ENGINEERING AND UTILITY OF FLUORESCENT NANODIAMOND PARTICLES (NDP-F) FOR DIAGNOSTICS AND TREATMENT OF BLOOD CLOTS

IN HUMAN AND VETERINARY MEDICINE

BACKGROUND OF THE INVENTION

Field of the Invention

[001] The present invention relates to the field of medical and veterinary diagnostics and treatment. More specifically, the invention relates to a diagnostic reagent, tool, and system that are specific for detection of platelets and blood clots. In addition, the invention relates to detection of internal body bleeding sites in a variety of diseases and trauma conditions.

Description of Related Art

[002] Cardiovascular diseases, such as stroke and heart attack, are the leading cause of mortality in developed countries. Death from strokes and heart attacks are predominantly the consequence of blood clots (thrombi) formed in the cerebral and cardiac vessels or thromboembolic events (TEE) associated with blood clots formed in remote vessels (e.g., peripheral venous system, cardiac atria appendixes). While several factors are well known to contribute to a fatal TEE (e.g., atherosclerosis vascular disease) there is a clear "diagnostic and prognostic gap" in the assessment of the specific and "total clot burden" in individuals that carry known risks factors (e.g., atherosclerotic vascular disease) let alone factors not yet fully vetted as predictive of TEE. Invariably, clinical presentation of consequences of blood flow occlusion by thrombi leading to stroke or heart attack command prompt medical

investigations in search for the TEE culprits. Such investigations are mostly hospital-based imaging tests, such as angiography, CAT scans, MRI, and ultrasound. The current technologies are no doubt critical for timely and successful management of strokes and heart attacks, yet several important limiting factors must be addressed. First, hospital-based technologies to assess blood clots in vessels are of limited access for the general population, especially elderly people who carry cardiovascular risks. Second, even in hospitals, access to these imaging technologies has a certain time requirement associated with the tests and their evaluation by specialists. In the case of a stroke, where the treatment window is limited to three to four and one half hours after the onset of the event, much of the time is spent in establishing patient eligibility for thrombolysis treatment, often to the extent of missing the critical window for treatment. [003] On the other extreme, once a diagnosis of cardiac arrhythmia, such as atrial fibrillation (chronic, relapsing) is made, the risk of TEE mandates lifelong treatment with anti -coagulants even though a presence of clots in the cardiac chambers (appendixes) is unknown. These few examples point to a major "diagnostic gap" of TEE risks, which due to lack of early diagnosis and preventative measures often results in fatal outcomes. Early assessment of whole body clot burden or TEE risk, in ambulatory settings that allows easy and broad access and affordable cost is needed.

DESCRIPTION OF THE INVENTION

[004] The present invention provides diagnostic agents and methods for risk assessment of subjects, including both humans and non-human animals at risk of TEE, which are the main cause of cardiovascular death from strokes and heart attacks. The diagnostic agents and methods of the invention enable assessment of the total body burden of intravascular clots, the propensity to detect nascent thrombi at common predilection sites for clot formation using non-radiation (e.g., X-ray), non-MRI, and non-ultrasound techniques. The technology is a non-invasive, telemetry-based fluorescent recording suitable for use in a fast and affordable ambulatory setting. The invention includes multiple innovative scientific and engineering breakthroughs.

[005] The invention is based, at least in part, on the recognition that fluorescent nano- diamond particles (NDP-F) tagged with the disintegrin Bitistatin (Bt) have the innate ability to bind avidly to activated platelet fibrinogen receptors (PFR). Data presented herein demonstrate successful coupling of Bt to NDP-F and retention of Bt bioactivity. The methods, reagents, and de novo protocols used to accomplish the invention are not known to the inventors as having been reported by others before.

[006] The methodology of coupling proteins/peptides to carboxyl-functionalized NDP-F is well described. Preservation of the active domains responsible for the biological action of the coupled proteins/peptides remains challenging, and is generally considered to be a trial and error endeavor. A major innovation of the present invention is the demonstration of a concentration-dependent association of the engineered NDP-F-Bt agent to purified PFR. The selection of Bt was based on the high selectivity/specificity of Bt to the PFR, concomitant with negligible interactions with other RGD-dependent integrins, such as receptors for vitronectin (avb3) and fibronectin (a5bl) (Marcinkiewicz, 2013). The present strategy to utilize Bt for clot imaging differs significantly from previous studies aimed at demonstrating the utility of Tc99-Biti statin to map blood clots in vivo. (Knight et al, 1998, 2000, 2007). The present imaging strategy is based on the innate near infrared (NIR) fluorescence emitted upon excitation of the DP-F, thereby eliminating high radioactivity exposure required by other imaging techniques. It is also envisioned that coupling Bt to a nanoparticle will be beneficial for the extension of the lifetime of the Bt at the site of its biological target.

[007] The present invention enables broad scale survey of TEE risks that is applicable in many medical emergency and life-threatening conditions beyond strokes and heart attacks. The technology can be used not only for individuals suspected to be at risk for TEE but also be periodically deployed as part of primary health care office assessments, no different than annual mammography, lipids tests, or physical examination. The technology monitors fluorescent light emittance and is expected to be highly affordable, minimally invasive (requiring only a single injection of a safe dose of nanoparticles), and can be conducted and interpreted under an ambulatory setting by a trained emergency medical technician or a primary care physician, similar to ECG monitoring.

[008] One general aspect of the invention is an imaging agent for detection of a thrombus in a subject. The agent is composed of three elements, as follows: i) a fluorescent nano-diamond particle (NDP-F); ii) a ligand that functionalizes the NDP-F, such as a

-COOH, -OH, -NH ₂ , or -C=0 moiety; and iii) a protein attached to the ligand. The three can be bonded together in any order and by any type of chemical bonds, but are typically covalently bonded in the order described. The objective of the imaging agent is to specifically bind the agent to a discrete biological target in a human or non-human animal body.

[009] Yet another aspect of the invention is a diagnostic method for detection of activated platelets. The diagnostic method is based in large part on the ability of the Bt to bind to PFR with high affinity and/or avidity. The method thus relies on the Bt to target the imaging agent to activated platelets, and thus sites of thrombus formation, and on the fluorescence of the NDP-F to allow non-invasive and relatively harmless imaging of the location and size of the thrombus, or multiple thrombi. In embodiments, the diagnostic method is qualitative, and in other embodiments it is semi-quantitative or quantitative. In general, the method comprises: i) administering to a subject suspected of having, or potentially having, one or more sites of thrombus, a detectable amount of the diagnostic agent of the invention, ii) allowing sufficient time for the diagnostic agent to localize to the site(s) of thrombus, and iii) detecting the diagnostic agent by detecting fluorescence emission after excitation with a suitable electromagnetic stimulus (e.g., excitation light, such as emission from a hand held device). It is to be understood that, in some embodiments, in step iii) the act of excitation can be omitted if the fluorescent tag is intrinsically fluorescent in the subject's body. The step of administering can be any action that results in introduction of the imaging agent into the systemic blood stream of the subject. It thus may be, for example and without limitation, via intravenous injection or infusion.

[010] Yet further, and in accordance with the method described immediately above, the invention includes a method of detecting clots or clot formation in subjects. As with the method described above, this method can be considered a method of detecting or imaging clots, clot formation, platelet activation, or pathological zones that form a risk for clot formation, such as an atherosclerotic vascular plaque or inflammation. The method steps are those described above.

[Oil] As those of skill in the art will immediately recognize, the diagnostic and imaging methods of the present invention, by virtue of introduction of a non-natural bio-active substance into a subject's body, do not relate solely to collection of data regarding a biological event, but instead relate to physical and physiological changes to the subject's body. For example, introduction of the non-naturally occurring imaging agent into the blood stream of a subject physically alters the make-up of the blood stream. In addition, binding of the imaging agent to activated platelets alters the body's natural ability to interact with the activated platelets, and thus the clotting cascade. Other physical and physiological changes upon administration of the imaging agent of the invention will be apparent to the skilled artisan.

[012] The present invention also encompasses kits for practicing the methods of the invention. Broadly speaking, a kit according to the invention includes the imaging agent of the invention in packaged form suitable for distribution, delivery, and/or storage for use in a method of the invention. In customary fashion, the package is made of a suitable material, such as, but not limited to, a cardboard or plastic box and the like, a metal container and the like, or a foil pouch or the like. In some embodiments, the kit includes sufficient imaging agent in a container for a single administration, whereas in other embodiments, the kit includes sufficient imaging agent for two or more administrations. In embodiments, where the kit includes sufficient imaging agent for two or more administrations, the imaging agent can be supplied in a single container for multiple uses or in two or more containers, each containing sufficient imaging agent for a single use. Of course, a combination of single-use and multiple-use containers can be included in a kit. In embodiments, the kit (regardless of how many containers of imaging agent are provided in the kit) can be provided in packaged combination with one or more reagents or devices for administration of the imaging agent to a subject. As such, and without limitation, a kit of the invention can include, in packaged combination, the imaging agent with an antiseptic (e.g., ethanol swabs or pads or iodine swabs or pads), one or more syringes, one or more needles adapted to connect with a syringe, and/or one or more pieces of gauze and/or adhesive to facilitate closure and healing of the site of administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate data supporting embodiments of the invention, and together with the written description, serve to explain certain principles of the invention.

[014] Fig. 1 is a fluorescence scan result showing two dimensional screening of excitation vs. emission wavelengths to optimize fluorescence of NDP-Fs used in the exemplary embodiment. The figure shows optimization of the maximal fluorescence for NDP-Fs. The heat map represents the results of fluorescence screening in the entire spectrum of excitation vs. emission wavelengths. The experiment was performed using Tecan plate reader. NDP-Fs were applied on a 96-well plate (0.3 mg/0.1 ml) as a suspension in PBS. The blank, or negative control, was established using PBS alone.

[015] Fig. 2 presents a line graph indicating detection of Bt on NDP-Fs in a "semi- ELISA" assay. NDP were coupled to Bt or BSA, which were used in concentrations as indicated on the X-axis, per 1 mg of NDP-Fs. 0.2 mg of each NDP sample was used for semi-ELISA in three replicates. The experiment was performed on a 96-well plate (U-shape bottom) with gentle rotation during incubations. NDPs were blocked with 10% goat serum before primary antibody against Bt was added. NDP samples were incubated with anti-Bt antibody for one hour at 37°C, and the plate was washed three times with PBST by centrifugation (1,000 x g) at room temperature. Goat anti-rabbit IgG AP conjugated (Sigma Inc.), diluted 1 :2000, was added and incubated for one hour as above. Final washing was performed as above and substrate (pNPP) was added to AP. Color was developed for 30 minutes, and NDP samples were centrifuged. The supernatant was transferred to a 96-well plate and read using an ELISA plate reader under 405 nm wavelength. Error bars represent SD from three independent samples applied for the semi-ELISA procedure. [016] Fig. 3 is a line graph showing interaction of purified integrin with DPs in semi- ELISA. The experiment was performed using a U-shape bottom 96-well plate. The plate was blocked overnight with 5% BSA, whereas NDPs were blocked with 3% BSA by incubation for 1 hour at 37°C before application on the plate. Blocked NDPs were added to the wells (0.2 mg per well) and the plate was centrifuged (10,000 x g) at room temperature. Platelet Fibrinogen Receptor (PFR) at the indicated concentrations was added in 0.1 ml of Hanks' Balanced Salt Solution (HBSS) containing Ca ²⁺ (as CaCl ₂ ) and Mg ²⁺ (as MgCl ₂ ) at physiological concentrations to each well and incubated for 1 hour at 37°C. NDPs were washed three times by centrifugation of the plate (1,000 x g) at room temperature and primary polyclonal antibody against the fibrinogen receptor was added (2 μg/ml). Incubation was performed for one hour at 37°C, and samples were washed three times, as described above. Goat anti-rabbit IgG AP conjugated (Sigma Inc.), diluted 1 :3000, was added and incubated for one hour as above. Final washing was performed as above and substrate (pNPP) was added to AP. Color was developed for 30 minutes, and NDP samples on the plate were centrifuged at 10,000 x g at room temperature to collect the NDP as a pellet. Supernatant was transferred to flat bottom 96-well (0.1 ml) plates and read using an ELISA plate reader at 405 nm wavelength.

[017] Fig. 4 presents a line graph showing adhesion of NDP-F-Bt and NDP -BSA to immobilized PFR. PFR was immobilized on a 96-well plate by overnight incubation at 4°C in PBS. The plate and NDPs were blocked with 3% BSA. NDPs coupled to 1 mg protein (Bt or BSA) were used in the experiment. NDPs (300 mg) were added to the wells. Incubation was performed for one hour at 37°C in HBSS buffer containing calcium and magnesium at physiological concentrations. Unbound NDPs were intensively washed out six times using the same buffer with vacuum aspiration. Finally, HBSS (100 μΐ) was added to the wells and fluorescence was read using a fluorescence plate reader with 485 nm (excitation) and 530 (emission) wavelengths.

[018] Fig. 5 presents a line graph showing quantitation of adhesion of NDP-F-Bt and NDP -BSA to immobilized fibrinogen receptor. Fibrinogen receptor was immobilized on 8- well glass chamber slides, by overnight incubation at 4°C. The wells were blocked with 3% BSA, and NDPs previously also blocked by 3% BSA were added (50 mg per well per 200 ml) in HBSS containing calcium and magnesium at physiological concentrations. The adhesion procedure was performed as per Fig. 4. In the final step, the slide was prepared with mounting buffer (Vector Lab). Images were analyzed under fluorescence microscope (400x) using an oil objective. The numbers of DPs were calculated using ImageJ software. Error bars represent SD for three independent pictures taken for each concentration of fibrinogen receptor.

[019] Fig. 6 depicts representative images of adhered DP-F-Bt to immobilized PFR. PFR (concentrations indicated) was immobilized on an 8-well chamber slide, and the experiment was performed as described in Fig. 5. In the legend, the bars represent 20 μπι.

[020] Fig. 7 shows pictures of fluorescent images taken by rVIS and confocal microscopy of carotid artery clots after treatment with FNDP via an external carotid artery infusion. Fig. 7A shows in situ carotid bifurcation region image indicating fluorescence of carotid arterial clot after treatment visible via IVIS imaging after exposure of the carotid bifurcation zone. Fig. 7B and 7C are high magnification images of fluorescence emanating from the carotid bifurcation in vivo suggesting accumulation of FNDP in the clot. Fig. 7D shows ex vivo fluorescence of carotid artery bifurcation denoting one branch that shows fluorescence corresponding to the clot location within the carotid bifurcation. Figs. 7E and 7F show confocal images taken on an Olympus 1X83, showing that FNDP fluorescence is detected at an excitation of 543nm and an emission of 655-755nm.

[021] Fig. 8 shows Fluorescent images taken by rVIS and confocal microscopy of carotid artery clots after intravenous treatment with FNDP. Fig. 8A shows an ex vivo fluorescent image of a carotid artery from saline-treated control. Fig. 8B shows an ex vivo fluorescent image of a carotid artery from an IV FNDP -treated animal showing fluorescence localized to the branch with clot. Fig. 8C-F show confocal images taken on Olympus ΓΧ83. Fig. 8G is a graph showing the number of FNDPs present in carotid clot lysates from animals treated locally via the external carotid artery or intravenously as compared with saline treated controls.

[022] Fig. 9 shows fluorescent microscopy of the specificity of the interaction of F- NDP-Bt for clot generation from rat blood plasma by thrombin (1 U/ml). Panel 9A shows images of plasma clots obtained from fluorescence microscope Olympus 1X81 analysis, under lOOx magnification. Panel 9B shows images of plasma clots obtained in IVIS 50 imaging system. Wavelengths used for measurement: excitation Cy5.5 BkG (580-610 nm), emission Cy5.5 (695-770 nm). For background subtraction: excitation GFP (445-490 nm), emission Cy5.5 (695-770 nm). Exposure time: 1 minute. Arrows point the localization of the clot.

[023] Fig. 10 shows images of vessels filled with F-NDP-Bt implanted subcutaneously in a rat (post-mortem). Panel 10A shows an image of the implanted glass capillaries filled with F- DP-Bt (4 mg/ml) or PBS (control). Exposure time was 5 seconds. Panel 10B shows an image of a rat aorta filled with F-NDP-Bt. The rat aorta was dissected from a euthanized female rat, washed with PBS to remove residues of coagulated blood, and filled with 300 μΐ of F-NDP-Bt suspension (2 mg/ml) in PBS.

DETAILED DESCRIPTION OF VARIOUS EMBODFMENTS OF THE INVENTION

[024] Reference will now be made in detail to various exemplary embodiments of the invention, data supporting those embodiments being illustrated in the accompanying drawings.

[025] Fluorescent nano-diamond particles (NDP-F or FNDP), functionalized by carboxyl groups (-COOH) were purchased from Adamas Nanotechnologies, Inc. (Raleigh, NC). Size distribution analysis revealed the peak of the diameter of the NDP as 734.5 (± 223.6, SD) nm. Optimization of fluorescence for the NDPs was performed by 2D screening of excitation vs. emission wavelengths (Fig. 1). Two areas of optimal fluorescence were established for NDPs, which should be useful for application in medical imaging (circled on Fig. 1). Detailed screening revealed two optimal correlations of excitation vs. emission wave lengths: 480 nm vs. 520 nm, and 565 nm vs. 700 nm. The first correlation represents the typical green fluorescence, whereas the second is characterized by the long stokes shift of fluorescence with near IR emission. The near IR emission is very useful for detection in vivo because it is in the optical therapeutic window of autofluorescence of factors present in human and non-human animal non-invasive imaging environments {e.g., water, hemoglobin, oxyhemoglobin, melanin).

[026] The NDP-Fs were found to be resistant to photobleaching. Exposure of the slides containing NDPs samples for intense fluorescence light resulted in no changes in the intensity of their fluorescence in the time points up to 5 hours (data not shown).

[027] Bitistatin is derived from snake venom and belongs to the disintegrin family of proteins. It has previously been investigated as potential reagent for detection of deep venous thrombosis (DVT) using radioactive tags (Knight et al, 1998, 2000). This RGD-disintegrin showed the best parameters for detection of DVT when compare with other snake venom disintegrins such as kistrin and barbourin. Therefore, we selected this fibrinogen receptor- binding ligand for coupling to NDP-Fs for the purpose of detecting activated platelets (or their aggregates) in clots present in the venous circulation (vein thrombus). [028] Bitistatin is an 83-amino acid polypeptide, which originally was isolated from venom of Bitis arietants (Shebuski et al, 1989). We purified this naturally occurring polypeptide from the same snake venom using methodology developed in our laboratory for purification of other snake venom disintegrins (Marcinkiewicz et al, 1999; incorporated herein by reference). This method includes two steps of reverse phase HPLC with application of C ₁₈ column and a linear gradient of acetonitrile as a protein elution agent. Purity of the obtained Bt was tested by SDS-PAGE and quantified by digitalization of bands on standard protein gels by Coomassie blue staining. The content of Bt was estimated at over 98% in the final protein preparation. This preparation was suspended in PBS for coupling to NDPs.

[029] The active site of Bt was characterized and found to include an RGD motif, which is important for ligand binding to certain integrins, such as αΙ¾β3, ανβ3, and α5β1. Bt is quite selective in that it has been shown to bind only the PFR, αΙ¾β3 integrin, which is exclusively expressed on circulating platelets. The aspartic acid in the RGD sequence was recognized by site-directed mutagenesis of recombinant proteins and short peptide synthesis as the most essential amino acid for disintegrin binding to the fibrinogen receptor. We hypothesized that coupling of Bt to NDPs that were functionalized by an attached amine group could affect the activity of disintegrin by engagement with carboxyl groups present in the side chain of the aspartic acid. Therefore, we selected NDPs functionalized by a carboxyl group to attach the Bt on NDPs surfaces.

[030] Coupling of carboxyl (-COOH) groups present on NDPs to NH ₂ groups present on Bt was performed using a standard protocol developed previously (Grabarek and Gergely, 1990). This method is based on the application of the cross-linker l-ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride (EDC), which activates carboxyl groups to bind amine groups by forming an O-acylisourea intermediate that quickly reacts with an amino group to form an amide bond. O-acylisourea undergoes quick hydrolysis. Therefore, to increase the efficiency of coupling, sulfo-N-hydroxysuccinimide (sulfo-NHS) was added to the reaction mixture to generate an amine-reactive sulfo-NHS ester.

[031] BSA was coupled in parallel to the control NDPs.

[032] A detailed description of a suitable coupling procedure follows:

[033] Two samples of NDP, each containing 2 mg in 1.8 ml of 0.1M MES, 0.5M NaCl, pH 6.0, were prepared. Sample A contained 1 mg Bt per 1 mg NDPs. Sample B contained 1 mg BSA per 1 mg NDPs, and served as a control. [034] To each sample, 0.72 mg EDC was added and the mixture incubated at room temperature (about 21°C to 25°C). with gentle rotating for 15 minutes. After incubation, 2.5 μΐ of 2-mercaptoethanol (to give a final concentration of 20 mM) was added to quench the EDC and stop the carboxyl group activation.

[035] The activated NDPs were transferred to 1.5 ml centrifuge tubes and centrifuged at 10,000 x g for 5 minutes at room temperature. The NDP pellets were washed twice with 1 ml PBS and suspended in coupling buffer (PBS). One milligram of either Bt or BSA in 1 ml of PBS was added to each sample tube. The tubes were incubated for two hours at room temperature with gentle rotating. Ten μΐ of 1M Hydroxylamine-HCl (350 mg in 5 ml PBS) was added to each tube for quenching (final concentration of Hydroxylamine-HCl should be about 10 mM).

[036] The sample tubes were centrifuged at 10,000 x g for 5 minutes at room

temperature. The supernatants were saved for protein concentration determination to check coupling efficiency. The NDP-F-Bt particles were washed three times with 1 ml PBS each time. The washed particles were suspended in a volume of PBS to achieve a concentration of 1 mg/ml.

[037] Coupling efficiency was evaluated using immune detection of Bt on the surface of NDPs (NDP-Bt). For ease of reference in this document, the detection method is called semi- ELISA. Broadly speaking, polyclonal antibody against Bt was developed commercially in rabbits (Chemicon/Millipore Inc.) using purified native polypeptide as the antigen. The optimal (saturated) concentration of Bt was estimated at 1 mg per 1 mg NDPs. A

representative plot from three independent experiments is presented in Fig. 2.

[038] A suitable assay for determining coupling efficiency follows:

[039] Using a 96-well plate having a U-shaped bottom and that has previously been blocked by incubation overnight at 4°C with 5% BSA in PBS, 200 μΐ of an NDP-F-Bt or NDP-F-BSA solution (1 mg/ml NDP in PBS) was each applied to a separate well. The plate was centrifuged for 10 minutes at 1,000 x g. The supernatant was removed and the plate gently shaken by vortexing to disperse and re-suspend the pellet of NDP -F.

[040] 200 μΐ of 10% goat serum in PBST (PBS + 0.05% Tween-20) was added and mixed a few times by intensive aspiration and releasing from a pipet tip. The mixture was incubated for one hour at 37°C with slow agitation. The mixture was centrifuged as above and washed three times with 200 μΐ of PBST. After each centrifugation, the pellet was dispersed by vortexing. After the final wash, 100 μΐ of anti-bitistatin (5 μg/ml in PBST) was added to the wells and incubated for one hour at room temperature. The plate was then washed three times with PBST as described above. To each pellet, 100 μΐ of a 1 :2000 dilution of goat anti-rabbit IgG AP conjugate (from Sigma) in PBST was added to the wells and the mixture incubated for one hour at 37°C. The mixture was centrifuged at 1,000 x g for 10 minutes, and the pellet was washed three times with PBST as above.

[041] 150 μΐ of the AP substrate p PP (from Sigma) was added and color was developed for approximately 30 minutes (until the intensity of the yellow color was visible) at room temperature with gentle agitation. The reaction was blocked by adding 100 μΐ of 3 M NaOH (this step is optional). The plate was then centrifuged and 100 μΐ of the supernatant was transferred to a 96-well plate (flat bottom). The absorbance of the supernatant was measured at 405 nm using an ELISA plate reader.

[042] The activity of DP-Bt was tested using PFR purified from platelets, αΙ¾β3 integrin (Millipore Inc.). Two types of assays were applied to confirm the presence of disintegrin on NDP-Bt, including binding of soluble PFR to NDP-F-Bt and adhesion of NDP- F-Bt to fibrinogen receptor immobilized on the surface (plastic or glass). Binding of soluble PFR was determined on the basis of the functional "sandwich" ELISA, which is referred to herein as a functional semi-ELISA. In this case, immune-detection of αΙ¾β3 integrin bound to the NDP-Bt was performed using a polyclonal antibody against the αΠβ integrin subunit (purchased from Santa Cruz Inc.).

[043] A suitable protocol for a functional semi-ELISA for detecting binding of fibrinogen receptor to NDP-F-Bt follows:

[044] Using a 96-well plate having a U-shaped bottom and that has previously been blocked by incubation overnight at 4°C with 5% BSA in PBS, 200 μΐ of an NDP-F-Bt or NDP-F-BSA suspension (1 mg/ml NDP in PBS) was each applied to a separate well. The plate was centrifuged for 10 minutes at 1,000 x g. The supernatant was removed and the plate gently shaken by vortexing to disperse the pellet of NDP-F. 200 μΐ of 3% BSA PBST (PBS + 0.05% Tween-20) was added and the composition was mixed a few times by intensive aspiration and releasing from a pipet tip. The mixture was incubated for one hour at 37°C with slow agitation. Alternatively, the blocking step may be performed in a tube before application on the 96-well plate. The mixture was centrifuged as above and washed three times with 200 μΐ of PBST. After each centrifugation, the pellet was dispersed by vortexing.

[045] Fibrinogen receptor was then added at the required concentrations (i.e., the amounts required to generate a dose-response; see Fig. 3) in 200 μΐ of HBSS containing physiological concentrations of Ca ²⁺ and Mg ²⁺ and incubated for one hour at 37°C. The plate was then washed three times with PBST as above. Next, 100 μΐ of anti -fibrinogen receptor (2 μg/ml from Santa Cruz Inc.) polyclonal antibody in PBST was added to the wells and incubated for one hour at room temperature. The plate was washed three times with PBST as above. To the pellets, 100 μΐ of a 1 :3000 dilution of goat anti-rabbit IgG AP conjugate (from Sigma) in PBST was added to the wells and incubated for one hour at 37°C. The mixture was centrifuged at 1,000 x g for 10 minutes, and the pellets washed three times with PBST as above.

[046] 150 μΐ of the AP substrate pNPP (from Sigma) was added and color was developed for approximately 30 minutes at room temperature with gentle agitation. The reaction was blocked by adding 100 μΐ of 3 M NaOH (this step is optional). The plate was centrifuged and 100 μΐ of supernatant was transferred to the well of a 96-well plate (flat bottom). The absorbance of the supernatant was measured at 405 nm using an ELISA plate reader.

[047] As can be seen from Figure 3, the PFR binds to the NDP-Bt in a dose-dependent manner, whereas the control NDP-BSA were not active in this assay.

[048] Adhesion of NDP-Bt to immobilized fibrinogen receptor was performed in two formats based on the estimation of the fluorescence of attached NDPs. First, integrin was immobilized on a 96-well plate and adhered NDPs were detected using a fluorescence plate reader. The results obtained showed linear progression of the adhesion of NDP-Bt to increased concentration of immobilized purified receptor (Fig. 4).

[049] Adhesion of NDP-Bt was also monitored under fluorescence microscopy. The number of adhered NDPs was quantified using computer software (Fig. 5). Representative images of adhered NDP-Bt are presented in Fig. 6.

[050] To further characterize the effects of the FNDP, fluorescent images were taken by rVIS and confocal microscopy of carotid artery clots after treatment with FNDP via external carotid artery infusion. The results are shown in Fig. 7. In two independent experiments, infusion of FNDP via the external carotid artery commenced 3-5 minutes after FeCl ₃ application and continued over 15 minutes (5 minutes beyond the end of FeCl ₃ infusion). The FNDP solution consisted of 1.5 ml of PBS where 5 mg/ml of FNDP was suspended (after vortexing of the solution). This route of infusion was selected so as to avoid possible "first pass" elimination of the particles by peripheral organs. Following completion of FNDP infusion, the rat was euthanized and subjected to imaging by rVIS and/or fluorescent microscopy. Figures 7A-D show imaging of fluorescence that was performed on an IVIS scanner designed for whole animal imaging using a 580-610 nm excitation and a 695-770 nm emission passband with a 2 second exposure. Auto-fluoresence was subtracted based on excitation at 445-490 nm. Figure 7A shows an in situ carotid bifurcation region image, indicating fluorescence of carotid arterial clot after treatment visible via rVIS imaging after exposure of the carotid bifurcation zone. Figures 7B and C are high magnification images of fluorescence emanating from the carotid bifurcation in vivo, suggesting accumulation of FNDP in the clot. Figure 7D is an ex vivo photograph of fluorescence of carotid artery bifurcation denoting one branch showing fluorescence corresponding to the clot location within the carotid bifurcation. Figure 7E and F are confocal images taken on Olympus ΓΧ83 of FNDP in which fluorescence is detected at an excitation of 543 nm and an emission of 655-755 nm. Background fluorescence was collected from the same excitation, with emissions of 555-625 nm and was subtracted from the foreground to reduce auto- fluorescence. Figure 7E shows ex vivo fluorescence of the carotid artery at 4x magnification. Figure 7F shows FNDP treated carotid arteries after they are flushed with RIPA lysis buffer and replicates were combined together to form a lysate. Lysate was then deposited onto a cover-glass and imaged at 20x magnification. Large numbers of FNDP at various aggregate sizes around platelets are visible.

[051] Figure 8 shows fluorescent images taken by rVIS and confocal microscopy of carotid artery clots after intravenous treatment with FNDP. After clot formation by ferric chloride, treatment with saline control or FNDP by intravenous infusion via tail vein or femoral vein was performed in three rats. FNDP were infused (over 10 min) as a suspension in PBS at 1 ml solution containing 1 mg/ml FNDP. Carotid arteries were removed from the animal for imaging and placed in 70% denatured ethanol for preservation until imaging. FNDP were conspicuously identified at the site of clot formation. FNDP were identified in each of the three specimens obtained following intravenous infusion, yet the three specimens were treated together as one for lysate inspection. Figures 8A and 8B show imaging of fluorescence as performed on an rVIS scanner designed for whole animal imaging using a 580-610 nm excitation and a 695-770 nm emission passband with a 2 second exposure. Autofluorescence was subtracted based on excitation at 445-490 nm. Figure 8A shows an ex vivo fluorescent image of a carotid artery from saline-treated control. Auto-fluorescence could not be entirely eliminated, but is evenly distributed across control specimen. Figure 8B shows an ex vivo fluorescent image of a carotid artery from an IV FNDP -treated animal, showing fluorescence localized to the branch with a clot. Figures 8C-F show confocal images taken on Olympus 1X83. The figures show that F DP fluorescence is detected at an excitation of 543 nm and an emission of 655-755 nm. Background fluorescence was collected from the same excitation, with emissions of 555-625 nm and was subtracted from the foreground to reduce auto-fluorescence. Figures 8C and D show ex vivo fluorescence of carotid artery at 4x magnification of saline treated and FNDP treated animals, respectively. Auto-fluorescence could not be entirely eliminated, but is evenly distributed across control specimens, while fluorescence is localized to the branch with clot in the IV treated animal. (See panels E and F.) Treated carotid arteries were flushed with RIPA lysis buffer and replicates were combined together to form a lysate. Lysate was then deposited onto a cover- glass and imaged at 20x magnification. In order to increase contrast for visual inspection, images were processed with an un-sharp mask in ImageJ.

[052] Figure 8E shows that saline-treated control shows no detectable fluorescence. Figure 8F shows that FNDP appear as frequent fluorescent spots in the treated samples.

Figure 8G presents a graph showing the number of FNDPs present in carotid clot lysates from animals treated locally via the external carotid artery or intravenously as compared with saline treated controls. FNDPs were counted in replicate images after thresholding in ImageJ.

* denotes p<0.01 by T-test.

[051] Fig. 9 depicts images of rat blood plasma clots following treatment with F-NDP- Bt and F-NDP-BSA. Rat blood was collected by heart puncture and centrifuged at 100 x g for 20 minutes at room temperature to obtain platelet rich plasma (PRP). A thrombus was generated by adding thrombin (1 U/ml) and incubating for 15 minutes at 37°C. The clot that was formed was washed 3x by decanting with HBSS containing calcium and magnesium, and then sliced. Pieces of the clot were incubated with a suspension of F-NDP-Bt and F-NDP- BSA (50 μg/ml) in HBSS containing calcium and magnesium for 60 minutes at 37°C, and washed 3x with the same buffer as above, then applied on the glass slide for imaging. Images of plasma clots obtained from fluorescence microscope Olympus ΓΧ81 analysis, under lOOx magnification are shown in Fig. 9A. Images of plasma clots obtained using an rVIS 50 imaging system are shown in Fig. 9B. Wavelengths used for measurement: excitation Cy5.5 BkG (580-610 nm), emission Cy5.5 (695-770 nm). For background subtraction: excitation GFP (445-490 nm), emission Cy5.5 (695-770 nm). Exposure time: 1 minute. Arrows in Fig. 9B point the localization of the clot. [052] Fig. 9 shows the specificity of interaction of F- DP-Bt with clot generated from rat blood plasma by thrombin (1 U/ml). Analysis of clot under fluorescence microscope (Fig. 9A) revealed that F-NDP-Bt accumulated on the surface of the thrombus to a high extent, although this accumulation was not evenly distributed. Fluorescence microscopy imaging identified areas with high green fluorescence intensity (represented by bright spots in the black and white image), which may indicate zones of the condensation of activated platelets. Fluorescence live imaging system (IVIS 50) also exhibited binding of F-NDP-Bt to the plasma clot (Fig. 9B). However, in this system the near infrared (NIR) detection was set up, based on the optimization performed as presented in Fig. 1. Control nanoparticles, containing coupled BSA to the surface (F-NDP-BSA), interacted with the clot to a negligible level in both imaging assays. Detection of F-NDP-Bt by NIR suggested a usefulness of functionally active F-NDP-Bt for imaging in living organisms, because emission wavelength is localized within an "optical therapeutic window" (600 - 1300 nm).

[053] Therefore, detection of F-NDP-Bt was performed in a rat model for verification of that hypothesis. The results are shown in Fig. 10. The skin areas of observation fields were prepared for implantation by hair shaving. The incision was made by scalpel and vessels were inserted under the skin of dead rats. The dead rats were placed in IVIS 50 Imaging System, and measurement of fluorescence under NIR spectrum was performed. Wavelengths used for measurement: excitation Cy5.5 BkG (580-610 nm), emission Cy5.5 (695-770 nm). For background subtraction: excitation GFP (445-490 nm), emission Cy5.5 (695-770 nm). A representative image of the implanted glass capillaries filled with F-NDP-Bt (4 mg/ml) or PBS (control) are shown in Fig. 10A. Exposure time was 5 seconds. Figure 10B shows an image of a rat aorta filled with F-NDP-Bt. The rat aorta was dissected from a euthanized female rat, washed with PBS to remove residues of coagulated blood, and filled with 300 μΐ of F-NDP-Bt suspension (2 mg/ml) in PBS. The aorta was secured from both ends by knots of surgical sutures. Exposure time was 1 minute. Fig. 10A demonstrates NIR imaging of F- NDP-Bt, experimentally implanted under rat skin. Suspensions of F-NDP-Bt were infused into glass capillaries and into dissected rat aorta (Fig. 10B), before subcutaneous

implantation. Clear images showed precise localization of both artificial (capillary) and natural (aorta) vessels in the rats.

[054] As should be evident, in exemplary embodiments, the present invention provides a diagnostic agent for detection or imaging of thrombotic events in a human or non-human animal, where the agent comprises a fluorescent nanodiamond particle chemically bonded to disintegrin Bitistatin (Bt). In certain embodiments, the fluorescent nanodiamond particle and the Bt are covalently bonded. The diagnostic agent is fluorescent as a result of an intrinsic property of the nanodiamond particle. The diagnostic agent emits a detectable

electromagnetic signal when excited by an electromagnetic source.

[055] Additional exemplary embodiments relate to a method for diagnosis or prognosis of a thrombo-embolic event. In these embodiments, the method comprises: administering to a subject suspected of having suffered from or suspected of being at risk of, a thromboembolic event, a diagnostically effective amount of the diagnostic agent of the invention; allowing sufficient time for the diagnostic agent to localize to the site(s) of thrombus; and detecting the diagnostic agent by detecting fluorescence emission of the diagnostic agent. The method can be practiced as a method of detection of activated platelets and/or a method of detecting clots or clot formation in subjects.

[056] Another exemplary embodiment of the invention relates to a kit. The kit includes the diagnostic agent of the invention in packaged form suitable for distribution, delivery, and/or storage for use in a diagnostic method for detection of a thrombus. The packaged form includes a suitable material for distribution, delivery, and/or storage of the diagnostic agent. In certain embodiments, the kit further comprises, in packaged combination, one or more reagents or devices for administration of the diagnostic agent of the invention to a subject. The kit can also include a device that emits excitation energy for the diagnostic agent, and preferable further includes a detector for detection of emission response from the diagnostic agent. In a particular exemplary embodiment, the device is a hand-held device.

[057] It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. REFERENCES

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