WO2012109755A1 - Fatty ester-based particles and methods of preparation and use thereof - Google Patents

Abstract

Fluorescent particles and methods of preparation and use thereof are provided in which a fatty matrix comprising fatty ester is employed for the encapsulation of a fluorescent payload. The fluorescent payload may be a fluorescent nanoparticle or a fluorophore. In one embodiment, the fluorescent payload is a plurality of quantum dots, which may be incorporated without a passivation capping layer while preserving the quantum efficiency of the quantum dot. The compositions can be used in biological imaging in vitro, in vivo and ex vivo. Whole body imaging may be achieved by selecting a fluorescent payload (such as a PbSe quantum dots) that has an emission bandwidth that is suitable for imaging of a tissue.

Description

FATTY ESTER-BASED PARTICLES AND METHODS OF PREPARATION AND

USE THEREOF

FIELD OF THE INVENTION

The present invention is directed to fluorescent particles and related compositions, and methods for their use in imaging.

BACKGROUND OF THE INVENTION

Medical imaging has become a staple in a clinician's arsenal for the detection, prevention and treatment of human diseases including cancers.¹'² Current imaging modalities applied to oncology, such as magnetic resonance imaging and positron emission tomography (PET), require expensive instrumentation and long subject exposure times. In the case of PET, potentially genotoxic radioisotope contrast agents are employed.³ In contrast, optical imaging methods utilize non-ionizing radiation, are relatively less expensive and less hazardous, and can be performed quickly with brief exposure times.

However, traditional optical imaging in medicine has been severely limited by light scattering and autofluorescence of tissue in the ultraviolet and visible light range, and by poor biocompatibility and physiological instability of fluorophores.³'⁴ Images have only been acquired at depths of a few millimeters below the surface of the skin, either due to limited penetration distance of incident light or emission light³' ⁴ Though the tissue interference may be avoided at wavelengths longer than 900nm, water in the body can interfere with the fluorescence signals. Therefore, for in vivo optical imaging of deep tissue, the fluorophore should have excitation and emission wavelengths in the near- infrared (NIR) region ranging from 650 nm to 900 nm. In this region the light scattering, absorbance, and autofluorescence of tissue are at a minimum, providing an opportunity for deeper optical interrogations in vivo with a predicted tissue penetration depth of 1 to 2 cm.⁴'⁵

Ideally, an NIR fluorophore for medical applications should have a large absorption extinction coefficient, high NIR quantum yield, low photobleaching, and lack of toxicity.⁶ Organic fluorophores are poor candidates for deep optical medical imaging because of their poor circulation half-lives, poor NIR quantum yields, rapid photobleaching, and spectral shifting due to rapid plasma protein binding, metabolism, and/or chemical degradation.^6"8 For this reason, NIR-tuned semiconductor quantum dots (QD) have been developed and applied to medical imaging for sentinel lymph node mapping ^" and tumor imaging. ' The properties of QDs that render them effective fluorophores for medical fluorescence imaging stem from their high quantum yield with NIR excitation and their large effective Stake's shifts, resulting in the potential for optical image acquisition at greater tissue depths.³' ¹⁴' ¹⁵ However, the development of QDs for medical-grade fluorophores faces major hurdles owing to the inherent toxicity and poor colloidal and photostability of QDs.¹⁶ Heavy metal precursors are required for the synthesis of QDs,¹⁷ and ligands are employed for their surface functionalization.¹⁸ Despite surface modification of QDs with less toxic element or hydrophilic compounds to make them more biocompatible and more stable in aqueous media, their extensive liver uptake and poor clearance rates result in unsafe pharmacokinetic profiles.¹⁹' ²⁰ Therefore, many efforts have been made to encapsulate them in biocompatible materials such as lipid micelles, polymeric micelles, or liposomes.

Recently, solid lipid nanoparticles comprising fatty acids have been shown to be a suitable matrix for encapsulating fluorophores such as quantum dots, as taught by Shastri in US Patent Application Publication No. US2006/0083781, and Liu et al.²¹ While the fatty acid matrix is compatible with quantum dots capped with an inorganic, wide-bandgap layer, such as core-shell CdSe/ZnS quantum dots, the use of quantum dots capped by organic ligands typically results in undesirable quenching of the fluorescence quantum yield. Accordingly, such solid fatty acid nanoparticles are not suitable as fluorophore encapsulating vehicles for a wide variety of quantum dots. US Patent Application Publication No. 2008/0089836 of Hainfeld describes coating of a nanoparticle (e.g. gold, silver, iron oxide) with bilayer molecules.

SUMMARY OF THE INVENTION

A first aspect of the invention is a fluorescent particle. The particle includes a fatty matrix that includes hydrophobic portions of a plurality of fatty ester molecules. A fluorescent payload encapsulated in the matrix.

The fluorescent payload of the particle can be a plurality of fluorescent molecules, a plurality of fluorescent nanoparticles, or a mixture thereof. In an embodiment where the payload is a plurality of nanoparticles, the nanoparticles may be of the same chemical constitution, for example, PbSe quantum dots (QDs) as exemplified below. The payload can be a plurality of organic molecules. There can be single type of organic molecule or more than one type, as described further below.

In a preferred aspect, a substantial portion of the particle is the fatty ester, usually more than 5% by weight of the particle.

Where fluorescent nanoparticles make up the payload (or at least a part of the fluorescent payload), there are several such nanoparticles encapsulated by the matrix of the composite particle. There can thus be e.g., five or more encapsulated. It is thus typical that the size of such component nanoparticles is smaller than the particle itself. The diameter of the particle can thus be at least about 5 times the average diameter of encapsulated nanoparticles.

In another aspect, the particle itself is dimensionally a nanoparticle, and is referred to here as a fatty nanoparticle.

The fatty ester may be selected from the group consisting of tristearin, tripalmitin, trilaurin, ethyleicosanoate, glyceryl behenate, and cetylpalmitate, and hard fats, synthetic waxes, natural waxes, and beeswax. The fatty ester may be an ester of a substance selected from the group consisting of glycerol, sucrose, C10-C54 saturated fatty acids with straight chains, C10-C54 unsaturated fatty acids with straight chains, C10-C54 saturated fatty acids with branched chains, C10-C54 unsaturated fatty acids with branched chains, C10-C54 saturated fatty alcohols with straight chains, C10-C54 unsaturated fatty alcohols with straight chains, C10-C54 saturated fatty alcohols with branched chains, and C10-C54 unsaturated fatty alcohols with branched chains.

The fluorescent payload may comprise a fluorescent nanoparticle, which may comprise a hydrophobic capping ligand.

The hydrophobic capping ligand may be selected from the group consisting of oleic acid, trioctyl phosphine, butanoic acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 9-hexadecenoic acid, octadecanoic acid, 9-octadecenoic acid, 12-hydroxy-9-octadecenoic acid, 11-octadecenoic acid, 9,12- octadecadienoic acid, 9,12,15-octadecatrienoic acid, 6,9,12-octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid, 5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, docosanoic acid, 13-docosenoic acid, 4,7, 10,13, 16, 19-docosahexaenoic acid, and

tetracosanoic acid, alkylphosphenes.

The particle, or fatty nanoparticle, may further comprise a surface PEGylating block copolymer. The surface PEGylating block copolymer may comprise poly(ethylene glycol) (PEG) linked with a fatty chain or a hydrophobic polymer chain. The surface PEGylating block copolymer may be at least one block of poly(ethylene oxide) and at least one block of hydrophobic polymers selected from the group consisting of poly (propylene oxide), polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic acid-co-glycolic acid), polydioxanone, polyurathans, polystyrene, polycaprolactone, and polycarbonates.

The particle, or fatty nanoparticle, may include a surface coating selected from the group consisting of Tween® family surfactants, Triton® family surfactants, Tergitol® family surfactants, Pluronic® family surfactants, Span® family® surfactants, polyvinyl alcohol), poly(vinyl pyrrolidone), linear starch, modified starch, egg phosphatidylcholine, egg lecithine, and soy phosphatidylcholine.

A particle of the invention may include a surface coating comprising activated groups adhered to a surface of the fluorescent nanoparticle, the activated groups having a spacer chain grafted thereto. The spacer chain may comprise PEG.

A fluorescent nanoparticle may be a quantum dot, such as a PbSe quantum dot, CdSe quantum dots, CdS quantum dots, CdTe quantum dots, CdSe/ZnS quantum dots, CdSe/ZnSe quantum dots, InAs quantum dots, InP quantum dots, and PbS quantum dots.

The quantum dot may comprise a core coated with the hydrophobic capping ligand. A fatty nanoparticle may further comprise a fluorophore encapsulated in the fatty matrix. The fluorescent nanoparticle may be a first fluorescent nanoparticle, where the fatty matrix further encapsulates at least one additional fluorescent nanoparticle. The emission spectrum of the additional fluorescent nanoparticle may be spectrally distinct from the emission spectrum of the first fluorescent nanoparticle.

The fluorescent nanoparticle may be a semiconductor nanocrystal, such as a carbon- based nanocrystal or a silicon-based nanocrystal.

The fluorescent nanoparticle may be selected from the group consisting of silica- organic dye hybrid nanoparticles, calcium phosphate-organic dye hybrid nanoparticles, and fluorophore doped polymer nanoparticles.

The fatty matrix may further comprise one or more of a fatty acid and a fatty alcohol, and may further comprise an additional lipid compound.

A diameter of the fatty nanoparticle may be between approximately 20 and 1000 nm, and more preferably between approximately 50 and 250 nm.

The emission spectrum of the fluorescent payload may be selected to be suitable for fluorescent imaging through a tissue, and may be between approximately 400 and 950 nm. The emission spectrum of the fluorescent payload may be in the near-infrared spectral region.

The fluorescent payload may be a fluorophore, such as xanthene derivatives, fluorescein, rhodamine, cyanine derivatives, cyanine, indocarbocyanine, napthalene derivatives, dansyl, prodan derivatives, coumarin derivatives, oxadiazole derivatives, pyrene and its derivatives, oxazine derivatives, Nile red, cresyl violet, acridine derivatives, arylmethine derivatives, tetrapyrrole derivatives, porphyrin, phthalocyanine, and

polymethines. The fluorophore may be a first fluorophore, where the solid fatty matrix may further encapsulate an additional fluorophore, and where an emission spectrum of the additional fluorophore is spectrally distinct from an emission spectrum of the first fluorophore.

A particle, or fatty nanoparticle, of the invention may further comprise one or more of a binding moiety, label, reporter, hapten, and conjugate.

In another aspect, there is provided a method of producing fatty nanoparticles comprising a solid fatty matrix encapsulating a fluorescent payload, the method comprising the steps of: providing a fluorescent nanoparticle coated with a hydrophobic capping ligand; forming a suspension by suspending the fluorescent nanoparticle in a hydrophobic solvent; heating a fat beyond a melting point and obtaining molten fat, wherein the molten fat comprises a fatty ester; mixing the suspension with the molten fat; evaporating the solvent and obtaining a first dispersion comprising the fluorescent nanoparticles dispersed in the molten fat; forming an emulsion by heating a quantity of water above the melting point and adding the water to the dispersion; and homogenizing the emulsion to form nanodroplets comprising a fatty matrix encapsulating the fluorescent nanoparticles; and forming a second dispersion comprising the fatty nanoparticles by combining the emulsion with an isotonic and inert aqueous suspending medium.

The quantity of water may be approximately 0.001: 1 to 2:1 by volume ratio to the quantity of the fat. A quantity of surfactant may be added prior to the step of adding the water.

The quantity of surfactant may be provided as an aqueous solution, and where a quantity of the surfactant in the entire emulsion is approximately 0.05% to 10% by weight.

The step of forming the second dispersion may be performed within a suitable time interval of the step of homogenizing the emulsion to prevent coalescence of the nanodroplets. The step of homogenizing the emulsion may comprise one of ultrasonicating the emulsion, passing the emulsion through a microfluidizing device, and directly introducing the emulsion to a high pressure homogenizer.

The isotonic and inert aqueous suspending medium may be a solution comprising one of saline, dextrose and buffer salts. A quantity of one or more block copolymers may be added for stabilization of the fatty nanoparticles.

In yet another aspect, there is provided a method of producing fatty nanoparticles comprising a solid fatty matrix encapsulating a fluorophore, the method comprising the steps of: heating a fat beyond a melting point and obtaining molten fat, wherein the molten fat comprises a fatty ester; adding a quantity of the fluorophore to the molten fat; forming an emulsion by heating a quantity of water above the melting point and adding the water to the molten fat; and homogenizing the emulsion to form nanodroplets comprising a fatty matrix encapsulating the fluorophore; and forming a dispersion comprising the fatty nanoparticles by combining the emulsion with an isotonic and inert aqueous suspending medium.

The step of forming the dispersion may be performed within a suitable time interval of the step of homogenizing the emulsion to prevent coalescence of the nanodroplets. The step of homogenizing the emulsion may comprise one of ultrasonicating the emulsion, passing the emulsion through a microfluidizing device, and directly introducing the emulsion to a high pressure homogenizer.

The isotonic and inert aqueous suspending medium may be a solution comprising one of saline, dextrose and buffer salt. A quantity of one or more block copolymers may be added for stabilization of the fatty nanoparticles. The step of adding a quantity of one or more block copolymers may be performed prior to the step of adding the quantity of water.

In still another aspect, there is provided a method of imaging, the method comprising the steps of: providing fatty nanoparticles as described above, wherein an emission bandwidth of the fluorescent payload is selected to be suitable for fluorescent imaging through a tissue of a subject; administering the fatty nanoparticles to the subject; optically exciting the fluorescent payload; and imaging fluorescent emission produced by the fluorescent payload.

A diameter of the fatty nanoparticles may be selected to be between approximately 50 and 250 nm. The step of administering the fatty nanoparticles may comprise one of intravenous, intra-arterial, intraperitoneal, subcutaneous, intratumoral, intrapleural, or intramuscular injection of the fatty nanoparticles.

The step of imaging the fluorescent emission may comprise imaging a substantial portion of a body of the subject. The fluorescent payload may comprise fluorescent emission characterized by two or more distinct emission bands, the method further comprising spectrally separating the fluorescent emission to resolve the distinct emission bands. The fatty nanoparticle may comprise a conjugated molecular species for selectively targeting a tissue type, a cell type or a molecule type, the method further comprising the step of identifying a presence of the tissue type, cell type or molecule type in the subject based on the fluorescent emission.

A further understanding of the functional and advantageous aspects of the invention can be realized by reference to the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described, by way of example only, with reference to the drawings, in which:

Figure 1 illustrates dispersion of PbSe QDs throughout a matrix of fatty ester-based nanoparticles (FEN), showing a schematic illustrating PbSe QDs dispersed throughout a matrix of fatty ester nanoparticles (FEN) having a corona of poly(ethylene glycol) (PEG).

Figure 2 is a flow chart illustrating a method of preparing QD-FEN.

Figure 3 is a schematic representation of the utility of fluorophores in the visible versus the near infrared spectrum for deep tissue fluorescence imaging. In the visible region, due to light scattering and absorption by living tissue, poor penetration of the excitation photon into the skin or the penetration of the emission photon out of the skin prevents the detection of the fluorophore signal in vivo. Photons in the near infrared spectrum are capable of deep tissue penetration.

Figure 4 is a graph depicting the wavelength range that provides the least tissue interference for fluorescent imaging, and within which near infrared fluorophores are effective.

Figure 5 illustrates (a) proton and (b) carbon NMR spectra of eicosanoic acid and ethyl eicosanoate.

Figure 6 provides (a) a schematic and (b) a flow chart that illustrate methods of construction of tissue phantoms for quantification of fluorescence penetration depth.

Figure 7 (a) and (b) provide transmission electron micrographs of QD-FEN that clearly demonstrate the encapsulation of the metal QDs (dark spots) within the fatty ester matrix. The absence of dark spots in (b) reflects the absence of QDs in the FEN.

Transmission electron micrograph of PbSe QDs on their own is shown in the top right inset of (a). Scale bars in (a) and (b) are 100 nm, scales bars in insets of (a) are 10 nm.

Figure 8(a) is a graph illustrating the absence of photoluminescence quenching of PbSe QDs encapsulated in solid fatty ester-based nanoparticles compared to QD-loaded myristic acid nanoparticles. The absence of quenching with QD-FEN is determined by comparison of the emission spectra of free PbSe QDs (no quench, circle dotted line) to the emission spectra of QD-loaded myristic acid nanoparticles (quenched, solid line), and the reversal of the quenching effect with the use of fatty ester in QD-FEN (rectangle dotted line).

Figure 8(b) is a graph illustrating the presence of quenching when QDs were dispersed in 1-octanoic acid (dotted line) but not in n-octanol (solid line).

Figure 9 illustrates quantitative, continuous measurement of penetration depth of fluorophore signal in a tissue phantom model, (a) Schematic diagram of the tissue phantom constructed for depth penetration measurements, showing the 1-mm thick gel containing QD- FEN encased at an angle within the tissue-like phantom. The depth measurements account for excitation photon (λ_εχ) and emission photon ( _em) penetration, (b) The light scattering and light absorption characteristics of the tissue phantom characterized by the scattering (μ«, dotted) and absorption (Ua, solid) coefficients, respectively, as measured by two integrating spheres in series, (c) Fluorescent images taken from the top of a 1.5 cm (1 mg QD-FEN/mL, top) and 2.7 cm (5 mg QD-FEN/mL, bottom) maximal QD-FEN gel depth phantom, illustrating disappearance of detectable signal as a function of gel depth, (d) A plot of the pixel intensity versus tissue phantom depth of the phantoms depicted in Figure 9(c), quantifying the maximal penetration of the 1 mg QD-FEN/mL gel to -1.3 cm (solid) and of the 5 mg QD-FEN/mL gel to -2.5 cm (dotted). Background fluorescence levels are indicated by a horizontal solid line for the 1 mg QD-FEN/mL phantom and a horizontal dotted line for the 5 mg QD-FEN/mL phantom.

Figure 10 is a graph illustrating the toxicity of QD-FEN or PbSe QDs in free, water- soluble form (MTPEG-PbSe QD). Toxicity was assessed in vitro in a rat hepatocyte assay for accelerated assessment of cytotoxicity. Saline, fatty ester nanoparticles, QD-FEN, and MTPEG-PbSe QD were incubated at a low (60 μg) and high (300 ng) dose in 10 mL of cell suspension, and cytotoxicity was assessed at 30 minutes (black), 60 minutes (dark grey), and 120 minutes (light grey). Statistical comparisons of survival data of QD-FEN and MTPEG- PbSe QD against fatty ester nanoparticles for each dosing level were performed using a Nested ANOVA and post-hoc Tukey's Test.†Significant differences (p<0.05) in survival rates between low dose fatty ester nanoparticles, and QD-FEN or MTPEG-PbSe QD.†† Significant differences (p<0.05) in survival rates between low dose QD-FEN and MTPEG- PbSe QD. *Significant differences (p<0.05) in survival rates between high dose fatty ester nanoparticles, and QD-FEN or MTPEG-PbSe QD. ** Significant differences (p<0.05) in survival rates between high dose QD-FEN and MTPEG-PbSe QD. Data represent the mean and standard deviation of three trials.

Figure 11 illustrates QD-FEN utility for deep, real-time, in vivo fluorescence imaging following intravenous administration, providing favorable biodistribution and clearance of the nanocrystal cargo. The biodistribution of QD-FEN in a green fluorescent protein- expressing MDA435 model of human breast cancer was followed over 7 days with whole animal fluorescent imaging, (a) Fluorescent image of the breast tumor grown in the right leg of a mouse, (b) Longitudinal biodistribution and elimination of the QD-FEN. (c) Necropsy analysis of the tissue distribution showed significant accumulation of QD-FEN in the spleen and gall bladder, without accumulation in the liver after 3 hours, (d) Tumor accumulation of the QD-FEN was noted by 30 minutes after intravenous administration. Near complete elimination of the QD-FEN was observed 7 days after administration.

Figure 12 is a set of scan images of mice containing bilaterally xenografted MDA435 human breast cancer tumour cells expressing EGFP. (a) Tumor cells were coinjected with a medium without Matrigel® (left) and with Matrigel® (right) into the mammary fat pads, (b) PbSe QD-FEN were injected intratumorally into each of the bilateral tumor and tumor leakage of QD-FEN was assessed over time.

Figure 13 is a graph illustrating the binding affinity of pyrene-loaded cRGDf -FEN for the integrin α_νβ3 receptor-coated 96-well microplate. The fluorescence intensity originates from the fluorophore pyrene encapsulated in the FEN.

Figure 14 presents fluorescent stereomicroscopic images showing QD-FEN and cRGDf -QD-FEN accumulation at the tumor tissues and neovasculature. The images were taken at 12 hours following injection of the nanoparticle formulation. These images show the vascular distribution (left column, bright field image), location of tumor growth (middle column) imaged at Ex/Em 465 nm/540 nm for green fluorescent protein-transfected cancer cells, and location of untargeted QD-FEN (right column, top row) or targeted cRGDfK-QD- FEN (right column, bottom row) imaged at Ex/Em 710 nm/840 nm for PdSe QDs. The white arrows indicate the co-localization of cRGDf -QD FEN to areas of neovascular beds of the tumor known to highly express the cognate RGD receptor (integrin 0^3). The results suggest that while the untargeted QD-FEN are useful for imaging the tumor tissue, the targeted QD- FEN are particularly useful for imaging areas with high integrin α_νβ3 expression.

Figure 15 is a graph showing emission spectrum of pyrene- and PbSe QD-co-loaded

FEN. Figure 16 shows that the fluorescence of CdSe QDs formulated in myristic acid- based nanoparticles (myristate-QD, solid line) is completely quenched, whereas the CdSe QDs formulated in beeswax-based nanoparticles (beeswax-QD, dotted line) emitted a very strong fluorescence peak at 610 nm wavelength after excitation at 450 nm.

Figure 17 shows number- weight distributions of various encapsulating nanoparticles:

(A) tristearin, (B) tribehenin, (C) glyceryl behenate, (D) glyceryl palmitostearate, (E) glyceryl monostearate 40-55, (F) phosphotidylcholine, (G) beeswax (H) lauric acid, (I) myristic acid, (J) stearic acid, (K) 50:50 tribehenin: stearic acid, and (L) 75:25 tribehenin: stearic acid.

Figure 18 shows fluorescence spectra of quantum dots encapsulated within different lipids (A- J) having various lipid structures. *: indicates that quantum dots were dispersed within these chemicals.

Figure 19 shows the effect of carboxylic acid concentration on the quantum dot emission spectra.

Figure 20 shows the effect of lipid chain length on the emission spectra of the quantum dots. *: indicates that quantum dots were dispersed within these chemicals.

DETAILED DESCRIPTION OF THE INVENTION

Generally speaking, the systems described herein are directed to fatty ester-based solid nanoparticles and methods of use thereof. As required, embodiments of the present invention are disclosed herein. However, the disclosed embodiments are merely exemplary, and it should be understood that the invention may be embodied in many various and alternative forms. The Figures are not to scale and some features may be exaggerated or minimized to show details of particular elements while related elements may have been eliminated to prevent obscuring novel aspects. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention. For purposes of teaching and not limitation, the illustrated embodiments are directed to fatty ester-based solid nanoparticles comprising a fluorescent payload and imaging methods of use thereof.

As used herein, the terms, "comprises" and "comprising" are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in this specification including claims, the terms, "comprises" and "comprising" and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other configurations disclosed herein.

As used herein, the terms "about" and "approximately", when used in conjunction with ranges of dimensions of particles, compositions of mixtures or other physical properties or characteristics, are meant to cover slight variations that may exist in the upper and lower limits of the ranges of dimensions so as to not exclude embodiments where on average most of the dimensions are satisfied but where statistically dimensions may exist outside this region. It is not the intention to exclude embodiments such as these from the present invention.

Embodiments provided herein disclose formulations of fatty ester-based nanoparticles (FEN) for delivery of fluorophore pay loads, and methods of forming and utilizing such nanoparticles for imaging and other applications. The novel formulations are particularly useful for encapsulating near infrared (NIR) PbSe QDs resulting in lower in vitro

cytotoxicity, improved physiological stability, in vivo biodistribution and clearance of the semiconductor nanocrystals, and enhanced aqueous QD optical properties resulting in deep- tissue imaging capabilities.

As further described below, the encapsulation of QDs and other fluorophores with clinically relevant optical properties in fatty ester-based nanoparticles yields improved aqueous quantum yield of QDs, and improved imaging depth, resulting in significant progress towards enhanced fluorophore biocompatibility and utility. Accordingly, the embodiments provided herein enable non-invasive, non-ionizing, and low cost deep tissue fluorescent imaging, supporting rapid, high spatial and temporal resolution images. In addition, the nanoparticle formulations are compatible with most emulsifiers (e.g. Poloxamer 188, polysorbate 80, lecithin, sodium glycocholate, and Pluronic® types) approved by drug regulatory agencies. This versatility as a carrier of a variety of payload makes the fatty acid- ester based nanoparticles a potential generic platform for the delivery of diverse therapeutic and diagnostic agents.

Referring to Figure 1, a nanoparticle 100 is shown comprising fluorescent nanoparticles 110 residing in a fatty matrix 120 comprising a fatty ester. Unlike known lipid encapsulation nanoparticles and methods, the fatty ester nanoparticles (FEN) of the present embodiment employ a fatty matrix containing fatty esters, which provide a suitable environment for protecting organic ligand-capped quantum dots without compromising quantum efficiency. The solid fatty matrix comprising a fatty ester encapsulates the fluorescent particles and shields them from an external environment. The shielding property of the fatty ester matrix maintains a high quantum efficiency of the fluorescent particles and provides an effective transport mechanism for numerous applications, including in vivo imaging.

The fluorescent particle may be one or more particles or a collection or cluster thereof, including, but not limited to, semiconductor nanocrystals, quantum dots (including CdSe, CdS, CdTe, CdSe/ZnS, CdSe/ZnSe, InAs, InP, PbSe, PbS); silicon nanocrystals, carbon nanocrystals, silica-organic dye hybrid nanoparticles, calcium phosphate-organic dye hybrid nanoparticles; and fluorophore-doped polymer and glass particles, such as fluorescent beads.

Quantum dot passivation techniques known to those skilled in the art include the use of organic capping ligands, such as oleic acid, and inorganic wide-bandgap materials. In the latter case, the inorganic shell surrounds the quantum dot core to protect the internal exciton from quenching surface states, and must be carefully formed. On the other hand, the passivation function of organic capping ligands is compromised due to steric hindrance and photo-degradation. In contrast, the solid fatty ester-based matrix according to the present embodiments can encapsulate the quantum dot and shield it from an external environment. Accordingly, a wide variety of quantum dots can be encapsulated, including those capped by organic ligands and inorganic layers.

The size limitations of quantum confinement limit the size of quantum dots that retain fluorescence. Generally, QDs are less than 10 to 12 nm (usually less than 5 nm) and as such can be loaded into fatty ester particle of the invention having a size in the range of 20 to 3000 nm. The FEN 100 preferably has a diameter ranging from 20 to 1000 nm. A diameter of 50 to 250 nm has been found to be suitable for long circulation in the body.

The fluorophores may include, but are not limited to, xanthene derivatives (including fluorescein, rhodamine), cyanine derivatives (including cyanine, indocarbocyanine), napthalene derivatives (including dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives, pyrene and derivatives, oxazine derivatives (including Nile red, cresyl violet), acridine derivatives, arylmethine derivatives, tetrapyrrole derivatives (including porphyrin, phthalocyanine), and polymethines. The fatty matrix preferably comprises a concentration of fatty ester ranging from approximately 5% to 100% by weight. In one embodiment, the fatty matrix may be formed substantially from fatty esters. In another embodiment, the fatty matrix may comprise both fatty ester and fatty acid. In another embodiment, the fatty matrix may comprise fatty esters, fatty alcohols, hydrocarbons, fatty acid and other lipid soluble compounds. As will be described further below, the inventors have found that a fatty ester-based nanoparticle formed according to embodiments provided herein can effectively avoid quenching of encapsulated uncapped quantum dot fluorophores.

Many biocompatible and biodegradable lipids (e.g. fatty esters of glycerol such as tristearin, tripalmitin, trilaurin, glyceryl behenate; fatty esters of sucrose; hard fats such as Witepsol® series, cetylpalmitate, C10-C54 saturated and unsaturated fatty acids with straight and branched chains including but not limited to stearic acid and palmitic acid), and C10-C54 saturated fatty alcohols with straight and branched chains are available for the preparation of fatty ester-based nanoparticle according to embodiments provided herein.

The fluorescent particle is preferably capped with a capping ligand in order to be encapsulated in the fatty ester nanoparticle. Suitable capping ligands for forming the particle cap include, but are not limited oleic acid, trioctyl phosphine, butanoic acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid hexadecanoic acid, 9- hexadecenoic acid, octadecanoic acid, 9-octadecenoic acid, 12-hydroxy-9-octadecenoic acid, 11-octadecenoic acid, 9,12-octadecadienoic acid, 9,12,15-octadecatrienoic acid, 6,9,12- octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid, 5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, docosanoic acid, 13-docosenoic acid, 4,7,10,13,16,19- docosahexaenoic acid, tetracosanoic acid, and alkylphosphenes.

FEN 100 may be coated with block co-polymers 130 such as poly(ethylene glycol) (PEG) linked with a fatty chain or a hydrophobic polymer chain, such as Myrj™ 52, Myrj™ 56, Brij™ 58, or any member of the Myrj™or Brij™ copolymer families, Tween® family surfactants, Tergitol® family surfactants and Pluronic® family surfactants that provide surface PEGylation of the FEN, imparting unique biodistribution and biocompatibility properties. Other examples of surface PEGylating block copolymers contain a block or more blocks of poly(ethylene oxide) and a block or two blocks of hydrophobic polymers such as poly(propylene oxide), polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic acid- co-glycolic acid), polydioxanone, polyurathans, polystyrene, and polycarbonates. The PEGylation serves two important functions: (1) it makes the particles invisible to the cells of the body that would otherwise scavenge them from the blood and reduce their circulation times; and (2) it ensures a stable nanoemulsion is formed and it provides for longer term storage of the FEN once formed. By providing a PEG corona, one can sterically stabilize individual particles, preventing irreversible aggregation in aqueous media. Upon storage in a refrigerated environment, PEGylated FEN can be readily redispersed with manual shaking.

Other hydrophilic polymers can be used in place of PEG, including, but not limited to, poly (vinyl alcohol), poly (vinyl pyrrolidone), linear or modified starch, Tween® family surfactants, Triton® family surfactants , Tergitol® family surfactants, and Pluronic® family surfactants. Other nanoparticle stabilizing compounds include Span® family surfactants, egg phosphatidylcholine, egg lecithin, and soy phosphatidylcholine. Another alternative involves including the addition of activated groups on the surface of the FEN and then post-grafting of a spacer chain such as PEG to the surface.

Referring now to Figure 2, a method is provided for preparing QD-FEN. In step 200, quantum dots are provided for encapsulation in a FEN and suspended in a suitable solvent, preferably a hydrophobic low boiling point (less than 60°C) solvent, such as a solvent comprising hexanes, chloroform, acetone, methanol, ethanol, acetate, and dichloromethane. In step 210, one or more fatty esters are provided and heated until melted and in step 220, the quantum dot suspension is added to the molten fatty ester. The solvent is evaporated in step 230, preferably under agitation, to obtain a dispersion of the QDs in the molten fatty ester.

An aqueous solution of at least a surfactant or a copolymer is added to the molten mixture in step 240. For example, approximately 0.01 % to 10% of total emulsion volume of Pluronic® F68 may be added, preferably 0.05% to 2%, to achieve more uniform

nanoparticles in terms of shape and size. This amount was rationally chosen as it was found to provide uniformly spherical FEN without forming micelles in the continuous phase.

A quantity of water (for example, approximately 0.001:1 to 2:1 ratio of water to the quantity of fatty ester; preferably distilled and deionized) is added in step 250, where the water is pre-heated above the melting temperature of the fatty ester.

The emulsion is subsequently ultrasonicated (or alternatively microfluidized or homogenized) in step 260. Following sonication, the entire emulsion is dispersed in an aqueous suspending medium that is biologically inert and isotonic, such as cold 0.9% saline, a buffer solution or 5% dextrose in step 270. This step is performed within a suitable time interval to prevent the still molten nanoemulsion droplets from coalescing, which can otherwise result in the nanoemulsion droplets not forming uniform or nano-sized FEN.

Block copolymers are preferably included in the formulation to stabilize the molten nanoemulsion droplets, and to coat the surface of the solidified nanoparticles with poly(ethylene glycol) chains, imparting enhanced biocompatilibity to the QD-FEN. The block copolymers are preferably added prior to performing step 250. Suitable block copolymers are listed above.

In another embodiment, the FEN may encapsulate one or more fluorophores.

Fluorophores that can be delivered using the fatty ester nanoparticles according to embodiments disclosed herein include organic and particulate fluorophores. Exemplary organic fluorophores include: xanthene derivatives, including fluorescein, and rhodamine; cyanine derivatives, including cyanine and indocarbocyanine; napthalene derivatives, including dansyl and prodan derivatives; coumarin derivatives; oxadiazole derivatives;

pyrene derivatives; oxazine derivatives including Nile Red and cresyl violet; acridine derivatives; arylmethine derivatives; and tetrapyrrole derivatives including porphyrin and phthalocyanine. Multiple fluorophores may be encapsulated within a given FEN to enable multi-window or multi-band spectral applications. In one embodiment, a FEN may encapsulate one or more fluorophores and one or more fluorescent particles, and the emitted signals from the multiple fluorophores or fluorescent particles may be spectrally resolved.

Fatty ester nanoparticles with encapsulated fluorophores may be fabricated in a manner similar to that disclosed above for the encapsulation of nanoparticles. However, the step of providing a hydrophobic cap need not be performed in the case of the fluorophores, as will be readily understood by those skilled in the art. The fluorophores may be dissolved or dispersed in a suitable organic solvent (as in the case of the fluorescent nanoparticles) prior to mixing with the molten ester-containing fat, or may be directly added in power form to the molten fat. After incorporating the fluorophore into the molten fat, the process steps disclosed above in the case of fluorescent nanoparticles may be followed to obtain the fatty ester-based nanoparticles with encapsulated fluorophores.

Fatty ester-based nanoparticles are beneficial in their low toxicity and high biocompatibility. Compared to synthetic polymers, fatty esters are more attractive as they are natural chemicals existing or as metabolic byproducts in the body. They are also found in large quantity in dietary food including fats and oils of plant and animal origins. Therefore, they are generally recognized as safe (GRAS) materials by the regulatory agencies such as US Food and Drug Administration (FDA) and Health Canada.

In addition to high safety and biocompatibility, nanoparticulate carriers comprising fatty esters can exhibit advantageous properties which polymeric micelles and liposomes lack. Polymer micelles have a core-shell structure while liposomes have a lipid bilayer structure. These structures are formed only under certain conditions and can dissociate in the physiological media upon injection and/or being diluted by the body fluid, thus, prematurely releasing the payload.

In contrast, the fatty ester-based nanoparticles disclosed herein have a solid matrix structure in vivo owing to a melting temperature higher than the body temperature. Thus, these particles are more stable than liposomes and micelles, providing improved and prolonged protection of the payload, e.g. fluorophore. They have less storage and payload leakage problems compared to systems such as liposomes. They are easy to manufacture and do not carry the relatively high cost required for large scale production of liposomes.

Moreover, the significant toxicity and acidity associated with a number of biodegradable polymeric materials are also not expected in the fatty ester-based nanoparticles.

Fluorescent FEN as described above are useful for a wide range of applications, including imaging. In a preferred embodiment, fluorescent FEN are prepared to enable deep tissue imaging. Referring to Figure 3, visible fluorophores, when employed for tissue imaging applications, suffer from (a) poor excitation and/or (b) a lack of detectable emission. The poor applicability of visible fluorophores results from the high absorption and scattering cross sections of tissue in the visible spectrum. As shown in Figure 3(c) and Figure 4, near infrared fluorophores, preferably with emission and excitation wavelengths within the range of 650 nm to 950 nm, are much better suited to in vivo imaging applications.

Furthermore, fluorescent FEN may include a binding moiety, label, reporter, hapten and/or conjugate for use in various applications, such as diagnostic assays and imaging applications. For example, FEN may be functionalized with ligands, receptors, and/or targeting moieties such as proteins, peptides, aptamers, nucleic acids, carbohydrates, and antibodies for specifically binding FEN to selected analytes and/or tissues, such as tumor cells.

Accordingly, in one embodiment, shown in Figure 4, a fluorescent FEN comprises an infrared fluorescent payload (i.e. a fluorophore and/or infrared fluorescent particle(s)), enabling deep tissue imaging. The emission spectrum of the fluorescent payload is preferably selected to be suitable for fluorescent imaging through a tissue. The fluorescent payload is preferably one or more PbSe nanocrystals, such a quantum dot or clusters thereof. The combination of stealth functionality, biocompatibility, high quantum efficiency, and infrared excitation and emission makes such nanoparticles uniquely well suited for imaging applications, including whole-body imaging, as further described below.

While the methods and systems described below demonstrate single photon excitation fluorescence imaging, the excitation of the fluorophores may be achieved through a multi- photon absorption process. Other fluorescent modalities that can be used include

photoacoustic imaging, single and dual photon, fluorescent molecular tomography, and hyperspectral imaging.

The following examples are presented to enable those skilled in the art to understand and to practice the present invention. They should not be considered as a limitation on the scope of the invention, but merely as being illustrative and representative thereof.

EXAMPLES EXAMPLE 1: QD-FEN

Synthesis of Fatty Esters

25

Fatty esters were synthesized as described in Hoshi et al. (1973). Briefly, eicosanoic acid was dissolved in chloroform, to which an ethanolic solution of cupric acetate, as well as ethanolic HCl, was added. The solution was stirred for 1 hour, after which time the reaction was quenched with distilled deionized water, and the fatty ester extracted with chloroform. The chloroform was evaporated under nitrogen gas, yielding a white solid. The solid was dissolved in CDCI₃ and the structure was confirmed by standard ^lH- and ¹³C-NMR performed on a Varian Mercury 300 MHz instrument. The proton and carbon NMR spectra and molecular structures of the fatty ester, ethyl eicosanoate, and the fatty acid, eicosanoic acid, are given in Figures 5(a) and 5(b). No residual eicosanoic acid was observed in either NMR spectrum, indicating complete conversion from acid to ester.

Various lipids were evaluated for their ability to form FEN including the esters of fatty acids of carbon chain lengths ranging from C1-C54. The nature of the other lipid constituents can vary greatly with the source of the waxy material, but they include hydrocarbons, sterol esters, aliphatic aldehydes, primary and secondary alcohols, diols, ketones, β-diketones, and many more. Formulation of QD-FEN

Lead selenide (PbSe) nanocrystals were employed for the formulation of QD-FEN due to their relative ease of synthesis, the tunability of their emission within the NIR range, their high resultant quantum yields with NIR excitation, and the demonstrated photostability of this semiconductor system. ^{H 15}' ²² The synthesis of PbSe QDs was based on a room temperature scheme under ambient atmospheric conditions described in Evans et al. (2008)¹⁵, resulting in hydrophobic, oleic acid-capped nanocrystals ~7 nm in diameter further suspended in hexanes.¹⁵

Briefly, lead oxide was dissolved in octadecene and oleic acid under a nitrogen atmosphere at 150°C. Once dissolved, the solution was cooled, opened to ambient air, and a solution of selenium in trioctylphosphine was added. The solution was stirred for 4 hours, after which time the quantum dots were isolated through the addition of butanol and methanol. For preparation of water-soluble QD for toxicity assessment, the exchange of oleic acid for MTPEG on the PbSe QD surface was accomplished as described in Evans et al. (2008).¹⁵ The fluorescence spectra of the PbSe QDs were recorded on an Ocean Optics spectrofluorimeter.

Fatty ester (e.g. ethyleicosanoate) (50 mg), as well as the copolymer surfactants Myrj™ 52 ( 12.5 mg) and Myrj™ 56 ( 1.5 mg), were heated to 80°C until melted. The molten mixture was stirred and PbSe QDs suspended in hexanes were added. The molten mixture was stirred and PbSe QDs suspended in hexanes were added. The mixture was stirred for 20 minutes to allow for the evaporation of hexanes and dispersion of the QDs in the molten fatty ester. An aqueous solution of Pluronic® F68 was then added to the molten mixture, followed by distilled deionized water pre -heated to 80°C. The emulsion was ultrasonicated using a Hielscher UP-100S probe sonicator (100 Watts, 30 kHz) for 5 minutes. Immediately following sonication, the entire emulsion was dispersed in cold 0.9% saline.

Particle size and zeta potential were measured on a Nicomp 380 Zetasizer by dynamic light scattering and electrophoretic light scattering, respectively. Particle morphology and QD loading were assessed by transmission electron microscopy of unstained samples using a Hitachi H-7000 microscope.

The formulated QD-loaded fatty acid or fatty ester nanoparticles were 110+15 nm in hydrodynamic diameter, as measured by dynamic light scattering, with the PbSe QD being trapped within the now solid matrix (as shown in Figure 1). The measured zeta potential (ζ = -20+1.3 mV) was favorable to both a stable emulsion and good biocompatibility. Myrj™ block copolymers (Myrj™ 52 and Myrj™ 56) were included in the formulation to stabilize the molten nanoemulsion droplets, and to coat the surface of the solidified nanoparticles with poly(ethylene glycol) chains, imparting enhanced biocompatibility to the QD-FEN (as shown in Figure 1).

The coating of nanoparticles with a poly(ethylene glycol) corona was previously shown to impart a stealth property to liposomal formulations, evading the recognition and uptake by the reticuloendothelial system, thus, extending the circulation time of the particles in the body.²³ Under electron microscopic imaging, the PbSe QDs were clearly seen as black spots within the matrix of the QD-FEN, seen in Figure 7(a). A transmission electron micrograph of PbSe QDs on their own is shown in the top right inset of (a). The electron density characteristic of metal particles was absent in electron micrographs of QD-free fatty ester nanoparticles, as shown in Figure 7(b).

Spectral Characteristics of QD-FEN

PbSe QDs showed an emission maximum at 860 nm (Figure 8(a)) and a quantum yield in hexanes of ~ 15% at an excitation wavelength ke_X of 710 nm, as determined using l, r-diethyl-4,4'-carbocyanine iodide as the quantum yield standard ( x = 710 nm, X_em = 735 nm, quantum yield = 3.6% 24 ). PbSe QDs encapsulated in myristic acid-based nanoparticles demonstrated near complete quenching of the QD emission (Figure 8(a)). Without intending to be limited by theory, the QD-quenching phenomenon has been attributed to either the physical transition of the QD environment from a liquid to a solid phase as the nanoemulsion was cooled and the fatty droplets were solidified, or to the presence of the carboxylic acid headgroups of the fatty acid chains.

To investigate each potential cause separately, PbSe QDs were synthesized and suspended in n-octanol or 1-octanoic acid, and the fluorescence emission spectra were recorded, and are shown in Figure 8(b). Since both dispersing media were liquid at room temperature, any fluorescence quenching would only be due to the presence of the carboxylate of the octanoic acid and not the physical phase of the medium itself. The photoluminescence curves obtained provided strong evidence that the fluorescence quenching of the QDs by the myristic acid nanoparticles was due to the carboxylate groups of the fatty acid matrix.

To mask the carboxylate groups of the fatty acid matrix, fatty acid ethyl esters of eicosanoic acid were synthesized as described in Hoshi et al. 25 and QD-FEN were formulated. The return of the strong photoluminescence at 860 nm was observed (Figure 8(a)), providing further evidence that the previously observed QD fluorescence quenching was due to the interaction of the carboxylate groups of the fatty acid with the surface of the PbSe QDs and not due to the liquid-to-solid phase transition of the nanoparticulate carrier matrix.

Use of ethyl eicosanoate as the carrier matrix resulted in a 3 -fold increase the quantum yield of QD-FEN suspended in water compared to free PbSe QDs suspended in hexanes, increasing from -15% to -45%, determined at λ_εχ = 710 nm with 1, l '-diethy 1-4,4'- carbocyanine iodide as the quantum yield standard. The previously published quantum yield for water-soluble PbSe QDs stabilized by (1-mercaptoundec-l l-yl)tetra(ethylene glycol)

(MTPEG) was 30%, ¹⁵ indicating an increased efficiency of 50% for the QD-FEN formulation determined spectrally compared to free water-soluble QDs.

It is believed that the enhanced fluorescence efficiency of QD-FEN can be ascribed to the protection of QDs in a hydrophobic nanoenvironment of fatty ester matrix. The preparation of water-soluble QDs involves exchange of hydrophobic ligand with hydrophilic ones and direct dispersion of QDs in aqueous medium, which have previously been shown to decrease the quantum yield of PbSe QDs.¹⁵ In the present formulation, the PbSe QDs were maintained in their oleic acid cap and encapsulated in the hydrophobic matrix of QD-FEN and were never in direct contact with the aqueous medium, leading to high quantum yields. Imaging Depth of Penetration of QD-FEN

One limitation of medical fluorescence imaging is the shallow distance of image acquisition, often to below 10 mm from the surface of the skin.⁶ In order to quantitatively measure the depth of image acquisition possible using QD-FEN, a novel system for the continuous measurement of biologically relevant imaging depth was developed and implemented, as shown in Figure 9.

Tissue phantoms were prepared based on the compositions described in De Grand et al. (2006).²⁶ Tris-buffered saline was poured on top of a known amount of sodium azide and gelatin to make a final concentration of 15mM and 10w/v% respectively. The mixture was heated to 50°C using a water bath (Haake D8 Immersion heater) with constant stirring by a Caframo BDC 1850 overhead stirrer at 650-800 rpm. Once the gelatin was dissolved, the mixture was cooled to 37°C with constant stirring. Hemoglobin and intralipid were added to reach concentrations of 170μΜ and 1 v/v%, respectively. The construction methods of the tissue phantom are provided in Figure 6. In step 400, a gel cassette intended for electrophoresis is used as a mold to form a 1 mm thick slab of tissue phantom containing QD-FEN. QD-FEN in saline are mixed with warm phantom mixture at 1 mg QD-FE/mL or 5 mg QD-FEN/mL concentrations. For the 5 mg QD-FEN/mL slab, extra gelatin is added to make a final gelatin solution of 10 wt % to ensure proper slab solidification. Glassware is warmed to 37°C to prevent premature solidification of the phantom mixture. The warm QD-FEN mixture is then pipetted into the space between the two glass plates almost to the brim. The entire set up is then refrigerated at 4°C until solidified.

Prior to phantom construction, the mold is refrigerated at 4°C to ensure rapid solidification of the phantom and prevent intralipid separation. As shown in step 410, to construct the bottom layer of the phantom, the mold is tilted such that the bottom is inclined to 45°. Warm tissue phantom mixture is poured to a desired depth and tapped to remove any air bubbles. The bottom layer is refrigerated until solid. The remainder of the tissue phantom mixture is kept warm at 37°C with constant stirring.

In step 420, the QD-FEN slab is removed from the electrophoresis gel cassette and trimmed to fit the mold. The slab is then laid midline across the length of the bottom tissue phantom layer, and any air bubbles are smoothed out.

Warm tissue phantom mixture is poured over top to fill the mold in step 430, and the mold is refrigerated until solidified. For depths approximately 3 cm or deeper, warm tissue phantom is poured and solidified layer-by-layer to prevent dissolution of the QD-FEN slab layer.

In step 440, solidified tissue phantom is removed from the mold by running a thin spatula around the edge. Once complete, the tissue phantom is stored at 4°C until needed. Excess tissue phantom mixture or QD-FEN slab mixtures are also stored at 4°C, to be melted at 37°C for re-use.

In order to ensure that the composition of the tissue phantom produced a gel with optical properties comparable to that of human tissue, the scattering coefficient (μ₈) and absorption coefficient (Ua) were quantified using integrating spheres. The sample was mounted between 2 BK7, 1mm thick optical windows (Esco Products), which were separated by 1 mm thick spacer. The spacer had a cutout at the top so that the phantom mixture could be poured in before it solidified. A halogen white light source (HL-2000, Ocean Optics) was used, via focusing lenses, to illuminate the sample when it was mounted in a Reflectance - Transmittance (RT) integrating sphere system. The 6 inch diameter integrating spheres (SphereOptic) were mounted one at a time to eliminate cross talk. The R sphere was mounted in front of the sample position and reflectance, background, and reference signal spectra were recorded. The R sphere was then removed and the T sphere was mounted behind the sample. The transmittance, background and reference spectra were recorded. A Monte-Carlo simulation using 10⁶ photons and an asymmetry parameter = 0.9 generated a lookup table which matched the R and T values with Ua and μ₈ values.

Tissue phantoms were imaged using a Xenogen IVIS Spectrum instrument in epifluorescence mode. The phantom was placed on the stage and images were taken with automatic exposure and aperture settings in field of view C. One image was taken from the top of the gel phantom, and a second image was taken of the cross section of the gel. Intensity scales were set to a monochromatic (red) type with units of radiant efficiency and the range was set to full.

ImageJ²⁷ was used for image analysis, which consisted of four steps:

i. Using the monochromatic scale, a calibration of pixel intensity versus the intensity value in radiant efficiency was performed. The dimensions of the intensity scale were determined in pixels, allowing the dimensions of one pixel to be determined in terms of the intensity scale dimensions. ImageJ was then used to determine the pixel-by-pixel intensity of the intensity scale bar, allowing for a plot of pixel intensity versus radiant efficiency to be generated.

ii. Using the top view image of the gel phantom, the pixel intensity versus the distance in millimeters from the origin (selected to be the top-most point of the QD-FEN slab in the gel phantom) was determined. The determination of the dimensions of a pixel allowed for a plot of pixel intensity versus distance in millimeters from the origin to be generated.

iii. Using the cross-sectional image, the depth of the QD-FEN gel slab was determined from the top of the gel phantom, resulting in a plot of distance from the origin versus QD-FEN slab depth

iv. Combining the plot of pixel intensity versus distance from the origin (Step 2) with the plot of distance from the origin versus QD-FEN slab depth (Step 3), a plot of pixel intensity in radiant efficiency versus QD-FEN slab depth was generated, as shown in Figure 9(d). Measures of the distance a photon could travel in the phantom before the photon was absorbed (μ₃) or scattered (μ₈) were performed on 1-mm thick samples of the tissue phantom using two integrating spheres in series to prevent cross-talk between absorption and scattering measurements (Figure 9(b)), and were found to be in agreement with the same parameters of real tissue.²⁶ The 1-mm thick slab containing the QD-FEN continuously and nearly linearly decreased in depth from one side of the phantom to the other, permitting the quantification of fluorescence signal strength as a function of phantom depth from a single fluorescent image acquired with a Xenogen I VIS Spectrum whole animal imager (Figure 9(c)). Quantification of the signal intensity versus depth was performed with ImageJ,²⁷ yielding the plot of signal intensity versus penetration depth in Figure 9(d). From this plot, it can be seen that the QD- FEN showed strong signal penetration beyond 20 mm, reaching nearly 25 mm in depth with 5 mg QD-FEN/mL phantom containing a total of 1.6 mg of PbSe QDs. Previous quantitative investigations of the penetration depth of fluorescence of NIR fluorophores reported limits up to 9.7 mm,²⁶ less than half of the penetration depth of the QD-FEN.

Determination of Cytotoxicity of QDs and QD-FEN

The main limitation preventing the application of semiconductor nanocrystals to medical fluorescent imaging is the toxicity arising from their heavy metal constituents,¹⁷ their surface capping and functionalizing agents,¹⁸ and their poor pharmacokinetic performance,¹⁹ e.g. accumulation mainly in the liver without being cleared.²⁰ In the case of PbSe QDs, the well known toxicity of Pb²⁸ and the potential toxicity of Se at high doses²⁹ limits the biomedical applications of this nanocrystal in its free form.

The toxicity of QD-FEN was assessed against water soluble MTPEG-capped PbSe QDs in isolated rat hepatocytes. Hepatocytes were isolated from rats by perfusion of the liver with collagenase as described in Moldeus et al. (1978).³⁴ Isolated hepatocytes (10⁶ cells/mL) (lOmL) were suspended in Krebs-Henseleit buffer (pH 7.4) containing 12.5mM HEPES in continually rotating 50mL round-bottomed flasks, under an atmosphere of 95% 0₂ and 5% C0₂ in a water bath of 37°C for 30 min. Hepatocyte viability was assessed microscopically by plasma membrane disruption as determined by the trypan blue (0.1% w/v) exclusion test.

35

Hepatocyte viability was determined at 30, 60, and 120 min, and the cells were at least 80- 90% viable before use.

This model has been used for rapid toxicity screening and has demonstrated in vitro- in vivo toxicity extrapolation.³⁰ Water soluble, MTPEG-PbSe QDs showed significant hepatocyte toxicity, resulting in less than 40% hepatocyte survival upon exposure to 6 μg/mL of PbSe QDs after 120 min (Figure 10). Exposure of hepatocytes to the same dose of PbSe QDs in QD-FEN for 120 min resulted in the survival of 80% of hepatocytes. Even after exposure to 30 μg/mL of QD-FEN for 120 min, approximately 70% of hepatocytes still survived.

The fatty ester nanoparticle itself has been shown to be biocompatible in vitro³¹ and in vivo³², a result that was confirmed with the hepatocyte toxicity screening assay presented herein. Without intending to be limited by theory, it is believed that the reduced toxicity of QD-FEN may arise from the mitigation of two processes involved in QD toxicity: the loss of free heavy metal ions from QD degradation,¹⁷ and the aggregation of the QDs in tissue due to loss of the water-soluble capping ligand from the nanocrystal surface.⁶' ¹⁸ The degradation of the nanocrystal into its free metal ions is thought to occur in the endolysosomal compartment of the cells following QD uptake, a compartment that is acidic and promotes metal ion loss. Since PbSe QDs are trapped within the solid matrix core of the fatty ester carrier, the endolysosomal acidification is unlikely to reach the fatty ester-encased QDs, resulting in maintenance of structural integrity and prevention of free Pb²⁺ leaching. Since PbSe QDs in a hydrophobic, oleic acid-capped state favorably interact with the hydrophobic core of the fatty ester nanoparticles stabilized by PEG and surfactant, their aggregation becomes moot. As a result, the encapsulation of PbSe QDs in the unique hydrophobic, biocompatible matrix of fatty ester nanoparticles significantly reduces the toxicity of PbSe QDs in vitro.

In Vivo Whole Animal Imaging with QD-FEN

Human MDA435 breast adenocarcinoma cells were stably transfected to express enhanced green fluorescent protein (EGFP). The EGFP plasmid (pEGFP-Nl, Clontech) was linearized by digestion with the restriction endonuclease Dra III (New England Biolabs), and was transfected into the cells by incubation with CaPC . After 24 hours of incubation, cells were grown in selection media containing minimal essential medium supplemented with 10% fetal calf serum and 600 μg/mL of the selection antibiotic G418. Cell colonies that formed and that were fluorescent were lifted and plated in selection medium in individual wells of a 24-well microplate. The three best colony expansions, determined by fluorescent intensity and number of fluorescent cells, were selected for sorting by fluorescence activated cell sorting (FACS). The brightest 20% of each of the three expanded colonies were selected by FACS, and were further expanded in regular growth medium. Two hundred μΕ of QD-FEN in saline suspension (64 μg of PbSe QD total) were injected intravenously in the tail vein of nude mice.

The mice were inoculated in the inguinal mammary fat pad with the human breast tumor cell line (MDA435) stably expressing green fluorescent protein. To establish orthotopic xenograft breast tumors, 1 million human MDA435 cells expressing EGFP were injected into the inguinal mammary fat pads of nude mice using the subiliac lymph node as an injection landmark to ensure greater mouse-to-mouse reproducibility of the tumor model. Tumors were monitored for growth using fluorescent imaging during the course of the three week growth period (Figure 11(a)). Mice were injected with QD-FEN formulation into the lateral tail veins and anaesthetized with isoflurane. Images were acquired using a Xenogen IVIS Spectrum whole animal imager with λ_εχ = 710 nm and X_em = 840 nm. Mice were imaged intact for 7 days to determine both the distribution of the fluorescent hybrid nanoparticle, as well as the clearance of the fluorophore over time (Figure 11(b)). Background fluorescence was detected as largely intestinal background due to porphyrins in the mouse chow.

Following the intravenous injection of QD-FEN, clear, bright fluorescence was observed. Within the first 2 hours of administration, broad fluorescence was observed throughout the animal with bright spots in the abdominal region. Abdominal fluorescence was observed not to arise from the liver, the main site of uptake of free, water-soluble QDs and the main site of QD retention that results in the poor biocompatibility of the Fluorophore in such cases.²⁰ From 4 to 8 hours after injection, the QD-FEN were seen to accumulate in the spleen, and this accumulation could be seen to decrease to nearly pre-injection levels by 96 hours after injection (Figure 11 (b)). Within 7 days (168 hr), whole animal fluorescence decreased to nearly background levels, evidence that the encapsulation of PbSe QDs in FEN can enhance the biocompatibility of QDs by enabling near complete clearance of the fluorophore from the body. In contrast, free QDs in vivo are retained long term and demonstrate poor clearance, the major cause of concern regarding the application of semiconductor nanocrystals in a clinical setting. 19 ' 20 ' 33

The fatty ester nanoparticle fluorophore delivery system of the present invention facilitates fluorescence that maximizes the utility of whole animal fluorescence imaging instrumentation, providing excitation (710 nm) and emission (840 nm) wavelengths as far towards the near infrared as the excitation sources and cameras can accommodate. This is important as the further in the near infrared the fluorophore can excite and emit, the greater the tissue penetration depth of the exciting and emitting photons, and the greater the depth of the structures that can be seen in vivo. The fluorescence signal of the QD-loaded FEN in a simulated tissue were detected up to a 25 mm depth, more than double the penetration depth reported previously (e.g. 9.7 mm) [26]. In live mice, signals from the QD-FEN in deep tissue were clearly seen by the imager. QD-FEN injected intravenously into nude mice were shown to achieve deep tissue imaging without signs of toxicity for up to 3 weeks, following repeated injections (once a week for 3 weeks).

By zooming in on the mammary fat pad area of the animal, tumor fluorescence above tissue background was noted by 1 hour (60 min) after QD-FEN injection, indicating the passive accumulation of QD-FEN within the tumor tissue (Figure 11(d)). Organs identified in Figure 11(c) include the heart 300, lungs 305, liver 310, gall bladder 315, kidneys 320, spleen 325, in addition to the tumor 330. Post-mortem examination of nude mice 3 hours after injection with saline, 3 hours after QD-FEN injection, and 7 days following QD-FEN injection revealed accumulation of the QD-FEN primarily in the spleen and gall bladder at 3 hours, with some accumulation in the kidneys (Figure 11(c)). The renal accumulation decreased to background and the QD-FEN were almost completely cleared from the spleen and gall bladder by 7 days. No hepatic, heart, or lung accumulation was noted even at 3 hours after injection, further demonstrating that the danger of hepatic uptake of the PbSe QDs was mitigated by encapsulation in the solid fatty ester nanoparticles. QD-FEN were shown to passively accumulate in the tumor by 3 hours after injection and retention of some of the QD- FEN was observed at 7 days post-administration.

EXAMPLE 2: Studies of Tumor Leakage and Tumor Imaging with FEN

Application of Near Infrared PbSe Quantum Dot-Loaded Solid Lipid Nanoparticles for the Assessment of Tumor Leakage in Live Animals

Xenograft tumors resemble spontaneous human tumors in morphology and thus are often grown in animals for evaluation of efficacy and toxicity of therapies including chemotherapy. To grow these tumors quickly and efficiently, cancer cells are commonly cultured on Matrigel® in preparation for inoculation. However, there are no data indicating the growth of solid tumors grown on Matrigel® will have the same morphology as those without Matrigel® with respect to assessment of enhanced permeability and retention (EPR) effect of nanoparticulate carriers and leakage of tumor tissue. QD-FEN were assessed for their ability to differentiate differences in tumor leakage from Matrigel®-based and

Matrigel®-free xenograft tumors. Xenograft tumors were generated by inoculation of human breast cancer cells bilaterally in the mammary fat pads (pars inguinalis) of female nude mice (Taconic Inc., USA). The subiliac lymph node was used as a landmark for the injection of lxlO⁶

MDA435/LCC6 human breast cancer cells expressing enhanced green fluorescent protein. In all mice, the right-side tumor contained cells suspended in oc-minimal essential medium, while the tumor on the left of the mouse contained cells diluted in Matrigel® (BD

Biosciences, USA). Tumors were grown for 3 weeks and imaged using a Xenogen IVIS Spectrum (Caliper Life Sciences, USA) system to detect the green fluorescent protein photoluminescence (excitation = 465 nm, emission = 540 nm, slit widths = 10 nm). To assess the differential rate of tumor leakage of nanoparticles, 50 \L (0.5 mg of particles) of PbSe QD-loaded FEN were injected intratumorally in the middle of each tumor, approximately 0.25 centimeters from the skin surface. Images of the mouse, ventral side up such that both tumors were visible simultaneously, were taken over the course of 6 hours using the Xenogen Ivis Spectrum to track nanoparticle residence in the tumors (excitation = 710 nm, emission = 840 nm, slit widths = 10 nm). Differential leakage of nanoparticles was clearly evident between the tumor with and without Matrigel®, with the Matrigel®-containing tumor showing more rapid nanoparticle clearance (Figure 12(b)). As more nanoparticles leaked from the tumor, there was less fluorescence from the tumor and more accumulation in the spleen, which was observed to fluoresce 2 hours after nanoparticle injection.

Application of Near Infrared PbSe Quantum Dot-Loaded Solid Lipid Nanoparticles for Microscopic Imaging of Solid Tumor Tissue

EXAMPLE 3: Preparation and Application of Co-encapsulation of Multiple

Fluorophores

50 mg of ethyleicosanoate, 4 mg of Myrj™ 56 and 8 mg of Myrj™ 52 were added to a 15 mL conical tube. The tube was heated at 65°C in a circulating water bath until the fatty ester and surfactants were melted. 4 mg of Pyrene was added in the molten fatty ester followed by addition of PbSe QD and stirred. 50μΙ. of lOOg/L of Pluronic® F68 and 388μΙ. of water are added to the solution. The solution is stirred for 20 minutes and then

ultrasonicated using a Hielscher UP100H probe ultrasonicator (Hielscher USA, Inc., Ringwood NJ, USA) at 80% peak amplitude and 5 mm probe depth in solution for 5 minutes. The entire emulsion is immediately transferred into 5 mL of distilled deionized water. The fluorescence spectrum of the dual fluorophore (pyrene and QD)-loaded FEN was analyzed with an Ocean Optics spectrofluorimeter. Figure 15 shows two distinct emission peaks respectively from pyrene and PbSe FEN. The same procedure can be applied to load other fluorescent dyes together with QDs in FEN including, but not limited to Nile red, fluorescein isothiocyanate, Indocyanine Green. QDs with different emission wavelengths can also be loaded in FEN using this method.

The applications of co-encapsulation of multiple fluorophores include sequential imaging of the same tissue in vivo macroscopically or microscopically, and ex vivo microscopically or visually. The NIR QD fluorophores are necessary for detecting the location of interest in living body.

EXAMPLE 4: Targeted cRGDfk-PbSe QD encapsulated FEN

Preparation of cRGDfk-PbSe QD Encapsulated FEN

50 mg of ethyleicosanoate, 4 mg of Myrj™ 56 conjugated to the targeting moiety (in this example, it was conjugated to either cRGDf or a blocked targeting moiety

c(CBQCA)RGDfK) and 8 mg of Myrj™ 52 were added to a 15 mL conical tube. The tube was heated at 80°C in a circulating water bath until the fatty ester and surfactants were melted. 400 μΕ of PbSe QD in hexanes or 4 mg of Pyrene was added to the molten mixture and stirred. 50μΕ of lOOg/L of Pluronic® F68 and 388μΕ of water are added to the solution. The solution is stirred for 20 minutes and then ultrasonicated using a Hielscher UP100H probe ultrasonicator (Hielscher USA, Inc., Ringwood NJ, USA) at 80% peak amplitude and 5 mm probe depth in solution for 5 minutes. The entire emulsion is immediately transferred into 5 mL of 0.9% sodium chloride solution.

Assay of Binding Capacity of cRGDfK-FEN on Integrin Receptor

The binding capability of the targeted FEN was assessed in vitro by incubating untargeted FEN, targeted cRGDfK-conjugated FEN, and blocked c(CBQCA)RGDfK- conjugated, pyrene-loaded FEN in a 96-well plate that was previously bound with integrin ανβ3, the cognate receptor for cRGDfK. As seen in Figure 13, without previous binding of the integrin receptor to the 96-well plate, no binding of untargeted, targeted, or blocked FEN occurred. However, with wells coated with the integrin receptor, binding was evident with targeted FEN. This binding was abolished when the targeting moiety was blocked

[(CBQCA)RGDfK] , when non-fluorescent cRGDfK-FEN were pre-incubated with the integrin receptor to occupy all binding sites, or when the well was pre-incubated with free RGDfK. Application of cRGDfk-PbSe QD-FEN for Optical Imaging of Tumor Neovasculatur

The active targeting of QD-loaded FEN was assessed in vivo using human

MDA435/LCC6 WT-EGFP tumors grown in dorsal skin fold window chambers. One million MDA435/LCC6-WT-EGFP human tumor cells were injected subcutaneously into the backs of nude mice and were let grow for 3 days. Dorsal skin fold window chambers were then assembled to reveal the now formed tumors. Untargeted QD-FEN and actively targeted RGDf -QD FEN were formulated, containing PbSe QD. QD-FEN were injected

intravenously into the mice (tail vein) and images were acquired using a fluorescent stereomicroscope at 12 hours following injection. The images in Figure 14 show the vascular distribution (left column, bright field), location of tumor growth (middle column, tumor), and location of untargeted (top row) or RGDf -targeted (bottom row) QD-FEN (right column, PbSe QD). The white arrows indication the co-localization of RGDf -QD FEN to areas of neo vascular beds of the tumor known to highly express the cognate RGD receptor (integrin α_νβ3). It is clear that the untargeted QD-FEN are more disperse and diffuse through the tumor, whereas the targeted QD-FEN are more punctate and localized to areas of high tumor expression of its cognate receptor.

EXAMPLE 5: QD Embodiment in Beeswax FEN

Formulation of CdSe QD-FEN in beeswax

Beeswax (50 mg) and copolymer surfactants Myrj™ 52 (12.5 mg) and Myrj™ 56 (1.5 mg) were heated to 80°C until melted. The molten mixture was stirred and

trioctylphosphine-capped CdSe QDs suspended in hexanes were added. The mixture was stirred for 20 minutes to allow for the evaporation of hexanes and dispersion of the QDs in the molten. An aqueous solution of Pluronic® F68 was then added to the molten mixture, followed by distilled deionized water pre-heated to 80°C. The emulsion was ultrasonicated using a Hielscher UP-100S probe sonicator (100 Watts, 30 kHz) for 5 minutes. Immediately following sonication, the entire emulsion was dispersed in cold 0.9% saline. The same method was applied to prepare CdSe QD embedded in myristic acid in place of beeswax.

Particle size and zeta potential were measured on a Nicomp 380 Zetasizer by dynamic light scattering and electrophoretic light scattering, respectively. The formulated QD-loaded fatty acid or beeswax nanoparticles were 110+15 nm in hydrodynamic diameter. Spectral Characteristics of CdSe QD-FEN

The spectra in Figure 16 shows that the fluorescence of CdSe QD formulated in myristic acid-based nanoparticles is completely quenched (solid line), whereas the CdSe QD formulated in beeswax -based nanoparticles emitted very strong fluorescence peak at 610 nm wavelength after excited at 450 nm (dashed line). This result suggests that presence of a certain proportion of fatty esters is essential for maintaining the fluorescence signal of the CdSe QDs.

Beeswax is a complex mixture of several chemical compounds with high proportion of wax esters (e.g. 35 to 80%). These compounds include straight-chain monohydric alcohols with even-numbered carbon chains from C¾ to C₃₆ and straight-chain acids also having even numbers of carbon atoms up to C₃₆ (including some Ci₈ hydroxy acids) e.g., esters, diesters and triesters. The free acids and alcohols occur in minor amounts. It contains also hydrocarbons having odd-numbered carbon chains from C₂i to C₃₃ and a minor amount of a colouring matter (3-hydroxy-flavone).

The composition of beeswax varies depending on its geographical origin. A typical composition of one product of yellow beeswax is (NRC, 1981):

Total esters 70-71 % (w/w)

Free alcohols 1-1.5% (w/w)

Free acids 9.6-10.9% (w/w)

Hydrocarbons 12.1-15.1 (w/w)

3-Hydroxyflavone 0.3 (w/w)

The foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents.

EXAMPLE 6: Effects of lipid structure on QD quenching

Due to their determined excitation and emission spectra and their nanometer size, QDs have been widely used in studies to determine various characteristics of nanoparticle drug delivery. Our previous experiments have suggested that the use of certain lipids in the encapsulation of QDs quench the signal of QDs. Experiments were performed to determine which lipids cause such an effect and to screen the lipid composition based upon the differences in chemical structure between the QD-quenching and non-quenching lipids. Materials and Methods

A 15mL conical tube containing 5mL of DDI water was refrigerated. In a second 15mL conical tube, 50mg of a lipid candidate, 4mg of PEG100SA, and 8mg of PEG40SA were melted at 80°C. While stirring, 400μΕ of QDs were added to the melted lipid and the conical tube left uncapped for 10 minutes to allow for the evaporation of hexane. In the case of phosphotidylcholine, 500μΕ of hexane was added in addition to the lipid candidate, 4mg of PEG100SA and 8mg of PEG40SA and the mixture vortexed to dissolve the

phosphotidylcholine. The conical tube was then left uncapped and stirred for 10 minutes to allow the hexane to evaporate.

While continuing to stir, 50μΕ of Pluronic® F68 (lOOmg/mL) and 388μΕ of DDI water were added to the mixture and stirred for 30 minutes. The solution was then ultrasonicated using a Hielscher UP100H probe ultrasonicator (Hielscher USA, Inc.

Ringwood NJ, USA) at 100% peak amplitude and 5mm probe depth in solution for 5 minutes at the same temperature. The entire solution was then pipetted into the conical tube containing the 5mL of DDI water and stirred for 5 minutes. Particle size was measured on a Nicomp 380 Zetasizer by dynamic light scattering. The fluorescence spectra of the undiluted particle were measured on an Ocean Optics spectrofluorimeter.

Polyoxyethylene 40 stearate, dodecanoic acid, beeswax, lead (II) oxide,

trioctylphosphine, 1-octadecene, selenium, hexane, tribehenin, tristearin, myristic acid, methanol, ethylenediaminetetraacetic acid, and L-a-phosphatidylcholine were purchased from Sigma- Aldrich. Stearic acid and 1-butanol were purchased from Fisher Chemicals. Precirol ATO 5, Compritol 888 ATO and glyceryl monostearate 40-55 type 1 were purchased from Gattefosse. Polyethylene (100) stearate (Myrj 59) was purchased from Spectrum, and Pluronic® F-68 was purchased from BASF. Chromic/sulphuric acid solution was purchased from Anachemia and ethyl arachidate was purchased from Tokyo Chemical Industry Co., LTD. Propionic acid and formic acid were purchased from BDH, and acetic acid from Caledon Laboratory Chemicals.

Synthesis of PbSe Quantum Dots

PbSe quantum dots were synthesized as previously reported. Briefly, within a nitrogen environment, lead (II) oxide was dissolved in 1-octadecene and oleic acid at a temperature 150°C and stirred for 1 hour. The solution was then removed from heat and allowed to cool to room temperature while remaining in a nitrogen environment. Once cooled, the solution was then exposed to open air, and a solution made from selenium dissolved in trioctylphosphine was added. This solution was left to stir overnight. The next day the solution was purified through the use of methanol and 1-butanol, after which it was dispersed in hexane.

Synthesis and characterization of SLN Encapsulated Quantum Dots

The lipid candidate, PEG40SA (8 mg) and PEG100SA (4 mg) were melted at 80°C.

Where phosphatidylcholine was used as the lipid candidate, phosphatidylcholine (50 mg), PEG40SA and PEG100SA were first dissolved in hexane. The mixture was then stirred for 10 minutes to allow the hexane to evaporate. The melted mixture was stirred as the QDs suspended in hexane were added, and then left to stir for 10 minutes to allow for hexane evaporation. Aqueous Pluronic® F-68, followed by distilled deionized water, both preheated to 80°C, was then added to the mixture. The molten mixture was left to stir for 30 minutes, after which it was subjected to ultrasonication using a Hielscher UP100H probe ultrasonicator (Hielscher USA, Inc. Ringwood NJ, USA) for 5 minutes at the same temperature. The entire emulsion was immediately dispersed in cold DDI water. Particle size was measured on a Nicomp 380 Zetasizer by dynamic light scattering and the fluorescence spectra of the undiluted particle were measured on an Ocean Optics spectrofluorimeter.

Dispersion of Quantum Dots Within Chemicals Containing Carboxylic Acid Head Groups

Eighty μΕ of QDs were dispersed in lmL of a carboxylic acid-containing chemical and then vortexed until homogenized. The fluorescence spectra of the undiluted mixture were then measured using an Ocean Optics spectrofluorimeter.

Results

The fluorescence emission spectra of PbSe QDs encapsulated within various lipid candidates were analyzed and compared. The chemical structures of the lipid candidates analyzed varied in terms of composition and chain length. As shown in Table 1, the average size of the resultant particles was found to be between 70 and 250nm, with the exception of the particles formed with phosphatidylcholine. The nanoparticles formed with phosphatidylcholine were found to be in the range of 15-30nm. Table 1. Lipid candidate chemical structure, solid lipid nanoparticle size and quantum dot fluorescence results. *=as melted at 88°C; **=QDs dispersed in these chemicals

Figure 17 provides number-weight distribution data for each of: (A) tristearin, (B) tribehenin, (C) glyceryl behenate, (D) glyceryl palmitostearate, (E) glyceryl monostearate 40- 55, (F) phosphotidylcholine, (G) beeswax, (H) lauric acid, (I) myristic acid, (J) stearic acid, (K) 50:50 tribehenin: stearic acid, and (L) 75:25 tribehenin: stearic acid encapsulating nanoparticles.

When the chemical structures of the different lipids listed in Table 1 were compared, it was found that PbSe QDs encapsulated within solid lipid nanoparticles (SLNs) made with a carboxylic acid (-COOH) head group-containing lipid resulted in lower photoluminosity intensity values (Figures 181 and 18 J) compared to QDs encapsulated within solid lipid nanoparticles lacking -COOH head group-containing lipids (Figures 18B-H).

From the results shown in Figure 181 and those depicted in Figures 20A-B and D-F, a decrease in photoluminescence intensity was also observed in cases where PbSe QDs were dispersed within chemicals which were liquids at room temperature. Figure 20 indicates that the carbon tail chain length of a lipid can also impact QD photoluminescence intensity. In cases where SLNs were made, longer lipid chain length corresponded to lower

photoluminescence intensity values. The impact of carboxylic acid group concentration on SLNs made from lipids derived from mixtures that differed in terms of the ratio between tribehenin (which does not contain a carboxylic acid group) and stearic acid (which does possess a carboxylic acid group) was also examined. The fluorescence spectra of QDs encapsulated within SLNs formed from varying mixture ratios is shown in Figures 19C and D. When the fluorescence spectra of Figure 19C is compared to that of Figure 19A, it can be clearly seen that in the case where an SLN was made from a mixture containing a 50:50 ratio of tribehenin: stearic acid, a decrease in photoluminescence intensity was observed. However, when the fluorescence spectra of a mixture consisting of a 75:25 ratio of tribehenin: stearic acid (shown in Figure 19D) is compared to the emission spectra of QDs encapsulated within SLNs made without stearic acid (Figure 19A), only a small decrease in fluorescence intensity was observed.

The foregoing examples illustrate the invention.

As mentioned above, an aspect of the invention is a fluorescent particle, the particle comprising: a fatty matrix comprising hydrophobic portions of a plurality of fatty ester molecules; and a fluorescent pay load encapsulated in the matrix.

Preferably, there are multiple fluorescent molecules or fluorescent nanoparticles encapsulated within the fatty matrix of the particle. There can be one or more types of nanoparticles, one or more types of organic fluorophores, mixtures of nanoparticles, mixtures of organic fluorophores, or mixtures of both.

Typically, the fatty ester makes up at least 5% by weight of the particle. In embodiments, the fatty ester makes up at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60% of the weight of the particle. A plurality of fatty ester molecules having hydrophobic tails can define a hydrophobic matrix of a composite particle of ester molecules, making up between 5% and 99% by weight of the hydrophobic elements of the composite particle, particularly the encapsulated portion of the particle. The range can be 10 to 90%, 20 to 80%, 30 to 80%, 40 to 80%, 50 to 90%, 60 to 90%, 70% to 90% or such makeup can be at least 20, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

The hydrophobic portions of the fatty ester molecules are preferably hydrocarbon chains of from 10 to 54 carbons in length, wherein a chain may be saturated or unsaturated, linear or branched. The 10 to 54 carbons can be 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 carbons in length. Less usual, but included here, are carbon chains having an odd number of carbons within the CIO to C54 range. A chain can contain double bonds, for example, 1, 2, 3 or 4 double bonds, and can be linear or unbranched. The chain can be saturated. The chain can make up the "R-O", the "R-C=0" part of an ester portion of the molecule, or there can be a chain in each such location, as for, example, the beeswax constituent shown in Table 1. In the case of glycerol derivatives, there is preferably at least one fatty acid in ester linkage with a hydroxyl group of the glycerol. The glyceryl moiety may have further substituents as in the case of phosphatidyl choline, which provides the fatty ester with a zwiterionic hydrophilic head while the fatty acid portion provides a hydrophobic portion which contributes to the matrix of the fluorescent composite particle. In the case of e.g., monoglycerides e.g., glyceryl behenate, the fatty ester is nonionic, the glyceryl moiety being hydrophilic. Also useful in this invention are diglycerides and triglycerides.

As exemplified, a fatty nanoparticle of the invention comprising beeswax was found to fluoresce with a maximum near 610 nm despite the presence of free acids. It is preferred that if there are fatty acid molecules present, the molar ratio of ester/carboxyl groups in a particle of the invention be at least 4, preferably greater than 5, or greater than 6, or greater than 7, or greater than 8. In this context, reference is being made to ester/carboxyl groups directly bound to hydrophobic groups that form part of the matrix of the particle.

Certain preferred particles of the invention having in vivo applications, described previously, are dimensioned in the nano-range e.g. 20 to 250 nm. Such particles, if they contain fluorescent nanoparticles or quantum dots, can contain least 5 said nanoparticles, or at least 10, or at least 15, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70 said fluorescent nanoparticles per particle.

A particle can be at least about 5 times the average diameter of the nanoparticles, and the diameter of the particle can be up to about 250 times the average diameter of the nanoparticles. For example, a fatty nanoparticle in which fluorescent nanoparticles having an average diameter of about 5 nm are encapsulated in the matrix has a diameter of 200 nm, then the diameter of that particle is about 40 times the average diameter of the fluorescent nanoparticles. The diameter of the particle can be between about 10 and 250 times the average diameter of the nanoparticles, or between about 10 and 200, or between about 10 and 180, , or between about 10 and 160, , or between about 10 and 140, or between about 10 and 120, or between about 10 and 100, or between about 10 and 80, or between about 10 and 60, or between about 10 and 50, or between about 15 and 200, or between about 15 and 50, or between about 15 and 40, or between about 15 and 30, or about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 times the average diameter of the

nanoparticles.

A particle of the invention can have a diameter of at least 20 nm, or have a diameter of up to 3000 nm. A particle can have a diameter of between 20 nm and 2000 nm, or between 20 nm and 1000 nm. The particle can be a fatty nanoparticle having a diameter between 20 nm and 900 nm, or between 20 and 800 nm, or between 20 and 700 nm, or between 20 and 600 nm, or between 20 and 700 nm, or between 20 and 600 nm, or between 20 and 500 nm, or between 20 and 400 nm, or between 20 and 300 nm, or between 20 and 250 nm, 20 and 200 nm, or between 30 and 250 nm, or between 20 and 150 nm, or between 30 and 150 nm, or between 40 and 250 nm, or between 40 and 150 nm, or between 40 and 100 nm. The particle can have a diameter of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm.

The invention includes fluorescent compositions containing the fluorescent particles and/or fatty nanoparticles of the invention.

The invention may also be said broadly to be composed of the parts, elements and features referred to or indicated herein, individually or collectively, in their various possible combinations. It is to be understood that those combinations are described as though each is explicitly described herein. A particle containing the combination of cetylpalmitate (fatty ester) and a plurality of CdTe quantum dots (fluorescent payload), and having a diameter between 20 and 600 nm is an example of such possible combination.

As mentioned above, in a preferred aspect, a substantial portion of the particle is the fatty ester, usually more than 5% by weight of the particle. The particle can be up to 95% or more by weight the fatty ester, or it can be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% fatty ester, by weight. In embodiments, the particle, or a composition of the particles, can be in the range of 5% to 95% fatty ester by weight, or such range could be 5% to 90%, 5% to 80%, 10% to 75%, 10% to 60%, 15% to 60%, 15% to 50%, 20% to 60%, 25% to 70%, 30% to 90%, 35% to 90%, 40% to 95%, 50% to 90%, 60% to 90%, or 70% to 90%.

Fluorescent particles of the invention can find use in a broad range of applications including those described above. The skilled person would recognize the advantage(s) of coupling a fluorescent particle of the invention with a variety of types of effector agents. Such an agent could be covalently linked or otherwise coupled to the particle, and could include, for example, one or more of a protein, a protein fragment, a binding domain, a target-binding domain, a binding protein, a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody (a VHH for example), a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab')₂ antibody fragment, a Fab' antibody fragment, a single-chain Fv (scFv) antibody fragment, an antibody binding domain (a ZZ domain for example), an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, biotin, a biotin derivative, an avidin, a streptavidin, a substrate, an enzyme, an abzyme, a co-factor, a receptor, a receptor fragment, a receptor subunit, a receptor subunit fragment, a ligand, an inhibitor, a hormone, a lectin, a polyhistidine, a coupling domain, a DNA binding domain, a FLAG epitope, a cysteine residue, a library peptide, a reporter peptide, an affinity purification peptide, a diagnostic agent, a therapeutic agent, a chemotherapeutic agent, an anti-angiogenic agent, a cytokine, a chemokine, a growth factor, a drug, a prodrug, a binding molecule, a ligand for a cell surface receptor, a chelator, an immunomodulator, an oligonucleotide, a hormone, a toxin, a contrast agent, or a pro- apoptotic agent, etc.

It will be understood that recitations of numerical ranges by endpoints include all numbers subsumed within that range. Also, a recited range having an endpoint within a different recited range is a disclosure of any other range having endpoints of those recited ranges. For example, recitation of the ranges 20 to 350 and 10 to 300 is a disclosure of the ranges 20 to 300 and 10 to 350.

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Claims

THEREFORE WHAT IS CLAIMED IS:

1. A fluorescent particle, the particle comprising:

a fatty matrix comprising hydrophobic portions of a plurality of fatty ester molecules; and a fluorescent payload encapsulated in the matrix.

2. The particle of claim 1, wherein the fluorescent payload comprises a plurality of fluorescent molecules, a plurality of fluorescent nanoparticles, or a mixture thereof.

3. The particle of claim 1 or claim 2, wherein the fatty ester makes up at least 5% by weight of the particle, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%.

4. The particle of any preceding claim, wherein hydrophobic portions of the fatty ester molecules are hydrocarbon chains of from 10 to 54 carbons in length, wherein a chain may be saturated or unsaturated, linear or branched.

5. The particle of claim 4, wherein a said chain is 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 carbons in length.

6. The particle of claim 5, wherein a said chain contains 1, 2, 3 or 4 double bonds.

7. The particle of claim 5, wherein the chain is unbranched.

8. The particle of claim 7, wherein the chain is saturated.

9. The particle of any preceding claim, wherein the ester molecule is non-ionic.

10. The particle of any one of claims 1 to 8, wherein the ester molecules include a zwitterionic head, over a pH range of at least 2 to 7.

11. The particle of claim 10, wherein the ester molecules comprise a phosphatidyl choline.

12. The particle of any one of claims 1 to 5, wherein the hydrophobic portion comprises the C10-C50 tail portion of the alcohol portion of the ester molecules.

13. The particle of claim 12, wherein the ester is a glyceryl ester.

14. The particle according to any one of claims 1 to 5 wherein said ester molecules comprise tristearin, tripalmitin, trilaurin, ethyleicosanoate, glyceryl behenate, or

cetylpalmitate.

15. The particle according to any one of claim 1 to 5 wherein said ester molecules comprise hard fats, synthetic waxes, natural waxes, or beeswax.

16. The particle according to claim 1, 2 or 3 wherein said fatty ester is an ester of a substance selected from the group consisting of glycerol, sucrose, C10-C54 saturated fatty acids with straight chains, C10-C54 unsaturated fatty acids with straight chains, C10-C54 saturated fatty acids with branched chains, C10-C54 unsaturated fatty acids with branched chains, C10-C54 saturated fatty alcohols with straight chains, C10-C54 unsaturated fatty alcohols with straight chains, C10-C54 saturated fatty alcohols with branched chains, and C10-C54 unsaturated fatty alcohols with branched chains.

17. The particle according to any preceding claim wherein said fluorescent payload comprises a fluorescent nanoparticle.

18. The particle according to claim 17, comprising at least 5 said nanoparticles, or at least 10, or at least 15, or at least 20, or at least 30, or at least 40, or at least 50 said nanoparticles.

19. The particle of claim 18, wherein the diameter of the particle is at least about 5 times the average diameter of the nanoparticles.

20. The particle of claim 18, wherein the diameter of the particle is up to about 250 times the average diameter of the nanoparticles.

21. The particle of claim 18, wherein the diameter of the particle is between about 10 and 250 times the average diameter of the nanoparticles, or between about 10 and 200, or between about 10 and 100, or between about 10 and 80, or between about 10 and 60, or between about 10 and 50, or between about 15 and 50, or between about 15 and 40, or between about 15 and 30, or about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 times the average diameter of the nanoparticles.

22. The particle according to any one of claims 17 to 21 wherein said fluorescent nanoparticles comprise a hydrophobic capping ligand.

23. The particle according to claim 22 wherein said hydrophobic capping ligand is selected from the group consisting of oleic acid, trioctyl phosphine, butanoic acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 9- hexadecenoic acid, octadecanoic acid, 9-octadecenoic acid, 12-hydroxy-9-octadecenoic acid,

11-octadecenoic acid, 9,12-octadecadienoic acid, 9,12,15-octadecatrienoic acid, 6,9,12- octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid, 5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, docosanoic acid, 13-docosenoic acid, 4,7,10,13,16,19- docosahexaenoic acid, tetracosanoic acid, and alkylphosphenes.

24. The particle according to any preceding claim, wherein the fatty ester molecules form an emulsion, wherein the emulsion is preferably a nanoemulsion.

25. The particle according claim 24, further comprising an agent which stabilizes the emulsion.

26. The particle according to any one of claims 1 to 25 further comprising a surface PEGylating block copolymer.

27. The particle according to claim 26 wherein said surface PEGylating block copolymer comprises poly(ethylene glycol) (PEG) linked with a fatty chain or a hydrophobic polymer chain.

28. The particle according to claim 26 wherein said surface PEGylating block copolymer is at least one block of poly(ethylene oxide) and at least one block of hydrophobic polymers selected from the group consisting of poly(propylene oxide), polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic acid-co-glycolic acid), polydioxanone, polyurathans, polystyrene, polycaprolactone, and polycarbonates,

29. The particle according to any one of claims 26 to 28 further comprising a surface coating selected from the group consisting of Tween® family surfactants, Triton® family surfactants, Tergitol® family surfactants, Pluronic® family surfactants, Span® family surfactants, poly(vinyl alcohol), poly(vinyl pyrrolidone), linear starch, modified starch, egg phosphatidylcholine, egg lecithine, and soy phosphatidylcholine.

30. The particle according to any one of claims 26 to 28 further comprising a surface coating comprising activated groups adhered to a surface of said fluorescent nanoparticle, said activated groups having a spacer chain grafted thereto.

31. The particle according to claim 30 wherein said spacer chain comprises PEG.

32. The particle according to claim 27 or 28 wherein said fluorescent nanoparticle is a quantum dot.

33. The particle according to claim 32 wherein said quantum dot is a PbSe quantum dot.

34. The particle according to claim 32 wherein said quantum dot is selected from the group consisting of CdSe quantum dots, CdS quantum dots, CdTe quantum dots, CdSe/ZnS quantum dots, CdSe/ZnSe quantum dots, InAs quantum dots, InP quantum dots, and PbS quantum dots.

35. The particle according to any one of claims 32 to 34 wherein said quantum dot comprises a core coated with said hydrophobic capping ligand.

36. The particle according to any one of claims 2 to 35 wherein said fluorescent nanoparticles are first fluorescent nanoparticles, and wherein said fatty matrix further encapsulates second fluorescent nanoparticles.

37. The particle according to claim 36 wherein an emission spectrum of said second fluorescent nanoparticles is spectrally distinct from an emission spectrum of said first fluorescent nanoparticles.

38. The particle according to claim 2 wherein said fluorescent nanoparticles are semiconductor nanocrystals.

39. The particle according to claim 2 wherein said fluorescent nanoparticles carbon-based nanocrystals or silicon-based nanocrystals.

40. The particle according to claim 2 wherein said fluorescent nanoparticles are silica- organic dye hybrid nanoparticles, calcium phosphate-organic dye hybrid nanoparticles, or fluorophore-doped polymer nanoparticles.

41. The particle according to any one of claims 1 to 40 wherein said fatty matrix further comprises one or more of a fatty acid and a fatty alcohol.

42. The particle according to any one of claims 1 to 41 wherein said fatty matrix further comprises a lipid compound.

43. The particle of any one of claims 1 to 19, wherein the particle has a diameter of at least 20 nm.

44. The particle of any one of claims 1 to 19, wherein the particle has a diameter of up to 3000 nm.

45. The particle of any one of claims 1 to 19, wherein the particle has a diameter of between 20 nm and 2000 nm, or between 20 nm and 1000 nm, or between 20 nm and 900 nm, or between 20 and 800 nm, or between 20 and 700 nm, or between 20 and 600 nm, or between 20 and 700 nm, or between 20 and 600 nm, or between 20 and 500 nm, or between 20 and 400 nm, or between 20 and 300 nm, or between 20 and 250 nm, 20 and 200 nm, or between 30 and 250 nm, or between 20 and 150 nm, or between 30 and 150 nm, or between 40 and 250 nm, or between 40 and 150 nm, or between 40 and 100 nm.

46. The particle of claim 45, wherein the particle has a diameter of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm.

47. The particle according to any one of claims 1 to 26 wherein a diameter of the particle is between approximately 20 and 1000 nm.

48. The particle according to claim 47 wherein a diameter of the particle is between approximately 50 and 250 nm.

49. The particle according to any preceding claim wherein an emission spectrum of said fluorescent payload is selected to be suitable for fluorescent imaging through a tissue.

50. The particle according to any one of claims 1 to 48 wherein an emission spectrum of said fluorescent payload is between approximately 400 and 950 nm.

51. The particle according to any one of claims 1 to 48 wherein an emission spectrum of said fluorescent payload is in the near infrared spectral region.

52. The particle according to claim 1 or 2 wherein said fluorescent payload is an organic fluorophore.

53. The particle according to claim 53 wherein said a fluorophore is one or more of a xanthene derivative, a fluorescein, rhodamine, cyanine derivatives, cyanine,

indocarbocyanine, or napthalene derivative, a dansyl or prodan derivative, a coumarin derivative, an oxadiazole derivative, pyrene or a derivative thereof, an oxazine derivative, Nile red, cresyl violet, an acridine derivative, an arylmethine derivative, a tetrapyrrole derivative, a porphyrin, a phthalocyanine, or a polymethine.

54. The particle according to claim 53 wherein said fluorophore is a first fluorophore, wherein said solid fatty matrix further encapsulates an additional fluorophore, and wherein an emission spectrum of said additional fluorophore is spectrally distinct from an emission spectrum of said first fluorophore.

55. The particle according to any preceding claim wherein said particle further comprises one or more of a binding moiety, label, reporter, hapten, and conjugate.

56. A method of producing fatty nanoparticles comprising a solid fatty matrix encapsulating a fluorescent payload, said method comprising the steps of:

providing a fluorescent nanoparticle coated with a hydrophobic capping ligand; forming a suspension by suspending said fluorescent nanoparticle in a hydrophobic solvent;

heating a fat beyond a melting point and obtaining molten fat, wherein said molten fat comprises a fatty ester;

mixing said suspension with said molten fat;

evaporating said solvent and obtaining a first dispersion comprising said fluorescent nanoparticles dispersed in said molten fat;

forming an emulsion by heating a quantity of water above said melting point and adding said water to said dispersion; and

homogenizing said emulsion to form nanodroplets comprising a fatty matrix encapsulating said fluorescent nanoparticles; and

forming a second dispersion comprising said fatty nanoparticles by combining said emulsion with an isotonic and inert aqueous suspending medium.

57. The method according to claim 56 wherein said quantity of water is approximately 0.001:1 to 2:1 by volume ratio to said fat.

58. The method according to claim 56 further comprising the step of adding a quantity of surfactant prior to said step of adding said water.

59. The method according to claim 58 wherein said quantity of surfactant is provided as an aqueous solution, and wherein a quantity of said surfactant in the entire emulsion is approximately 0.05% to 10% by weight.

60. The method according to claim 56 wherein said step of forming said second dispersion is performed within a suitable time interval of said step of homogenizing said emulsion to prevent coalescence of said nanodroplets.

61. The method according to claim 56 wherein said step of homogenizing said emulsion comprises one of ultrasonicating said emulsion, passing the emulsion through a

microfluidizing device, and directly introducing the emulsion to a high pressure

homogenizer.

62. The method according to claim 56 wherein said isotonic and inert aqueous suspending medium is a solution comprising one of saline, dextrose and buffer salts.

63. The method according to any one of claims 56 to 62 further comprising the step of adding a quantity of one or more block copolymers for stabilization of said fatty

nanoparticles.

64. A method of producing fatty nanoparticles comprising a solid fatty matrix encapsulating a fluorophore, said method comprising the steps of:

heating a fat beyond a melting point and obtaining molten fat, wherein said molten fat comprises a fatty ester;

adding a quantity of said fluorophore to said molten fat;

forming an emulsion by heating a quantity of water above said melting point and adding said water to said molten fat; and

homogenizing said emulsion to form nanodroplets comprising a fatty matrix encapsulating said fluorophore; and

forming a dispersion comprising said fatty nanoparticles by combining said emulsion with an isotonic and inert aqueous suspending medium.

65. The method according to claim 64 wherein said step of forming said dispersion is performed within a suitable time interval of said step of homogenizing said emulsion to prevent coalescence of said nanodroplets.

66. The method according to claim 64 wherein said step of homogenizing said emulsion comprises one of ultrasonicating said emulsion, passing the emulsion through a

microfluidizing device, and directly introducing the emulsion to a high pressure

homogenizer.

67. The method according to claim 64 wherein said isotonic and inert aqueous suspending medium is a solution comprising one of saline, dextrose and buffer salt.

68. The method according to any one of claims 64 to 67 further comprising the step of adding a quantity of one or more block copolymers for stabilization of said fatty

nanoparticles.

69. The method according to claim 68 wherein said step of adding a quantity of one or more block copolymers is performed prior to said step of adding said quantity of water.

70. A method of imaging, said method comprising the steps of:

providing fatty nanoparticles are according to any one of claims 1 to 33, wherein an emission bandwidth of said fluorescent payload is selected to be suitable for fluorescent imaging through a tissue of a subject;

administering said fatty nanoparticles to said subject;

optically exciting said fluorescent payload; and

imaging fluorescent emission produced by said fluorescent payload.

71. The method according to claim 70 wherein a diameter of said fatty nanoparticles is selected to be between approximately 50 and 250 nm.

72. The method according to claim 70 or 71 wherein said step of administering said fatty nanoparticles comprises one of intravenous, intra-arterial, intraperitoneal, subcutaneous, intratumoral, intrapleural, or intramuscular injection of said fatty nanoparticles.

73. The method according to any one of claims 70 to 72 wherein said step of imaging said fluorescent emission comprises imaging a substantial portion of a body of said subject.

74. The method according to any one of claims 70 to 73 wherein said fluorescent payload comprises fluorescent emission characterized by two or more distinct emission bands, said method further comprising spectrally separating said fluorescent emission to resolve said distinct emission bands.

75. The method according to any one of claims 70 to 74 wherein said fatty nanoparticle comprises a conjugated molecular species for selectively targeting one of a tissue type, a cell type and a molecule type, said method further comprising the step of identifying a presence of said one of a tissue type, a cell type and a molecule type in said subject based on said fluorescent emission.

76. A composition comprising a plurality of particles as defined by any one of claims 1 to 55.