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Traceable Drug Delivery: Lighting the Way with Qdots Xiaohu Gao University of Washington Seattle, Washington Semiconductor nanocrystals, also known as quantum dots (Qdots), have become an indispensable tool in biomedical research, especially for multiplexed, quantitative, and long-term fluorescence imaging and detection (Alivisatos, 2004; Medintz et al., 2005; Michalet et al., 2005; Smith et al., 2006). The basic rationale for using Qdots is their unique optical properties are generally not available in individual molecules or bulk semiconductor solids. In comparison with conven- tional organic dyes and fluorescent proteins, Qdots have distinctive characteristics, such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescent colors. Recent advances in nanoparticle-surface chemistry have led to the develop- ment of polymer-encapsulated probes that are highly fluorescent and stable under complex biological conditions (Dubertret et al., 2002; Gao et al., 2004; Wu et al., 2003). This new generation of water-soluble Qdots has solved the problems of quantum yield decrease, chemical sensitivity, and short shelf-life, which were previously encountered in the ligand exchange-based Qdot solubilization method (Chan and Nie, 1998). As a result, Qdots linked with bioaffinity molecules have created new opportunities for multicolor molecular imaging in living cells and animal models, as well as for traceable drug delivery (Dahan et al., 2003; 33

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34 FRONTIERS OF ENGINEERING Dubertret et al., 2002; Gao et al., 2004; Larson et al., 2003; Lidke et al., 2004; Wu et al., 2003). TRACEABLE DRUG DELIVERY Traceable drug delivery has the potential to elucidate the pharmacokinet- ics and pharmacodynamics of drug candidates and to provide design principles for engineering drug carriers. Due to concerns about long-term in vivo toxicity and degradation, however, Qdots are currently limited to use in cells and small animals. However, because both cells and small animals are used extensively in testing drug candidates, even in these limited studies, traceable therapeutics have had a significant impact on life-science research, such as in drug discovery, validation, and delivery. Following drug molecules or drug carriers noninvasively in real time in live organisms requires specialized imaging techniques. Compared with traditional imaging modalities, such as magnetic resonance imaging (MRI) and positron emission tomography (PET), optical imaging is highly sensitive, quantitative, and capable of multiplexing. In addition, it is significantly cheaper than MRI and PET; thus it will substantially reduce the cost and shorten the time of new drug develop- ment. Therefore, for the development and optimization of nanocarriers, Qdots can be an excellent “prototype” from which biocompatible carriers of similar sizes and with similar surface properties can be made for clinical uses. The importance of the structural properties of Qdots for drug-delivery research has only recently been realized. First, the size of Qdots can be continu- ously tuned from 2–10 nm in diameter, which, after polymer encapsulation, gener- ally increases to 5–20 nm. Particles smaller than 5 nm are quickly cleared by renal filtration (Choi et al., 2007), whereas bigger particles are more likely to be taken up by the reticuloendothelial system before reaching the targeted disease sites. In addition, larger particles have limited penetration into solid tissues. Recent advances in Qdot nanocrystal synthesis will enable scientists to systematically assess the effects of size on delivery efficiency and specificity and identify the optimal dimensions of drug carriers. Second, because of the high surface-to-volume ratio of nanomaterials, it is possible to link multiple functionalities on single Qdots while keeping the overall size in the optimal range. For example, the Qdot core can serve as the structural scaffold and the imaging contrast agent; and small-molecule hydrophobic drugs can be embedded between the inorganic core and the amphiphilic polymer coat- ing. Hydrophilic therapeutic agents (e.g., small interfering RNA [siRNA] and antisense oligodeoxynucleotide [ODN]) and targeting biomolecules (e.g., anti- bodies, peptides, and aptamers), in turn, can be immobilized onto the hydrophilic side of the amphiphilic polymer via either covalent or noncovalent bonds. The fully integrated nanostructure may behave like a magic bullet that not only can

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TRACEABLE DRUG DELIVERY 35 identify, bind to, and treat diseased cells, but can also emit detectable signals for real-time monitoring of its trajectory. DELIVERY OF siRNA USING QDOTS RNA interference (RNAi), which is emerging as one of the most powerful technologies for sequence-specific suppression of genes, has potential applications ranging from functional gene analysis to therapeutics. Because of the relatively low immunogenic and oncologic effects of RNAi, the development of nonviral delivery methods in vitro and in organisms is generating considerable interest. In recent years, a number of strategies have been developed based on liposomes, gold and silica nanoparticles, cationic and biodegradable polymers, and peptides (Bielinska et al., 1999; Boussif et al., 1995; Chesnoy and Huang, 2000; Kneuer et al., 2000; Niidome et al., 2004; Roy et al., 2005; Rudolph et al., 2003; Sandhu et al., 2002; Takeshita et al., 2005; Tang et al., 1996; Zanta et al., 1999). The delivery efficiency, however, remains low, especially under in vivo conditions. Another limitation of existing delivery technologies is the lack of an intrinsic signal for long-term, real-time imaging of siRNA transport and release. We recently developed a new technology by combining Qdots with amphi- pol, another class of nanomaterial, for traceable and efficient delivery of siRNA molecules. Amphipols, linear polymers with alternating hydrophilic and hydro- phobic side chains, are widely used for solubilizing integral membrane proteins and delivering them into cell lipid bilayers (Gorzelle et al., 2002; Nagy et al., 2001; Pocanschi et al., 2006; Tribet et al., 1996, 1997). Unlike detergent-based micelles, amphipols belt around the transmembrane domain of membrane proteins and do not disrupt the integrity of cell membranes during delivery. To our surprise, however, when amphipols are mixed with nanoparticles coated with hydrophobic surface ligands, these two types of nanomaterials form stable complexes that are not only capable of carrying siRNA molecules into cytoplasm but can also protect them from enzymatic degradation. Compared with classic siRNA carriers, such as Lipofectamine, this new class of nanocarrier works in both serum-free and complete cell-culture media. The new nanocarrier also outperforms polyethyleneimine in gene silencing under both conditions with significantly reduced toxicity. In addition, Qdots provide a bright, stable fluorescent signal for intracellular siRNA imaging (Figure 1). CONCLUSION AND PERSPECTIVE As a powerful imaging probe, Qdots already play an important role in fun- damental biology, as well as in in vitro disease diagnostics and prognostics. The unique structural and surface properties of Qdots, such as tunable and uniform size, flexible drug-linking and doping mechanism, large surface-to-volume ratio, and a wide spectrum of surface-reactive groups, have recently opened a new

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scaled for portrait above landscape below 36 FIGURE 1  Time-dependent fluorescence imaging of the entry and transport of Qdot-siRNA nanoparticles in living cells. Images were obtained 15 minutes to 24 hours after the addition of Qdot-siRNA. Top panels are fluorescence images, and bottom panels are the corresponding bright- field images. (Figure can be viewed in color at

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TRACEABLE DRUG DELIVERY 37 avenue of research—targeted and traceable drug delivery. However, high-quality Qdots (visible and near infrared dots with a narrow emission profile and high quantum yield) are mainly made with heavy metals whose long-term toxicity is largely unknown. Despite this limitation, they have been used as drug carriers in cells and small animals and have proven to be an outstanding discovery tool for drug screening and validation, as well as a prototype material for drug-carrier engineering. If high-quality Qdots could be prepared from relatively nontoxic compounds (e.g., silicon and carbon), or if the toxic components could be inertly protected from exposure and subsequently cleared from the body, Qdots could become clinically relevant. Another primary challenge of drug delivery is maintaining a useful concen- tration of the drug in the targeted tissue while preventing toxicity. Achieving this therapeutic window has not been studied with Qdots thus far, but, ideally, engi- neered Qdots should be able to stabilize therapeutic compounds, increase their plasma-circulation time while reducing the concentration of free drug to minimize unwanted side effects, and release the drug with a well-controlled profile. In addi- tion, the targeting and therapeutic compounds might be covalently linked to the Qdot surface via cleavable chemical bonds so that the bioconjugates are initially large enough to avoid renal filtration, and later, after the ligands have been cleaved, small enough to be cleared out of the body. ACKNOWLEDGMENTS This work was supported by grants from NIH, NSF, DOD, and the University of Washington. The author thanks NSF for a Faculty Early Career Development Award (CAREER). REFERENCES Alivisatos, P. 2004. The use of nanocrystals in biological detection. Nature Biotechnology 22(1): 47–52. Bielinska, A.U., C. Chen, J. Johnson, and J.R. Baker, Jr. 1999. DNA complexing with polyamidoamine dendrimers: implications for transfection. Bioconjugate Chemistry 10(5): 843–850. Boussif, O., F. Lezoualc’h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, and J.P.A Behr. 1995. Versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America 92(16): 7297–7301. Chan, W.C.W., and S. Nie. 1998. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281(5385): 2016–2018. Chesnoy, S., and L. Huang. 2000. Structure and function of lipid-DNA complexes for gene delivery. Annual Review of Biophysics and Biomolecular Structure 29: 27–47. Choi, H.S., W. Liu, P. Misra, E. Tanaka, J.P. Zimmer, B. Itty Ipe, M.G. Bawendi, and J.V. Frangioni. 2007. Renal clearance of quantum dots. Nature Biotechnology 25(10): 1165–1170. Dahan, M., S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller. 2003. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302(5644): 442–445.

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