SUMMARYThe field of nanotechnology holds great promise for the diagnosis and treatment of human disease. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or their biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. Using quantum dots (QDs) as a model system, we have precisely defined the requirements for renal filtration and urinary excretion of inorganic, metal-containing nanoparticles. Zwitterionic or neutral organic coatings prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter (HD) by over 15 nm and prevented renal excretion. A final HD smaller than 5.5 nm resulted in rapid and efficient urinary excretion, and elimination of QDs from the body. This study provides a foundation for the design and development of biologically targeted nanoparticles for biomedical applications. KeywordsNanotechnology; Quantum Dots; Biodistribution; Clearance; Fluorescence Imaging Although targeted nanoparticles hold promise for the detection and treatment of human disease, toxicity -either potential or real -remains the major roadblock to clinical translation. 1 Historically, the U.S. Food and Drug Administration (FDA) has required that agents injected into the human body, especially diagnostic agents, be cleared completely, in a reasonable amount of time. This policy makes sense in that total body clearance minimizes the area under the exposure curve. It also minimizes the chance that the agent will interfere with other diagnostic tests. For example, gold, used extensively in the nanotechnology literature, has a linear attenuation coefficient 150-fold higher than even bone, and at doses injected intravenously would likely preclude accurate computed tomographic (CT) scanning, especially in organs such as the liver, where it eventually accumulates. Against this backdrop is the inherent stability of most nanoparticles. Indeed, a recent study suggests that quantum dots (QDs) with the appropriate organic coating are retained in the body for at least two years and remain fluorescent. 2 When considering that many nanometer-sized objects proposed for clinical use contain heavy metals, regulatory approval of such stable particles is unlikely, and the type of long-term toxicity studies that would be required for such approval will continue to discourage clinical translation.A potential solution to this conundrum is to focus on the physiology underlying biodistribution and clearance of agents injected intravenously into the body. For globular proteins, a hydrodynamic diameter (HD) of approximately 5-6 nm is associated with the ability to be cleared rapidly from the body via renal filtration and urinary excretion (Table 1). Nanoparticle toxicity would be minimized, if not eliminated, if there were a way to clear them from the body. However, currently it is unknown what the renal filtration threshold is for metal-based nanometer-sized objec...

DIAGNOSTIC AGENTS-Nanoparticles functionalized with ligands that target tumors can be cleared from the body through the kidneys if they have a hydrodynamic diameter of less than 5.5 nm. KeywordsNanotechnology; Quantum Dots; Nanoparticles; Diagnostic Imaging; Image-Guided Surgery; Biodistribution; Clearance; Fluorescence Imaging; Tumor Targeting INTRODUCTORY PARAGRAPHInorganic/organic hybrid nanoparticles are potentially useful in biomedicine but to avoid nonspecific background fluorescence and long-term toxicity, they need to be cleared from the body within a reasonable time scale. 1 Previously, we showed that rigid spherical nanoparticles such as quantum dots can be cleared by the kidneys if they had a hydrodynamic diameter less than 5.5 nm and a zwitterionic surface charge. 2 Here we show that quantum dots functionalized with high-affinity small molecule ligands that target tumors can also be cleared by the kidneys if their hydrodynamic diameter is less than this value, which sets an upper limit of 5-10 ligands per quantum dot for renal clearance. Animal models of prostate cancer and melanoma show receptor-specific imaging and renal clearance within 4 h post-injection. This study suggests a set of design rules for the clinical translation of targeted nanoparticles that can be eliminated through the kidneys.Although many classes of biocompatible, inorganic-based nanomaterials have been developed for medical diagnostics and therapeutics, 3-7 many presently available formulations require potentially toxic elements. 8 Efforts have been made to reduce toxicity by modulating the composition, particle shape, physical size, and surface coating of the nanoparticles. 9 One common strategy is to engineer nanoparticles using biocompatible and biodegradable polymeric coatings. 10-13 However, polymer coatings generally increase particle size over the * Corresponding Author: John V. Frangioni, M.D., Ph.D., Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Room SL-B05, Boston, MA 02215, Phone: 617-667-0692 Fax: 617-667-0981, jfrangio@bidmc.harvard.edu Author contributions H.S.C., W.L., F.L., K.N., and P.M. performed the experiments. H.S.C., M.G.B., and J.V.F. reviewed, analyzed, and interpreted the data. H.S.C., M.G.B., and J.V.F. wrote the paper. All authors discussed the results and commented on the manuscript. Competing financial interestsThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Nanotechnol. Author manuscript; available in PMC 2011 January 1. Published in final edited form as:Nat Nanotechnol. [20][21][22][23] However, most of these papers fail to consider the autofluorescence of living tissue, the passive targeting caused by leaky tumor vasculature, the use of a receptor-negative tumor as a control, and the high background accumulation of nanoparticles in the RES. Indeed, most tumor targeting of nanoparticles in animals described to date is likely the result of enhanced permeability and retention, 24 and not specific targeting.To render nanoparticles vi...

The signal-to-background ratio (SBR) is the key determinant of sensitivity, detectability, and linearity in optical imaging. As signal strength is often constrained by fundamental limits, background reduction becomes an important approach for improving SBR. We recently reported that a zwitterionic near-infrared (NIR) fluorophore, ZW800-1, exhibits low background. Here we show that this fluorophore provides much-improved SBR when targeted to cancer cells or proteins by conjugation with a cyclic RGD peptide, fibrinogen, or antibodies. ZW800-1 outperforms the commercially available NIR fluorophores IRDye800-CW and Cy5.5 in vitro for immunocytometry, histopathology and immunoblotting, and in vivo for image-guided surgery. In tumor model systems, tumor-to-background ratios of 17.2 are achieved after only 4 h post-injection, compared with 5.1 for IRDye800-CW and 2.7 for Cy5.5. Our results suggest that introducing zwitterionic properties into targeted fluorophores may be a general strategy for improving the SBR in diagnostic and therapeutic applications.

The field of biomedical optics has matured rapidly over the last decade and is poised to make a significant impact on patient care. In particular, wide-field (typically > 5 cm), planar, near-infrared (NIR) fluorescence imaging has the potential to revolutionize human surgery by providing real-time image guidance to surgeons for tissue that needs to be resected, such as tumors, and tissue that needs to be avoided, such as blood vessels and nerves. However, to become a clinical reality, optimized imaging systems and NIR fluorescent contrast agents will be needed. In this review, we introduce the principles of NIR fluorescence imaging, analyze existing NIR fluorescence imaging systems, and discuss the key parameters that guide contrast agent development. We also introduce the complexities surrounding clinical translation using our experience with the Fluorescence-Assisted Resection and Exploration (FLARE™) imaging system as an example. Finally, we introduce state-of-the-art optical imaging techniques that might someday improve image-guided surgery even further.

SUMMARYNanoparticles (NPs) have the potential to revolutionize drug delivery, however, administering them to the human body without the need for intravenous injection remains a major challenge. In this study, a series of near-infrared (NIR) fluorescent NPs were systematically varied in chemical composition, shape, size, and surface charge, and their biodistribution and elimination were quantified in rat models after lung instillation. We demonstrate that NPs with hydrodynamic diameter (HD) less than ≈ 34 nm and a non-cationic surface charge translocate rapidly from lung to mediastinal lymph nodes. NPs of HD < 6 nm can traffic rapidly from the lungs to lymph nodes and the bloodstream, and then be subsequently cleared by the kidneys. We discuss the importance of these findings to drug delivery, air pollution, and carcinogenesis. KeywordsNanoparticles; nanomedicine; drug delivery; air pollution; lymph node uptake; biodistribution; renal clearance * Co-Senior Authors: Beth Israel Deaconess Medical Center 330 Brookline Avenue, Room SL-B05 Boston, MA 02215 Phone: 617-667-0692 Fax: 617-667-0981 jfrangio@bidmc.harvard.edu Harvard School of Public Health 665 Huntington Avenue Boston, MA 02115 Phone: 617-432-0127 Fax: 617-432-4710 atsuda@hsph.harvard.edu . AUTHOR CONTRIBUTIONS H.S.C., Y.A., J.H.L., S.H.K., A.M., N.I., and A.T. performed the experiments. H.S.C., M.G.B., M.S.B., A.T., and J.V.F. reviewed, analyzed, and interpreted the data. H.S.C., A.T., and J.V.F. wrote the paper. All authors discussed the results and commented on the manuscript. Nanoparticles (NPs) have been proposed as diagnostic, therapeutic, and theragnostic agents for a wide variety of human diseases. 1-3 Lung-based drug delivery of NPs is receiving increased attention due to the large surface area available and the minimal anatomical barriers limiting access to the body. 4 In this study, we explore whether it would be possible to administer NPs via the lung, and in so doing, attempt to define the key parameters that mediate lung to body NP translocation and subsequent elimination (i.e., clearance). COMPETING INTERESTS STATEMENTLung-administered NPs also have significant implications for air pollution. Recent toxicological studies have confirmed that nano-sized or ultrafine particles reach deep into the alveolar region of the lungs 5,6 and cause severe inflammation reactions due to their large surface areas per mass. 6 Inhalation of NPs is increasingly recognized as a major cause of adverse health effects, and has especially strong influence on the cardiovascular system and hemostasis, leading to increased cardiovascular morbidity and mortality. [6][7][8] The standard approach for studying the translocation of inhaled NPs and ultrafine air pollutants from the lungs to extrapulmonary compartments in animals is to perform postmortem analysis of tissues after inhalation of carbon-based particles, 9 radiotracers, 10 or neutron-activated metal particles. 11-13 Recently, Moller et al. reported that ultrafine NPs could pass from the lungs into bloodstream an...

Semiconductor nanocrystals (quantum dots, QDs) are usually described as fluorophores having remarkable photostability, large absorption cross sections, and tunable emission peaks. Equally important, QDs also serve as versatile nanoscale objects of precisely tunable size and morphology, having exceptionally narrow size distributions. 1, 2 Size is an especially important parameter in the design of nanomaterials for applications in biology, for both in vitro and in vivo applications. In cell labeling applications, for example, size can affect endocytosis or limit access to receptors of interest, such as those in the neuronal synapse. 3 In live animals, particle size can dramatically affect biodistribution and pharmacokinetics. 4, 5 The optimal size depends on the application. For example, we have previously shown that Type II CdTe(CdSe) core (shell) QDs with a hydrodynamic diameter (HD) of ∼19 nm can be used to selectively map sentinel lymph nodes. 6 More recently, we have also shown that 8.7 nm HD Type I InAs(ZnSe) QDs allowed the mapping of multiple lymph nodes and also showed the potential extravasation of QDs from the vasculature. 7 There has been a strong interest in reducing the HD of QDs for in vivo applications as this could increase bioavailability and lead to an improved understanding of clearance mechanisms. In this communication, we demonstrate bio-compatible fluorescent QDs with an exceptionally small HD of ∼6 nm using a CdSe(ZnCdS) core(shell) structure coated with DL-cysteine and show renal clearance of these QDs in rat models.Cysteine-coated nanocrystals have been demonstrated previously with CdS QDs synthesized directly from aqueous solution in the presence of L-cysteine hydrochloride. 8 However, these QDs suffered from low QY (6−8%) and broad fluorescence (FWHM >100 nm) due to emission from surface trap sites. We report the synthesis of high-quality cysteine-coated CdSe(ZnCdS) core(shell) QDs using well-developed nanocrystal synthetic procedures, in which cores are formed through the rapid injection of metal and chalcogenide precursors into hot solvent and then overcoated with a thin shell of a higher band gap material. Following overcoating, the native ligands are exchanged with DL-cysteine to yield water dispersible QDs (QD-Cys).Ligand exchange with cysteine was achieved using a biphasic exchange method in which QDs dispersed in chloroform were mixed with a solution of DL-cysteine in phosphate buffered saline (PBS). This biphasic mixture was stirred vigorously at room temperature, and phase transfer of QDs from the organic to the aqueous phase occurred over ∼2 h, leaving a colorless chloroform layer ( Figure 1a). The QDs were precipitated twice with ethanol and re-dispersed in PBS at pH 7.4 for analysis. QD-Cys as synthesized formed macroscopic aggregates upon standing at room temperature overnight. Storing the samples in the dark at 4 °C only extended jfrangio@bidmc.harvard.edu, mgb@mit.edu. Supporting Information Available: Experimental procedures, transmission electron microscopy data, and ad...

To address two fundamental and unsolved problems in optical imaging (nonspecific uptake of near‐infrared fluorophores by normal tissues and organs and incomplete elimination of unbound targeted fluorophores from the body), novel zwitterionic near‐infrared fluorophores (e.g., ZW800‐1) were synthesized and their performance compared in vivo to conventional molecules (e.g., ICG) as a function of charge, charge distribution, and hydrophobicity (see picture).

Because of their large size compared to small molecules and their multifunctionality, nanoparticles (NPs) hold promise as biomedical imaging, diagnostic, and theragnostic agents. However, the key to their success hinges on a detailed understanding of their behavior after administration into the body. NP biodistribution, target binding, and clearance are complex functions of their physicochemical properties in serum, which include hydrodynamic diameter, solubility, stability, shape and flexibility, surface charge, composition, and formulation. Moreover, many materials used to construct NPs have real or potential toxicity or may interfere with other medical tests. In this review, we discuss the design considerations that mediate NP behavior in the body and the fundamental principles that govern clinical translation. By analyzing those nanomaterials that have already received regulatory approval, most of which are actually therapeutic agents, we attempt to predict which types of NPs hold potential as diagnostic agents for biomedical imaging. Finally, using quantum dots as an example, we provide a framework for deciding whether an NP-based agent is the best choice for a particular clinical application.