Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo.

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 marrow-derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.

Renal clearance of quantum dots.

The 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 intravenously administered quantum dots in rodents 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 by >15 nm and prevented renal excretion. A final hydrodynamic diameter <5.5 nm resulted in rapid and efficient urinary excretion and elimination of quantum dots from the body. This study provides a foundation for the design and development of biologically targeted nanoparticles for biomedical applications.

Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector.

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 matrix-modifying treatments can significantly enhance the therapeutic response.

Compact cysteine-coated CdSe(ZnCdS) quantum dots for in vivo applications.

We have developed a versatile nanoparticle construct using a compact cysteine coating on a CdSe(ZnCdS) core(shell) nanocrystal (QD-Cys) that is biologically compatible, exceptionally compact, highly fluorescent, and easily functionalized. The small hydrodynamic diameter of QD-Cys ( approximately 6 nm) allows for renal clearance of these nanoparticles in rat models. Moreover, the ability to directly conjugate to QD-Cys opens up the possibility of functionalized nanocrystals for in vivo targeted imaging, in which small targeting molecules can be appended to QD-Cys, and unbound QDs can be rapidly cleared to achieve high signal/noise ratios and to reduce background toxicity.

Incorporation of iron oxide nanoparticles and quantum dots into silica microspheres.

We describe the synthesis of magnetic and fluorescent silica microspheres fabricated by incorporating maghemite (gamma-Fe2O3) nanoparticles (MPs) and CdSe/CdZnS core/shell quantum dots (QDs) into a silica shell around preformed silica microspheres. The resultant approximately 500 nm microspheres have a narrow size distribution and show uniform incorporation of QDs and MPs into the shell. We have demonstrated manipulation of these microspheres using an external magnetic field with real-time fluorescence microscopy imaging.

Synthesis of CdSe/CdTe nanobarbells.

Synthesis of monodisperse samples of CdSe nanorods with CdTe tips is achieved using the mechanism of rod nucleated growth to form CdSe/CdTe nanobarbells. This synthesis produces a nanocrystal displaying "type-II" behavior with a morphology that is particularly well suited for internal exciton separation and carrier transport.

Size series of small indium arsenide-zinc selenide core-shell nanocrystals and their application to in vivo imaging.

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.

A ratiometric CdSe/ZnS nanocrystal pH sensor.

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.

Engineering InAs(x)P(1-x)/InP/ZnSe III-V alloyed core/shell quantum dots for the near-infrared.

Quantum dots with a core/shell/shell structure consisting of an alloyed core of InAs(x)P(1-x), an intermediate shell of InP, and an outer shell of ZnSe were developed. The InAs(x)P(1-x) alloyed core has a graded internal composition with increasing arsenic content from the center to the edge of the dots. This compositional gradient results from two apparent effects: (1) the faster reaction kinetics of the phosphorus precursor compared to the arsenic precursor, and (2) a post-growth arsenic-phosphorus exchange reaction that increases the arsenic content. The cores have a zinc blend structure for all compositions and show tunable emission in the near-infrared (NIR) region. A first shell of InP leads to a red-shift and an increase in quantum yield. The final shell of ZnSe serves to stabilize the dots for applications in aqueous environments, including NIR biomedical fluorescence imaging. These NIR-emitting core/shell/shell InAs(x)P(1-x)/InP/ZnSe were successfully used in a sentinel lymph node mapping experiment.