**4. Biocompatibility and functionalization of cadmium-containing quantum dots**

Biocompatibility is a word that is used extensively in biomaterials science. The incorporation of QDs into biological systems often requires strategies for the manipulation of the ligands bound to the surface of the QDs surface in order to make them water-soluble and biocompat‐ ible, compatible with living tissue or a living system by not being toxic, injurious, or physio‐ logically reactive [43]. QDs must be rendered water-soluble through the modification of their surface in preparation for biological applications. An ideal water-soluble ligand should meet the following requirements: (1) provide QDs with stability and solubility in biological buffers; (2) maintain a high resistance to photobleaching and other photophysical properties in aqueous media; (3) have functional groups which are able to conjugate to biomolecules; (4) minimize overall hydrodynamic size. The stability of QDs in water can be obtained through either a complete ligand exchange procedure, or through steric stabilization where the native hydrophobic surface is coated with amphiphilic molecules and/or polymers [44, 45].

to this approach include the availability of antibodies, their selectivity and affinity, and the increased hydrodynamic radius of the quantum dot conjugate. Nonetheless, antibody-QD conjugates are often the method of choice and make up much of the QDs in biology literature.

Pharmacokinetic Properties and Safety of Cadmium-Containing Quantum Dots as Drug Delivery Systems

http://dx.doi.org/10.5772/58553

477

Peptides can also be conjugated to QDs in a direct approach by linking to thiol-rich domains. A direct binding approach was used to bioactivate and solubilize QDs with phytochelatinrelated peptides [55]. Peptide-functionalized quantum dots have been successfully used for targeting cellular proteins such as growth factor receptors, G protein-coupled receptors, integrins, and ion channels [56, 57]. In particular, ~30-50 arginine-glycine-aspartatic acid (RGD) peptides have been conjugated to NIR quantum dots to specifically target αvβ3 integrins in mouse tumor neovasculature *in vivo* [58], while other studies have relied on high-affinity peptide neurotoxin quantum dot nanoconjugates to image endogenous proteins in living cells and *ex vivo* tissue [59]. Overall, peptide-quantum dot nanoconjugates offer distinct advantages over antibody-mediated targeting, and their potential as biological probes is being actively explored. On the other hand, a single QD can be conjugated to multiple protein molecules, which can be similar or different depending on the intended application. Approximately 15-20 maltose binding proteins, a 44-kDa protein measuring 3 × 4 × 6.5 nm, can be attached to a single 6-nm QD [60]. Because of their brightness and photostability, water-stabilized QDs have been used to track many receptor-mediated endocytic trafficking events in live cells using fluores‐ cence microscopy [61]. For example, QDs conjugated to EGF have been used to track the dimerization of the EGF receptor (EGFR) and its ability to elicit downstream signal transduc‐ tion events [62]. Biotinylated α-bungarotoxin was bound to streptavidin-conjugated QDs to characterize the assembly dynamics of acetylcholine receptor clusters in postsynaptic mem‐ branes [63]. Recently, high-resolution imaging methods in the nanometer range have been developed to image the membrane transport and dynamics of tumor cell proteins during metastasis in living mice using antibody-conjugated QDs [64]. This technology can also be applied to detecting cancer cells in sentinel lymph nodes in whole animals using QDs conju‐ gated to tumor-specific molecules [65]. One can envision several ligand-conjugated quantum dots, with each ligand conjugated to a different size (color) quantum dot, allowing a multi‐

Different schemes have been developed to conjugate ssDNA and dsDNA to the surface of QDs. DNA-QD conjugates retain the selectivity of DNA and the photophysical properties of QDs, allowing detection of single or multiple DNA targets. DNA–QD conjugates require solubility in water, stability under physiological conditions and minimal nonspecific DNA binding to the QD surface. The thiol-modified oligonucleotide can be conjugated to QDs in a direct ligand exchange approach (native cap exchange) where the oligonucleotide displaces the surfacebound mercaptopropionic acid and yields aqueous stable and strongly fluorescent oligonu‐ cleotide bound QDs [66]. Applications of QDs include *in vitro* diagnostics, imaging and therapeutics. QDs are used as labels in immunoassays, immunohistochemical staining, cellular

plexed fluorescent assay for drug discovery.

imaging and multiplex diagnostics.

QDs have been adapted to the desired application by conjugation to a recognition moiety, e.g., antibodies, peptides, oligonucleotides or aptamers, or by coating with streptavidin. Several functionalizations have been adapted to the shell layer coating the core, making the core/shell QDs most adaptable for biological applications [46]. In order to suitably functionalize the QDs, there are several methods that have been successfully used for conjugation of QDs to the desired biomolecules. These include electrostatic attraction, covalent linkage, adsorption, and mercapto (-SH) exchange [47]. The choice depends on the features of the biomolecule of interest; for example, thiol-containing biomolecules can be conjugated to QDs via mercapto exchange. In contrast, simple small molecules such as oligonucleotides and various serum albumins were found to readily adsorb non-specifically to the surface of water-soluble QDs. The factors affecting the adsorption are pH, ionic strength, temperature, and surface charge of the molecules [48]. There are three primary ways to target a biocompatible and functional quantum dot: with antibodies, with peptides, or with small molecules. The simplest labeling strategy uses antibodies; the most complicated is that of small molecules, as this approach usually requires more synthetic chemistry. Each approach has its advantages and disadvan‐ tages, and no approach is a universal solution.

Chemical conjugation of antibodies to semiconductor QDs is attractive because the proteins of interest can be visualized. For conjugation of QDs to antibodies, the orientation of the antibody on the QD is important given its functionality as a targeting moiety. The conjugation strategy contributes to the control of antibody orientation. For example, the use of biotinylated antibodies and streptavidin-coated QDs provides no control over the orientation of the antibody on the surface of the QD owing to the presence of multiple biotinylation sites on the antibody. Antibody-quantum dot conjugates have been used in a myriad of applications [49, 50]. An immunoassay for the detection of hepatitis B surface antigen [51] conjugated CdTe/CdS QDs to anti-hepatitis B surface antigen antibodies using protein G as a linking bridge, instead of covalently linking the QDs to the antibodies. Other studies developed a microplate immunoassay for detection of the cardiovascular marker C-reactive protein in 104 serum samples, with a limit of quantification of 0.19 μg/l within 1.5 h [52]. A multiplex immunoassay for the simultaneous detection of staphylococcal enterotoxin B and chicken IgY (IgG) in the same well of a 96-well microtiter plate was also undertaken [53]. A multiplex fluoroimmunoassay for the detection of lung cancer markers—neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) in human serum—was recently developed [54]. A wide selection of antibody-quantum dot conjugates is also commercially available. Disadvantages to this approach include the availability of antibodies, their selectivity and affinity, and the increased hydrodynamic radius of the quantum dot conjugate. Nonetheless, antibody-QD conjugates are often the method of choice and make up much of the QDs in biology literature.

ible, compatible with living tissue or a living system by not being toxic, injurious, or physio‐ logically reactive [43]. QDs must be rendered water-soluble through the modification of their surface in preparation for biological applications. An ideal water-soluble ligand should meet the following requirements: (1) provide QDs with stability and solubility in biological buffers; (2) maintain a high resistance to photobleaching and other photophysical properties in aqueous media; (3) have functional groups which are able to conjugate to biomolecules; (4) minimize overall hydrodynamic size. The stability of QDs in water can be obtained through either a complete ligand exchange procedure, or through steric stabilization where the native

hydrophobic surface is coated with amphiphilic molecules and/or polymers [44, 45].

tages, and no approach is a universal solution.

476 Application of Nanotechnology in Drug Delivery

QDs have been adapted to the desired application by conjugation to a recognition moiety, e.g., antibodies, peptides, oligonucleotides or aptamers, or by coating with streptavidin. Several functionalizations have been adapted to the shell layer coating the core, making the core/shell QDs most adaptable for biological applications [46]. In order to suitably functionalize the QDs, there are several methods that have been successfully used for conjugation of QDs to the desired biomolecules. These include electrostatic attraction, covalent linkage, adsorption, and mercapto (-SH) exchange [47]. The choice depends on the features of the biomolecule of interest; for example, thiol-containing biomolecules can be conjugated to QDs via mercapto exchange. In contrast, simple small molecules such as oligonucleotides and various serum albumins were found to readily adsorb non-specifically to the surface of water-soluble QDs. The factors affecting the adsorption are pH, ionic strength, temperature, and surface charge of the molecules [48]. There are three primary ways to target a biocompatible and functional quantum dot: with antibodies, with peptides, or with small molecules. The simplest labeling strategy uses antibodies; the most complicated is that of small molecules, as this approach usually requires more synthetic chemistry. Each approach has its advantages and disadvan‐

Chemical conjugation of antibodies to semiconductor QDs is attractive because the proteins of interest can be visualized. For conjugation of QDs to antibodies, the orientation of the antibody on the QD is important given its functionality as a targeting moiety. The conjugation strategy contributes to the control of antibody orientation. For example, the use of biotinylated antibodies and streptavidin-coated QDs provides no control over the orientation of the antibody on the surface of the QD owing to the presence of multiple biotinylation sites on the antibody. Antibody-quantum dot conjugates have been used in a myriad of applications [49, 50]. An immunoassay for the detection of hepatitis B surface antigen [51] conjugated CdTe/CdS QDs to anti-hepatitis B surface antigen antibodies using protein G as a linking bridge, instead of covalently linking the QDs to the antibodies. Other studies developed a microplate immunoassay for detection of the cardiovascular marker C-reactive protein in 104 serum samples, with a limit of quantification of 0.19 μg/l within 1.5 h [52]. A multiplex immunoassay for the simultaneous detection of staphylococcal enterotoxin B and chicken IgY (IgG) in the same well of a 96-well microtiter plate was also undertaken [53]. A multiplex fluoroimmunoassay for the detection of lung cancer markers—neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) in human serum—was recently developed [54]. A wide selection of antibody-quantum dot conjugates is also commercially available. Disadvantages

Peptides can also be conjugated to QDs in a direct approach by linking to thiol-rich domains. A direct binding approach was used to bioactivate and solubilize QDs with phytochelatinrelated peptides [55]. Peptide-functionalized quantum dots have been successfully used for targeting cellular proteins such as growth factor receptors, G protein-coupled receptors, integrins, and ion channels [56, 57]. In particular, ~30-50 arginine-glycine-aspartatic acid (RGD) peptides have been conjugated to NIR quantum dots to specifically target αvβ3 integrins in mouse tumor neovasculature *in vivo* [58], while other studies have relied on high-affinity peptide neurotoxin quantum dot nanoconjugates to image endogenous proteins in living cells and *ex vivo* tissue [59]. Overall, peptide-quantum dot nanoconjugates offer distinct advantages over antibody-mediated targeting, and their potential as biological probes is being actively explored. On the other hand, a single QD can be conjugated to multiple protein molecules, which can be similar or different depending on the intended application. Approximately 15-20 maltose binding proteins, a 44-kDa protein measuring 3 × 4 × 6.5 nm, can be attached to a single 6-nm QD [60]. Because of their brightness and photostability, water-stabilized QDs have been used to track many receptor-mediated endocytic trafficking events in live cells using fluores‐ cence microscopy [61]. For example, QDs conjugated to EGF have been used to track the dimerization of the EGF receptor (EGFR) and its ability to elicit downstream signal transduc‐ tion events [62]. Biotinylated α-bungarotoxin was bound to streptavidin-conjugated QDs to characterize the assembly dynamics of acetylcholine receptor clusters in postsynaptic mem‐ branes [63]. Recently, high-resolution imaging methods in the nanometer range have been developed to image the membrane transport and dynamics of tumor cell proteins during metastasis in living mice using antibody-conjugated QDs [64]. This technology can also be applied to detecting cancer cells in sentinel lymph nodes in whole animals using QDs conju‐ gated to tumor-specific molecules [65]. One can envision several ligand-conjugated quantum dots, with each ligand conjugated to a different size (color) quantum dot, allowing a multi‐ plexed fluorescent assay for drug discovery.

Different schemes have been developed to conjugate ssDNA and dsDNA to the surface of QDs. DNA-QD conjugates retain the selectivity of DNA and the photophysical properties of QDs, allowing detection of single or multiple DNA targets. DNA–QD conjugates require solubility in water, stability under physiological conditions and minimal nonspecific DNA binding to the QD surface. The thiol-modified oligonucleotide can be conjugated to QDs in a direct ligand exchange approach (native cap exchange) where the oligonucleotide displaces the surfacebound mercaptopropionic acid and yields aqueous stable and strongly fluorescent oligonu‐ cleotide bound QDs [66]. Applications of QDs include *in vitro* diagnostics, imaging and therapeutics. QDs are used as labels in immunoassays, immunohistochemical staining, cellular imaging and multiplex diagnostics.
