Multifunctional Thio-Stabilized Gold Nanoparticles for Near-Infrared Fluorescence Detection and Imaging of Activated Caspase-3.

BACKGROUND: Gold nanoparticles (AuNPs) are commonly used in nanomedicine because of their unique spectral properties, chemical and biological stability, and ability to quench the fluorescence of organic dyes attached to their surfaces. However, the utility of spherical AuNPs for activatable fluorescence sensing of molecular processes have been confined to resonance-matched fluorophores in the 500 nm to 600 nm spectral range to maximize dye fluorescence quenching efficiency. Expanding the repertoire of fluorophore systems into the NIR fluorescence regimen with emission >800 nm will facilitate the analysis of multiple biological events with high detection sensitivity. OBJECTIVE: The primary goal of this study is to determine if spherical AuNP-induced radiative rate suppression of non-resonant near-infrared (NIR) fluorescent probes can serve as a versatile nanoconstruct for highly sensitive detection and imaging of activated caspase-3 in aqueous media and cancer cells. This required the development of activatable NIR fluorescence sensors of caspase-3 designed to overcome the nonspecific degradation and release of the surface coatings in aqueous media. METHOD: We harnessed the fluorescence-quenching properties and multivalency of spherical AuNPs to develop AuNP-templated activatable NIR fluorescent probes to detect activated caspase-3, an intracellular reporter of early cell death. Freshly AuNPs were coated with a multifunctional NIR fluorescent dye-labeled peptide (LS422) consisting of an RGD peptide sequence that targets αvß3-integrin protein (αvß3) on the surface of cancer cells to mediate the uptake and internalization of the sensors in tumor cells; a DEVD peptide sequence for reporting the induction of cell death through caspase-3 mediated NIR fluorescence enhancement; and a multidentate hexacysteine sequence for enhancing self-assembly and stabilizing the multifunctional construct on AuNPs. The integrin binding affinity of LS422 and caspase-3 kinetics were determined by a radioligand competitive binding and fluorogenic peptide assays, respectively. Detection of intracellular caspase-3, cell viability, and the internalization of LS422 in cancer cells were determined by confocal NIR fluorescence spectroscopy and microscopy. RESULTS: Narrow size AuNPs (13 nm) were prepared and characterized by transmission electron microscopy and dynamic light scattering. When assembled on the AuNPs, the binding constant of LS422 for αvß3 improved 11-fold from 13.2 nM to 1.2 nM. Whereas the catalytic turnover of caspase-3 by LS422-AuNPs was similar to the reference fluorogenic peptide, the binding affinity for the enzyme increased by a factor of 2. Unlike the αvß3 positive, but caspase-3 negative breast cancer MCF-7 cells, treatment of the αvß3 and caspase-3 positive lung cancer A549 cells with Paclitaxel showed significant fluorescence enhancement within 30 minutes, which correlated with caspase-3 specific activation of LS422-AuNPs fluorescence. Incorporation of a 3.5 mW NIR laser source into our spectrofluorometer increased the detection sensitivity by an order of magnitude (limit of detection ~0.1 nM of cypate) and significantly decreased the signal noise relative to a xenon lamp. This gain in sensitivity enabled the detection of substrate hydrolysis at a broad range of inhibitor concentrations without photobleaching the cypate dye. CONCLUSION: The multifunctional AuNPs demonstrate the use of a non-resonant quenching strategy to design activatable NIR fluorescence molecular probes. The nanoconstruct offers a selective reporting method for detecting activated caspase-3, imaging of cell viability, identifying dying cells, and visualizing the functional status of intracellular enzymes. Performing these tasks with NIR fluorescent probes creates an opportunity to translate the in vitro and cellular analysis of enzymes into in vivo interrogation of their functional status using deep tissue penetrating NIR fluorescence analytical methods.

Synthesis of dye conjugates to visualize the cancer cells using fluorescence microscopy.

The clinical diagnosis of most cancers is based on evaluation of histology microscopic slides to view the size and shape of cellular nuclei and morphological structure of tissue. To achieve this goal for in vivo and in-deep tissues, near infrared dyes-bovine serum albumin and immunoglobulin G conjugates were synthesized. The spectral study shows that the absorption and fluorescence of the dye conjugates are in the "tissue optical window" spectral ranges between 650 and 900 nm. The internalization and pinocytosis of the synthesized compounds were investigated at cell level using fluorescence microscopy to obtain the optimal concentration and staining time.

Automatic segmentation of fluorescence lifetime microscopy images of cells using multiresolution community detection--a first study.

Inspired by a multiresolution community detection based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Furthermore, using the proposed method, the mean-square error in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The multiresolution community detection method appeared to perform better than a popular spectral clustering-based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in mean-square error with increasing resolution.

Ultrabright NIR fluorescent mesoporous silica nanoparticles.

Near-infrared (NIR) water-dispersible fluorescent tags are of big importance for biomedical imaging. Bright, stable, biocompatible NIR fluorescent nanoparticles have great translation potential to improve diagnosis of early stages of different diseases. Here we report on the synthesis of exceptionally bright ("ultrabright") fluorescent meso(nano)porous silica nanoparticles of 28 ± 3 nm in diameter. The NIR fluorescent dye LS277 is encapsulated inside these silica nanoparticles. The wavelengths of the maximum excitation/fluorescence of the particles are 804/815 nm. The absorptivity coefficient of the particles is 2.1 × 108 M-1 cm-1 at 805 nm and the quantum yield of the dye increased by a factor of 5 after encapsulating to 1.5%. The fluorescent brightness of these particles is more than 2000× higher than the fluorescence of one molecule of LS277 in water. When exited in NIR spectral region (>700 nm), these particles are up to 4× brighter than QD800 commercial quantum dots emitting at 800 nm. We demonstrate that the synthesized NIR mesoporous silica nanoparticles easily internalize 4T1luc breast tumor cells, and remain bright for more than 9 weeks whereas the dye is completely bleached by that time.

Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.

Preclinical evaluation of Mab CC188 for ovarian cancer imaging.

Cancer stem cells (CSCs) have been successfully isolated from solid tumors and are believed to be initiating cells of primary, metastatic and recurrent tumors. Imaging and therapeutic reagents targeted to CSCs have potential to detect subclinical tumors and completely eradicate the disease. Previously, we have demonstrated that Mab CC188 binds to colon cancer CD133- and CD133+ (CSCs) cells. In this study, we examined the reactivity of Mab CC188 to ovarian cancer cells including CD133+ cells and primary tumor tissues using immunofluorescence staining methods and tissue microarray technique. We also explored the feasibility of using NIR dye-labeled Mab CC188 probe to image ovarian tumors in vivo. Mab CC188 stains both CD133- and CD133+ cells of ovarian cancer. Tissue microarray analysis reveals that 75% (92/123) of ovarian cancer cases are positively stained with Mab CC188. Weak positive (±), positive (+), strong positive (++) and very strong positive (+++) stains are 14.8, 3.7, 11 and 24.4%, respectively. In contrast, Mab CC188 staining is low in normal cells and tissues. In vivo study show that significant amounts of the probe accumulates in the excretion organs in the early period postinjection. At 24 hr, the imaging probes have largely accumulates in the tumor, while the intensity of the imaging probe decreases in the liver. The tumor uptake was still evident at 120-hr postinjection. Our work suggests that Mab CC188-based imaging and therapeutic reagents are capable of detecting early stage ovarian tumors and effectively treating the tumor.

Near infrared photonic finger imager for prostate cancer screening.

A portable rectal near infrared (NIR) scanning polarization imaging unit with an optical fiber-based rectal probe, designated as a Photonic Finger (PF), was designed, developed, built and tested. PF was used to image and locate the three dimensional (3D) positions of abnormal prostate tissue embedded inside normal prostate tissue. An inverse image reconstruction algorithm, namely Optical Tomography using Independent Component Analysis (OPTICA) was developed to unmix the signal from targets (cancerous tissue) embedded in a turbid media (normal tissue) in the backscattering imaging geometry. The Photonic Finger combined with OPTICA was ex vivo tested to characterize different target(s) inside different tissue medium, including cancerous prostate tissue embedded inside large pieces of normal tissue. This new developed instrument, Photonic Finger, may provide an alternative imaging technique, which is accurate, of high spatial resolution and non-or-less invasive for prostate cancers screening.

Using in-vivo fluorescence imaging in personalized cancer diagnostics and therapy, an image and treat paradigm.

The major goal in developing drugs targeting specific tumor receptors, such as Monoclonal AntiBodies (MAB), is to make a drug compound that targets selectively the cancer-causing biomarkers, inhibits their functionality, and/or delivers the toxin specifically to the malignant cells. Recent advances in MABs show that their efficacy depends strongly on characterization of tumor biomarkers. Therefore, one of the main tasks in cancer diagnostics and treatment is to develop non-invasive in-vivo imaging techniques for detection of cancer biomarkers and monitoring their down regulation during the treatment. Such methods can potentially result in a new imaging and treatment paradigm for cancer therapy. In this article we have reviewed fluorescence imaging approaches, including those developed in our group, to detect and monitor Human Epidermal Growth Factor 2 (HER2) receptors before and during therapy. Transition of these techniques from the bench to bedside is the ultimate goal of our project. Similar approaches can be used potentially for characterization of other cancer related cell biomarkers.

Time-resolved fluorescence polarization dynamics and optical imaging of Cytate: a prostate cancer receptor-targeted contrast agent.

Cypate-octreote peptide analogue conjugate (Cytate) was investigated as a prostate cancer receptor-targeted contrast agent. The absorption and fluorescence spectra of Cytate were ranged in the near-infrared "tissue optical window." Time-resolved investigation of polarization-dependent fluorescence emitted from Cytate in solution as well as in cancerous and normal prostate tissues was conducted. Polarization preservation characteristics of Cytate in solution and tissues were studied. Fluorescence intensity emitted from the Cytate-stained cancerous prostate tissue was found to be much stronger than that from the Cytate-stained normal prostate tissue, indicating more Cytate uptake in the former tissue type. The polarization anisotropy of Cytate contained in cancerous prostate tissue was found to be larger than that in the normal prostate tissue, indicating a larger degree of polarization preservation in Cytate-stained cancerous tissue. The temporal profiles of fluorescence from Cytate solution and from Cytate-stained prostate tissue were fitted using a time-dependent fluorescence depolarization model. The photoluminescence imaging of Cytate-stained cancerous and normal prostate tissues was accomplished, showing the potential of Cytate as a fluorescence marker for prostate cancer detection.

Spectral polarization imaging of human prostate cancer tissue using a near-infrared receptor-targeted contrast agent.

The Cypate-Bombesin Peptide Analogue Conjugate (Cybesin) was used as a prostate tumor receptor-targeted contrast agent. The absorption and fluorescence spectra of Cybesin were measured and shown to exist in the NIR tissue "optical window". The spectral polarization imaging of Cybesin-stained prostate cancerous and normal tissues shows that prostate cancerous tissue takes-up more Cybesin than that of prostate normal tissue, making Cybesin a potential marker of prostate cancer.

Metabolism-enhanced tumor localization by fluorescence imaging: in vivo animal studies.

We present a high-sensitivity near-infrared optical imaging system for noninvasive cancer detection and localization based on molecularly labeled fluorescent contrast agents. This frequency-domain system utilizes the interferencelike pattern of diffuse photon density waves to achieve high detection sensitivity and localization accuracy for the fluorescent heterogeneity embedded inside the scattering media. A two-dimensional localization map is obtained through reflectance probe geometry and goniometric reconstruction. In vivo measurements with a tumor-bearing mouse model by use of the novel Cypate-mono-2-deoxy-glucose fluorescent contrast agent, which targets the enhanced tumor glycolysis, demonstrate the feasibility of detection of a 2-cm-deep subsurface tumor in the tissuelike medium, with a localization accuracy within 2-3 mm.

Small animal imaging. current technology and perspectives for oncological imaging.

Advances in the biomedical sciences have been accelerated by the introduction of many new imaging technologies in recent years. With animal models widely used in the basic and pre-clinical sciences, finding ways to conduct animal experiments more accurately and efficiently becomes a key factor in the success and timeliness of research. Non-invasive imaging technologies prove to be extremely valuable tools in performing such studies and have created the recent surge in small animal imaging. This review is focused on three modalities, PET, MR and optical imaging which are available to the scientist for oncological investigations in animals.

Novel fluorescent contrast agents for optical imaging of in vivo tumors based on a receptor-targeted dye-peptide conjugate platform.

We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target over-expressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst(2)) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide-dye conjugates are effective as contrast agents for optical imaging of tumors.

Novel receptor-targeted fluorescent contrast agents for in vivo tumor imaging.

RATIONALE AND OBJECTIVES: To evaluate the efficacy of a novel tumor receptor-specific small-peptide-near-infrared dye conjugate for tumor detection by optical imaging. METHODS: A novel, near-infrared dye-peptide conjugate was synthesized and evaluated for tumor-targeting efficacy in a well-characterized rat tumor model (CA20948) known to express receptors for the chosen peptide. A simple continuous-wave optical imaging system, consisting of a near-infrared laser diode, a cooled CCD camera, and an interference filter, was used in this study. RESULTS: Tumor retention of two non-tumor-specific dyes, indocyanine green and its derivatized analogue, bis-propanoic acid cyanine dye (cypate), was negligible. In contrast, the receptor-specific peptide-cypate conjugate (cytate) was retained in the CA20948 tumor, with an excellent tumor-tonormal-tissue ratio in the six rats examined. CONCLUSIONS: Optical detection of tumors with a receptor-targeted fluorescent contrast agent has been demonstrated. This result represents a new direction in cancer diagnosis and patient management.

Stabilization of the optical tracer agent indocyanine green using noncovalent interactions.

Indocyanine green is a medically useful dye that absorbs and fluoresces in the near infrared and has been sporadically employed clinically as an optical tracer agent for liver function evaluation and cardiac output measurements. The poor stability of this dye in aqueous solution, especially at the high concentrations needed for bolus injection, has been a hindrance in clinical application. However, by using carefully chosen macromolecular additives, the stability of these aqueous dye solutions may be enhanced significantly. Such noncovalent binding between dye and carrier molecules was found to preserve substantially the dye in aqueous solutions for several weeks with no apparent changes in the measured in vivo biological properties.

Preparation and antibiotic activity of monobactam analogues of nocardicins.

A series of diverse beta-lactam analogues of nocardicins with interesting antimicrobial properties were prepared. Coupling of glucosamine to these compounds improved their water solubility. Aminoacid derivatives produced a stereoinduction on the quaternary enantiotopic carbon of the starting compound 1. Evaluation of their antimicrobial activity showed that the introduction of alpha-amninoacids to monobactams increased their activity. The importance of asymmetric carbon is exemplified by the higher antibiotic activity of L-alpha-aminoacids than the D-series. No significant difference was observed between fluorinated and non-fluorinated monobactams.