WO2010043053A1

WO2010043053A1 - Single-domain antibody functionalized quantum dots for cellular imaging of cancer cells

Info

Publication number: WO2010043053A1
Application number: PCT/CA2009/001490
Authority: WO
Inventors: Kui Yu; Md. Badruz Zaman; Toya Nath Baral; Dennis Whitfield; Jianbing Zhang
Original assignee: National Research Council Of Canada
Priority date: 2008-10-15
Filing date: 2009-10-15
Publication date: 2010-04-22

Abstract

The invention relates to water soluble photoluminescent nanoparticles comprising surface ligands comprising surface accessible carboxyl groups or amino groups, or both carboxyl and amino groups. The invention also relates to uses of the photoluminescent nanoparticles for cellular imaging of tumor cells.

Description

SINGLE-DOMAIN ANTIBODY FUNCTIONALIZED QUANTUM DOTS FOR CELLULAR IMAGING OF CANCER CELLS

Field of the Invention

[0001] The present invention relates to single-domain antibody-functionalized quantum dots for cellular imaging of tumor cells.

Background of the Invention

[0002] Photoluminescent (PL) semiconductor nanocrystals, which are known as quantum dots (QD) when spherical in shape, have attracted significant attention in bio-labeling and bio- imaging applications (Michalet et al, 2005; Rhyner et al, 2006; Jiang et al, 2008). Semiconductor quantum dots (QD) have the characteristics of size-tunable bandgaps, bright photoemission with broad absorption but narrow emission bands, and excellent photostability, all of which make them attractive as optical imaging agents for biological studies. To make the application possible, the surface of the colloidal photoluminescent QD must be made not only water compatible but also resistant to pH variance, salt and other normal components of biological fluids. To this end, a variety of hydrophilic groups have been used to coat QD (Medintz et al, 2005).

[0003] Usually, such bio-oriented applications require targeting to the site of interest, and the use of antibodies is one strategy for specific and compelling targeting (Goldman et al, 2002;

Chan & Nie, 1998). Conventional antibodies and some of their derivatives have been tested as targeting agents; however they suffer from disadvantages related to issues such as stability, aggregation, and production cost. Conventional immunoglobulin G (IgG) has a molecular weight of 150 kDa and is composed of two identical light chains and two identical heavy chains, as shown in Figure 1 (A). The light chain contains one variable (VL) domain and one constant domain (CL), whereas the heavy chain generally has four domains: one variable (VH) and three constant domains (CHs). The antigen-binding sites of IgG reside only in the variable regions (VL-VH, also referred to as Fv). Due to their size, IgGs are not suitable for targeting purposes. Furthermore, the conditions used for bio-conjugation can lead to their denaturation (Jayagopal et al, 2007; Wu et al, 2003).

[0004] Antibody fragments may also be used to provide the desired specificity for targeting; see, for example Ramakrishnan et al, 2009. The sizes of Fab (fragment, antigen binding) and single chain variable fragment (scFv) are approximately 50 and 25 kDa, respectively (Pathak et al, 2007). Despite of the advantage of relatively small size, their biophysical properties and production cost are still disadvantageous.

[0005] There remains a need for effective, specific imaging reagents allowing for better efficiency and targeting of specific molecules.

Summary Of The Invention

[0006] The present invention relates to single-domain antibody functionalized quantum dots for cellular imaging of tumor cells. In one embodiment, the present invention relates to QD bio-labeling, namely binding to cancer cells selectively and efficiently through a single- domain antibody ("sdAb") that is raised against a tumor marker. [0007] In one embodiment, the present invention provides a water-soluble photoluminescent nanoparticle comprising surface ligands comprising surface accessible carboxyl groups or amino groups, or both carboxyl and amino groups. The nanoparticles may be CdSe/ZnS nanoparticles. The nanoparticles as just described may be quantum dots.

[0008] The quantum dots as just described may further comprising a hydrophilic ligand which specifically binds to a biomolecule of interest. The hydrophilic ligand may be a single- domain antibody. In one embodiment, the sdAb may be a camelid sdAb; the sdAb may comprise EG2, which is raised against an antigenic tumor marker such as epidermal growth factor receptor (EGFR).

[0009] The nanoparticles described above may further comprise covalently attached polyethylene glycol molecules.

[0010] The nanoparticles as described herein may be used to selectively label and visualize a tumor cell. The tumour cell may be a breast cancer cell.

Brief Description Of The Drawings

[0011] In the drawings, like elements are assigned like reference numerals. The drawings are not necessarily to scale, with the emphasis instead placed upon the principles of the present invention. Additionally, each of the embodiments depicted are but one of a number of possible arrangements utilizing the fundamental concepts of the present invention. The drawings are briefly described as follows: [0012] Figure IA is a schematic drawing of conventional IgG, and Figure IB is a schematic drawing of a camelid heavy chain antibody.

[0013] Figure 2 is a graph showing optical properties of QD-a, QD-b, and QD-c dispersed in water. QD-a absorption is represented by the thin black line (right y axis), while the emission spectra (labeled as a, b, and c) were measured with the similar amount of QD-a, QD-b, and QD-c, respectively, with 350 nm excitation. With 500 nm excitation, QD-a exhibit QY ~ 8% in double-distilled water and FWHM ~ 38 nm; QD-b QY ~ 3%, and QD-c QY ~ 16%.

[0014] Figure 3 shows the images of targeted bio-labeling of breast cancer cells. Images on the left panel were taken for SK-BR3 cells, while those on the right panel for MDA-MB468 cells. The blue color represents nucleus staining with DAPI, green for surface staining with DiOCs(3), and red for staining of EGFR antigen by QD. In each panel from top to bottom, we used QD-a without sdAb, QD-b conjugated with sdAbs (EG2) only, QD-c conjugated with both EG2 and PEG, and QD-a together with non-conjugated EG2.

[0015] Figure 4 shows normalized optical spectra of absorption (thin line) and emission (thick line) of CdSe core QD in toluene ((1) and (3), with QY ~ 14% estimated with excitation wavelength of 500 nm) and CdSe/ZnS core/shell QD in toluene ((2) and (4), with QY ~ 8% estimated with excitation wavelength of 500 nm).

[0016] Figure 5 shows a TEM image of the CdSe QD (top with scale bar of 10 mn, optical properties shown in Figure 4) and CdSe/ZnS QD (bottom with scale bar of 5 nm, optical properties shown in Figure 4). [0017] Figure 6 shows a schematic drawing of water-soluble MUA-QD (left) and QD-a (right). Regarding the reaction between -COOH and lysine during the preparation of QD-a, it is necessary to point out that there are two amine groups in lysine, and both of them may be reacting with -COOH. However, due to the relatively high basicity of the terminal ε-NH2, ε- NH2 should be more reactive than α-NH2.

[0018] Figure 7 shows a schematic drawing of one embodiment of QD-c, namely PEGylated- and-sdAb-conjugated CdSe/ZnS QD.

Detailed Description Of Preferred Embodiments

[0019] The present invention relates to single-domain antibody functionalized quantum dots for cellular imaging of tumor cells. Any term or expression not expressly defined herein shall have its commonly accepted definition understood by those skilled in the art. As will be apparent to those skilled in the art, various modifications, adaptations and variations of the following description can be made without departing from the scope of the invention claimed herein. The various features and elements of the described invention may be combined in a manner different from the combinations described or claimed herein, without departing from the scope of the invention.

[0020] In one embodiment, the present invention provides a water-soluble nanoparticle comprising surface ligands comprising surface accessible carboxyl groups or amino groups, or both carboxyl and amino groups. [0021] As used herein, the term "nanoparticle" shall mean a particle having at least one dimension which is less than about 100 run. A "quantum dot" is a nanoparticle semiconductor whose excitons are confined in all three spatial dimensions. As a result, quantum dots have properties that are between those of bulk semiconductors and those of discrete molecules. QDs are well-known in the art, and have been described in many publications, for example but not limited to Michalet et al, (2005). The nanoparticles of the present invention may be "photoluminescent", also referred to herein using the abbreviation "PL"; thus, the nanoparticles absorb photons and re-emit photons.

[0022] The nanoparticles of the present invention may comprise any suitable material known in the art; see for example Yong et al (2009). For example, the nanoparticles or quantum dots may be, but are not limited to CdSe, CdS, CdTe, CdTeSe, PbS, and PbSe nanoparticles, or combinations thereof. In a specific, non-limiting example, the nanoparticles may comprise CdSe. Methods for synthesizing the various types of nanoparticles are known in the art, and thus are not reviewed here (for example, see Yu et al, 2005a; Aldana et al, 2001 ; Ouyang, 2009; Yu et al, 2005b; Ratcliffe et al, 2006; Li et al, 2009; Yu et al, 2004), and any such suitable method may be used. Without wishing to be bound by theory, varying the nanoparticle composition may vary the emission wavelength, thus allowing "tuning" of the nanoparticle.

[0023] The nanoparticles of the present invention have a size-dependent fluorescence spectrum, and thus the emission wavelength may also be varied ("tuned") by varying the size of the core. The quantum dots are generally obtained via wet-chemistry approach, thus resulting in thousands of nanocrystals of dimensions within a narrow size distribution. Varying the composition of the nanoparticles may also vary their size.

[0024] The nanoparticles of the present invention may further comprise an outer shell such as a ZnS shell (also referred to as "coating"). The ZnS outer shell aids in protecting the CdSe core from chemical reactions, and also passivates the CdSe QD surface such that the QD exhibits higher quantum yield. Methods for preparing the ZnS shell are also well-known to those of skill in the art (Li et al, 2003); for example, and without wishing to be limiting in any manner, the ZnS shell may be applied in successive monolayers over the CdSe core. Multiple ZnS monolayers may be applied to the nanoparticle; for example, and without wishing to be limiting 1, 2, 3, 4, or 5 ZnS monolayers may be applied; in another example, 2 to 4 ZnS monolayers may be applied to the nanoparticles. Varying the thickness of the ZnS coating may also vary the emission wavelength of the QD. Other types of outer shell may be applied to the nanoparticles of the present invention; for example, CdSe or CdS coatings may also be used. The monolayers comprising the outer shell of the nanoparticle of the present invention may be of the same or of varying composition. Methods of applying the monolayers to the nanoparticles are well-known to those skilled in the art.

[0025] The nanoparticles of the present invention may also comprise surface ligands. "Surface ligands" as used herein are molecules that render the CdSe/ZnS nanoparticle water- soluble. The surface ligands may comprise one or more than one type of hydrophilic functional groups, such as carboxyl (-COOH) or amino (-NH2) groups. The hydrophilic functional groups of the surface ligand should preferably be surface-accessible. Furthermore, the surface ligand should preferably not interfere with the photoluminescence of the QD. Any suitable surface ligand known in the art may be used in accordance with the present invention. For example and without wishing to be limiting, one or more than one amino acid residue may be used as a surface ligand. In a specific, non-limiting example, the surface ligand may be lysine. The CdSe/ZnS QD comprising the surface ligands may be prepared using any method known in the art; for example, and without wishing to be limiting, lysine may be conjugated to the QD using known methods (Jiang et al, 2006). The surface of the nanoparticle may need to be prepared in order to receive the surface ligand; for example, and without wishing to be limiting, -COOH groups on the surface of the nanoparticles may be obtained from ligand exchange with a suitable compound, for example mercaptoundecanoic acid (MUA). When the surface ligand is lysine, the reaction between -COOH on the nanoparticle surface and lysine during the preparation of nanoparticles according to the present invention, may occur at either of the two amine groups in lysine. However, due to the relatively high basicity of the terminal S-NH₂, ε-NH₂ should be more reactive than α-NH₂.

[0026] The nanoparticles as described herein may further comprise a hydrophilic ligand that specifically binds to a biomolecule of interest. The hydrophilic ligand may be a molecule that serves to target the desired cell type or tissue of interest. Various types of molecules may be used, for example and not limited to a single-domain antibody.

[0027] By the term "single-domain antibody", also referred to as "sdAb", "heavy chain antibody" (HCAb), or VHH, it is meant an antibody fragment comprising a single protein domain. sdAbs (Figure 1 (B) were discovered in camelids such as camels, llamas, alpacas and sharks (Hamers-Casterman et al, 1993; Matsunaga et al, 1990; Greenberg et al, 1995). Single- domain antibodies have also been observed in shark and are termed VNARs (Nuttar et al, 2001), and may be engineered based on human heavy chain sequences (Ward et al, 1989). Unlike conventional IgGs, these antibody molecules are naturally devoid of light chains, and comprise only the variable region (Arbabi-Ghahroudi et al, 1997). As the smallest antibody fragment, sdAb can be engineered to have very high affinity despite of its size of only about 13 kDa (Saerens et al, 2004).

[0028] In addition to their small size, sdAbs have several desirable features, including a low tendency to aggregate, which facilitates bio-conjugation reactions that are typically conducted at high protein concentrations. This feature is the consequence of change in their primary structure. In a conventional IgG, the heavy chain variable region (VH) is associated with the light chain variable region (VL) to form a dimeric antigen binding site (Padlan, 1994); the structural basis of this association is hydrophobic interaction between the two domains. In addition, VH is also associated with domain 1 of the heavy chain constant region (C_HI). AS a result of the hydrophobic residues on the surface of VH or VL, these molecules a tendency to aggregate when expressed alone. This is often true even when VHs and VLs are paired to form recombinant scFvs. In contrast, sdAbs identified in camelids are linked to the rest of the molecule through a flexible hinge region and are not required to pair with C _HI and VL (Muyldermans, 2001). The primary sequences of camelid sdAbs are altered to reflect this structural change: many hydrophobic residues at positions, where pairings are required, are replaced by more hydrophilic ones. These substitutions include Val37 to Tyr37 or Phe37, Gly44 to Glu44, Leu45 to Arg45 and Tyr47 to Gly47 (Hamers-Casterman et al, 1993; Conrath et al, 2003). As a result, sdAbs are more likely to exist as monomers and are resistant to many unfavorable conditions such as chemical detergents, extreme pH, and to certain extent heat denaturation and proteolysis (Dumoulin et al, 2002; Olichon et al, 2007; Harmsen et al, 2006). Complete refolding was observed after chemical-induced denaturation. In addition to these features, sdAbs can be produced inexpensively.

[0029] In one embodiment, the sdAb may be a camelid sdAb. In a specific, non-limiting example, the sdAb may comprise EG2, which is raised against epidermal growth factor receptor (EGFR), an antigen that is widely known as a tumor marker (Sato et al, 1983). The sequence of the EG2 sdAb may be:

QVKLEESGGGLVQAGDSLRVSCAASGRDFSDYVMGWFRQADYVNGWFRQAPGKER EFVAAISRNGLTTRYADSVKGRFTISRDNDKNMVYLQMNSLKPEDTAVYYCAVNSA GTYVSPRSREYDYWGQGTQVTVSS [SEQ. ID. NO. 1] as disclosed in Bell et al (2009), or a sequence substantially identical thereto.

[0030] A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered "substantially identical" polypeptides. Conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity).

[0031] In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term "basic amino acid" it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term "neutral amino acid" (also "polar amino acid"), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (GIn or Q). The term "hydrophobic amino acid" (also "non-polar amino acid") is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (He or I), phenylalanine (Phe or F), valine (VaI or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (GIy or G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (GIu or E), and aspartate (Asp or D). [0032] Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at http://ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.

[0033] The substantially identical sequences of the present invention may be at least 75% identical; in another example, the substantially identical sequences may be at least 70, 75, 80, 85, 90, 95, or 100% identical at the amino acid level to sequences described herein. Importantly, the substantially identical sequences retain the activity and specificity of the reference sequence.

[0034] The EG2 sdAb has been shown to have high affinitiy for EGFR, with a KD of 50 nM and a size of about 14 kDa (Bell et al, 2009).

[0035] The nanoparticles of the present invention may also comprise a combination of different sdAbs or a combination of sdAbs to produce a model system to deliver the cellular uptake and intracellular transport of QD in cancer cells for targeting and imaging.

[0036] The hydrophilic ligands may be linked to the nanoparticles by any suitable method known in the art. For example and without wishing to be limiting in any manner, the hydrophilic ligand may be linked to the hydrophilic particle via amide bonding; this may be done using synthetic approach called EDC method (Sehgal & Vijay 1994), in the presence of water-soluble carbodiimide, namely (l-ethyl-3-[3-dimethylarninopropyl]carbodiimide), or another suitable method. As would be known to a skilled person, varying the method of conjugation may improve water solubility of the nanoparticle.

[0037] In yet another embodiment, the nanoparticles of the present invention may further comprise (polyethylene)glycol (PEG molecules). For example, and without wishing to be limiting in any manner, the PEG molecules may be covalently attached amino terminated (polyethylene)glycol, NH2-PEG. The PEG may also be of various molecular weights, as described in Choi et al, 2009; for example, and without wishing to be limiting, the PEG may be of MW 5000. Without wishing to be bound by theory, the presence of PEG in the nanoparticles of the present invention may further enhance biological stability of the resulting nanoparticle. Methods for conjugating PEG on the nanoparticles of the present invention are known to those skilled in the art, for example, and without wishing to be limiting, conjugation of PEG to the nanoparticles may be achieved using the EDC (l-ethyl-3-[3- dimethylaminopropyl]carbodiimide) method, as described by Sehgal & Vijay, 1994.

[0038] Bio-conjugated CdSe/ZnS QD in accordance with the present invention may be prepared using various methods. In general terms, one embodiment of the invention comprises:

i) the synthesis of highly photoluminescent CdSe QD with narrow size distribution as the core; ii) the addition of a ZnS shell to form core-shell CdSe/ZnS QD;

iii) the exchange of surface ligands of the core-shell QD with mercaptoundecanoic acid followed by cross linking the surface through lysine conjugation (Jiang et al, 2006). The resulting CdSe/ZnS QD with their surface ligands comprising carboxyl groups and amino groups are water-soluble, highly fluorescent (with quantum yield (QY) of ca. 8% in water excited at 500 nm), and stable against flocculation in pH > 7.5. We monitored these QD (labeled as QD-a) dispersed in double-distilled water for more than three months and little aggregation was observed);

iv) covalently attached a hydrophilic ligand, a sdAb EG2, to the QD-a and the resulting is labeled as QD-b; in some cases further covalently attached amino terminated (polyethylene)glycol, MW 5000, NH2-PEG to further enhance biological stability and the resulting is labeled as QD-c. Both were achieved using the EDC (l-ethyl-3-[3-dimethylaminopropyl]carbodiimide) method.

[0039] In a preferred embodiment, each of the steps i) to iv) is optimized to satisfy wide- ranging criteria including aggregate-free, stability, and self-assembling leading to functional PEGylated QD-sdAb conjugates (QD-c) that have suitable optical properties for biological imaging and detection.

[0040] As described herein, sdAbs conjugated to QD can be used for targeting cancer cells for imaging and detecting purposes. As will be apparent to one skilled in the art, the sdAb chosen will be specific for a chosen marker, which will be indicative of a cancer cell. The QD-sdAb conjugates may then be used in standard immunoassay procedures, using visualization or detection techniques known in the art. When applied to a sample suspected of containing cancer cells, the QD-sdAb conjugates thus permit visualization or detection of any cancer cells present bearing the chosen marker. In alternative embodiments, the emission wavelength can be varied by varying the core material composition and their sizes, the thickness of the ZnS coating can be increased, and other hydrophilic ligands may be used to improve water solubility, including varying combinations of PEGylation and sdAb conjugation.

[0041] In another aspect, by using different sdAbs or even combinations of sdAbs, peptide- mediated QD may be used as a model system to deliver the cellular uptake and intracellular transport of QD in cancer cells for targeting and imaging.

Examples

Example 1 - Chemicals.

[0042] Cadmium oxide (CdO, Aldrich 99.99%), sulfur (S, Anachemia Chemicals Ltd. 99.98%, powder), selenium (Se, Aldrich 99.5%, 100 mesh powder), tributylphosphine oxide (TOPO, Aldrich 90%), hexadecylamine (HDA, Aldrich), zinc oxide (ZnO, Alfa Aesar 99.99% powder), 1-octadecene (ODE, Aldrich 90%), oleic acid (OA, Aldrich 90%), tri-n- octylphosphine (TOP, Aldrich 90%), tributylphosphine (TBP, Aldrich), and mercaptopropionic acid (MPA, Aldrich), DL-lysine (Sigma 98% TLC), α-aminopropyl-ω- methyl-(polyethylene)glycol (NH2-PEG-5000, Nektar Transforming Therapeutics MW 5000), N.N'-dicyclohexylcarbodiimide (DCC, Aldrich 99%), (l-ethyl-3-[3- dimethylaminopropyl]carbodiimide) (EDC, Fluka), 4',6-diamidino-2-phenylindol(DAPI).

Example 2 -Synthesis and characterization of CdSe Core QD and CdSe/ZnS Core/Shell QD.

[0043] TOP, TOPO or TOPO/HDA -capped CdSe nanocrystals were synthesized using published methods (Puiso et al, 2003), with some modification. For a typical reaction, the mixture of 2.5 mmol of CdO, 7.0 mmol of stearic acid, and 50 ml of ODE in a 250 rnL three- neck flask was heated to about 200 ~ 290⁰C to obtain a clear colorless solution. After this solution was cooled to 100 ~ 120⁰C temperature, HDA (18 g) and 10 g of TOPO was added into the flask. Under nitrogen flow, this system was reheated to 280⁰C. At this temperature, 2.1 ml of Se-TBP solution (made by dissolving 20 mmol of Se in 10 ml of TBP) was quickly injected. The growth temperature was then reduced to 260 ~ 280⁰C for 10 ~ 15 min. The reaction mixture was allowed to cool to room temperature. The reaction product was dispersed in hexanes or toluene and transferred into a separating funnel; about equal volume of MeOH was added and extracted vigorously for ca. 30 min. Afterwards, the funnel was kept still overnight to allow formation of two layers: the nanocrystals remained in the upper layer, namely the hexanes/ODE layer or toluene/ODE layer. Usually, three rounds of purification were performed to obtain a clear and transparent upper layer. The CdSe QD in this upper layer is used for the preparation of CdSe/ZnS QD, after the QD concentration was determined with the measurement of UV absorption. [0044] A ZnS shell with a few monolayers (ML) was grown on the purified CdSe QD, using a modified successive ion layer adhesion and reaction (SILAR) technique (Li et al, 2003). Zinc oxide (ZnO) and elemental sulfur (S) were used as Zn and S source compounds. To optimize the ZnS shell growth, the reaction temperature was varied from 200 to 240⁰C. A typical ZnS coating was performed as follows: 40 ml of ODE and 16 g of HDA were loaded into a 250 mL reaction vessel, heated to 45 ~ 80⁰C under vacuum, bubbled with nitrogen flow for 30 ~ 60 min, and cooled to room temperature. Afterwards, the CdSe QD (ca. 10-6 mol of ca. 3.5 nm in diameter) dispersed in hexane (ca 10 ml total) were added to the mixture of ODE and HDA. The reaction flask was kept at ca. 100⁰C under vacuum for ca. 30 min to remove hexane and other materials such as moisture; then, the solution was heated up to 200 ~ 24O⁰C under nitrogen flow for the addition of the materials for the ZnS shell growth.

[0045] Based on the CdSe nanocrystal concentration obtained, the assumption that the surface of the CdSe cores consists of Se and Cd atoms equally, and that the CdSe and CdSe/ZnS QD were spherical in shape, the ZnO and S needed for one ZnS ML were calculated. Two 0.1 M stock solutions were prepared: the Zn-source/ODE solution was made with ca 0.2g ZnO, 6.1 g OA, and 18 ml ODE heated up to ca. 300⁰C; the S/ODE solution was made with 0.16 g S in 50 ml ODE heated up to ca 100⁰C.

[0046] For the first ZnS ML coating, 1.1 ml of the Zn-source/ODE solution was injected at 200⁰C for ca. 5 min; then, the reaction temperature was increased to ca. 230⁰C for ca 5 min, and then cooled down to 210⁰C, followed by the injection of 1.1 ml the S/ODE solution for ca. 5 min. The reaction temperature was then increased to 240⁰C for ca 10 min. [0047] For growth of the second to the fifth ZnS ML, similar alternating additions of the Zn- source/ODE solution and S/ODE solution were performed, but with the amount of solution added being 1.5, 2.0, 2.5, 3.1 ml, respectively. In general, after the addition of the Zn- source/ODE and S/ODE solution for the growth of one ML, a growth period of 10 ~ 20 min at ca. 24O⁰C was allowed. Usually, it took ca. 5 hours to complete the addition of 5 MLs from the Zn and S source compounds. The reaction flask was kept overnight at 150 ~ 160⁰C under N₂, and finally cooled to room temperature.

[0048] The purification of the resulting CdSe/ZnS QD was carried out, in a manner similar to that of CdSe QD: the reaction product was dispersed in hexanes and transferred into a separating funnel; about equal volume of MeOH was added and extracted vigorously for ca. 30 min. Afterwards, the funnel was kept still overnight to allow formation of two layers, with the nanocrystals in the upper layer. At least five rounds of purification were performed to obtain a clear and transparent lower layer, namely MeOH layer; a transparent MeOH layer suggests there are little unreacted materials in the upper QD layer.

[0049] The optical properties of the CdSe and CdSe/ZnS QD were characterized. Absorption spectra were acquired on a Perkin Elmer Lambda 45 UV/vis spectrometer, and the photoluminescent (PL) emission spectra were acquired on a Fluoromax-3 spectrometer (Jobin Yvon Horiba, Instruments SA) with a 450 W Xe lamp as the excitation source using an excitation wavelength of 350 nm or 500 nm. The PL quantum yield (QY) was estimated as compared to that of Dye R590 (rhodamine 6G in EtOH, QY 95%). Figure 4 shows the optical properties of the resulting CdSe purified QD dispersed in toluene. The CdSe QD exhibited UV absorption peaking at 568 ran (blue thin line, suggesting a diameter of ca 3.5 nm) and photoemission peaking at 582 nm (blue thick line, with excitation wavelength at 350 ran). The CdSe/ZnS core/shell QD exhibited UV absorption represented by the red thin line and photoemission peaking at 602 nm (red thick line, with excitation wavelength at 350 nm).

[0050] The CdSe core and CdSe/ZnS core/shell QD were directly imaged by transmission electron microscopy (TEM). The TEM study was performed on a JEOL JEM-2100F electron microscope operating at 200 kV and equipped with a Gatan Ultrascan 1000 CCD camera. The TEM grids were prepared by drying dilute purified nanocrystal dispersions in hexanes on 300- mesh carbon-coated TEM copper grids in air. Figure 5 shows representative TEM images of the CdSe and CdSe/ZnS QD.

Example 3 - Preparation of water-soluble CdSe/ZnS QD.

[0051] The purified CdSe/ZnS QD (ca 1.4 x 10^"6 mole) of Example 2 were dispersed in 1 ml toluene in a 25 ml reaction flask. About 2 g of mercaptoundecanoic acid (MUA) was added with stirring. The mixture was heated up to ca 100⁰C under vacuum to remove toluene and moisture for a few minutes. Then the temperature was decreased to 80 ~ 9O⁰C under N2; after 3 hours, 2 ~ 3 ml of DMSO was added, with the temperature cooling to 40 ~ 50⁰C, and the reaction flask was stirred overnight at the same temperature. Afterward, the reaction was cooled to room temperature, and chloroform was added to precipitate the resulting MUA- coated CdSe/ZnS QD, hereafter referred to as MUA-QD. Centrifugation was performed at 3700 rpm for ca. 15 min with the precipitate consisting of the QD; the precipitate was further washed with THF to remove MUA. [0052] Next, MUA-QD were dissolved in 2 ~ 3 ml DMSO with stirring until a clear solution was obtained. For cross-linking with lysine, another two stock solutions were prepared: 3 - 4 mmol lysine in 1 ml water, and 3 - 4 mmol DCC in 0.5 ml DMSO. The DCC solution was added into the MUA-QD solution first with stirring. Afterwards, the lysine solution was added; immediately, the reaction mixture became cloudy. This reaction mixture was stirred for 2 hours at room temperature. Afterwards, the mixture was centrifuged at 3700 rpm for ca. 15 min; the solvent was decanted, while the solid was dissolved in double-distilled water. A cloudy dispersion was obtained and transferred onto a regenerated cellulose 5000 MWCO centrifuge filter device; after centrifuging, QD-a were obtained; at least three rounds of such purification (filtration), were performed. The resulting QD-a, whose surface ligands comprise primary amino groups and carboxylic acid groups, were stored at 4°C prior to further surface modification. The concentration of this stock solution (1.1 x 10^"3 mmol/ml) was determined by optical absorbance.

Example 4 - Conjugation of sdAb and PEG-5000 with water-soluble CdSe/ZnS QD.

[0053] The QD-a of Example 3 were conjugated with sdAb (QD-b), or sdAb and PEG (QD-c) via amide bonding; the synthetic approach is the so-called EDC method (Sehgal & Vijay 1994), with the presence of water-soluble carbodiimide, namely (l-ethyl-3-[3- dimethylaminopropyljcarbodiimide).

[0054] QD-b was synthesized by mixing 30 μl QD-a stock solution (3.4 x 10-5 mmole) with 1 ml PBS solution of EG2 (3.7 x 10-5mmole), with a QD-to-sdAb molar ratio of ca. 1 : 1. 50 ~ 60 μl stock solution of EDC (made of 9 mg EDC in 200 μl distilled water) was added at room temperature with vigorously stirring for three hours.

[0055J Similarly, QD-c was synthesized by mixing 30 μl QD-a stock solution (3,4 x 10-5 mmole) with 1 ml phosphate buffered saline solution (PBS consisting of 137 mM NaCl, 2.7 mM KCI, 10 mM Na2HP04, and 2mM KH2P04 in H20 with pH ~ 7.4) comprising sdAb EG2 (0.55mg/ml; with molecular weight of ca 15K, EG2 is estimated to be 3.7 x 10^'5 mmole), as well as 100 μl aqueous solution consisting of PEG- 5000 (1.8 x 10^"4 mmole). The molar ratio of QD-to-sdAb-to-PEG was maintained as ca. 1 : 1 : 5. 50 ~ 60 μl stock solution of EDC (made of 9 mg EDC in 200 μl distilled water) was added at room temperature with vigorously stirring for three hours.

[0056] The resulting two solutions were used for the optical measurements (Figure 2) and immunocytochemistry (Example 5, Figure 3).

[0057] Figure 2 shows the optical properties including absorption and emission of the water- soluble QD labeled as a, b, and c, dispersed in double-distilled water, with a similar QD concentration (-3.4 x 10^"8 M). Emission spectrum (a) was obtained from an aqueous solution of 30 μl QD-a stock solution diluted with 1.0 ml distilled water; emission spectrum (b) was obtained from a solution comprising the same amount of QD-b in a total 1.1 ml solution; and emission spectrum (c) was obtained from the same amount of QD-c in a total 1.2 ml solution. The UV absorption spectrum is from QD-a dispersion. It is evident that the coating with PEG and sdAb markedly enhanced the PL efficiency of the QD-c in water (Figure 2 line c), while it is decreased with EG2 surface modification alone (QD-b, Figure 2 line b). The original CdSe/ZnS QD-a in water have a quantum yield of ca 8% which increased to ca 16% after modification with both PEG and sdAb. No distinct difference is observed in the emission peak position and full width at half maximum (FWHM) of the PL spectra of the QD-a, QD-b, and

[0058] Each of the QD-b and QD-c was purified with 30000 MWCO centrifuge filter device first and then 10000 MWCO. The purified QD-b and QD-c were also used for immunocytochemistry (Example 5). No significant difference was observed in our bio- labeling experiments with the purified and un-purified QD samples.

Example 5 - Immunocytochemistry.

[0059] Breast cancer cell lines SK-BR3 and MDA-MB-468 were obtained from ATCC and cultured in RPMI (Gibco) medium supplemented with 10% fetal bovine serum (FBS, Roche) at 37°C, 5% CO2 in 6 well plate (Falcon) with a sterile cover slip in each well till about 70% confluence. The cells on the cover slip were first fixed in 10% formaldehyde in PBS for 10 minutes. The wells were washed once with PBS and incubated with 2% skimmed milk in PBS for 2 hours to block non-specific binding. Afterwards, the cells were incubated with the QD for one hour and washed three times, each wash taking three minutes using PBS. Competition assay was performed by adding an excess amount of unconjugated sdAb. Counter- staining was performed with DAPI (0.1 μg/ml, Molecular Probes, Eugene) and DiOC5(3) (10 μg/ml, Molecular Probes). Following immunochemical staining the cover slips were mounted using the Prolong Antifade Kit (Molecular Probes) and observed under an Olympus BX51 fluorescent microscope. [0060] Figure 3 shows the images of targeted bio-labeling of breast cancer cells. Cell nuclei were visualized in blue with 4',6-diamidino-2-phenylindole (DAPI) staining and cell surfaces in green with DiOC5(3) labeling. When binding of the bio-conjugated CdSe/ZnS QD shown in Figure 2 takes place on the cell surface, the cells should be lit up in red color. The CdSe/ZnS QD shown in Figure 2 were applied to SK-BR3 (Figure 3, left column) and MDA- MB468 (Figure 3, right column) breast cancer cells that express the EGFR. SK-BR3 cancer cells express a higher level of EGFR than MDA-MB468 cells. Shown in each column of Figure 3, from top to bottom, QD-a, QD-b, QD-c, and QD-a together with un-conjugated EG2 were applied, to evaluate the specific binding of EG2 to the cancer cell as well as to demonstrate the successful bio-conjugation of EG2 to the QD. Figure 3 demonstrates that the QD-a without sdAb are not targeted; QD-b stained SK-BR3 relatively more, compared to staining with MDA-MB468; QD-c stained both SK-BR3 and MDA-MB468 cells with similar signal intensity. QD-a with unconjugated EG2 showed relatively minor staining of the cell surface. A color version of Figure 3 is shown in Zaman et al., J. Phys. Chem. C, vol. 1 13, no. 2, pp. 496-499, 2009., the contents of which are incorporated by reference, where permitted.

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Claims

WHAT IS CLAIMED IS:

1. Water soluble photoluminescent nanoparticlss eomprising surface Uganda comprising surfaee accessible earboxyl groups or amino groups, or both carboxyl and amino groups.

2. The nanoparticles of claim 1, wherein the nanoparticles are CdSe/ZnS nanoparticles.

3. The nanoparticles of claim 1 or 2, wherein the nanoparticles are quantum dots,

4. The nanoparticles of any one of claims 1 to 3, further comprising a hydrophilic ligand that specifically binds to a biomolecule of interest.

5. The nanoparticles of claim 4, wherein the hydrophilic ligand comprises a single-domain antibody.

6. The nanoparticles of claim 5, wherein the single-domain antibody is a camelid antibody.

7. The nanoparticles of claim 5 or 6, further comprising covalently attached polyethylene glycol molecules.

8. The nanoparticles of any one of claims 5 to 7, wherein the single-domain antibody is EG2.

9. The use of the nanoparticles of any one of claims 5 to 8, to selectively label and visualize a tumor cell.

10. The use of claim 9, wherein the tumor cell is a breast cancer cell.