Project Four:

Development of Nanocrystals as Probes for Optical and Magnetic Resonance Imaging

Project References
 Maryann Fitzmaurice
Clemens Burda
Mark Pagel
Andrew Rollins
James Willson, Case Western Reserve University

We propose to develop novel nanocrystals (NC) as a platform technology for use as exogenous probes for optical and magnetic resonance (MR) imaging in vivo in humans. The imaging probe platform consists of a NC payload, solubilized and rendered biocompatible by encapsulation a phospholipid micelle. The encapsulated NC are targeted by conjugation to tumor-specific antibodies (Ab) or other targeting agents.

Specific Aims:

  1. Development of novel NC as exogenous probes for three imaging modalities: fluorescence (FL), MR and optical coherence tomography (OCT).
  2. Development of NC-Ab conjugates as targeted probes for molecular-based imaging of cancer.
  3. Incorporation of NC into targeted nanoparticles with other payloads to enable imaging of their biodistribution and pharmacokinetics in animal models.

Background and Significance: NC are nanometer sized particles whose properties result from quantum-size confinement, which occurs when the particles are smaller than their exciton Bohr radius (1-5 nm). Due to their small size and optical/magnetic properties, semiconductor (SNC) and metallic NC (MNC) have potential as exogenous imaging probes, if appropriately encapsulated to minimize toxicity and maximize solubility and targeted for molecular specificity.

FL imaging is used routinely in animal models, but is not yet in clinical use in humans. SNC are ideal probes for FL imaging due to their tunable narrow emission wavelengths, broad excitation ranges, high absorption cross-sections, high photoluminescence quantum yields, photostability, and long emission lifetimes, as compared to conventional fluorescent dyes [1, 2]. Until recently, FL imaging in humans was limited by poor light penetration through optically thick tissues. We propose to develop novel SNC probes that fluoresce in the near infrared (NIR) “optical window”, where there is minimal light scattering and absorption and excitation/emitted light penetrates to tissue depths required for FL imaging in humans [3].

NC of some metal alloys are superparamagnetic (SP), attracted to an applied field but retain no residual magnetism after field removal [4, 5], rendering them suitable for use as MR imaging probes. The pure transition metals Fe and Co have larger magnetic moments than current MR contrast agents such as SP iron oxide, allowing imaging at lower field strengths. However, Fe NC are unstable and will spontaneously combust when exposed to air, and although less combustible, Co NC form an oxide coating when exposed to air, which reduces their magnetic strength. To circumvent these problems, we propose to develop noble metal alloy analogues of these MNC as MR probes. We also propose to dope these noble metal alloys with photoluminescent SNC to create probes for dual modality MR-FL imaging.

OCT has μm-scale resolution, is the only optical technique now capable of true tomographic imaging, and is in clinical use [6, 7]. Improvements in OCT image contrast are needed, but unlikely without a technological quantum leap, such as development of new contrast agents. Recently several techniques, including excited state absorption [8] and second harmonic generation (SHG) [9-11], have shown potential. We propose to develop novel MNC contrast agents for OCT using SHG.

Preliminary Data: Our NC are in development in the Case Center for Chemical Dynamics and Nanomaterials Research (CCDNR), using novel surfactant-assisted organometallic synthetic methods developed in the Burda group [13]. CdSe/ZnS and CdSe/CdS NC (some magnetically doped) have been developed as prototypes that fluoresce in the visible spectral range [14, 15].

Achievements to date are:

  1. Development of a one-pot synthesis of uniform, 5-nm, highly monodispersed, high photoluminescence quantum yield NC, which is fast, inexpensive, and simple[13].
  2. Characterization of the morphology, size, and FL, magnetic, and Raman properties of the NC.
  3. Micelle encapsulation of the NC and assessment of spectral properties of the encapsulated NC.
  4. Conjugation of the NC to the model proteins, bovine serum albumin and streptavidin.

The Rollins group also has expertise in OCT and took part in the first demonstrations of molecular contrast in OCT using excited state absorption of methylene blue [8] and SHG of collagen [9].

Research Plan:

Aim 1.
Development of novel SNC and MNC optimized for in vivo optical and MR imaging We propose to synthesize 4 types of NC, in the CCDNR and new Inorganic Materials Core:

  1. InP and InAs SNC as FL imaging probes that have NIR excitation and emission orders of magnitude greater and farther in the NIR than conventional NIR emitting dyes.
  2. Fe- and magnetically-doped Cu-based MNC as MR probes more stable and with magnetic moments orders of magnitude greater than existing MR contrast agents.
  3. Fe- and Co-based MNC doped with photoluminescent SNC as dual modality MR-FL imaging probes.
  4. Colloidal gold and silver MNC as probes for OCT imaging using SHG.

Aim 2.
Development of NC-Ab conjugates for molecular-based imaging of cancer. All four types of NC payloads will be rendered soluble in aqueous media and biocompatible by encapsulation in the hydrophobic core of micelles composed of n-poly(ethyleneglycol)phosphatidylethanolamine (PEG-PE) and phosphatidylcholine. Encapsulated NC will be targeted by conjugation to anti-Her-2/neu Ab using streptavidin-biotin or a phospholipid-conjugation method, for imaging of Her-2/neu over-expression in breast cancer [14, 16-18]. Humanized anti-Her-2/neu Abs are commercially available and FDA-approved for human use, which should expedite translation of the NC imaging platform into clinical use. The NC platform technology can be applied to image other molecular targets, tumors or non-neoplastic diseases.

After characterization of the size, shape, charge, chemical bond structure, static and time-dependent FL, magnetic and SHG properties of the NC and NC-Ab conjugates in the new Inorganic Materials and Analytical Cores, in vitro imaging studies will be performed using human breast adenocarcinoma cell lines with differential expression of Her-2/neu. In vivo imaging studies will follow, first using explants of these cell lines in nude mice and then spontaneous breast cancers in an MMTV HerB2 transgenic mouse model, developed at the Case Comprehensive Cancer Center and maintained in the new Animal Core. In vivo imaging studies will be performed under addenda to an existing IACUC approved protocol using the micro-MR and FL imaging systems in the Case Small Animal Imaging Facility (SAIF). The FL imaging system currently planned for the SAIF must be upgraded and equipped with a CCD camera with suitable quantum efficiency in the NIR. An existing OCT system developed at Case will also need to be adapted for SHG imaging. Those NC-Ab conjugates that appear the most promising in the in vitro and in vivo studies in animals will ultimately undergo testing in the new GLP core in preparation for clinical trials.

Aim 3.
NC probes for imaging biodistribution and pharmacokinetics of other targeted nanoparticles. All four types of NC will be made available for incorporation into targeted nanoparticles with other payloads developed by other BRTT investigators to enable imaging of their biodistribution and pharmacokinetics in animal models. Due to their anticipated high sensitivity, NC probes should enable imaging of nanoparticles at low concentration in body fluids or tissues or bound to low abundance molecular targets. Optical and MR imaging techniques will also enable serial imaging of each animal, substantially reducing the number of animals and time required to these studies.