.

Resurrecting and sub-culturing fibroblasts:

[00116] A vial of cells was removed from liquid nitrogen ensuring that it was tightly screwed shut. The vial was sprayed with 70% IMS . The vial was carefully thawed out at 37 ^<C either in a water bath or in the incubator. Once thawed, the tightly-closed vial was immersed in 100% ethanol, and then opened carefully. Using a 1 ml pipette, the cell suspension was transferred to a 15 ml sterile universal tube containing 10 ml of fresh pre-warmed medium (DMEM + 10%FCS + 100IU/ml penicillin, 100μg/ml streptomycin and 0.625μg/ml amphotericin B 2mM L-glutamine) and centrifuged at 200 g for 5 min. The media (supernatant) was aspirated and the cells were resuspended in 8-10 ml of fresh pre-warmed medium. The cells were plated out in a T75 flask and the flask was placed horizontally in a 5% C02/95% air humidified incubator. The media was changed the next day to remove dead cells and replenished with 8- 10 ml of pre-warmed media. The flasks were checked daily and re-fed with complete media every two days. Cells were passaged at 80% confluency. Fibroblasts (80% confluency) were washed twice with PBS and 1 -2ml of trypsin/EDTA added to the flask. The flasks containing the cells were placed in the incubator for 5-10 minutes. After approximately 10 minutes the cells were visualised under the microscope to check if the cells were rounding up and detaching from the tissue culture plastic. The underside of the flask was tapped repeatedly to dislodge the cells. Media containing FCS was added to inactivate the trypsin/EDTA used to detach the cells. The cells suspension was pipetted into a universal tube and the cells spun at 200 g for 5 minutes. The supernatant was discarded and the cells resuspended in 3-5 ml of media. The cells were pipetted up and down 10-15X to ensure a single cell re-suspension was generated. At this point the cells were counted using a Neubauer haemocytometer.

MTT assay:

[00117] Fibroblasts (5000 cells /well) were seeded in a 24 well plate for 24 hours.

Polypyrrole (solid content 1 .44%) was added to the cells with various dilutions (1 :10-1 :10000) in media (DMEM with 10%FCS) and incubated for the next seven days. MTT assay was performed on day 3, 5 and 7. The media was removed from the wells and the cells washed twice with PBS. MTT solution (0.5mg/ml in PBS) was added to the wells and placed in the incubator at 37 ^<€ for 40 minutes. MTT solution was aspirated and acidified isopropanol (300μΙ) was added to each well. Two aliquots of 150μΙ of the acidified isopropanol was transferred to a 96 well plate and optical density was read using a plate reader set at wavelengths of 540nm and referenced at 630nm.

Polypyrrole incubation of fibroblasts:

[00118] Fibroblasts were incubated with PPy at various dilutions for 7 days. MTT assay was performed at day 3, 5 and 7. As seen in Figure 13, the PPy at 1 :10 dilution exhibit toxicity, i.e. with increasing concentration the number of viable cells decreased.

Example 3 - Assessment of the ability of OCT to detect changes in rabbit cornea post PPy incubation

[00119] Rabbit eyes (corneas) were removed from ex-vivo New Zealand white rabbits weighing from 2.4 to 2.6 kilograms destined for the food chain and transported in phosphate saline buffer. Rabbit corneas were incubated in 1 :20 PPy dilution in media (DMEM with

10%FCS) (-50 nm diameter, solid content 1 .44%) for 5 minutes and 10 min in a 5% C02/95% air humidified incubator. Post incubation the rabbit corneas were washed in PBS to remove any excess PPy. The PBS washed corneas were imaged using a SS-OCT at 1300nm. Figure 14 presents images that are representative of the rabbit cornea incubated with PPy. As seen in the "wash" images there appeared to be a change in the contrast in the OCT images. The change in contrast was more apparent in the PBS wash samples than the "wash and wipe" samples. The reason for this most likely is that after the PBS wash a very thin film PPy remained adhered on to the surface of the cornea, with the result that the entire image below this film became darkened due to beam attenuation. That this darkening could apparently be greatly reduced by a physical wipe after the wash unfortunately suggests that the agent did not diffuse greatly into the corneal epithelium.

Example 4 - The ability of OCT to detect changes in tissue engineered melanoma models post PPy incubation

[00120] Tissue engineered models of melanoma composites were prepared and used as models to detect changes using SS-OCT system post Polypyrrole incubation

Isolation and maintenance of keratinocytes and dermal fibroblasts:

[00121 ] Human skin keratinocytes and human dermal fibroblasts were harvested from split thickness skin grafts (STSGs) obtained from specimens following routine breast reduction and abdominoplasties. The STSGs were cut into 0.5cm² pieces using a scalpel blade and incubated overnight (12-24hrs) at 4°C in 0.1 % w/v trypsin. Media containing fetal calf serum (FCS) was added to neutralise the trypsin and the epidermal and dermal layers were carefully separated using a pair of forceps with fine points. A scalpel blade was used to gently scrape the keratinocytes from the undersurface of the epidermis and the papillary surface of the dermis. The cells were collected in media containing FCS in a sterile centrifuge tube. The cells suspension was centrifuged at 200 g for 5 min. The supernatant was discarded and the cells were re-suspended in keratinocyte medium (Greens) and plated into tissue culture flasks.

Keratinocytes were cultured in Green's medium consisting of Dulbecco's modified eagles medium (DMEM) and Ham's F12 medium in a 3:1 ratio supplemented with 10% (v/v) foetal calf serum (FCS), 100IU/ml penicillin, 100μg/ml streptomycin, 0.625μg/ml amphotericin B, 6.25μg/ml adenine, 10ng/ml transferrin, 5μg/ml insulin, O^g/ml hydrocortisone, and 8.5ng/ml cholera toxin. Human dermal fibroblasts were isolated as previously described. Dermal fibroblasts were cultured in fibroblast growth medium consisting of DMEM Dulbecco's modified eagles medium supplemented with 10% (v/v) FCS, 100IU/ml penicillin, 2mM L-glutamine, 100μg/ml

streptomycin and 0.625μg.ml amphotericin B.

[00122] The isolated cells were cultured at 37°C in a 5% C02/95% air humidified incubator. The cells were split 1 :3 every 6-7 days and subcultured as previously described. Keratinocytes were used up to passage 3 and passage 4-9 fibroblasts were used in all experiments.

Production of tissue engineered (TE) model:

[00123] Split thickness skin grafts were obtained during routine plastic surgery and breast reduction and abdominoplasty operations. The split thickness skin was immersed in 1 M sodium chloride solution for approx. 18hrs at 37^, which resulted in acellular de-epidermized human dermis (DED). The DED was washed in PBS and stored in Green's medium. DED was cut into 1 .5x1 .5 cm squares. The skin was placed papillary side up in a 6 well plate. A stainless steel ring was placed on the DED and melanoma TE models were produced using 1 x10⁵ dermal fibroblast, 3x10⁵ dermal ketainocytes.and 5x10⁵ melanoma cells. The melanoma cell line HBL was established in Professor Ghanem's laboratory from a lymph node metastasis of a nodular melanoma. Cells were maintained in melanoma culture media consisting of Ham's F10 medium supplemented with 5% FCS, 5% NBCS (new born calf serum), 2mM L-glutamine, 100IU/ml penicillin, 100μg/ml streptomycin and 0.625μg.ml amphotericin B. The constructs were submerged in media for two days and then raised to air liquid interface. The TE melanoma models were cultured in Green's medium for up to 4 weeks and the media was changed every two to three days. Polypyrroles incubation of TE model:

[00124] The TE melanoma models were incubated in 1 :20 PPy (-50 nm diameter, solid content 1 .44%) dilution in media (DMEM with 10%FCS) for 1 hr in a 5% C02/95% air humidified incubator. Post incubation the melanoma composites were washed with PBS to remove the PPy and imaged using a SS-OCT system. The OCT images were taken after washing and wiping the sample with PBS. A visible darkening of the composites is seen post incubation. As seen in Figure 15. The images were analysed using Matlab as seen in Figure 16.

References

[I] D. Huang, E. A. Swanson, C. P. Lin, J. S. Scguman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, J. G. Fujimoto, Science, 1991, 254, 1178.

[2] J. G. Fujimoto, M. E. Brezinski, G. J. Tearney, S. A. Boppart, B. Bouma, M. R. Hee, J. F. Southern, E. A. Swanson, Nature Medicine, 1995, 1, 970.

[3] G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Boppart, C. Pitris, J. F. Southern, J. G. Fujimoto, Science, 1997, 276, 2037.

[4] G. J. Jaffe, J. Caprioli, Am. J. Ophthalmol, 2004, 137, 156.

[5] M. A. Choma, M. V. Sarunic, C. Yang, J. A. Izatt, Opt. Express, 2003, 11, 2183.

[6] S. A. Boppart, W. Luo, D. L. Marks, K. W. Singletary, BreastCancer Res. Treat. , 2004, 84, 85.

[7] E. V. Zagaynova, O. S. Streltsova, N. D. Gladkova, L. B. Snopova, G. V. Gelikonov, F. I. Feldchtein, A. N. Morozov, . Urol., 2002, 167, 1492.

[8] A. B. Pardee, G. S. Stein, The Biology And Treatment Of Cancer, Wiley-Blackwell, 2009, Ch.l.

[9] S. A. Boppart, A. L. Oldenburg, C. Xu, D. L. Marks, . Biomed. Opt., 2005, 10, 041208.

[10] A. M. Zysk, F. T. Nguyen, A. L. Oldenburg, D. L. Marks, S. A. Boppart, . Biomed. Opt. , 2007, 12, 051403.

[I I] E. V. Zagaynova, M. V. Shirmanoval, M. Y. Kirillin, B. N. Khlebtsov, A. G. Orlova, I. V. Balalaeva, M. A. Sirotkinal, M. L. Bugroval, P. D. Agrba, V. A. Kamensky, Phys. Med. Biol. , 2008, 53, 4995.

[12] M. S. Shim, C. S. Kim, Y.-C. Ahn, Z. Chen, Y. J. Kwon, . Am. Chem. Soc , 2010, 132, 8316.

[13] A. L. Oldenburg, J. R. Gunther, S. A. Boppart, Opt. Lett., 2005, 30, 747.

[14] T. R. Carskim An Investigator's Brochure, "Indocyanine Green: History Chemistry Pharmacology Indication Adverse Reactions Investigation and Prognosis", Becton Dickinson and Company: Hunt Valley, 2004.

[15] Z. Yaqoob, E. McDowell, J. Wu, X. Heng, J. Fingler, C. Yang, . Biomed. Opt., 2006, 11, 054017.

[16] A. L. Oldenburg, D. A. Zweifel, C. Xu, A. Wei, S. A. Boppart, Proc. ofSPIE, 2005, 5703, 50.

[17] A. L. Oldenburg, M. N. Hansen, A. Wei, S. A. Boppart, Proc. ofSPIE, 2007, 6429, 64291Z-1.

[18] A. L. Oldenburg, M. N. Hansen, D. A. Zweifel, A. Wei, S. A. Boppart, Opt. Express, 2006, 12, 6724.

[19] J. Chen, F. Saeki, B. J. Wiley, H. Cang, M. J. Cobb, Z.-Y. Li, L. Au, H. Zhang, M. B. Kimmey, X. Li, Y. Xia, Nano Lett., 2005, 5, 473.

[20] H. Cang, T. Sun, J. Chen, B. J. Wiley, Y. Xia, X. Li, Opt. Express, 2005, 30, 3048.

[21] T. S. Troutman, J. K. Barton, M. Romanowski, Adv. Mater. , 2008, 20, 2604.

[22] H.-Y. Tseng, C.-K. Lee, S.-Y. Wu, T.-T. Chi, K.-M. Yang, J.-Y. Wang, Y.-W. Kiang, C.-C. Yang,

M.-T. Tsai, Y.-C. Wu, H.-Y. E. Chou, C.-P. Chiang, Nanotechnology, 2010, 21, 295102.

[23] C.-K. Lee, H.-Y. Tseng, C.-Y. Lee, S.-Y. Wu, T.-T. Chi, K.-M. Yang, H.-Y. Chou, M.-T. Tsai, J.-Y.

Wang, Y.-W. Kiang, C.-P. Yang, Bio. Opt. Express, 2010, 1, 1060.

[24] R. McNeill, R. Siudak, J. H. Wardlaw, D. E. Weiss, Aust. J. Chem., 1963, 16, 1056.

[25] S. P. Armes, B. Vincent, . Chem, Soc, Chem. Commun. , 1987, 288.

[26] J.-Y. Hong, H. Yoon, J. Jang, Small, 2010, 6, 679.

[27] H.-Y. Woo, W.-G. Jung, D.-W. Ihm, J.-Y. Kim, Synth. Met., 2000, 160, 588. [28] P. J. Tarcha, D. Misun, D. Finley, M. Wong, J. J. Donovan, Polymer Latexes (ACS Symposium Series), 1992, 492, 347.

[29] M. R. Pope, S. P. Armes, P. J. Tarcha, Bioconjugate Chem. , 1996, 7, 436.

[30] P. M. George, A. W. Lyckman, D. A. Lavan, A. Hegde, Y. Leung, R. Avasare, C. Testa, P. M. Alexander, R. Langer, M. Sur, Biomaterials, 2005, 26, 3511.

[31] J. M. Fonner, L. Forciniti, H. Nguyen, J. D. Byrne, Y.-F. Kou, J. Syeda-Nawaz, C. E. Schmidt, J.Biomed. Mater. , 2008, 3, 034124.

[32] A. Ramanaviciene, A. Kausaite, S. Tautkus, A. Ramanavicius, . Pharm. PharmocoL, 2007, 59, 311.

[33] S. D. Perrault, C. Walkey, T. Jennings, H. C. Fischer, W. C. W. Chan, Nano Lett, 2009, 9, 1909.

[34] H₂0 has a very strong NIR absorption band at around 1390 nm (L. Kow, D. Labrie, P. Chylek, Appl.

Opt., 1993, 32, 3531). To removethis undesirable feature, a spectrum was recorded for the polypyrrole nanoparticles dispersed in a 9: 1 D₂0/H₂0 mixture. The extinction coefficient at 1300 nm was calculated based on the spectra recorded in H₂0 to prevent isotopic effects affecting the result.

[35] P. Pfluger, G. B. Street, . Chem. Phys., 1984, 80, 544.

[36] R. B. Bjorklund, B. Liedberg, . Chem. Soc, Chem. Commun. , 1986, 1293.

[37] J. M. Schmitt, A. Knuttel, A. Gandjbakhche, R. F. Bonner, Proc. SPIE, 1993, 2989, 197.

[38] J. M. Schmitt, A. Knuttel, R. F. Bonner, Appl. Opt., 1993, 32, 6032.

[39] P. Lee, W. Gao, X. Zhang, Appl. Opt., 2010, 49, 3538.

[40] H. C. Van de Hulst, Light Scattering by Small Particles, Dover Publications, Inc. (Canada), 1981.

[41] As for previous studies [16,17,18,20], the μ^οστ value determined by OCT is comparable with the In_spect measured by spectroscopy after appropriate calibration.

[42] Backscattering albedo related to tissue phantom (a') was equivalent to the ratio of albedo of 2% intralipid tissue phantom containing different concentration of polypyrrole nanoparticles to the albedo of 2% intralipid tissue phantom (without polypyrrole nanoparticles).

[43] Complex refractive index is essential for Mie scattering calculation. In the absence of precise complex refractive index data for PPy at 1300 nm, it is assumed that complex dielectric function of PPy measured at 1818 nm (5500 cm^"1) by Kramers-Kronig technique [46] is comparable with that at 1300 nm. The real (n) and imaginary (k) parts of the complex refractive index were calculated from the complex dielectric function (ε = Z + ε₂ϊ = 0.80 + 2.74 ) [46] using the following relations: Re(e) = Z = n - k² and lm(s) = s₂ = 2nk [46,47].

[44] The density of polypyrrole bulk powder was found to be 1.440 g cm^"3 (measured by helium pycnometry). The density of gold is 19.32 g cm^"3 [45].

[45] CRC Handbook of Chemistry & Physics (Eds: 55th), Chemical Rubber Co., 1974-75: B-15.

[46] R. Turcu, M. Brie, G. Leising, V. Tosa, A. Mihut, A. Niko, A. Bot, Appl. Phys. A, Mater. Sci. Processing 1998, 67, 283.

[47] G. Leising, Phys. Rev. B, 1988, 38, 10313.

Claims

CLAIMS:

1 . A use of sterically-stabilised organic conducting polymeric nanoparticles as contrast agents in biophotonic imaging.

2. The use according to claim 1 , wherein the biophotonic imaging is optical coherence tomography (OCT).

3. The use according to any preceding claim, wherein the polymeric nanoparticles are sterically stabilised polypyrrole, poly(3,4-ethylenedioxythiophene) (PEDOT) or polyaniline nanoparticles.

4, The use according to any preceding claim, wherein the polymeric nanoparticles sterically stabilised polypyrrole nanoparticles.

5. The use according to claim 4, wherein the polypyrrole nanoparticles comprise a polypyrrole defined by formula I:

Formula I

wherein n is at least 2 and where X^" is a dopant anion, such as chloride or tosylate,

6. The use according to any preceding claim, wherein the sterically-stabilised poly nanoparticles comprise a polymeric core coated with a hydrophilic stabiliser polymer.

7. The use according to claim 6, wherein the hydrophilic stabiliser polymer is a polymer comprising hydrophilic polymer blocks.

8. The use according to claim 6, wherein the hydrophilic polymer is selected from the group including poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC), poly(glycerol

monomethacrylate) (PGMA), poly(N-vinylpyrrolidone), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), or polyvinyl alcohol) (PVA).

9. The use according to any of claims 6 or 8, wherein the stabiliser comprises PVA physically absorbed upon the polypyrrole core.

10. The use according to any preceding claim, wherein the polymeric nanoparticles absorb photons in the near infra-red spectrum.

1 1 . The use according to claim 10, wherein the polymeric nanoparticles absorb photons in the range of 1200 to 1350 nm.

12. The use according to any preceding claim, wherein the polymeric nanoparticles have an average particle size within the range of 5 to 200 nm.

13. A contrast agent composition, comprising sterically-stabilised polymeric nanoparticles as defined in any preceding claim, in admixture with a diluent or carrier.

14. A method of imaging a substrate, comprising contacting the substrate with sterically- stabilised polymeric nanoparticles as defined in any one of claims 1 to 12, or a contrast agent composition according to claim 13; and acquiring images of the substrate.