WO2009035770A2 - Cholera toxin subunit b conjugated quantum dots for live cell labelling - Google Patents

Abstract

Conjugates were developed for internal labelling and tracking of live cells having cell surface gangliosides. The conjugate is comprised of at least one, and preferably up to three Vibrio cholerae toxin subunits B, preferably in the pentameric form, attached to a quantum dot. The conjugates may be used as luminescent labels in a reagent system for labelling live cells. The conjugates are internalized into small vesicles dispersed throughout the cytoplasm. A method of labelling and method of detecting cells having cell surface gangliosides are described. The conjugates are particularly useful for the general labelling of mammalian cells.

Description

CHOLERA TOXIN SUBUNIT B CONJUGATED QUANTUM DOTS FOR LIVE CELL LABELLING

Inventors

Byron Ballou Marcel P. Bruchez Subhasish K. Chakraborty James AJ. Fitzpatrick

Susan Andreko Justin C. Crowley

GOVERNMENT RIGHTS:

This invention was made with government support under NIH Grant No. R01EB000364 and NIH Grant No. ROlEB 004343. The government has certain rights in this invention.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Patent Application Serial No. 60/961,635, filed 23 July 2007.

FIELD OF THE INVENTION

The invention relates to cellular labelling and more particularly, to the use of quantum dot conjugates for intracellular labelling and cell tracking.

Fluorescent labelling of cells allows tracking movement, cell division, and cellular interactions in vitro and in vivo. Cell labelling using semiconductor nanocrystal (also known as quantum dots) conjugates was originally reported by Bruchez, M., Jr.; et al., Science 1998, 281, (5385), 2013-2016 and Chan, W. C. et al., Science 1998, 281, (5385), 2016-2018. Both groups emphasized that the excellent fluorescence properties of quantum dots (brightness, choice of many emission maxima, chemical stability and photostability) would be well suited to cell labelling, especially cell tracking over long periods of time. Quantum dots are useful because of their size and optical properties. These semiconductor nanocrystals, whose radii are smaller than the bulk exciton Bohr radius, constitute a class of materials intermediate between molecular and bulk forms of matter. Quantum confinement of both the electron and hole in all three dimensions leads to an increase in the effective band gap of the material with decreasing crystallite size.

Consequently, both the optical absorption and emission of semiconductor nanocrystals shift to the blue (higher energies) as the size of the nanocrystals gets smaller.

Quantum dots are nanoparticles composed of an inorganic, crystalline semiconductive material. Because of their unique photophysical, photochemical and nonlinear optical properties, they have attracted a great deal of attention. As a result of the increasing interest in semiconductor nanocrystals in a variety of contexts, there is now a fairly substantial body of literature pertaining to methods for manufacturing such nanocrystals. Broadly, these routes may be classified as involving preparation in glasses (see Ekimov et al. (1981) JETP Letters 34:345), aqueous preparation (including preparation that involve use of inverse micelles, zeolites, Langmuir-Blodgett films, and chelating polymers; see Fendler et al. (1984) J. Chem. Society, Chemical Communications 90:90, and Henglein et al. (1984) Ber. Bunsenges. Phys. Chem. 88:969), and high temperature pyrolysis of organometallic semiconductor precursor materials (Murray et al. (1993) J. Am. Chem. Soc. 115:8706; Katari et al. (1994) J. Phys. Chem. 98:4109). A method for rendering hydrophobic semiconductor nanocrystals dispersible in aqueous media is disclosed in U.S. Patent No. 7,108,915, the relevant portions of which are incorporated herein by reference.

One attempt at applying quantum dots for a general use cell label resulted in the development of polyarginine-conjugated quantum dots. See, Lagerholm, B. C. et al., Nano letters 2004, 4, (10), 2019-2022. One of the most useful aspects of the polyarginine labelling is speed; cells rapidly take up the quantum dot conjugates, reaching half- saturation at 12 minutes after addition of the quantum dots. Cells are readily labelled at high levels (>100 quantum dots per cell); quantum dots are taken up into endosomes, and appear localized as very bright spots. This method allows considerable flexibility in concentration of quantum dot conjugates administered, level of polyarginine substitution, and easy coupling of quantum dots to both polyarginine and other biotinylated reagents that are to be internalized along with the quantum dots. A difficulty in using the polyarginine technique, however, is that the quantum dots are taken up into the cells as large aggregates. Fluorescence correlation spectroscopy (FCS) showed that aggregates are formed very rapidly on mixing quantum dots with polyarginine, with or without serum in the medium. Even larger aggregates appear to be formed on the cell surface. Internalization results in irregular labelling, and tracing cells through several generations is difficult due to unequal division of quantum dot contents.

Many other methods have been used to label living cells using quantum dots. Examples include microinjection, (see Dubertret, B. et al., Science 2002, 298, (5599), 1759- 1762) electroporation (see Chen, F.; Gerion, D., Nano Letters 2004, 4, (10), 1827-1832), internalization via cationic detergents (see Srinivasan, C. et al., J. MoI. Ther. 2006, 14, (2),

192-201; Hsieh, S. C; et al.,. J. Biomaterials 2006, 27, (8), 1656-1664.), non-specific binding of quantum dots followed by internalization (see Murasawa, S.; et al., Arterioscler. Thromb. Vase. Biol. 2005, 25, (7), 1388-1394), binding and internalization via cell-penetrating peptides (see Mattheakis, L. C. et al., Anal. Biochem. 2004, 327, (2), 200-208), and binding to many specific cell surface molecules via attached ligands, often followed by internalization (see Yu, W. et al., J. Am. Chem. Soc. 2007, 129, (10), 2871-2879; Xie, M. et al., Chem. Commun., (Camb) 2005, (44), 5518-5520). However, most of the published methods demand manipulations other than simple addition of quantum dots to the cells. Some methods require serum starvation before the cells will uptake the quantum dots. The polyarginine technique described above requires stressing the endocytic machinery of the cell population before uptake of the quantum dot into the cell can proceed. Observations of cellular activity following stress induced uptake of the label may alter the very activity to be observed.

Given the growth in both the synthesis and use of new quantum dot conjugates it has become essential to provide adequate characterization so that results from different laboratories can be compared effectively. One group, Larson, D. R. et al., Science 2003, 300, (5624), 1434-1436, used fluorescence correlation spectroscopy (FCS) to characterize water soluble quantum dots for use as multi-color probes in the multiphoton microscopy of live animals. They reported the effective hydrodynamic radii of the quantum dots and the problems due to excitation saturation effects that arise from large two-photon absorption cross-sections of quantum dots. Theoretical work on such saturation effects and the relation between nanocrystal size and two-photon absorption cross-section has since been reported. (see Davis, L. M.; et al.,. Current Pharmaceutical Biotechnology 2006, 7, (4), 287-301; and Pu, S. C. et al, Small 2006, 2, (11), 1308-1313). Effective sizes and degrees of aggregation have also been reported for quantum dots stabilized by different surface chemistries and Pons, T. et al., Journal of Physical Chemistry B 2006, 110, (41), 20308-20316, reported using dynamic light scattering methods in conjunction with transmission electron microscopy (TEM) and X-ray diffraction (XRD) to characterize the sizes of quantum dots modified with different surface ligands. The influence of blinking on FCS and imaging has been explored, and correctives were demonstrated. See, for example, Yao, J. et al., Proceedings of the National Academy of Sciences of the United States of America, 2005, 102, (40), 14284- 14289; Bachir, A. I. et al., Journal of Applied Physics, 2006, 99, (6), 064503/1-064503/7; Rochira, J. A. et al., Journal of Physical Chemistry C, 2007, 111, (4), 1695-1708. Uses of FCS for quantum dot quantitation, determining mobility, and aggregation have been demonstrated. See Thakur, A. et al., Canadian Undergraduate Physics Journal, 2005, 3, (3), 7-12; Chen, C. S.; et al., Journal of Nanoparticle Research, 2006, 8, (6), 1033-1038; Stavis, S. M. et al., Lab on a Chip 2005, 5, (3), 337-343; and Dong, C. Q. et al., Journal of Physical Chemistry C 2007, 111, (22), 7918-7923.

INVENTION SUMMARY

There is a need for a quantum dot label and a method of labelling that permits less aggregation of the label. There is also a need for a label and a method of labelling that allows the label to remain dispersed in cell cytoplasm for longer periods of time than has been the experience with conventional cell labels. There is a further need for a quantum dot label and a method of labelling that would be applicable to many cell types. Finally, there is a need for a quantum dot label and a method of cell labelling that is easy to use.

At least one or more of the foregoing needs is satisfied by a conjugate comprised of at least one Vibrio cholerae toxin subunit B attached to a quantum dot. In one embodiment, one or more of the foregoing needs may be satisfied by a reagent system comprised of a luminescent label that includes a quantum dot having at least one Vibrio cholerae toxin subunit B attached thereto. There may be from one to three, one to two, or two to three Vibrio cholerae toxin subunits B attached to each quantum dot.

The quantum dot may be any suitable known quantum dot. In one or more embodiments of the invention, the quantum dot may be selected from the group consisting of carboxylated quantum dots, amino-functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof.

A method of labelling cells is also provided. The method comprises adding to a plurality of live cells selected from cells having cell surface gangliosides, a luminescent label comprising at least one Vibrio cholerae toxin subunit B attached to a quantum dot, and exposing the cells to the luminescent label for a period of time sufficient to allow the cells to absorb the label. The method may further include the step of washing the cells, typically with normal growth media, to remove any unabsorbed label. In one or more embodiments of the method, the concentration of the luminescent label added to the cells may be in the range of from about 250 pM to 4 nM.

One advantage of the method is that the plurality of cells may be maintained in normal growth media before and during the labeling procedure. It was surprisingly found that the cells do not need to be placed under any stress to enable the uptake of the Vibrio cholerae toxin subunit B - quantum dot conjugate. The Vibrio cholerae toxin subunit B - quantum dot conjugate may be added to the media. After a period of time, the conjugate is absorbed into the cells. The luminescent conjugate may be in an unaggregated form or may form aggregates having a size of about 20 to 200 nm. The period of time may generally be at least about one quarter hour to over night, or a comparable period sufficient to allow the cholera toxin subunit B - quantum dot conjugate to be taken up by the cells. The period of time may more particularly range from about one half hour to 18 or more hours, or from about one to about 18 hours, or from 12 to about 18 hours or from about 12 to about 16 hours. Those skilled in the art will recognize that the period of time may vary depending on the particular cell species and the media in which the cells are grown and/or maintained.

The cells may be mammalian cells, dendritic cells, cells from primary human cell populations or any cell or cell population having gangliosides on the surface of the cell. The cells may be selected from the group of cells consisting of stem cells, bone marrow cells, immune cells, tumor cells and combinations thereof.

The method of labelling described herein provides a labelled cell population comprising a plurality of cells labelled internally with a luminescent label, each label comprised of up to three Vibrio cholerae toxin subunits B attached to a quantum dot. A method of detecting cells having cell surface gangliosides is also provided. The method may be used to track cells through two or more generations and to track cell differentiation. The method of detection may comprise adding to a plurality of cells, a luminescent label comprising a Vibrio cholerae toxin subunit B -quantum dot conjugate, exposing the cells to the luminescent label for a period of time sufficient to allow cells having cell surface gangliosides to absorb the luminescent label, and detecting the presence of cells having internal luminescence. The luminescence may be detected by any suitable means of detecting luminescence, including without limitation, fluorescent microscopy, flow cytometry, fluorescent imaging, or fluorimetry.

Other non-limiting aspects of the various embodiments described herein are provided in the following description

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments described herein may be understood by reference to the following description, taken with the accompanying drawings as follows

Figures l(A) and (B) represent chromatography graphs. Figure l(A) is a graph of (i) a column chromatography of unreacted carboxyl quantum dots; (ii) CTB-QD conjugates; (iii) and overlay of i and ii. Figure l(B) represents a graph of the rechromatography of the main fractions from the columns in Figure l(A), wherein (iv) is unreacted carboxyl quantum dots; (v) is CTB-QD conjugates and (vi) is an overlay of iv and v.

Figure 2 is an image of the gel electrophoresis of 655nm quantum dots (a-e) wherein unreacted carboxyl quantum dots are in lanes (a) and (e); a first fast protein liquid chromatography (FPLC) fraction is in lane (b); the main FPLC fraction is in lane (c); the FPLC tail peak is in lane (d). Only conjugates eluted in the main FPLC fraction (c) were used as labelling reagents.

Figure 3 illustrates the labelling of live cells with 655nm CTB-QD conjugates (a. differential interference contrast microscopy (DIC), b. confocal fluorescence and c. overlay), wherein row I is NIH 3T3 fibroblasts, row II is human mesenchymal stem cells (hMSC), row III is mouse muscle derived stem cells (MDSC), row IV is M21 human melanoma and row V is MH15 teratocarcinoma mouse tumor cells. All cells were imaged 18 hours post labelling. Figure 4 illustrates a comparison of images obtained 18 hours post labelling of NIH 3T3 fibroblasts labelled with 605nm QTracker® polyarginine conjugated quantum dots (a-c) and 655nm carboxyl quantum dots (d-f) wherein images a and d were obtained by differential interference contrast microscopy (DIC), images b and e were obtained by confocal fluorescence, and images c and f are overlays of a/b and d/e, respectively.

Figure 5 is a graph of correlation functions for the 655nm carboxyl (^π) and CTB-QD (Δ) conjugates. The inset highlights the differences in the two correlation functions.

Figures 6(A)-(D) represent hMSC cell behaviour after labelling with CTB-QD conjugates. Figure 6(A) illustrates hMSC labelled with 250 pM, 1, 4 or 16 nM 655 -CTB- QD overnight, trypsinized and replated onto glass-bottom dishes. Figure 6(B) illustrates 655- CTB-QD labelled (250 pM) hMSC exhibiting induction of ALP activity with 7 days of treatment with osteogenic medium. Figure 6(C) is a bar graph of qPCR analysis for Osx gene expression after 24 hr treatment in the presence (solid bars) or absence (open bars) of 100 ng/mL BMP-2. Bars represent mean ± SEM, n=3. The * indicates that it differs significantly from control (-BMP-2/0 nM CTB-QD-655), p<0.05. The ** indicates that it differs significantly from -BMP-2 group for each CTB-QD-655 dose, p<0.05. Figure 6(D) illustrates hMSC co-cultures of 4 nM CTB-QD-605, -655 or - 705 labelled hMSC.

Figures 7(A)-(E) represent mouse muscle derived stem cells (MDSC) cell behavior after labelling with CTB-QD conjugates. Figure 7(A) represents labelled and unlabeled MDSC analyzed by flow cytometry for Sca-1 and CD34 expression. Cells were labelled using 4 nM CTB-QD-655. Figure 7(B) shows that CTB-QD-655 labelled MDSC form myotubes under serum deprivation (2% serum) for 72 hr. Co-cultures of MDSCs labelled with 4 nM of either CTB-QD-655 or -705 under serum deprivation for 72 hr show fusion of two differently labelled cells to form a myotube that contains both labels. Figure 7(C) is a bar graph of the results of qPCR analysis for Osx gene expression after 24 hr treatment in the presence (solid bars) or absence (open bars) of 100 ng/mL BMP-2. Bars represent mean ± SEM, n=3. The * indicates that it differs significantly from the control (-BMP-2/0 nM CTB- 655), p<0.05. The ** indictaes that it differs significantly from -BMP-2 group for each CTB- 655 dose, p<0.05. Figure 7(D) is a bar graph of the results of qPCR analysis for expression of Alp gene after CTB-QD labelling, with the same significances as in bar graph (C). Figure 7(E) illustrates myotube formation in CTB-QD-655 labelled MDSC. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The articles "a," "an," and "the" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical objects of the article. By way of example, "a quantum dot" means one or more quantum dots, and thus, possibly, more than one quantum dot is contemplated and may be employed or used.

The term "attached," as in, for example, the "attachment" of a cholera toxin subunit B to a quantum dot, includes covalent binding, non covalent binding, adsorption, and physical immobilization. The terms "associated with," "binding" and "bound" are identical in meaning to the term " attached. "

As used herein, the term "comprising" means various components conjointly employed in the preparation of the conjugates, labels, systems or methods of the present disclosure. Accordingly, the terms "consisting essentially of and "consisting of are embodied in the term "comprising".

The term "cholera toxin" as used herein refers to a multimeric protein toxin from the gram-negative pathogenic bacterium, Vibrio cholerae which is an oligomeric complex made up of six protein subunits, one A subunit (an Ai peptide linked by a disulfide bond to an A₂ peptide) and five copies of a B subunit. Subunit A is toxic. Cholera toxin subunit B, which is not itself toxic, facilitates passage of the A subunit across cell membranes by interaction with a G_ml ganglioside on the cell surface. The five B subunits each weigh about 12 kDa and together form a pentameric (five-membered) ring.

As used herein "cholera toxin subunit B", "Vibrio cholerae toxin subunit B," "subunit B", and "CTB" may be used interchangeably and unless otherwise stated refer to the nontoxic, cell binding pentameric form of the cholera toxin of the subunit B protein from Vibrio cholerae. CTB binds to gangliosides on the surfaces of cells. Its pathway of uptake has been extensively studied. See, for example, De Haan, L. et al., MoI. Membr. Biol, 2004, 21, (2), 77-92; Lencer, W. L, Int. J. Med. Microbiol, 2004, 293, (7-8), 491-494; and Lord, J. M. et al., Curr. Top. Microbiol. Immunol, 2005, 300, 149-168. CTB has been used to promote internalization of many conjugated and associated materials, including antigens for immunization. See, Holmgren, J. et al., Immunol. Lett., 2005, 97, (2), 181-188; Lycke, N., Curr. MoI. Med., 2005, 5, (6), 591-597; Stevceva, L. et al., Curr. Pharm. Des., 2005, 11, (6), 801-811; and Vajdy, M. et al., Expert Opin. Biol. Ther., 2005, 5, (7), 953-965.

The term "dendritic cells" pertains to a heterogeneous group of multifunctional leukocytes rather than a distinct cell type. Dendritic cells are found in the interstitial spaces of many organs. Subpopulations of dendritic cells differ in phenotype, functions, and tissue localization. Dendritic cells undergo phenotypic and functional changes during their maturation and migration.

The term "gangliosides" means any of a group of glycosphingolipids found on cell surfaces. They are comprised of an oligosaccharide chain containing at least one acidic sugar, called sialic acids, attached to a ceramide. The acidic sugar is N-acetylneuraminate or N-glycolylneuraminate. A basic composition comprises a ceramide-glucose-galactose-iV - acetylneuraminate, with the 2 hydrocarbon chains of the ceramide moiety embedded in the plasma membrane of the cell and the oligosaccharides on the extracellular surface. Gangliosides are found predominantly in central nervous system tissues where they constitute 6% of all phospholipids. Gangliosides are complex glycosphingolipids in which oligosaccharide chains containing N-acetylneuraminic acid (NeuNAc) are attached to a ceramide. NeuNAc, an acetylated derivative of the carbohydrate sialic acid, makes the head groups of gangliosides anionic. The 40+ known gangliosides differ mainly in the position and number of N-Acetylneuraminic acid (NeuNAc) residues. Their structural diversity results from variation in the composition and sequence of the sugar residues. In all gangliosides the ceramide is linked through its C-I to a β-glucosyl residue which in turn is bound to a β-galactosyl residue. They are involved in diverse roles including cell to cell contact, ion conductance, and as receptors. The form G_M i accumulates in tissues in G_M i gangliosidoses and the form G_M2 in G_M2 gangliosidoses.

The term "luminescence" or "luminescent" as used herein means the process of emitting electromagnetic radiation (light) from an object or an object that emits or is capable of emitting such electromagnetic radiation. Luminescence results when a system undergoes a transition from an excited state to a lower energy state with a corresponding release of energy in the form of a photon. These energy states can be electronic, vibrational, rotational, or any combination thereof. The transition responsible for luminescence can be stimulated through the release of energy stored in the system chemically or added to the system from an external source. The external source of energy can be of a variety of types including chemical, thermal, electrical, magnetic, electromagnetic, and physical, or any other type of energy source capable of causing a system to be excited into a state higher in energy than the ground state. For example, a system can be excited by absorbing a photon of light, by being placed in an electrical field, or through a chemical oxidation-reduction reaction. The energy of the photons emitted during luminescence can be in a range from low-energy microwave radiation to high-energy x-ray radiation. Typically, luminescence refers to radiation in a range including ultraviolet, visible, and near infrared radiation, from 300 to 2000 nm.

The term "nanoparticle" refers to a particle, generally a semiconductive or metallic particle, having a diameter in the range of about 1 nm to about 1000 nm, preferably in the range of about 2 nm to about 50 nm, more preferably in the range of about 2 nm to about 20 nm. Semiconductive and metallic "nanoparticles" generally include a passivating layer of a water- insoluble organic material that results from the method used to manufacture such nanoparticles.

The term "normal growth medium" as used herein refers to any of the media designed to support long term expansion and growth of the cells of or from a particular species; that is, the media that the cells of a particular species are typically cultured in to allow expansion of the cells for a term of at least one, and preferably two, and more preferably three or more generations of daughter cells in the media, as opposed to a media designed specifically for labelling or imaging or designed to alter the cells in some predetermined manner.

The terms "quantum dot" "Qdot", "QD", and "semiconductor nanocrystal," are used interchangeably herein to refer to luminescent (i.e., capable of emitting electromagnetic radiation upon excitation) semiconductor nanoparticles composed of an inorganic semiconductive material, an alloy or other mixture of inorganic semiconductive materials, an organic semiconductive material, or an inorganic or organic semiconductive core contained within one or more semiconductive overcoat layers. Semiconductor nanocrystals include an inner core of one or more first semiconductor materials that is optionally contained within an overcoating or "shell" of a second semiconductor material.

A semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a "core/shell" semiconductor nanocrystal. The surrounding shell material will preferably have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the core substrate. Suitable semiconductor materials for the core and/or shell include, but are not limited to, the following: materials comprised of a first element selected from Groups 2 and 12 of the Periodic Table of the Elements and a second element selected from Group 16 (e.g., ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like); materials comprised of a first element selected from Group 13 of the Periodic Table of the Elements and a second element selected from Group 15 (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like); materials comprised of a Group 14 element (Ge, Si, and the like); materials such as PbS, PbSe, Bi₂S₃, and the like; and alloys and mixtures thereof. As used herein, all reference to the Periodic Table of the Elements and groups thereof is to the new IUPAC system for numbering element groups, as set forth in the Handbook of Chemistry and Physics, 81^st Edition (CRC Press, 2000).

Other than in the examples herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values, and percentages, such as those for amounts of materials, elemental contents, times and temperatures of reaction, ratios of amounts, and others, in the specification and attached claims may be read as if prefaced by the word "about," even though the term "about" may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains error necessarily resulting from the deviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (i.e., end points may be used). Also, it should be understood that any numerical range recited herein is intended to include all values and subranges subsumed therein. For example, a range of "1 to 10" is intended to include all subranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

All percentages, parts and ratios are based upon the total weight of the compositions of the present invention, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore, do not include solvents or byproducts that may be included in commercially available materials, unless otherwise specified.

All patents, publications, or other disclosure material referenced herein are incorporated by reference in their entirety. Any patent, publication, or other disclosure material, in whole or in part, that is incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

II. Description of Various Embodiments of the Invention

While the specification concludes with claims which particularly point out and distinctly claim the present invention; it is believed that the present invention will be better understood from the following description of various non-limiting embodiments.

It is to be understood that certain descriptions of various embodiments have been simplified to illustrate only those elements and/or features that are relevant to a clear understanding of the present invention, while eliminating, for purposes of clarity, other elements and/or features. Those of ordinary skill in the art, upon considering the present description of the various non-limiting embodiments of the present invention, will recognize that other elements and/or features may be desirable in order to implement the present invention. However, because such other elements and/or features may be readily ascertained by one of ordinary skill upon considering the present description of various embodiments of the invention, and are not necessary for a complete understanding of the present invention, a discussion of such elements and/or features is not provided herein. As such, it is to be understood that the description set forth herein is merely exemplary to the present invention and is not intended to limit the scope of the claims.

In one embodiment of the invention, cholera toxin subunits B (CTB) attached to quantum dots to form CTB-quantum dot conjugates were developed for labelling cells. Those cells having gangliosides on the surface of the cell membrane, including without limitation, mammalian cells, and in particular, human primary cells, are particularly well suited for labelling with CTB-quantum dot conjugates. In comparison tests described in more detail below, the CTB-quantum dot conjugates were internalized by all tested cell lines into small vesicles dispersed throughout the cell cytoplasm, while commercially available polyarginine conjugates rapidly accumulated into aggregates in large perinuclear endosomes. Although a large proportion of CTB-quantum dot conjugates eventually also accumulated in perinuclear endosomes, this accumulation required several days, and even then many CTB conjugated quantum dots remained in small vesicles dispersed throughout the cytoplasm, thus maintaining a dispersed luminescent pattern. The tests described herein demonstrate that CTB-quantum dot conjugates are a practical and advantageous improvement over the polyarginine conjugates for the general labelling of mammalian cells.

By using fluorescence correlation spectroscopy and gel filtration chromatography techniques for comparison, it has been found that the CTB-quantom dot conjugates are less prone to aggregation than their polyarginine counterparts and, further, that they remain less aggregated after cellular uptake and, it is believed based on some preliminary data, that they remain less aggregated for several generations of cell division.

In one embodiment, the quantum dots are carboxyl quantum dots, which are shown herein to readily conjugate to CTB. Other forms of quantum dots referred to above may also be used for conjugation with CTB. Nonlimiting examples include quantum dot is selected from the group consisting of carboxylated quantum dots, amino -functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof.

Luminescent labels may be made which are comprised of a plurality of quantum dots, each having at least one cholera toxin subunit B attached thereto to form the conjugates described herein. A reagent system may be used which is comprised of such luminescent labels. In a reagent system kit, for example, a supply of luminescent labels including the CTB-QD conjugates, suitable controls, buffers, standard wash solutions, and optional equipment, such as micro wells, plates and other containers for cells and suitable growth media may be provided.

A method of labelling cells is also provided. The method, which is described in more detail in the examples herein, generally comprises adding to a plurality of live cells selected from cells having cell surface gangliosides, a luminescent label comprising at least one Vibrio cholerae toxin subunit B attached to a quantum dot, and exposing the cells to the luminescent label for a period of time sufficient to allow the cells to absorb the label. The method may further include the step of washing the cells to remove any unabsorbed label. In one or more embodiments of the method, the concentration of the luminescent label added to the cells may be in the range of about 250 pM to 4 nM.

One significant advantage of the method is that the cells may be maintained in normal growth media before and during labelling. It was surprisingly found that, unlike methods heretofore used to label cells with quantum dots, the cells do not need to be placed under any stress to enable the uptake of the cholera toxin subunit B - quantum dot conjugates. After a period of time, the conjugate is absorbed into the cells. Those skilled in the art will recognize that the period of time may vary depending on the particular cell species and the media in which the cells are grown and/or maintained. The period of time may generally be at least about one quarter hour to over night, or a comparable period sufficient to allow the cholera toxin subunit B - quantum dot conjugate to be taken up by the cells. The period of time may more particularly range from about one half hour to 18 or more hours, or from about one to about 18 hours, or from 12 to about 18 hours or from about 12 to about 16 hours.

The luminescent conjugate may be in an unaggregated form or may form small aggregates having a size of about 20 to 200 nm.

The cells that may be labelled with the conjugate described herein may be any cell that has cell surface gangliosides. Of particular interest are mammalian cells, dendritic cells or cells from primary human cell populations. The cells may be selected from the group of cells consisting of stem cells, bone marrow cells, immune cells, tumor cells and combinations thereof. The method of labelling described herein provides a labelled cell population comprising a plurality of cells labelled internally with a luminescent label, each label comprised of up to three Vibrio cholerae toxin subunits B attached, or conjugated, to a quantum dot.

A method of detecting cells having cell surface gangliosides is also provided. The method of detection may comprise adding to a plurality of cells, a luminescent label comprising a Vibrio cholerae toxin subunit B-quantum dot conjugate, exposing the cells to the luminescent label for a period of time sufficient to allow cells having cell surface gangliosides to absorb the luminescent label, and detecting the presence of cells having internal luminescence. The luminescence may be detected by any suitable means of detecting luminescence recognized in the art, including without limitation, fluorescent microscopy, flow cytometry, fluorescent imaging, or fluorimetry to detect fluorescence intensity, for example in a micro well plate or a spectrometer.

The method also lends itself well to cell tracking. As stated above, the cholera toxin subunit B-quantom dot conjugates remain dispersed throughout the cytoplasm for days following the initial uptake into the cells. During that time, cells may undergo cell division and in some cases differentiation. The CTB-QD conjugates internalized in the cells allow tracking of the cells' growth, expansion, migration and differentiation until the quantity of CTB-QD conjugates are too diluted by cell divisions for useful observation.

The following examples are intended to more clearly illustrate aspects of the compositions and methods described herein, but are not intended to limit the scope thereof.

NON-LIMITING EXAMPLES

Materials and Methods

ITK-CARBOXYL™ quantum dots emitting at 605, 655 and 705nm were purchased from Invitrogen Corporation (Portland, OR). (605 and 655 are CdSe core with ZnS shell; 705nm emitters are mixed CdTe-CdSe core with ZnS shell; the carboxyl coating was originally described by Wu, X. et al, Nat Biotechnol, 2003, 21, (1), 41-46).

Cholera toxin B (CTB) and triethylammonium bicarbonate buffer (1.0M, pH 8.5; TEAB) were purchased from Sigma (St. Louis, MO) and EDC [(l-Ethyl-3(3-dimethylamino propyl) carbodiimide HCl] was purchased from Pierce Chemical Company (Rockford, IL); all were used as purchased.

EXAMPLE 1.

Conjugation: The lyophilized CTB product was reconstituted in water to a final concentration of 1 mg CTB/ml. The manufacturer's buffer was 0.05M Tris buffer, with 0.2M NaCl, 3mM NaN₃ and ImM Na₂EDTA at pH 7.5. The buffer was exchanged to TEAB buffer (0.01M, pH 8.5) using CENTRICON-10™ centrifugal filters (Millipore, Billerica, MA); the final concentration was estimated by absorbance at OD 280nm (based on the calculated value E₂₈o = 9.77 x 10³ for the 11,600 molecular weight subunit B monomer; this differs by 9% from the value of 11.02 x 10³ measured by the procedure disclosed in Lai, C. Y., J. Biol. Chem. 1977, 252, (20), 7249-57246.) Because CTB is normally a pentamer, the calculated pentamer molar absorption would be 4.8 x 10⁴.

A solution of carboxyl quantum dots (50μL, 8μM, 605, 655 or 705nm emitting quantum dots) was diluted by adding TEAB buffer (400μl of 0.01M, pH 8.5) containing 20μl of EDC (lmg/ml) and mixed at room temperature for 10 min. CTB (70μL of 17.6 μM as pentamer) in 0.0 IM TEAB buffer was then added to the reaction mixture, thus giving an input ratio of 3CTB/QD. After 2 hours constant mixing at room temperature, the CTB conjugate (CTB-QD) was either resuspended in 0.0 IM sodium borate buffer by three cycles of centrifugation and resuspension using CENTRICON- 100™ centrifugal ultrafilters (Millipore, Billerica, MA) or purified by gel filtration chromatography using a Pharmacia FPLC system coupled with a UV-M II detector set at 280nm, as described below.

The purified CTB-QD conjugates showed few or no visible aggregates or precipitated substances, and retained the spectroscopic properties of the original purchased quantum dots. Column chromatography, agarose gel electrophoresis and two-photon fluorescence correlation spectroscopy were used to characterize the effectiveness of the conjugation reaction and monodispersity of both the original carboxyl and CTB-QD preparations.

Alternative methods to activate and attach, or conjuagte, the choera toxin subunit B and the quantum dots may be used. EXAMPLE 2

Conjugation. In one alternative method, cholera toxin B subunit material would be activated by reacting cholera toxin B subunit material at 1 mg/mL in phosphate buffered saline (PBS) with 1 mM EDTA, with a sufficient quantity of 2-iminothiolane (Traut's reagent) at pH 7.5. This step would be followed by desalting and buffer exchange into 50 mM MES, pH 6.0, 1 mM EDTA buffer using Sephadex G25 resin to remove unreacted, and uncoupled iminothiolane. The excluded material, thiol-modified cholera toxin B, would be collected and immediately used in a coupling reaction with maleimide activated quantum dots.

To activate the quantum dots, QD ITK Amino-PEG quantum dots (1 micromolar concentration nanoparticles) in PBS, pH 7.4 would be combined with sulfo-SMCC (1 mM concentration), freshly prepared. After 1 hour, the activated quantum dots would be purified from excess crosslinker and buffer exchanged into 50 mM MES, pH 6.0, 1 mM EDTA buffer using G25 resin, and mixed with the thiol-modified cholera toxin B subunit material described above. This reaction proceeds at room temperature or 4 ⁰C for 2 hours, followed by gel filtration, as described for the carboxyl conjugated cholera toxin B reaction of Example 1.

Those skilled in the art will recognize that other suitable means of attaching cholera toxin subunits B to quantum dots of choice may be used to form the conjugates of the present invention.

EXAMPLE 3

Gel filtration chromatography: Carboxyl quantum dots or CTB-quantum dot conjugates (0.4nmoles, typically 500 μl) were loaded onto a prepacked Superose 6 column (GE Healthcare, Piscataway, NJ), then chromato graphed using an FPLC system (GE Healthcare). Buffer was 0.1M NaCl, 0.05M sodium borate, pH 8.5; flow rate 0.5ml/min; fraction volume 2ml; optical density monitored at 280nm. A sample run is shown in Figure l(A), (i), unreacted carboxyl quantum dots; (ii), CTB-QD conjugates; and (iii), an overlay of (i) and (ii). The absence of material eluting in the void volume in Figure l(A)(i) and the decreased retention volume of the conjugate peak in Figure 1 (A)(U), demonstrates that most of the CTB-QD conjugates do not aggregate. Figure l(B), (iv)-(vi) show a rerun of the peak fractions from the columns in Figure l(A). The trailing OD280 peaks in all columns in Figure l(A) are unidentified, but did not appear to have any toxic effects on cells.

EXAMPLE 4

Gel Electrophoresis: The conjugation of CTB to QDs was easily followed by gel electrophoresis, as shown in Figure 2. Electrophoresis was in 1% agarose (Fisher, Molecular Biology Grade) in tris-acetate-EDTA (TAE) buffer, pH 8; 12.5V/cm, typical run-time 1 hr. Only the main fraction of the CTB-QD conjugates from the FPLC was used as a labelling reagent (see, Figure 2, column c), and given the small increase in hydrodynamic radius in the conjugate, as described below, it is believed that there are two to three CTB protein molecules coupled to each quantum dot. Little or no material was retained at the origin.

EXAMPLE 5

Fluorescence Correlation Spectroscopy (FCS): The two-photon FCS instrument used in these experiments is based on the system described by Berland, K. M. et al., Biophysical Journal, 1995, 68, (2), 694-701, and will not be described in detail herein. Briefly, a femtosecond titanium sapphire laser (Mira 900D, Coherent, Santa Clara, CA) operating at 76 MHz with a pulse width of ~120fs and λ = 800nm was used as an excitation source. The laser output was directed into a 1.4NA 63X Plan-Apochromat oil immersion objective mounted in an inverted microscope (Axiovert 200M, Zeiss, Jena, Germany). Liquid samples were prepared within COVER WELL™ perfusion chambers (PC8R-1.0, Grace Bio-Labs, Bend, OR) mounted on type 1 coverslips. Fluorescence emission was collected through the objective and passed through an appropriate two-photon bandpass filter (Chroma, Rockingham, VT) to block any stray excitation light. Detection was provided by two avalanche photodiodes (SPCM AQR- 14, Perkin-Elmer, Quebec, Canada) whose TTL outputs were cross-correlated by an external correlator module (ALV5000/EPP, ALV Laser, Langen, Germany) connected to a data acquisition computer. Excitation volume calibration was performed using a 5OnM solution of fluorescein dye in phosphate buffered saline (PBS) resulting in values for ro and zo of 0.2781 + 0.0046 μm and 0.6275+ 0.0065 μm respectively (numbers denote one standard deviation). These spatial dimensions are in general agreement with those reported by Berland, K. M. et al., Biophysical Journ., supra, using similar laser intensities. Samples were prepared by passivating both the perfusion chamber and the coverslip with a 1% solution of bovine serum albumin for 15 minutes. This solution was carefully removed and a InM solution of the quantum dot preparation from Example 1 was added. In order to circumvent the artificial expansion of the excitation volume due to excitation saturation effects reported by Larson, D. R. et al, Science, 2003, 300, (5624), 1434-1436, very low excitation powers (~150μW) were used in conjunction with small quantum dot concentrations (-InM).

EXAMPLE 6

Cell growth: Tests were done on several cell samples as follows.

(1) NIH 3T3 mouse fibroblast cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (CS) and antibiotics (100 units/ml penicillin, and 100 μg/ml streptomycin).

(2) Mouse muscle-derived stem cells (MDSC) were isolated according to the procedure described in Qu-Petersen, Z. et al., J Cell Biol, 2002, 157, (5), 851-864 and cultured in DMEM (high-glucose), 10% FBS, 10% horse serum, 1% chick embryo extract, 20OmM glutamine and 1% penicillin/streptomycin including prophylactic for mycoplasma (Invitrogen, San Diego, CA).

(3) Human mesenchymal stem cells (hMSC) were obtained from Lonza, Inc. (Walkersville, MD). Cells were certified positive for adipogenic, chondrogenic and osteogenic potential assays by the manufacturer. Cells were maintained in mesenchymal stem cell basal medium (MSCBM), mesenchymal cell growth supplement, L-glutamine, penicillin and streptomycin obtained from Lonza, Inc. (Walkersville, MD).

(4) M21 human melanoma cells were obtained from Scripps, La Jolla, CA and maintained in RPMI 1640 medium, supplemented with 10% FCS and antibiotics as described above for the NIH 3T3 cells.

(5) MH15 mouse teratocarcinoma cells were obtained from Jackson Laboratories, Bar Harbor, ME, and maintained in RPMI 1640 medium, supplemented with 10% FCS and antibiotics as described above for the NIH 3T3 cells. Cell labelling: For labelling, CTB-QD conjugates were used at a concentrations between 25OpM and 4nM in the normal cell growth medium, and labelling was overnight, generally for 12-18 hours at 37°C. Both the labelling duration and QD conjugate concentration may be adjusted to change the extent of labelling; however, aggregation at the cell surface becomes evident at higher concentrations (>4nM) in some of the cell lines tested.

Although not required in all cases, the labelled cells may be washed, typically with the growth media normal for the particular cell population, to remove an excess label.

EXAMPLE 7

Imaging: All labelled cells were imaged 18 hours after labelling. Figure 3 illustrates the images obtained for the five different cell types of Example 6 labelled with the CTB-QD conjugates. It was surprisingly and unexpectedly discovered that the quantum dots were completely dispersed throughout the cytoplasm in each cell type when labelled with the CTB- QD conjugates of the invention, which is in stark contrast to what is seen when cells are labelled by polyarginine conjugates. It is believed that the quantum dots are in vesicles in the cytoplasm.

EXAMPLE 8

Comparative Example. For comparison, NIH 3T3 fibroblast cells were labelled with 605 nm QTRACKER™ polyarginine conjugated quantum dots (InM; Invitrogen, Portland, OR) and 605 nm carboxyl quantum dots (Invitrogen) alone. Figures 4(a-c) illustrate an example of the NIH 3T3 fibroblast cell labelled with the 605nm polyarginine conjugated quantum dots showing the aggregation of the quantum dots and their preferential localization in perinuclear endosomes. Figures 4(d-f) illustrate the same cell line, but labelled instead using 655nm carboxyl quantum dots. The lack of fluorescence in panel e demonstrates that the unconjugated carboxyl quantum dots are not able to pass through the cell membrane and label the cytoplasm under the same labelling conditions which generated the intracellular fluorescence shown for the conjugated quantum dots.

EXAMPLE 9

Fluorescence Correlation Spectroscopy (FCS) Analysis: The theory behind FCS is well established and need not be discussed in any great detail. See, Magde, D. et. al., Biopolymers, 1974, 13, (1), 29-61; Webb, W. W., Quarterly Reviews of Biophysics, 1976, 9, (1), 49-68. Briefly, each of the experimentally obtained correlation functions was fitted to the standard model of three-dimensional diffusion with a dark state contribution (as detailed in Equation 1)

wherein G(τ) is the correlation factor, G(O) is the correlation factor at lag time zero, τ is the lag time, τ_D is lateral diffusion time, S² is the volume parameter of the laser excitation, and T is the triplet state fraction, tT is the triplet state relaxation time, and e is the mathematical constant. In each of the fits to the experimental data, the values for GO and τ_D were initially estimated by eye and the value of S was kept fixed at the value determined in the calibration fits to the fluorescein data (S²= 0.1964).

Figure 5 shows the correlation data obtained from the 655nm emitting carboxyl quantum dots and those that were conjugated with CTB. The fitted parameters and extracted diffusion constants and hydrodynamic radii are shown in Table 1. The decrease in the diffusion constant measured by FCS translates into an increase in hydrodynamic radius by 2.8nm. As such, the overall diameter of the CTB-QD conjugate was increased by 5.6nm. These data confirm that the both the initial and the modified quantum dot solutions were mono-disperse and void of aggregates in solution. Both gel electrophoresis and column chromatography show one principal fraction in the conjugated product; the thickness of the CTB pentamer is ~3.1nm and the diameter is -6.2 nm as determined by X-ray diffraction using well known techniques; these measured conjugate sizes would be compatible with as little as one CTB protein with its long axis oriented on edge, or two proteins flattened on the quantum dot surface in various orientations with respect to each other, or three pentamers in a pyramidal orientation flattened on the quantum dot surface. Thus, in one embodiment there may be from one to three CTB pentamers for each quantum dot, or from one to two CTB pentamers for each quantum dot or from two to three CTB pentamers for each quantum dot. Those skilled in the art will recognize that CTB can be made to exist as a monomer. It is believed that the monomeric CTBs would conjugate with the quantum dots in the same manner as the pentameric form of CTB and that the monomeric form would be taken up by the cells.

Table 1 below shows the fitted diffusion times and extracted diffusion constants and hydrodynamic radii of 655nm carboxyl and CTB conjugated quantum dots.

TABLE 1 quantum dot τ _D /ms D / cm ²S^{" 1} τ_H / nm

655-carboxyl 0 4345 2. 2245 x lO^"7 9.8

655-CTB 0 5618 1. 7208 x lO^"7 12. 6

EXAMPLE 10

Effect on stem cell gene expression: To specifically address the issue of toxicity, the effects of the CTB QD conjugates on differentiation and marker expression in both human and mouse muscle-derived and mesenchymal stem cell lines were studied. In all cases described below, cells were labelled in growth medium as described above overnight (from 12-18 hr) using the indicated concentrations of quantum dots, then trypsinized and replated under the described conditions.

Stem cells labelled with CTB-QD conjugates appear to maintain their differentiation potential as well as stem cell properties. Human mesenchymal stem cells (hMSC) were labelled successfully with a range of doses of CTB-QD -655, from 16nM down to 250 pM (Figure 6A). After labelling, hMSC were placed under osteogenic conditions for 7 days; they then exhibited up-regulation of alkaline phosphatase (ALP) activity, which is an early marker of osteogenic lineage progression in vitro. hMSCs exhibiting positive staining for ALP activity also contained the quantum dot label indicating that CTB-QD-labelled hMSCs maintained their differentiation potential (Figure 6B). It has been previously reported that hMSC express osteogenic genes such as Osterix (Osx) in response to BMP-2 stimulation in vitro. (Celil, A. B. et al, J. Biol. Chem., 2005, 280, (36), 31353-9; Spangler, B. D., Microbiol. Rev., 1992, 56, (4), 622-647) Using the method of the present invention, it has been shown that hMSCs induce Osx gene expression within 24 hr of treatment with BMP-2 (Figure 6C) after labelling with CTB-655. Interestingly, the quantum dot label appears to have a synergistic effect with BMP-2 for induction of Osx gene expression. hMSC cells were also labelled with three different color CTB-QD conjugates as individual cultures and created 2-color co-cultures of hMSC (Figure 6D).

Mouse muscle derived stem cells (MDSCs) also appear to maintain their stem cell properties under CTB-QD labelling. CTB-QD-labelled MDSC maintain similar percentages of expression for surface markers indicative of the stem cell phenotype such as stem cell antigen 1 (Sca-1) and CD34, compared to non-labelled cells (Figure 7A).

MDSCs are inherently myogenic and readily fuse to form myotubes under serum deprivation. See, Qu-Petersen, Z. et al, J. Cell. Biol, 2002, 157, (5), 851-864. The data herein demonstrates that CTB-QD-655 labelled MDSC can also form myotubes under serum deprivation for 72 hr, hence maintaining their myogenic potential following labelling with CTB-QD conjugates. Fusion of individual cells, each labelled with a different color CTB- QD conjugate, to form myotubes was observed after serum deprivation for 72 hr (see, Figures 7B, E). Note the presence of both colors in the myotube connecting the fused cells in Figure 7B. Finally, as MDSCs are also known to be osteogenic. CTB-QD labelled MDSC have been shown to retain their osteogenic potential and up-regulate Osx and Alp gene expression under BMP-2 stimulation (see Figures 7C, D). Although BMP-2-induced Osx gene expression was slightly reduced in CTB-655 labelled cells compared to non-QD labelled cells, the fold induction is several orders of magnitude over cells not treated with BMP-2. The difference is mathematically significant, but this slight decrease is not considered to be biologically significant because the behavioral studies demonstrate that the CTB-QD labelled cells functioned in the same manner as unlabeled cells. Moreover, the decrease was not correlated with dose. In the case of Alp, BMP-induced gene expression was equal to or higher than that in unlabeled cells, and again was not correlated with QD dose. Taken together with the gene expression analysis of Osx in hMSC, the effect of CTB-QD conjugates on gene expression during differentiation is minimal; labelling of either hMSC or MDSC with CTB-QD conjugates does not appear to drastically influence their differentiation potential.

The foregoing experimental results may be contrasted with studies of the effect of EviTag quantum dots internalized using either Lipofectamine or TAT-protein mediated cell uptake. (See, Hsieh, S. C; et. al., J. Biomed. Mater. Res. B Appl. Biomater. 2006, 79B, (1), 95-101; and Hsieh, S. C. et. al., J. Biomaterials, 2006, 27, (8), 1656-1664). In one paper, the osteogenic potential of immortalized human mesenchymal stem cells appeared unimpaired, but alkaline phosphatase activity and the expression of mRNA for osteopontin and osteocalcin were reduced relative to cells exposed to the internalizing agent only. In the second paper, quantum dot-labelled mesenchymal stem cells showed reduced chondriogenesis and reduced expression of chondriogenesis-associated proteins and their mRNAs (type II collagen and aggrecan). In neither case was viability or growth affected. There was no observed consistent impairment in cell differentiation or impairment of marker expression. Unfortunately, the differences in origin of quantum dots (Quantum Dot Corporation (now part of Invitrogen) vs Evident), mode of internalization, cells used, and markers tested make a rigorous comparison of the results shown herein to the results of the referenced papers difficult. The most extensive test of gene expression after quantum dot internalization was by Zhang et al., Small, 2006, 2, (6), 747-751, who found minimal effects from internalization of PEG-silica-clad quantum dots. It is noted, however, that the gene expression or toxicity relative to the amount of internalized quantum dots used has not been assessed, (cp Chang, E. et al., Small, 2006, 2, (12), 1412-1417).

The experiments conducted and described herein demonstrate that the CTB-QD conjugates of the present invention are a useful alternative method for labelling several different cell types. Further, with the method of labelling and the conjugates of the present invention, labelling was more uniform than that given by polyarginine conjugates. Although cell labelling with CTB-QD conjugates has not been at the very high levels seen using polyarginine conjugates, cells labelled using either conjugate were readily imaged. It is believed that the more uniform cytoplasmic distribution given by CTB-QD conjugates would allow better following of cells through several generations, as the distribution of the quantum dots between daughter cells should be more uniform. It also has been observed that the CTB conjugated quantum dots remain in cells over several generations with no obvious toxicological effects.

Without wishing to be bound by theory, it is believed that the uptake of CTB-QD conjugates is by a pathway similar to that of the uptake of CTB labelled using conventional fluorophores; that is, mainly mediated by uptake in caveolae and retrograde transport through the Golgi apparatus and thence into the endoplasmic reticulum. This mechanism would explain the initial uniform labelling observed. However, CTB internalization may occur by several alternate pathways. The high sensitivity and stability conferred by the CTB-QD conjugates of the present invention may aid in exploring the minor uptake pathways. The advantages of the CTB-QD conjugates are that they should be usable with essentially all cell types having cell surface gangliosides; there are no reagents to mix; conjugates may be used in presence or absence of serum; aggregation is minimal; and the resultant cytoplasmic labelling is far more uniform than that obtained using polyarginine conjugates. Thus the CTB-QD conjugates are likely to be more suitable for long-term, multi- generation cell tracking.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

What is claimed is:

1. A conjugate comprising:

at least one Vibrio cholerae toxin subunit B attached to a quantum dot.

2. The conjugate recited in claim 1 wherein the quantum dot is selected from the group consisting of carboxylated quantum dots, amino-functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof.

3. The conjugate recited in claim 1 wherein there are from one to three Vibrio cholerae toxin subunits B attached to one quantum dot.

4. The conjugate recited in claim 1 wherein there are from one to two Vibrio cholerae toxin subunits B attached to one quantum dot.

5. A luminescent label comprising:

at least one Vibrio cholerae toxin subunit B attached to a quantum dot.

6. The luminescent label recited in claim 5 wherein the quantum dot is selected from the group consisting of carboxylated quantum dots, amino-functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof.

7. A reagent system comprising a luminescent label, said label comprising a plurality of quantum dots, each having at least one Vibrio cholerae toxin subunit B attached thereto.

8. The reagent system recited in claim 7 wherein the quantum dot is selected from the group consisting of carboxylated quantum dots, amino-functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof.

9. The reagent system recited in claim 7 wherein there are from one to three Vibrio cholerae toxin subunits B attached to each quantum dot.

10. A method of labelling cells comprising: adding to a plurality of cells selected from cells having cell surface gangliosides, a luminescent label comprising at least one Vibrio cholerae toxin subunit B attached to a quantum dot; and,

exposing the cells to the luminescent label for a period of time sufficient to allow the cells to take up the label.

11. The method recited in claim 10 further comprising washing the cells to remove any excess label.

12. The method recited in claim 10 wherein the cells are mammalian cells.

13. The method recited in claim 10 wherein the cells are dendritic cells.

14. The method recited in claim 10 wherein the cells are from primary human cell populations.

15. The method recited in claim 10 wherein the cells are selected from the group consisting of stem cells, bone marrow cells, immune cells, tumor cells and combinations thereof.

16. The method recited in claim 10 wherein the quantum dot is selected from the group consisting of carboxylated quantum dots, amino-functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof

17. The method recited in claim 10 wherein the quantum dot is a carboxylated quantum dot.

18. The method recited in claim 10 wherein there are from one to three Vibrio cholerae toxin subunits B attached to one quantum dot.

19. The method recited in claim 10 wherein there are from one to two Vibrio cholerae toxin subunits B attached to one quantum dot.

20. The method recited in claim 10 wherein there are from two to three Vibrio cholerae toxin subunits B attached to one quantum dot.

21. The method recited in claim 10 wherein the plurality of cells is maintained in normal growth media and the label is added to the media.

22. The method recited in claim 10 wherein the label is absorbed into the cells in an unaggregated form.

23. The method recited in claim 10 wherein the label absorbed into the cells form aggregates having of about 20 to 200 nm in hydrodynamic diameter.

24. The method recited in claim 10 wherein the concentration of label added to the cells ranges from about 250 pM to 4 nM.

25. The method recited in claim 10 wherein the period of time ranges from about 12 to 18 hours.

26. The method recited in claim 10 wherein the label absorbed into the cells is dispersed throughout the cells.

27. A labelled cell population comprising:

a plurality of cells labelled internally with a luminescent label, each label comprised of from one to three Vibrio cholerae toxin subunits B attached to a quantum dot.

28. The labelled cell population recited in claim 27 wherein the cells are those having cell surface ganglio sides.

29. The labelled cell population recited in claim 27 wherein the cells are mammalian cells.

30. The labelled cell population recited in claim 27 wherein the cells are dendritic cells.

31. The labelled cell population recited in claim 27 wherein the cells are from primary human cell populations.

32. The labelled cell population recited in claim 27 wherein the cells are selected from the group consisting of stem cells, bone marrow cells, immune cells, tumor cells, and combinations thereof.

33. The labelled cell population recited in claim 32 wherein the cells are mammalian.

34. The labelled cell population recited in claim 27 wherein the quantum dot is selected from the group consisting of carboxylated quantum dots, amino -functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof.

35. A method of detecting cells having cell surface gangliosides comprising:

adding to a plurality of cells a luminescent label comprising a Vibrio cholerae toxin subunit B -quantum dot conjugate;

exposing the cells to the luminescent label for a period of time sufficient to allow cells having cell surface gangliosides to absorb the luminescent label;

detecting the presence of cells having internal luminescence.

36. The method of detection recited in claim 35 wherein the luminescence is detected by fluorescent microscopy.

37. The method of detection recited in claim 35 wherein the luminescence is detected by flow cytometry.

38. The method of detection recited in claim 35 wherein the luminescence is detected by fluorescent imaging.

39. The method of detection recited in claim 35 wherein the luminescence is detected by fluorimetry.

40. The method of detection recited in claim 35 wherein the cells are exposed to the luminescent label for a period of at least 12 hours.

41. The method of detection recited in claim 35 wherein the conjugate comprises at least one Vibrio cholerae toxin subunit B attached to the quantum dot.

42. The method of detection recited in claim 35 wherein the quantum dot is selected from the group consisting of carboxylated quantum dots, amino -functional quantum dots, amino and polyethylene glycol functional quantum dots, and combinations thereof.

43. The method of detection recited in claim 35 wherein there are from one to three Vibrio cholerae toxin subunits B attached to one quantum dot.

44. The method of detection recited in claim 35 wherein there are from one to two Vibrio cholerae toxin subunits B attached to one quantum dot.

45. The method of detection recited in claim 35 wherein there are from two to three Vibrio cholerae toxin subunits B attached to one quantum dot.

46. The method of detection recited in claim 35 wherein the plurality of cells is maintained in normal growth media and the label is added to the media.

47. The method of detection recited in claim 35 wherein the luminescent label is absorbed into the cells in an unaggregated form.

48. The method of detection recited in claim 35 wherein the label absorbed into the cells form aggregates of about 20 to 200 nm in hydrodynamic diameter.

49. The method of detection recited in claim 35 wherein the concentration of luminescent label added to the cells ranges from about 250 pM to 4 nM.

50. The method of detection recited in claim 35 wherein the luminescent label absorbed into the cells is dispersed throughout the cells.

51. The method of detection recited in any of claims 35 to 50 wherein detecting the presence of cells having internal luminescence further comprises tracking the luminescence for one or more cycles of cell divisions.