Title of Invention: A Multimodal Fluorescent Probe

Technical Field The present invention relates to a nanoparticle comprising a fluorophore, an amphiphilic copolymer and a cyclic chelating reagent for detecting vascular integrity and leakage. The present invention also relates to a method of preparing such a nanoparticle, the use of such a nanoparticle as well as a method for in vivo imaging of blood vessels comprising such a nanoparticle. Background Art

The blood vessels serve as a physical barrier and function in a highly responsive and dynamic manner, allowing the regulation and rapid control of the transport rate of solutes, macromolecules and cells such as leukocytes between blood and tissue. The regulation of vascular permeability is a highly coordinated process that is fundamental for the maintenance of homeostasis in the body. While this regulation is important for the initiation and resolution of immune responses as well as for tissue repair, the loss of barrier function and increased vascular leakage are often associated with the pathogenesis of inflammatory diseases. It is therefore important to be able to image such phenomenon both in vivo and ex vivo.

Blood endothelium in the brain forms the blood brain barrier (BBB), which is a highly specialized interface between the vascular space and the brain parenchyma, governing the entry of substances into the central nervous system (CNS). Thus, the integrity and proper functionality of the BBB is crucial for maintaining the health of the CNS, and the loss of barrier function or malfunction of the BBB is commonly associated with neurological disorders. While this is an important field of research, it has been hampered by the lack of suitable tools and methods for studying brain barrier functionality.

Traditionally, organic dyes such as Evans Blue are the most commonly applied reagents to assess BBB integrity. For example, Evans Blue is intravenously injected into the animals prior to the end of the experiments, followed by dye extraction of the harvested brain using organic solvents. Consequently, the absorbance of Evans Blue is measured as a quantitation of the extent of vascular leakage in the brain. However, this approach is an end-point assay that requires the experimental animals to be sacrificed and brain tissues to be destructed, making it difficult to precisely localize haemorrhage position and actively track the changes of BBB permeability over time. Recently, using multiphoton microscopy, Evans Blue has been used for the qualitative visualization of blood vascular networks in vivo. However, Evans Blue fluorescence characteristics vary widely depending on their extent of binding to albumins in the blood plasma and even the type of buffer they are in. There remain limitations and pitfalls that hamper the progression of BBB assessment, which include the interference of readily diffusible free Evans Blue dye that is not observable by fluorescence imaging but detectable by end-point absorbance quantification, the binding specificity of Evans Blue (plasma proteins vs tissue) and its potential long term toxicity, which severely confound the interpretation of the results.

Although there are other known contrast agents for imaging blood vessels, they are known to have drawbacks such as low retention in blood, leakage from blood vessels, weak fluorescence signal, non-ideal spectral properties, and low sensitivity. Moreover, such contrast agents often only allow single platform analysis, and thus may only be capable of obtaining limited information.

There is therefore a need to provide a nanoparticle that overcomes or at least ameliorates, one or more of the disadvantages described above.

Summary

In an aspect, there is provided a nanoparticle comprising: a fluorophore; an amphiphilic copolymer; and a cyclic chelating agent, wherein the fluorophore and amphiphilic copolymer form a nanodot core, and wherein the cyclic chelating agent is conjugated onto the nanodot core.

The inventors of the present application have surprisingly found that the nanoparticle defined above may be advantageously suited for use as vascular imaging agents. In particular, the disclosed nanoparticles may demonstrate high fluorescence and gadolinium loading, making them easier to image using fluorescence and/or magnetic resonance imaging. The nanoparticles may have advantageously high quantum yield and photostability during fluorescence imaging, which may be comparable if not superior to known vascular imaging agents such as Evans Blue. Additionally, the nanoparticles defined above may emit strong visible fluorescence from low- energy irradiation in the near infrared region, minimizing the absorbance of biosubstrates. This may be beneficial for high resolution intravenous imaging. The disclosed nanoparticle may also advantageously be water-soluble, and have a high two-photon absorption (TP A) cross section which may be desirable for high resolution imaging. The nanoparticles may therefore be able to provide real-time fluorescent visualization of blood vessel integrity and leakage in vivo as well as sensitive end-point quantitation of vascular leakage. More advantageously, the presence of the gadolinium may facilitate vascular imaging by magnetic resonance imaging. Further, advantageously, any leakage in the vasculature may be quantified by measuring the level of gadolinium in the blood vessel or organ.

Importantly, the fluorophore may be capable of aggregation induced emission (AIE). Advantageously, in contrast to conventional fluorescent dyes suffering from aggregation-caused quenching (ACQ) effects, AIE fluorophores may be non-emissive in molecular state but may instead by induced to emit strong fluorescence in their aggregated state, facilitating the fabrication of ultrabright nanoparticles. Further advantageously, the amphiphilic copolymer may be used as a matrix to load the fluorophore so that a high density of the fluorophore may be achieved which in turn may result in high fluorescence due to AIE. In addition to their significantly high photostability, the AIE fluorophores may have excellent colloidal stability in aqueous media and biological buffers, making them highly promising candidates as flurorescent trackers in vivo. Importantly, the use of a cyclic chelating agent in the disclosed nanoparticle may facilitate loading of higher levels of gadolinium on the nanoparticle, in comparison to when non-cyclic chelating agents such as diethylenetriaminepentaacetic acid (DTP A) or its derivative is used to chelate the gadolinium. Further advantageously, the gadolinium chelation may show negligible effects on the optical properties of the nanoparticles. The nanoparticles may be encapsulated by an outer layer of amphiphilic copolymer, which may provide both colloidal and biological stability, as they would normally prevent the interactions of the organic fluorophores with the host biological molecules, such as receptors present on the cells of the host immune system, making them inert inside blood vessels. As such, the nanoparticles may display very high signal to background ratios for the purpose of detecting changes in vascular integrity.

Advantageously, the nanoparticles may be intensely bright under one -photon or multiphoton excitations. The nanoparticles may not undergo quenching or photobleaching easily, and may not show fluorescence intermittency. Advantageously, AIE molecules having long wavelength emission in the far-red/near-infrared (FR/NIR) region were selected for making the nanoparticles to ensure it further reduces light scattering in biological tissues and increases penetration depths for multiphoton microscopy. In another aspect, there is provided a method for preparing the nanoparticle as defined above, comprising the steps of: contacting a fluorophore with an amphiphilic polymer to form a nanodot core; and conjugating the cyclic chelating agent onto the nanodot core. Advantageously, the method may provide a facile and cost-effective method for preparing a nanoparticle as defined above.

In another aspect, there is provided the use of the nanoparticles as defined above for vascular imaging.

In use, the nanoparticles may exhibit stable and bright far-red/near infrared (FR/NIR) emission under one-photon and multi-photon excitation with excellent colloidal stability in biological environments. Advantageously, the nanoparticle may be used as tracers to systematically characterize the onset, progression and duration of the vascular leakage in various inflammatory diseases. This nanoparticle may allow cross-platform measurement of vascular integrity and leakage by intravital multiphoton microscopy (IV-MPM), fluorescence reflectance imaging (FRI) and magnetic resonance imaging (MRI) techniques.

Further advantageously, the high stability of the nanoparticles towards different sample treatment protocols, such as fixation and permeabilization of the harvested tissues further makes them a reliable tool to assess the consistence between real-time imaging information and end- point post-processed information. In contrast, Evans Blue (molecular weight approximately 1 kDa) requires binding to proteins to achieve fluorescence, which can significantly complicate the interpretation of results. In the disclosed nanoparticles, there is a choice of using the fluorescence from the AIE cores or the inert inorganic Gd ³⁺ ions as a stable dual modal indicator, which may greatly increase the detection reliability even after multiple downstream sample processing protocols.

In another aspect, there is provided a method of in vivo imaging of blood vessels in a subject, comprising a step of administering to the subject a nanoparticle as defined above; and exposing the subject to (i) visible light to elicit a fluorescence signal, (ii) radio waves to elicit magnetic resonance, or a combination thereof.

The nanoparticle as defined herein may advantageously be able to detect loss of vascular integrity in a human or animal subject. Advantageously, the nanoparticle may be multimodal, allowing visualization of the loss of vascular integrity as well as vascular leakage in vivo or ex vivo, by fluorescence imaging and/or magnetic resonance imaging. More advantageously, the nanoparticle may allow ex vivo measurements of vascular leakage by measuring the level of gadolinium in the blood vessel or organ. Advantageously, the nanoparticle may be applied topically or intravenously, depending on the application. Importantly, the nanoparticle may facilitate non-invasive imaging of vasculature in a human or animal subject.

Advantageously, when injected intravenously into mice, not only may the nanoparticles exhibit intense FR/NIR fluorescence suitable for in vivo multiphoton fluorescence imaging and ex vivo whole -brain imaging of blood vascular networks and haemorrhages, but they may also be used for the precise quantification of vascular leakage by measuring the gadolinium contend using inductively-coupled plasma mass spectrometry (ICP-MS). More advantageously, the nanoparticles may serve as an excellent predictor for vascular leakage and severity of experimental cerebral malaria (ECM), and may provide a conveniently broad spectrum of interrogation modalities for the visualization of blood vessel integrity, leakage and localization of inflammation in the brain.

In addition to their advantageous fluorescence properties, in resting conditions, the nanoparticles may flow smoothly inside blood vessels, and display minimal vascular leakage in tissues. Yet, during skin inflammation or ECM, whereby vascular integrity may be compromised, the nanoparticles may leak into the tissues, and rapidly aggregated around the affected blood vessels, forming distinctly brighter patches of signal that may remain stably retained for extended periods of time, even after the subject is perfused and treated with different buffers. Despite their persistence in the inflamed regions, the nanoparticles may be removed relatively rapidly from the circulation, but may still allow hours of in vivo real time imaging by virtue of their intense brightness. Hence, the nanoparticles may be uniquely suited for both in vivo realtime and deep tissue multiphoton imaging, as well as other ex vivo fluorescence microscopy techniques, including optically cleared brain for light sheet ultramicroscope. The end-point Gd ³⁺ content measurement may further provide sensitive quantification of changes in the vascular integrity.

The disclosed nanoparticle may advantageously exhibit low toxicity and may be safely administered to humans or animal subjects for enhancing imaging resolution. In addition, the robust chemical- and photo-stability of the nanoparticles may allow for prolonged monitoring of its biodistribution.

Definitions

The following words and terms used herein shall have the meaning indicated:

The terms "fluorophore" and "fluorogen" may be used interchangeably for the purpose of this disclosure, and generally refer to a chemical compound that can re-emit visible light upon excitation by visible light.

The term "aggregation-induced emission" for the purpose of this disclosure refers to a phenomenon whereby the photoluminescence efficiency or the quantum yield of a fluorophore is higher when the fluorophores are aggregated or in crystalline form compared to when the fluorophores are in solution phase.

"Acyl" means an R-C(=0)- group in which the R group may be an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl group as defined herein. Examples of acyl include acetyl, benzoyl and amino acid derived aminoacyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon.

"Acylamino" means an R-C(=0)-NH- group in which the R group may be an alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.

"Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a Q-C50 alkyl, preferably a Q-Q2 alkyl, more preferably a Q-Qo alkyl, most preferably Q-Ce unless otherwise noted. Examples of suitable straight and branched Ci-Ce alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like. The group may be a terminal group or a bridging group.

"Alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. The group may be a terminal group or a bridging group.

"Alkynyl" as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl. The group may be a terminal group or a bridging group.

"Amino" refers to groups of the form -NR _a R _b wherein R _a and R _b are individually selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl groups.

"Aminoalkyl" means an NH ₂ -alkyl- group in which the alkyl group is as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.

"Halogen" represents chlorine, fluorine, bromine or iodine. "Alkyloxy" refers to an alkyl-O- group in which alkyl is as defined herein. Preferably the alkyloxy is a Ci-Cealkyloxy. Examples include, but are not limited to, methoxy and ethoxy. The group may be a terminal group or a bridging group.

"Cycloalkyl" refers to a saturated monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane. A cycloalkyl group typically is a C ₃ -C ₁₂ alkyl group. The group may be a terminal group or a bridging group.

"Heterocyclic" refers to a saturated or unsaturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered.

"Heterocycloalkyl" refers to a saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1,3- diazapane, 1 ,4-diazapane, 1 ,4-oxazepane, and 1,4-oxathiapane. A heterocycloalkyl group typically is a C ₂ -Cn heterocycloalkyl group. A heterocycloalkyl group may comprise 3 to 8 ring atoms. A heterocycloalkyl group may comprise 1 to 3 heteroatoms independently selected from the group consisting of N, O and S. The group may be a terminal group or a bridging group.

"Aryl" as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C5 7 cycloalkyl or C5 7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl. The group may be a terminal group or a bridging group. Typically an aryl group is a Ce-Qs aryl group.

"Heteroaryl" either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, lH-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-, 3- or 4- pyridyl, 2-, 3-, 4-, 5-, or 8- quinolyl, 1-, 3-, 4-, or 5- isoquinolinyl 1-, 2-, or 3- indolyl, and 2-, or 3-thienyl. A heteroaryl group is typically a C Qs heteroaryl group. A heteroaryl group may comprise 3 to 8 ring atoms. A heteroaryl group may comprise 1 to 3 heteroatoms independently selected from the group consisting of N, O and S. The group may be a terminal group or a bridging group. The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or unless otherwise specified, may be substituted with one or more groups independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl, optionally substituted amine, optionally substituted acylamino, optionally substituted alkyloxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or any mixture thereof.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention. Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5 , from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Detailed Disclosure of Optional Embodiments

A multimodal hybrid nanoparticle probe, which allows direct visualization of the loss of vascular integrity as well as leakage in vivo has been developed. This hybrid nanoparticle allows cross-platform measurement of vascular integrity and leakage by intravital multiphoton microscopy (IV-MPM) and fluorescence reflectance imaging (FRI). Moreover, this hybrid nanoparticle is functionalized with gadolinium (Gd) that can be detected by magnetic resonance imaging (MRI), as well as enable ex-vivo measurement of vascular leakage by measuring gadolinium levels.

There is provided a nanoparticle comprising: a fluorophore; an amphiphilic copolymer; and a cyclic chelating agent, wherein the fluorophore and amphiphilic copolymer form a nanodot core, and wherein the cyclic chelating agent is conjugated onto the nanodot core.

The nanoparticle may further comprise a gadolinium ion (Gd ³⁺ ) chelated by the cyclic chelated agent.

The fluorophore may be capable of aggregation-induced emission (AIE). The fluorophore may be planar and conjugated.

The fluorophore may be selected from the group consisting of 2,3-bis[4- (diphenylamino)phenyl]fumaronitrile (TPAFN), 2,3-bis(4-(phenyl(4-(l ,2,2- triphenylvinyl)phenyl)amino)phenyl) fumaronitrile (TPETPAFN), tetraphenylethylene (TPE), tetraphenylpyrazine (TPP), distyrylanthracene (DSA), Boron Ketoiminate, Boron Diiminate- Based Polymers, and tetraphenylpyrrole and any mixture thereof.

The fluorophore may be present in the range of about 25 wt% to about 35 wt% of the nanoparticle, about 25 wt% to about 27.5 wt%, about 25 wt% to about 30 wt%, about 25 wt% to about 32.5 wt%, about 27.5 wt% to about 30 wt%, about 27.5 wt% to about 32.5 wt%, about 27.5 wt% to about 35 wt%, about 30 wt% to about 32.5 wt%, about 30 wt% to about 35 wt%, about 32.5 wt% to about 35 wt% of the nanoparticle.

The amphiphilic copolymer may be a functionalized l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE). Advantageously, the amphiphilic copolymer may provide a hydrophobic end for association with the fluorophore and a hydrophilic end for conjugation with the cyclic chelating agent. Further advantageously, the amphiphilic copolymer may act as a matrix for loading the fluorophores to form a nanodot core. By the amphiphilic copolymer acting as a matrix for loading the fluorophores and consequently bringing the fluorophores into close proximity with each other (i.e. causing aggregation), the amphiphilic copolymer may facilitate the enhancement of aggregation-induced emission.

The l,2-distearoyl-sn-glycero-3-phosphoethanolamine may be functionalised with polyethylene glycol repeat units. The l ,2-distearoyl-sn-glycero-3-phosphoethanolamine may be functionalised with polyethylene glycol repeat units in the range of about 10 to about 500, about 10 to about 20, about 10 to about 20, about 10 to about 50, about 10 to about 100, about 10 to about 200, about 20 to about 50, about 20 to about 100, about 20 to about 200, about 20 to about 500, about 50 to about 100, about 50 to about 200, about 50 to about 500, about 100 to about 200, about 100 to about 200 or about 200 to about 500 .

The polyethylene glycol units may be capped with a terminal group selected from the group consisting of amine, maleimide, folate, carboxylic acid, ester, halide, azide, hydroxyl, alkene, alkyne, and sulfhydryl.

The functionalized l,2-distearoyl-sn-glycero-3-phosphoethanolamine may be selected from 1,2- distearoyl-5 ^, «-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) -2000], 1,2- distearoyl-5 ^, «-glycero-3-phosphoethanolamine-N-[maleimide(polyethy lene glycol) -2000], or a mixture thereof. The cyclic chelating agent may comprise an optionally substituted heterocyclic ring.

The heterocyclic ring may be selected from the group consisting of 1,4,7,10- tetraazacyclododecane (cyclen), 1,4,8,11-tetraazacyclotetradecane (cyclam), 1,4,7- triazacyclononane, 1,5,9-triazacyclododecane, l,7-dioxa-4,10-diazacyclododecane, and any mixture thereof. The heterocyclic ring may be optionally substituted with a functional group selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl, optionally substituted amine, optionally substituted acylamino, optionally substituted alkyloxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and any mixture thereof,

The heterocyclic ring may optionally substituted with a functional group selected from the group consisting of hydrogen, halogen, carboxylic acid, acetic acid, 2-(aminoethyl)ethanamide, methyl acetate, methyl propanoate, ethyl acetate, ethyl propanoate, isopropyl acetate, isopropyl propanoate, tert-butyl acetate, tert-butylpropanoate, ester, amide, propiolamide, acetic acid N- hydroxysuccinimide (NHS) ester, and any mixture thereof.

The cyclic chelating agent may be an optionally substituted 1,4,7, 10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOT A).

The 1,4,7, 10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), before conjugation to the nanodot core, may be optionally substituted with amine reactive groups, such as for instance an ester of N-hydroxysuccinimide (NHS) or an imidoester, or with sulfhydrol -reactive groups, such as for instance maleimide, haloacetlyl or pyridyldisulfide. The 1,4,7, 10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), before conjugation to the nanodot core, may be optionally substituted with amine reactive groups, such as for instance an ester of N-hydroxysuccinimide (NHS) or an imidoester, or with sulfhydrol -reactive groups, such as for instance maleimide, haloacetlyl or pyridyldisulfide, at one or more of the pendant acetic acid groups. A linker may or may not be present between any one of the acetic acid groups and the amine reactive groups, such as for instance an ester of N-hydroxysuccinimide (NHS) or an imidoester, or with sulfhydrol-reactive groups, such as for instance maleimide, haloacetlyl or pyridyldisulfide. If present, the linker may be a Ci to Ci ₀ alkyl.

The cyclic chelating, agent, before conjugation to the nanodot core, may be 2,2',2"-(10-(2-((2,5- dioxopyrrolidin-l-yl)oxy)-2-oxoethyl)-l,4,7,10-tetraazacyclo dodecane-l,4,7-triyl)triacetic acid.

The particle may comprise about 1 to about 10,000 cyclic chelating agents. The particle may comprise about 1 to about 2, about 1 to about 5, about 1 to about 10, about 1 to about 20, about 1 to about 50, about 1 to about 100, about 1 to about 200, about 1 to about 500, about 1 to about 1000, about 1 to about 2000, about 1 to about 5000, about 2 to about 5, about 2 to about 10, about 2 to about 20, about 2 to about 50, about 2 to about 100, about 2 to about 200, about 2 to about 500, about 2 to about 1000, about 2 to about 2000, about 2 to about 5000, about 2 to about 10000, about 5 to about 10, about 5 to about 20, about 5 to about 50, about 5 to about 100, about 5 to about 200, about 5 to about 500, about 5 to about 1000, about 5 to about 2000, about 5 to about 5000, about 5 to about 10000, about 10 to about 20, about 10 to about 50, about 10 to about 100, about 10 to about 200, about 10 to about 500, about 10 to about 1000, about 10 to about 2000, about 10 to about 5000, about 10 to about 10000, about 20 to about 50, about 20 to about 100, about 20 to about 200, about 20 to about 500, about 20 to about 1000, about 20 to about 2000, about 20 to about 5000, about 20 to about 10000, about 50 to about 100, about 50 to about 200, about 50 to about 500, about 50 to about 1000, about 50 to about 2000, about 50 to about 5000, about 50 to about 10000, about 100 to about 200, about 100 to about 200, about 100 to about 500, about 100 to about 1000, about 100 to about 2000, about 100 to about 5000, about 100 to about 10000, about 200 to about 500, about 200 to about 1000, about 200 to about 2000, about 200 to about 5000, about 200 to about 10000, about 500 to about 1000, about 500 to about 2000, about 500 to about 5000, about 500 to about 10000, about 1000 to about 2000, about 1000 to about 5000, about 1000 to about 10000, about 2000 to about 5000, about 2000 to about 10000 or about 5000 to about 10000.

The cyclic chelating agent may be conjugated on the surface of the nanodot core.

The gadolinium may be present in the range of about 2 wt% to about 5 wt% of the nanoparticle. The gadolinium may be present in the range of about 2 wt% to about 3 wt%, about 2 wt% to about 4 wt%, about 3 wt% to about 4 wt%, about 3 wt% to about 5 wt%, or about 4 wt% to about 5 wt% of the nanoparticle.

The nanoparticle may have an average hydrodynamic size in the range of about 35 nm to about 42 nm. The nanoparticle may have an average hydrodynamic size in the range of about 35 nm to about 37 nm, about 35 nm to about 37 nm, about 35 nm to about 40 nm, about 37 nm to about 40 nm, about 37 nm to about 42 nm or about 40 nm to about 42 nm.

There is also provided a method for preparing the nanoparticle as defined above, comprising the steps of: contacting a fluorophore with an amphiphilic copolymer to form a nanodot core; and conjugating the cyclic chelating agent onto the nanodot core. The method may further comprise the step of chelating gadolinium ion (Gd ³⁺ ) with the cyclic chelated agent.

The amphiphilic copolymer may be l,2-distearoyl-OT-glycero-3-phosphoemanolamine-N- [amino(polyethylene glycol)-2000] .

The cyclic chelating agent may be 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-l-yl)oxy)-2-oxoethyl)- l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid.

The conjugating step may be done by reacting the N-hydroxysuccinimide (NHS) group of 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-l-yl)oxy)-2-oxoethyl)-l ,4,7,10-tetraazacyclododecane- 1 ,4,7-triyl)triacetic acid with an amino group of l,2-distearoyl-s7i-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] . It would be known to a person skilled in the art that any combination of complementary reactive groups on the amphiphilic copolymer and the cyclic chelating agent may be used to conjugate the cyclic chelating agent onto the nanodot core. For example, if the amphiphilic copolymer is functionalized with maleimide (i.e. if the amphiphilic copolymer is l,2-distearoyl-s7i-glycero-3- phosphoethanolamine-N-[maleimide(polyethylene glycol) -2000]), then the cyclic chelating agent may comprise a thiol group so that upon reaction, a stable carbon-sulfur bond is formed. Another example would be if the amphiphilic copolymer is functionalized with amine, then the cyclic chelating agent may comprise a thiocyanate group, so that upon reaction, a stable carbon- nitrogen is formed.

There is also provided the use of the nanoparticles as defined above for vascular imaging. The imaging may be fluorescence imaging, magnetic resonance imaging or a combination thereof.

The fluorescence imaging may be multi-photon fluorescence imaging or fluorescence reflectance imaging. The imaging may be performed in vivo or ex vivo.

The imaging may be for detection of vascular integrity and leakage.

There is also provided a method of in vivo imaging of blood vessels in a subject, comprising a step of administering to the subject a nanoparticle as defined above; and exposing the subject to (i) visible light to elicit a fluorescence signal, (ii) radio waves to elicit magnetic resonance, or a combination thereof.

The subject may be a human or an animal.

The nanoparticle may be administered topically or intravenously.

There is also provided the use of a nanoparticle as defined in any one of the preceding claims for the preparation of a vascular imaging agent or composition. Brief Description of Drawings

The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention. Fig. 1

[Fig. 1] is a schematic representation of the formation of AIE-Gd dots and the chemical structures of DSPE-PEG ₂₀ oo-NH ₂ , TPETPAFN and DOTA.

Fig. 2

[Fig. 2] refers to A) particle size distribution of AIE-Gd dots in aqueous solution measured by dynamic light scattering (DLS), B) HR-TEM image of AIE-Gd dots, insert is an enlarged TEM image of a single AIE-Gd dot, C) absorption and emission spectra of AIE-Gd dots aqueous solution, before and after Gd chelation, the emission spectrum was obtained upon excitation at 512 nm, and D) hydrodynamic size (measured by dynamic light scattering) and emission intensity changes of AIE-Gd dots upon continuous incubation in l x PBS buffer for 10 days. Fig. 3

[Fig. 3] refers to A) Luminescence image, B) luminescence intensity histogram, C) representative luminescence intensity time-traces, and D) Two-photon absorption (TPA) cross section spectra of AIE-Gd dot aqueous solution and Evan blue dye in 1 x PBS buffer in the presence of albumin.

Fig. 4

[Fig. 4] refers to two-photon images of ear skin vessels acquired at 3 and 0 hours post inflammation (treated with croton oil) or without inflammation (control). (A) shows images when AIE-Gd dots or Evans Blue were intravenously injected at 3 hours of inflammation induction and just prior to imaging. (Al) shows AIE-Gd dots in the control, (A2) shows AIE_Gd dots in the inflammation model, (A3) shows Evans Blue in the control, and (A4) shows Evans Blue in the inflammation model. (B) shows images when AIE-Gd dots of Evans Blue were intravenously injected at 0 hours of inflammation induction and imaged 3 hours later. (Bl) shows AIE-Gd dots in the control, (B2) shows AIE-Gd dots in the inflammation model, (B3) shows Evans Blue in the control, and (B4) shows Evans Blue in the inflammation model. Sclae bar represents 50 μπι.

Fig. 5 [Fig. 5] refers to A) Vascular leakage detection by measurement of Evans Blue dye absorbance comparing mouse ears treated with croton oil (inflamed) with control ears (healthy). B) Vascular leakage detection by using AIE-Gd dots, showing superior signal to background ratios.

Fig. 6

[Fig. 6] refers to A) Comparison of vascular leakage visualized by AIE-Gd dots in malaria parasite-infected mouse brains (A2) and uninfected mouse brains (Al) at day 7 after infection where cerebral malaria symptoms peak. B) Measured levels of Gd in total mouse brains, comparing malaria-infected mouse brains (day 7) and uninfected.

Fig. 7

[Fig. 7] refers to (A) naive C57BL/6 female mice and (B, C) ECM mice with neurological symptoms that were intravenously injected with (Al, Bl) AIE-Gd dots or (CI) Evans Blue 3 hours prior to mouse sacrifice and brain harvesting. (Dl) shows images of a control (uninfected) mouse brain stained with Evans Blue. A2, B2, C2 and D2 are enlarged images, respectively. The mouse brains were cleared with serial dehydration in methanol grades (50%, 70%, 95%, and 100%), followed by lipid removal by benzyl alcohol/benzyl benzoate (BABB) solution, prior to ultramicroscopic imaging by light sheet microscopy.

Fig. 8

[Fig. 8] refers to (A) IVIS images of mouse brains with different ECM parasitemia levels (Al : 2.54, A2: 3.30, A3: 3.34 and A4: 5.46). (B) Correlation of Luciferase radiance with parasitemia levels. (C) Multiphoton fluorescence images of mice brains with different ECM parasitemia levels (CI : 0.00, C2: 2.29, C3: 5.06, C4: 6.77). (D) Correlation of AIE-Gd dot fluorescence intensity with parasitemia levels. (E) Correlation of Evans Blue absorbance with parasitemia levels.

Fig. 9

[Fig. 9] refers to (A) Correlation of Gd3+ level with parasitemia levels. (B) Distribution of Gd ³⁺ levels in mouse brain among the different groups of mice. Naive: uninfected, Non-ECM: no neurological symptoms, and ECM: display prominent neurological symptoms indicative of BBB disruption. Student's t test was utilized for statistical contrast, ** refers P < 0.01, *** refers to P < 0.001.

Examples Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Example 1: Materials and Methods

Materials l,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(pol yethylene glycol)-2000] (DSPE-PEG2000-NH2) was a commercial product of Avanti Polar Lipids, Inc (US). Gadolinium(III) chloride hexahydrate and N,N-dimethylformamide (DMF) was purchased from Sigma-Aldrich (St. Louis, Missouri, United States of America). 2,2',2"-(10-(2-((2,5- dioxopyrrolidin-l-yl)oxy)-2-oxoethyl)-l,4,7,10-tetraazacyclo dodecane-l,4,7-triyl)triacetic acid (DOTA-NHS-ester) was purchased from CheMatech (France). Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under dry nitrogen immediately prior to use. All reactions and manipulations were carried out under nitrogen gas with the use of standard inert atmosphere. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). Ultrapure grade lOxphosphate buffered saline (PBS) buffer with pH = 7.4 was purchased from 1st BASE Singapore.

Methods

Characterization of AIE-Gd dots

Uv-vis absorption spectra were recorded on a Shimadzu UV-1700 spectrometer. Emission spectra were recorded on a Perkin-Elmer LS 55 spectrofluorometer. Hydrodynamic size and size distribution were measured by dynamic light scattering with a particle size analyser (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at room temperature, and atomic force microscopy (AFM). The morphology was studied by transmission electron microscopy (TEM, JEM-2010F, JEOL, Japan). Single particle fluorescence imaging was performed according to our previous reports. Wavelength Scan

The two-photon absorption (TPA) spectrum of AIE-Gd dots in aqueous solution was measured with a TPIF microscope with a tunable Ti: sapphire laser, using Rhodamine 6G in methanol as the standard. As a comparison, Evans Blue, a commonly used blood vascular imaging contrast and inflammation detection probe, was also selected for the TPA measurement. The emission signals of AIE-Gd dots and Evans Blue were collected upon excitation from 800 to 920 nm.

In vivo skin blood vasculature imaging

Prior to imaging, right ears of mice were pre -treated with croton oil with different lengths of time to induce inflammation with different severities. Hair was removed using Veet hair removal cream (Reckitt Benckiser), before placing the ear on a heating platform that maintains the physiological temperature of the ear at 35 °C. For two-photon fluorescence imaging experiments, mice were anesthetized (150 mg/kg ketamine and 10 mg/kg xylazine) and placed on a heating pad to maintain a core body temperature of 37 °C throughout each imaging procedure. AIE-Gd dots (80 nM, 4 μΕ/gram of mouse weight) or Evans Blue (Sigma) (20 mg/mL, 4 μΕ/g mouse bodyweight) were administered via retro-orbital injection at designated timings prior to imaging. All procedures were performed under the institution's IACUC (Institutional Animal Care and Use Committee) guidelines. A TriM Scope II single -beam two- photon upright microscope (LaVision BioTec) with a tuneable 680-1080 nm laser (Coherent) was used to acquire the images. Both AIE-Gd dots and Evans Blue were visualized using excitation wavelength of 950 nm and emission bandpass filters of 655/40 (Semrock).

Whole brain imaging

C57BL/6 female mice 5 to 6 weeks old were infected intraperitoneally (i.p.) with 10 ⁶ infected red blood cells (iRBCs) with P. berghei ANKA (231cll) (PbA) expressing luciferase and green fluorescent protein (GFP) controlled by efl-a promoter parasites. These mice typically develop neurological symptoms (paralysis, ataxia, deviation of the head and convulsion and/or coma) at day 7-8 after infection. AIE-Gd dots (80 nM, 4 μΕ/g mouse bodyweight) or Evans Blue (Sigma) (20 mg/mL, 4 μΕ/g mouse bodyweight) was injected intravenously under isoflurane anesthesia. The mice were then sacrificed 3 hours later, and the brains were harvested. Brains were firstly cleared using serial dehydration in methanol with grades of 50%, 70%, 95%, and 100% (2 h for every methanol grade). The brains were then followed by overnight incubation with 50% benzyl alcohol/benzyl benzoate solution (BABB) solution and 2 h incubation in 100% BABB solution. The cleared brains were imaged using light sheet ultramicroscope I (La Vision Biotec) equipped with the Olympus Zoombody MVX10 with 2XC and Imager 3QE as the detector using the Supercontinuum white light laser for excitation. For imaging AIE-Gd dots, bandpass filters used were 640/40 nm (Semrock) for excitation and 685/40 nm (Semrock) for emission. For Evans Blue, excitation bandpass was 470/40 nm (Semrock) and emission bandpass was 620/60 nm (Semrock), respectively. Bioluminescence imaging

To quantify the amount of parasite accumulation in the brain, ex vivo imaging was performed on day 7 post-infection on infected mice anesthetized with ketamine (150mg/kg) / xylazine (lOmg/kg) for 10 minutes and 200 μΐ of luciferin was injected intraperitoneally and allowed for circulation for 2 minutes. Intra-cardiac perfusion was performed to drain blood out from the circulation. Brains were removed and placed on the petri dish and bioluminescence imaging was acquired with a 10 cm FOV and exposure times of 5, 10, 30, and 60 s. Uninfected mouse injected with luciferin was used for background subtraction. Images with the highest bioluminescence signal, but lower than 62000 units, was analyzed using Living Imaging 3.0 by drawing region of interest (ROI) to obtain an average radiance (p/s/cm ² /sr).

Two-photon fluorescence imaging of BBB

C57BL/6 female with neurological symptoms were tail-bled to measure their levels of blood parasitemia. AIE-Gd dots (80 nM, 4 μΙ-Vg mouse bodyweight) or Evans Blue (20 mg/mL, 4 μΕ/g mouse bodyweight) were intravenously injected via retro-orbital injection. The mice were then sacrificed 3 hours later and perfused. The harvested intact brains were exposed to luciferin for ex vivo imaging (IVIS, Perkin Elmer) as previously described to measure the amount of parasite sequestered. The brains were further imaged by TriM Scope II single -beam two-photon upright microscope (LaVision BioTec). The fluorescence intensities were access by Image J software. Quantitative analysis

The fluorescence intensity of brain or ear tissues was accessed by Image J software. After fluorescence imaging, the brain or ear tissues were collected, displayed in DMF for 2 days for Evans Blue dye extraction. The contents of Evans Blue in each ear or brain were determined by its absorbance at 620 nm. For Gd ³⁺ content detection, the obtained brain or ear tissues were nitrified by 70% nitric acid and heating. After ear or brain tissues were completely decomposed, the remaining residues were dissolved in 5% nitric acid, and analyzed by ICP-MS (Agilent Technologies, CA, US). The contents of Gd ³⁺ in ear or brain tissues were then quantitatively determined by a standard curve. Quantitative data were expressed as means ± standard deviation. Student's t test was utilized for statistical contrast. P < 0.01 was deemed statistically significant.

Example 2: Synthesis of the Hybrid Nanoparticle

The hybrid nanoparticle was prepared as shown in Fig. 1. TPETPAFN shows negligible fluorescence in its unimolecular state in good solvents such as tetrahydrofuran (THF), while its aggregates in aqueous solution or solid state show intense FR/NIR emission centered at 655 nm. Such typical AIE character makes TPETPAFN ideal for the fabrication of ultra-bright organic dots. An amphiphilic lipid block copolymer, 1 ,2-distearoyl-snglycero- 3-phosphoethanolamine- N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000-NH2) was used as the matrix to load TPETPAFN molecules to form AIE dots. Upon AIE dots formation, the hydrophobic DSPE segment of the matrix intertwines with TPETPAFN molecules to form compact aggregate as the core, while amine group-terminated polyethylene glycol (PEG) chains extend outwards towards the water phase, stabilizing the resultant AIE dots, and rendering them with excellent colloidal stability and surface amino groups for further functionalization with gadolinium (Gd ³⁺ ). After obtaining the AIE dots, 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-l-yl)oxy)-2-oxoethyl)-l ,4,7,10- tetraazacyclododecane-l,4,7-triyl)triacetic acid (DOTA-NHS ester) was conjugated to the AIE dot surface, and further chelated with Gd ³⁺ to generate AIE-Gd dots.

Specifically, to fabricate AIE dots with amine group present at the surface, a homogeneous THF solution (1 mL) containing TPETPAFN (1 mg) and DSPE-PEG ₂₀ oo-NH ₂ (2 mg) was poured into MilliQ water (10 mL), immediately followed by sonication using a microtip probe sonicator (XL2000, Misonix Incorporated, NY) at 12 W output for 120 s. The hybrid nanoparticle suspension was further placed in the dark in a fume hood under stirring at 600 rpm overnight for THF evaporation. The AIE dots (5 mL) with surface amine groups were further mixed and reacted with DOTA-NHS-ester (1.5 time molecular excess) overnight. After reaction, the nanoparticle suspension was dialysed against MilliQ water using membrane with molecular cut-off of 6-8 kDa for 2 days to eliminate the unreacted and excess DOTA-NHS-ester. The obtained AIE dot suspension was further chelated with Gadolinium(III) chloride hexahydrate (1.5 time molecular excess). After overnight reaction, the resultant AIE-Gd dot suspension was further dialysed against MilliQ water using membrane with molecular cut-off of 6-8 kDa for 2 days to eliminate the unreacted and excess components. AIE-Gd dot suspension was further purified by filtering through a 0.2 μπι syringe driven filter, concentrated by a centrifugal filters with a molecular cut-off of 10 kDa (Merck Millipore, Germany), and stored at 4 °C until further use.

Example 3: Characterization of the Hybrid Nanoparticle Freeze-drying of the hybrid nanoparticle stock solution (1 mL) yielded 0.82 mg of powders. As the AIE-Gd dots are stable in water, the density of the dot suspension could be estimated to be approximately 1 g/cm ³ .

The encapsulated TPEPTAFN was calculated to be -31.7 wt% TPEPTAFN per dot, equivalent to approximately 4400 TPETPAFN molecules per dot. This high AIE fluorogen density ensures the high brightness of the AIE dots.

Ion coupled plasma-mass spectrometry (ICP-MS) was used to quantify the Gd(III) content in the obtained AIE-Gd dots. The result revealed that the AIE-Gd dots contain 2.3 wt% of Gd ³⁺ , which is estimated to be equivalent to approximately 2100 Gd ³⁺ ions per AIE-Gd dot.

Dynamic light scattering (DLS) results revealed that the AIE-Gd dots have an average hydrodynamic size of 38.9 + 2.6 nm with narrow size distribution and a polydispersity of -0.109 (Fig. 2A).

High-resolution transmission electron microscopy (HR-TEM) shows the morphologies of the AIE-Gd dots, which are spherical in shape with a size of approximately 36 nm (Fig. 2B). Atomic force microscopy (AFM) images indicates a similar size distribution. The apparently smaller size compared to what was measured by DLS may be due to the shrinkage effect of the organic dots in dry state.

The AIE-Gd dots in water suspension showed absorption and emission peaks centred at 512 and 668 nm, respectively, with a high fluorescence quantum yield (η) of 24% measured with 4- (dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyr an in methanol as the standard (Fig. 2C). Such an impressive η value in the FR/NIR region is most likely due to the AIE feature of TPETPAFN in the nanodots. This, together with the high molar absorptivity (9.6 x

10 7' M ^" 1'cm2 ¹ at 510 nm based on dot concentration) yields high brightness (2.2 x 107 M 1 cm 2 _ for AIE-Gd dots which is over 10-fold higher than commercially available Qtracker 655 (1.3 x 10 ⁶ M cm ) under the same excitation wavelength of 510 nm.

It should be noted that Gd ³⁺ chelation showed negligible effects on the optical properties of the nanodots as the absorption and emission spectra of the AIE dots were hardly affected by the chelation process, indicating high stability of the dots towards surface functionalization and stimuli change.

Moreover, the size and fluorescence of the AIE-Gd dots remained almost unchanged after 10 days incubation in 1 X phosphate -buffered saline (PBS) buffer at 37 °C (Fig. 2D), further indicating the high colloidal stability of AIE-Gd dots in biological buffer. Such a high colloidal stability may be attributed to the existence of the PEG chains, which suppress the nonspecific interaction between the TPETPAFN-Gd dots and the surrounding molecules, making them promising for in vivo vascular imaging.

The brightness and photostability of the individual AIE dots were further studied by wide- fluorescence microscope. Individual AIE dots showed bright fluorescence as shown in Fig. 3A, and the corresponding histograms of total number of photons emitted by 1216 representative AIE dots within 100 seconds show an average emitted photon number of 1.18 X 10 ⁶ counts (Fig. 3B), which is much higher than the selected benchmark (QD655, 2.8 X 10 ⁵ counts), indicating the high brightness of the AIE dots.

Continuous laser irradiation at a power of 525 μ\¥ over 100 seconds showed negligible effects to the fluorescence intensity of 4 representative AIE dots with different brightness levels (Fig. 3C), indicating high resistance of AIE dots towards photobleaching. It is worth noting that intermittency, the characteristic blinking which happens on conventional inorganic quantum dots, was not observed on AIE dots, making AIE dots highly desirable for real-time single dot tracing, red blood cell (RBC) flow measurement and other applications.

Materials with high two-photon absorption (TP A) cross section are able to emit strong visible fluorescence from low-energy irradiation in the far-red/near-infrared (FR/NIR) region, which minimizes the absorbance of biosubstrate, and is beneficial for clear intravital imaging of deep biological tissues at high resolution. The TPA spectrum of AIE_Gd dots in aqueous solution was measured using a multiphoton microscope equipped with a tuanable Ti:sapphire pulsed laser, using Rhodamine 6G in methanol as the standard. For comparison, that of Evans Blue in place of albumin was also measured. As shown in Fig. 3D, the maximum TPA cross section (δ) value of TPETPAFN-Gd dots was measured to be 6.7 X 10 ⁴ GM (based on dots concentration) upon excitation at 810 nm, giving rise to a δη value of 1.61 X 10 ⁴ GM, which is much higher than that of Evans Blue which needs to be bound to albumin to be emissive (δη = 18.2 GM based on Evans Blue molecular concentration). Such an intrinsically high TPA cross section of AIE-Gd dots in aqueous media under two-photon excitation is highly desirable for high resolution intravital imaging.

Example 4: Application of the Hybrid Nanoparticles

The hybrid nanoparticles were employed to visualize vascular leakage in the croton oil-induced inflammation in mouse ear skin by two-photon intravital microscopy. The ear skin of LysM- EGFP mice after intravenous administration of AIE-Gd dots (80 nM, 4 μΕ/g mouse bodyweight) was measured. The LysM-EGFP mice are able to express enhanced green fluorescent protein (EGFP) in myeloid cells, and neutrophils in particular expressing high EGFP levels. This reporter mouse strain enables the detection of tissue injury through visualization of neutrophil accumulation/recruitment at sites of inflammation or injury. Consequently, it provides a way to directly examine how inflammation may affect the intravascular activity of AIE-Gd dots.

To this end, 40 μΐ _^ of croton oil was applied to induce inflammation in the mouse ear skin, and acetone (vehicle) was applied to the contralateral ear as control. AIE-Gd dots were injected either at the start or 3 hours after the induction of inflammation, and imaging was performed thereafter at 3 hours post croton oil treatment.

As shown in Fig. 4, the nanoparticles were able to detect the loss of vascular integrity and leakage in the inflamed skin.

In control ears, the AIE-Gd dots showed excellent colloidal stability and flowed smoothly after being administered intravenously, with negligible aggregates present on the blood vessel walls, and was able to highlight the blood vessels (Fig. 4A1). This was likely due to the PEG segment of the AIE-Gd dots blocking non-specific interactions and protein adsorption. Moreover, no obvious leakage of the AIE-Gd dots from the blood vessels or increase of background signals in the interstitium was observed during the tested period, revealing that the AIE-Gd dots have excellent vascular retention under resting conditions. Notably, the intensity of the circulating AIE-Gd dots were observed to decline drastically over the experimental period of 3 hours as the blood vessels were less visible over time (Fig. 4B1), suggesting that the AIE-Gd dots were being fast cleared from the circulation.

In contrast, under inflammatory conditions, AIE-Gd dots leaked, formed aggregates (punctates structures) around the affected blood vessels and entered into the interstitium as shown by increased intensity in the interstitium (Fig. 4A2). This phenomenon was independent of the timing of AIE-Gd dots administration (at 0 hour or 3 hours after the induction of inflammation) (Figs. 4A2 and 4B2). On the other hand, when Evans Blue was injected just prior to imaging the skin after 3 hours croton oil treatment, there did not appear to be any significant leakage observed in the inflamed tissue (Fig. 4A4) compared to the control (Fig. 4A3). This could be due to the confounding dependence of Evans Blue fluorescence to albumin binding, it was likely that when the dye was freshly injected, the drastic differential brightness of the Evans Blue within the blood vessels completely masked the dim signals present in the tissues when viewed at normal contrast. Instead, when Evans Blue was injected at the start of the induction of inflammation and imaged 3 hours thereafter (Fig. 4B4), it was able to enter the interstitium progressively in the inflamed ear tissue (as seen in comparison to the control in Fig. 4B3), but now the widely-diffused signals emitted by the dye obscured most of the affected tissues (poor background to noise signals), making it difficult to even locate the smaller vessels, much less to assess the exact locations of vascular leakage.

Taken together, these results provide evidences to show that the AIE-Gd dots display superior sensitivity for the detection of changes in vascular functions with high contrast and low background noise. While fluorescence detection provides direct visualization of the changes in blood vessel functions, it can only serve at most as a semi-quantitative analysis of vascular leakage. AIE-Gd dots have been conjugated with Gd ³⁺ on their surfaces in order to measure with very high sensitivity (using ICP-MS) the amount of vascular leakage and/or accumulation in the tissues.

To further examine the sensitivity of the nanoparticle was then compared against that of using Evans Blue dye, the current de facto probe of choice. In order to do this, equal volumes of AIE- Gd dots (80 nM, 4 mouse bodyweight) with Evans Blue dye (20 mg/mL, 4 μh/g mouse bodyweight) were mixed and injected simultaneously into mice receiving the croton oil ear treatment. Mice were sacrificed at 1, 3, 5 or 7 h post-injection and were perfused before their ears were cut for analysis. N,N-dimethylformamide (DMF) was used to extract Evans Blue from the intact whole ears, and the amount of Evans Blue extracted was evaluated by measuring its absorbance at 620 nm. The ear tissues that remained after DMF extraction were then further dissolved in strong (70%) nitric acid under heat to decompose all organic compounds, and subsequently the Gd ³⁺ contents were assessed by ICP-MS. To compare the results from these two methods, the signal (croton oil-treated ears) to background (contralateral control ears) ratios were compared. Using the AIE-Gd dots method, croton oil-treated ears showed an approximately 4-fold increase in signal over control ears even at 1 h of croton oil treatment, rising to almost 9-fold at 7 h (Fig. 5A). On the other hand, using the Evans Blue method, it only managed to achieve a maximum of less than 3 -fold increase at 7 h (Fig. 5B).

The progressively increasing Evans Blue content in control ears demonstrated its undesirable leakage (possibly unbound to albumin) in normal blood vessel conditions (Fig. 5B), which was undetected by fluorescence imaging. In comparison, the background levels of the AIE-Gd dots remained consistently constant throughout the period of study (Fig. 5A). These results clearly indicate that the AIE-Gd dots method has a much higher sensitivity than the traditional Evans Blue measurements for qualitative and quantitative analysis of changes in vascular functions. Example 5: In Vivo Imaging

Having shown that the hybrid nanoparticles were able to detect the loss of vascular integrity in a simple prototypic skin inflammation model, the hybrid nanoparticles were then applied in studying a complex disease model such as experimental cerebral malaria in mice. The loss of vascular integrity and leakage in the brain of malaria infected mice was observed by multiphoton microscopy (Fig. 6A1 and 6A2). AIE-Gd dots were found to form punctate AIE- Gd accumulations along infected micro vessels for ECM brain (Fig. 6A1), clearly indicating the location of hemorrhage. But no AIE-Gd dots were found in the control brain after perfusion. To complement the imaging data, the Gadolimium (Gd) content in the control and infected brains were measured (Fig. 6B). It was observed that the infected brain had a significantly higher Gd concentration than the control brain (Fig. 6A2). Collectively, this further supported the fact that AIE-Gd dots are useful for detection of vascular integrity.

Having demonstrated these useful properties that make the AIE-Gd dots suitable for the detection of vascular integrity and leakage, the suitability of the AIE-Gd dots for assessing BBB function was tested. C57BL/6 female mice were inoculated by direct intravenous injection of red blood cells infected with P. berghei ANKA (PbA) parasites. These mice typically developed neurological symptoms (paralysis, ataxia, deviation of the head and convulsion and/or coma) at day 7-8 after infection. In the initial set of experiments, mice with observable neurological symptoms were injected with AIE-Gd dots (80 nM, 4 mouse bodyweight) or Evans Blue (20 mg/mL, 4 μh/g mouse bodyweight) intravenously. The mice were then sacrificed and the whole intact brains of the mice were harvested and analyzed with light sheet ultramicroscopic imaging. To assess the localization of the AIE-Gd dots in the brain, whole organ imaging on the optically cleared ECM brain was performed. As shown in Fig. 7A1, it was observed that AIE- Gd dots were able to highlight the brain vasculature of control mice. Upon enlarging the brain images (Fig. 7A2), it was observed that even the smaller capillaries in the pia mater could also be visualized by AIE-Gd dots with sharp resolution and negligible background signals from outside of blood vessels for controlled naive mice. AIE-Gd dots also formed punctate AIE-Gd accumulations along infected micro vessels for ECM brain (Fig. 7B1 and 7B2), clearly indicating the location of hemorrhage. However, Evans Blue failed to reveal changes in the BBB of ECM brain in this set of experiments as compared to the naive uninfected brains, where both ECM and control brains gave similar results (Figs. 7C1 and 7C2, and Fig. 7D1 and 7D2). Conceivably due to the lack of sensitivity. Together, the data show that the AIE-Gd dots are suitable and possess a high sensitivity and specificity for the detection of BBB disruptions during malaria infection. Having shown that the AIE-Gd dots were highly sensitive in detecting changes of BBB in malaria infected mice, the next step was to examine whether these probes were able to quantitatively detect the extent of ECM parasitemia levels during malaria infection. In this set of experiments, PbA-infected mice were tail-bled to measure their levels of parasitemia on day 7. Concurrently, these mice were injected with AIE-Gd dots and sacrificed 3 hours later. After perfusion, brains from these animals were harvested for bioluminescence analysis (IVIS) by exposure to luciferin and then imaged by two-photon microscopy. From the bioluminescence study, only the hotspot regions where PbA parasites aggregate can be identified with relatively poor spatial resolution as there is high background noise signals (Fig. 8A1 to Fig. 8A4), and correlation between parasite sequestration in the brain and systemic parasitemia could not be observed (Fig. 8B). In contrast, intense fluorescent signals from accumulated AIE-Gd dots in the vessels and interstitium of infected brain could be easily detected (Fig. 8C1 to Fig. 8C4). Further analyses of the image datasets showed increasing fluorescence intensities with parasitemia levels and with excellent correlation (Fig. 8D), which was not observed in Evans Blue treated mice (Fig. 8E), suggesting that accumulation/leakage of AIE-Gd dots could be a better indicator of ECM parasitemia levels as compared to the conventional bioluminescence technique or Evans Blue methods.

To further examine this hypothesis, the harvested brains were subjected for destructive ICP-MS analysis. It was observed that the Gd ³⁺ content was also a good predictor for the blood parasitemia levels (Fig. 9A). When additional analysis was performed by grouping the mice according to whether they displayed classical neurological symptoms of ECM, it was found that the Gd3 ⁺ levels were significantly higher in infected mice that exhibited signs of ECM when compared to those which did not have the symptoms (Fig. 9B). It is also noteworthy that the non-ECM brains contained similar Gd ³⁺ levels to those from naive mice (i.e. uninfected), suggesting that the AIE-Gd dots had almost negligible accumulation/leakage in the brains of non-ECM mice. Hence, the results not only demonstrate the multimodality of the AIE-Gd dots, but also further highlight the sensitivity and specificity of the AIE-Gd dots in detecting changes in brain vasculature during ECM development.

Industrial Applicability

The nanoparticle as disclosed herein may be useful in systematically detecting and characterizing the onset, progression and duration of the vascular leakage in various inflammatory diseases. The nanoparticle may be useful in cross-platform measurement of vascular integrity and leakage by intravital multiphoton microscopy (IV-MPM), fluorescence reflectance imaging (FRI) and magnetic resonance imaging (MRI) techniques. The nanoparticle may be useful in detecting and measuring loss of vascular integrity as well as vascular leakage in vivo or ex vivo, by fluorescence imaging and/or magnetic resonance imaging. The disclosed nanoparticle may be suitable for studying BBB function and for visualizing the blood vasculature and its permeability in dengue, encephalitis and lung inflammation.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.