Multi-Modal Molecular Imaging Agents

Each year about 31,000 Americans develop oral cancer. Squamous cell carcinoma accounts for 95% of all malignant oral lesions. Oral squamous cell carcinoma is a condition that will kill approximately half of patients afflicted within five years of diagnosis. Early diagnosis is stressed, because this could increase the survival rate from 50% to about 80%. Contrast enhanced diagnostic applications of fluorescence spectroscopy may offer an effective means for early, non-invasive and rapid detection of oral cancer. Underlying our aims are the hypotheses that spectral information from dye-enhanced fluorescence of oral lesions can be used as a reliable approach toward accurate and early detection of pre-malignant and malignant lesions in the oral cavity. This proposal will focus on the topical application of Lanthanide Chelate-based contrast agent to induce selective accumulation of a fluorescing dye in malignant transformed epithelial tissue for the purpose of early lesion detection.

Our preliminary work has shown:

(1) promising correlation between the spectral information observed in contrast-enhanced fluorescence and pathological state of the lesion in the early stage of development,

(2) the ability of malignant oral lesions to selectively accumulate the contrast agent as compared to benign lesions or normal tissue.

We propose to perform cellular and pre-clinical animal studies to:

(1) optimize the chemical structure and spectroscopic properties of the dye for safe and practical applications

(2) better understand the cellular and morphological basis for the observed selective uptake of the dye by pre-malignant and malignant oral tissues, and

(3) in pre-clinical animal studies, explore the diagnostic power of the proposed contrast enhanced approach and compare that to conventional techniques.

The knowledge gained from this study may lead to the development of a simple, inexpensive and non-invasive diagnostic tool for detection and screening of oral cancer in a dentist's office, guiding biopsy and follow up suspicious oral lesions by otolaryngologists and oral surgeons.

Abstract

The further development of a new class of multi-modal contrast agent is proposed. These unique molecular imaging agents exhibit bright luminescence, give MRI contrast, have long emission lifetimes for detection in the zero noise regime and can be tuned structurally for specific and sensitive molecular recognition. The polyazamacrocyclic lanthanide chelates complexes have been found to be non-toxic and useful for in-vitro diagnostics and in-vivo disease detection. Agents proposed here represent are molecular imaging agents that will be useful in bi-modal imaging and therapy tracking.

By preparing cocktails of both the Gd3+ and Eu3+ or Tb3+ complexes, it will be possible to obtain non-invasive anatomic-scale images by MRI and perform microscopic-scale imaging by fluorescence on the same cell or tissue. Under an expanded synthetic effort, several new agents will be created.

These include:

1) a near-infrared (NIR) complex with absorption and emission in the spectral region where tissue is relatively transparent

2) a long circulating bi-metallic with enhanced relaxivity for improved MRI and

3) a multi-signature a multimeric compound providing enhanced targeting and S/N. In-vitro and in-vivo studies will show the safety and efficacy of these new molecular imaging agents.

This proposal also seeks to capitalize on the observations that many cancer cells display a high density of peripheral benzodiazepine receptor (PBR) binding sites and that when the PBR ligand, PK-11195, is conjugated to our trifunctional lanthanide chelate, the resulting compound (Ln-PK-11195) can serve as a molecular signaling agent for PBR. Bi-modal imaging (fluorescence and MRI) of PBR expressing cells has been possible with Ln-PK-11195, thus, a new imaging agent has been created for the further study of mitochondrial function and its relationship to cancer cell proliferation.

Since PBR is upregulated by human glioblastoma, it has been possible to selectively label this disease ex-vivo. Under the proposed investigations we quantify Kd for Ln-PK-11195 in-vitro, investigating the influence of chemical structure on signaling and cellular uptake.

Finally, we will evaluate the efficacy of using Ln-PK-11195 as a bi-modal imaging contrast agent for anatomic-scale in-vivo brain cancer registration by MRI and the subsequent cellular-level characterization by fluorescence.

Abstract

Abstract

Time-Resolved Solute Detection Development - An expanded effort will be devoted to characterizing the temporal spectroscopic properties of the fluorescent chelates and implementing gated detection. As resources are made available for the purchase of a commercial system, the Texas Tech group will work on implementation of an endoscope to perform time-resolved detection of the lanthanide chelates in a remote format. This research effort will be ongoing to include the evaluation of new compounds as they are synthesized and new applications as they are realized (for example in-vitro diagnostics or chip-scale biosensing).

Screening of Anionic and Neutral, Highly Water Soluble Chelates - To ensure that Tb-PCTMB (lipophilic and neutral) is the best and most efficacious choice for clinical trials, Tb-PCTA (neutral and hydrophilic) and Tb-PCTMP (anionic and hydrophilic) will be evaluated in the colorectal cancer animal model. It is anticipated that this work can be completed within the next 3 months.

Mechanistic Aspects of Chelate Interaction with Cancerous Tissue - Evaluate the mode of chelate specificity using Tb-PCTMB, Tb-PCTA, an Tb-PCTMP Investigate the feasibility of using the fluorescent chelates in a cervical cancer model. {PCTA = 3,6,9-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid; PCTMP=3,6,9-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-trimethylenephosphonic acic}

Screening of Absorption Red-Shifted Chelates in Colon Cancer Model - To evaluate the potential of using Eu-Q-(F,Me)-CTMB (the lipophilic butyl half ester that is cleared through the liver) and Eu-Q-(F,Me)-CTME (the less lipophilic ethyl half ester that is cleared renally) as a contrast agent for detecting early columnar epithial disease or colorectal cancer. The best and most efficacious choice for for clinical trials will be evaluated in the AOM or DMH Sprague Dowley rat model. It is anticipated that this work will be completed within 6 months.

Attempt to Synthesize IR Absorbing and Emitting Complexes - Using newly identified antenna and alternate lanthanide chelates, complexes with absorption and emission in the near to far IR will be prepared. One goal of this effort is to make molecules that could be excited and detected deep in tissue, or have spectroscopic properties coincident with tissue transparent wavelengths.

Investigate the Feasibility of Using the Fluorescent Chelates for Novel Applications - These include in-vitro screening, chip-scale sensing, molecular - cellular signaling and cancer cell collection and detection (point-of-care diagnostics).

Under the proposed research, we will further develop a new contrast agent that specifically labels glioma cells and that provides both MRI and fluorescence signatures. This proposal seeks to capitalize on the observations that human glial tumors display a high density of peripheral benzodiazepine receptor (PBR) binding sites and that the conjugable form of the PBR ligand PK-11195 will preferentially bind to human primary brain tumors. Thus, through conjugation of our unique polyazamacrocyclic lanthanide chelating agents (Ln-Q-Y-CTMR) to the peripheral benzodiazepine receptor ligand, PK-11195, a marker (Ln-Q-Y-CTMR-PK11195) that will directly and selectively label viable glioma cells will be possible. We plan to prepare cocktails of both the Gd3+ complex to provide macroscopic scale images by MRI, and the Eu3+ or Tb3+ species to give microscopic scale detection by fluorescence imaging from the same tissue.

After the synthesis has been accomplished, we will establish specific uptake by PBR expressing glioma cells in-vitro, investigating the influence of linker chain length on complex stability, fluorescence and NMR signal and glioma cell uptake properties. In-vitro and in-vivo toxicity studies will also be conducted on the new contrast agent. Finally, we will demonstrate the efficacy of using the contrast agents for in-vivo brain cancer registration by MRI, visual detection of glioma lesions by fluorescence imaging and subsequent cellular-level characterization by fluorescence microscopy.

Micro-Fluidics and Nanosensing

On-Chip Interferometry [NSF]

We propose the optimization and implementation of a new, laser-based optical analysis scheme, on-chip interferometric backscatter detection (OCIBD) for universal on-chip solute determinations, particularly protein quantification in chip-scale CE. A series of preliminary investigations have shown OCIBD facilitates sub-nanoliter volume refractive index measurements in planer substrates. The detection system consists of a simple, folded optical train based on the interaction of a low power laser beam and an etched channel in a silica substrate. The channels, are 50-90 mm wide and 20-50 mm deep and consist of two radii joined by a flat portion at the base. They have the general shape of a half cylinder. The direct backscattered light from the channel takes on the form of a high contrast interference pattern that contains information related to the bulk properties of the fluid contained within the probe volume.

Positional changes of the interference pattern extrema (fringes) allow for the determination of Dn at the 10^-6 level (10^-7 noise level). The detection limit for sucrose is 206 mM or 5.3 x 10^-13 moles or 1.8 x 10^-10 grams. The detection volume typically ranges from 47-188 pL. Using OCIBD with CE, the proteins Egg Albumin, Lysozyme and Cytochrome C have been detected at the level of 15, 23 and 27 mM, respectively. In other words, 0.2-1.0 mg/ml of native protein could be quantified. Furthermore, a preliminary theoretical model for OCIBD has been developed, evaluated, and found to be in agreement with experimental data. It has also been shown that this model can be used to predict general system performance as a function of optical train modifications such as the chip's wall thickness and channel diameter. Improvements realized for OCIBD, under the proposed investigations, are predicted to result in refractive index sensitivity of Dn=10^-9 or detection limits for proteins in the range of 150-250 nM.

The result of this research will be a sensitive, picoliter volume, universal detection system that will be broadly applicable to on-chip bioanalysis schemes. It will be particularly useful in on-chip CE or HPLC and for quantifying non-fluorescent solutes from biological matrices.

MIBD for Capillary-Scale Universal and Polarimetric Detection [NSF]

A void exists in capillary separations for a simple, nanolilter volume, optical detection that has high sensitivity and can be used universally. The need for such a detector is particularly evident in the area of biological analysis, where the desired solute has little native absorbance or fluorescence. The micro-interferometric backscatter detection (MIBD) system has the potential to fill this void. MIBD has been employed for refractive index (RI) detection in CE and will be used to perform RI and polarimetric detection of poorly absorbing or non-fluorescent biologically important solutes in capillary electrochromatography (CEC).

MIBD is a significant advance in biological research instrumentation for several reasons. The folded, yet simple and inexpensive, optical train is easy to align; the fused silica capillary is directly probed without modification; and multi-pass signal enhancement affords ultra-high sensitivity (μmolar) in nanoliter volumes.

Preliminary results also indicate MIBD can be used as a capillary-scale polarimetric detector, allowing optical activity detection (polarimetry) in sub-nanoliter volumes with CEC. CEC is a powerful hybrid separation technique that uses features of both high-performance capillary electrophoresis (HPCE) and liquid chromatography (HPLC). Doing CEC on etched and functionalized capillaries, with inner diameters ranging from 20 to 100 mm, rapid and efficient separation of anionic and neutral compounds is possible, while avoiding some of the limitations encountered in packed capillaries.

The power of MIBD with CEC will allow new insights into the functionality of biologically significant species such as hormones, messengers and membrane constituents. As a first step to providing enhanced knowledge about these important species, CEC-RI and CEC-polarimetry will be used to quantify angiotensins, inositol phosphates and phospholipids.

Chemical Bonding Investigations by Micro-Interferometry [Welch Foundation]

The goal of our ongoing micro-volume detection research is to understand properties such as cold denaturation of proteins, solute aggregation and reaction kinetics in fluid systems bound by volumetric constraints. These properties are not well understood and are important in chemical and biochemical systems spanning the spectrum of disciplines from biophysics to separation science.

Unlike larger volume systems, which are less problematic to study, little is known about the special solute-solvent and solute-capillary interactions unique to these micro-volume environments. For example, when water is contained in a capillary tube, the solvation properties of water can be perturbed, yet the range and magnitude of these effects has not been clearly defined. These effects can now be studied using the micro-interferometeric backscatter detector (MIBD), which allows refractive index changes (density) at Dn=10^-7 to be measured in capillaries from 10 to 1000 mm in diameter. In another example, direct measurement of cold denaturation of proteins is made possible because fluids can be super-cooled in capillaries. Thus, using MIBD, which has temperature resolution of DC0=10^-4 over a range of -12^o C to 60^o C, folding process can be studied without modifying the protein.

Finally, the determination of reaction kinetics for species that are optically active and are found in small amounts within biological systems is made possible with MIBD which can be used to detect sub-picogram quantities of chiral species within picoliter volumes. In short we will use MIBD to study and solve previously intractable problems in chemical science.

On-Chip Backscattering Interferometry for Space-Based Biosensing and Diagnostics [NASA]

Abstract

On-Chip Detection of Proteins by MIBD [Applied Biosystems Inc.]

Abstract

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