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...

The development of a reversible chemical sensor based on a CdSe/ZnS nanocrystal (NC) is described. Signal transduction is accomplished by fluorescence resonance energy transfer (FRET) between the NC and a fluorescent pH-sensitive squaraine dye attached to the surface of the NC. The efficiency of FRET, and consequently the relative intensity of NC and dye emissions, is modulated with the pH-dependent absorption cross section of the squaraine dye. The design of a NC sensor based on FRET results in a ratiometric sensor since the emission intensities of dye and NC may be referenced to the isosbestic point between NC and dye emissions. The ratiometric approach allows sensing to be performed, regardless of issues surrounding collection efficiency (scattering environment, light fluctuations, etc.) and dye:NC loadings.

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...

Oncolytic viral therapy provides a promising approach to treat certain human malignancies. These vectors improve on replication-deficient vectors by increasing the viral load within tumors through preferential viral replication within tumor cells. However, the inability to efficiently propagate throughout the entire tumor and infect cells distant from the injection site has limited the capacity of oncolytic viruses to achieve consistent therapeutic responses. Here we show that the spread of the oncolytic herpes simplex virus (HSV) vector MGH2 within the human melanoma Mu89 is limited by the fibrillar collagen in the extracellular matrix. This limitation seems to be size specific as nanoparticles of equivalent size to the virus distribute within tumors to the same extent whereas smaller particles distribute more widely. Due to limited viral penetration, tumor cells in inaccessible regions continue to grow, remaining out of the range of viral infection, and tumor eradication cannot be achieved. Matrix modification with bacterial collagenase coinjection results in a significant improvement in the initial range of viral distribution within the tumor. This results in an extended range of infected tumor cells and improved virus propagation, ultimately leading to enhanced therapeutic outcome. Thus, fibrillar collagen can be a formidable barrier to viral distribution and matrixmodifying treatments can significantly enhance the therapeutic response. (Cancer Res 2006; 66(5): 2509-13)

We have developed a size series of unusually small, water-soluble (InAs)ZnSe (core)shell quantum dots (QDs) that emit in the near infrared and exhibit new behavior in vivo, including multiple sequential lymph node mapping and extravasation from the vasculature. The biological utility of these fluorescent probes resulted from our intentional choice to match the semiconductor material and water soluble ligand with a desired final hydrodynamic diameter and emission wavelength.Semiconductor nanocrystals (or quantum dots, QDs) are excellent fluorophores due to their continuous absorption profiles at wavelengths to the blue of the band edge, high photostability, and narrow, tunable emission peaks. For in vivo biological imaging applications, the QD emission wavelength should ideally be in a region of the spectrum where blood and tissue absorb minimally but detectors are still efficient, approximately 700-900 nm in the near infrared (NIR). 1 In addition, the hydrodynamic size of the QD should be appropriately matched to the biological experiment of interest. 2 In previous work, for example, we described the efficacy of Type II QDs with hydrodynamic diameters (HD) of 15.8-18.8 nm to map sentinel lymph nodes selectively. 2a Here we report the synthesis of a size series of (InAs)ZnSe (core) shell QDs that emit in the near infrared and exhibit HD < 10 nm. We demonstrate their utility E-mail: mgb@mit.ed. Supporting Information Available: Experimental procedures, transmission electron microscopy data, and additional optical characterization, including emission stability in serum. in vivo by imaging multiple, sequential lymph nodes and showing extravasation from the vasculature in rat models, neither of which has been achieved before with QDs to our knowledge. NIH Public AccessWhile InAs QDs are known, most studies report emission wavelengths longer than 800 nm. 3 Until now, only Battaglia and Peng have shown well defined InAs first absorptioin peaks at wavelengths below 800 nm. 4 Their work, however, primarily concerned InP QDs. We have developed a procedure for the synthesis of a well-characterized size series of small InAs cores (diameters < 2 nm). Moreover we have extended the work to show the overcoating of these very small cores with a second, higher bandgap semiconductor shell. Zinc selenide was chosen as the ideal shell material due to its reasonably small lattice mismatch with zinc blende InAs (6.44%), its high bulk band offsets (1.26 and 0.99 eV for the conduction and valence bands, CB and VB, respectively), and its reported ability to increase the quantum yield (QY) of InAs cores by more than an order of magnitude. 3e Longer emission wavelengths, particularly the biologically desirable 800-840 nm range, can be achieved by (1) increasing the core size or (2) the shell thickness, or (3) by altering the band offsets between core and shell such as by adding a small amount of Cd to the ZnSe shell. Therefore, by varying the core size, and the shell thickness or composition, a wide tunability of the final emissi...

The ability to synthesize semiconductor nanocrystals with narrow size distributions and high luminescent efficiencies has made quantum dots an attractive alternative to organic molecules in applications such as optoelectronic devices [1,2] and biological fluorescence labeling. [3][4][5] Not only are quantum dots (QDs) more stable to photooxidation relative to organic molecules, but their fluorescence is also more saturated (narrow emission bandwidths). Their size-tunable optical properties, which are independent of their chemical

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrowderived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.Intravital microscopy has provided unprecedented molecular, cellular, anatomical and functional insight into tumor biology and response to treatment 1 . This technique captures fluorescence from molecules that are injected into a host or expressed by cells 2,3 . Additionally, intrinsic signals such as second harmonic generation (SHG) emanating from collagen can be imaged using multiphoton microscopy 4,5 . Traditional fluorophores are prone to photobleaching, compromising the ability to image the same region repeatedly, and have relatively narrow excitation and broad emission spectra. Also, several excitation wavelengths may be required to excite all fluorophores and intrinsic signals, and overlapping emissions may obscure the delineation between multiple probes. Quantum dots, colloidal semiconductor nanocrystals 6 , have the potential to overcome these limitations: they are photostable, tunable to a desired narrow emission spectrum, relatively insensitive to the wavelength of excitation Correspondence should be addressed to R.K.J. (E-mail: jain@steele.mgh.harvard.edu). Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. light, and are especially bright fluorophores 7 . Recent studies exploit these optical properties for imaging of cells 8 or whole tumors 9 . The ability of quantum dots to show crucial information at the length scale between these two extremes has yet to be established 10 . Here, we present studies that highlight the synergy of quantum dots and multiphoton intravital microscopy for tumor pathophysiology studies: differentiating tumor vessels from both perivascular cells and matrix, assaying the ability of microparticles to access the tumor, and monitoring the trafficking of precursor cells. NIH Public Access RESULTS Customizing quantum dot emissionBecause quantum dot emissions are tunable by both size and chemical composition 6 , we prepared ...

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