We have successfully printed an anatomically shaped hand model with properties similar to in vivo investigations with a fluorescence hand imager using indocyanine green (ICG). By adding the fluorescent dye Lumogen IR 765, titanium dioxide powder and black ink to a 3D printing methacrylate photopolymer we realized phantoms with physiologically relevant absorption and reduced scattering coefficients as well as fluorescence properties similar to the widely applied contrast agent ICG. The phantoms show an excellent long-term photostability making them well suited for device performance monitoring and comparison. In contrast, fluorescence of phantoms printed with ICG continuously decreases over time.
Significance: Fluorescence imaging of rheumatoid diseases with indocyanine green (ICG) is an emerging technique with unique potential for diagnosis and therapy. Device characterization, monitoring of the performance, and further developments of the technique require tissue-equivalent fluorescent phantoms of high stability with appropriate anatomical shapes.
Aim: Our investigations aim at the development of a three-dimensional (3D) printing technique to fabricate hand and finger models with appropriate optical properties in the near-infrared spectral range. These phantoms should have fluorescence properties similar to ICG, and excellent photostability and durability over years.
Approach: We modified a 3D printing methacrylate photopolymer by adding the fluorescent dye Lumogen IR 765 to the raw material. Reduced scattering and absorption coefficients were adjusted to values representative of the human hand by incorporating titanium dioxide powder and black ink. The properties of printed phantoms of various compositions were characterized using UV/Vis and fluorescence spectroscopy, and time-resolved measurements. Photostability and bleaching were investigated with a hand imager. For comparison, several phantoms with ICG as fluorescent dye were printed and characterized as well.
Results: The spectral properties of Lumogen IR 765 are very similar to those of ICG. By optimizing the concentrations of Lumogen, titanium dioxide, and ink, anatomically shaped hand and vessel models with properties equivalent to in vivo investigations with a fluorescence hand imager could be printed. Phantoms with Lumogen IR 765 had an excellent photostability over up to 4 years. In contrast, phantoms printed with ICG showed significant bleaching and degradation of fluorescence over time.
Conclusions: 3D printing of phantoms with Lumogen IR 765 is a promising method for fabricating anatomically shaped fluorescent tissue models of excellent stability with spectral properties similar to ICG. The phantoms are well-suited to monitor the performance of hand imagers. Concepts can easily be transferred to other fluorescence imaging applications of ICG.
KEYWORDS: Magnetic resonance imaging, Luminescence, Collagen, Signal to noise ratio, Inflammation, Animal model studies, Statistical analysis, Plasma, Absorption, Imaging systems
Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (I NF ) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. I NF of moderate and high-grade arthritic joints were significantly higher (p<0.005 ) than the values of control and low-grade arthritic joints between 5 and 30 min after TSC-injection. This result correlated well with post-contrast MRI signal intensities at about 5 min after gadodiamide administration. Furthermore, I NF and signal increase on contrast-enhanced MRI showed high correlation with clinical and histopathological scores. Sensitivities and specificities for detection of moderate and high-grade arthritic joints were slightly lower for NIRF imaging (89%/81% ) than for MRI (100%/91% ). NIRF imaging using TSC, which is characterized by slower plasma clearance compared to indocyanine green (ICG), has the potential to improve monitoring of inflamed joints.
We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1′-bis-(4-sulfobutyl) indotricarbocyanine-5,5′-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.
KEYWORDS: Luminescence, Scanners, Imaging systems, Magnetic resonance imaging, In vivo imaging, Scattering, Near infrared, Spatial resolution, Animal model studies, Head
We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR)
spectral range following intravenous administration of contrast agents. The new imaging system has been characterized
with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments
were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint
inflammation have been compared with findings from contrast enhanced MR imaging and histology.
Diffuse optical tomography is a non-invasive method aiming at the detection of breast cancer. The sensitivity and
specificity of the method can be increased if a fluorescent contrast agent is used that accumulates in malignant
lesions. Recently, Philips developed an optical scanner, where the patient is lying on a bed, with one breast
hanging freely in a cup containing an optical matching fluid. 507 optical fibers are mounted in the surface of
the measurement cup. The breast is illuminated sequentially by half of these fibers while the other half is used
to collect the light that is emanating from the breast. The system uses near-infrared light of continuous wave
solid-state lasers to illuminate the breast at four different wavelengths. A complete measurement takes less than
ten minutes and involves five breast scans: transmission data are collected for four wavelengths, and fluorescence
data for excitation at one wavelength. Here, we present the image reconstruction scheme and a novel method to
assess the system performance in terms of lesion detectability. This method uses a statistical significance test on
simulated data with and without a lesion. It allows the quantification of the detectability of lesions for different
size, position, or contrast of the lesion. It also allows to analyze the potential impact of system improvements
or to judge the performance of an image reconstruction algorithm.
Kai Licha, N. Debus, S. Emig-Vollmer, B. Hofmann, M. Hasbach, D. Stibenz, S. Sydow, Michael Schirner, Bernd Ebert, Diethard Petzelt, C. Bührer, W. Semmler, R. Tauber
We develop a highly specific antibody-dye conjugate for optical imaging of peripheral lymph nodes. The contrast agent consists of the monoclonal antibody recognizing endothelial ligands for the lymphocyte homing receptor L-selectin, MECA-79, and a near-infrared (near-IR) fluorescent indotricarbocyanine dye. The targeting and biodistribution behavior of MECA-79 is studied after radio-iodination and intravenous injection into mice demonstrating specific uptake in lymph nodes and accumulation in high endothelial venules (HEV). After conjugation of MECA-79 with indotricarbocyanine dye, the fluorescence imaging properties of the MECA-79 dye conjugate are examined by intravenous injection in nude mice and laser-induced fluorescence whole-body imaging in vivo. The MECA-79 antibody-dye conjugate accumulates in peripheral lymph nodes, whereas an isotype antibody-dye conjugate does not. Specific lymph node near-IR fluorescent signals become detectable within minutes after injection, and stable imaging persists for more than 24 h. The results demonstrate that vascular targeting of endothelial expression of glyocproteins is feasible to visualize the accumulation of near-IR fluorescent MECA-79 in lymph nodes, making this technology potentially useful to characterize processes of inflammation.
The use of exogenous probes to gain a deeper understanding
of physiological and molecular processes in vivo through the
acquisition of optical signals, particularly via enhanced contrast
using molecular probes ~physiologically transported,
site-directed, or via reporter genes! has emerged with tremendous
vigor in the past few years. One such area of expanded
activity is in the area of early cancer detection, in great part
because it is so critical to the clinical outcome in the
treatment.1–3 As an example, in colon cancer, which accounts
for 15% of all U.S. cancer-related deaths, only 37% are found
early enough for moderate treatment1 and once these types of
cancer reach metastatic activity the survival rate is only 7%.
Oral and brain cancer represent other examples where a need
exists for early detection or improved imaging during treatment.
Each year about 31 000 Americans develop oral cancer.
Squamous cell carcinoma ~SCC! accounts for 95% of all malignant
oral lesions with SCC having a survival rate of only
50%. Yet when this type of cancer is detected in its earliest
stages, the survival rate becomes approximately 80%.4 In cancers
of the esophagus, the five-year survival rate is only listed
at 5%. In contrast, if these cancers are detected when it is still
contained in the mucosa the five-year survival rate becomes
90%.5 For brain cancer the survival rate is abysmal—less than
2 years for younger patients and just weeks for those that are
older—and is critically dependent on the imaging technique
used during treatment.
Many gastroenteropancreatic tumors express receptors for somatostatin (SST) and/or vasoactive intestinal peptide (VIP). These receptors can be used as molecular targets for the delivery of contrast agents for tumor diagnostics. We have synthesized conjugates consisting of a cyanine dye and an SST analogue or VIP for use as contrast agents in optical imaging. Receptor binding and internalization of these compounds were examined with optical methods in transfected RIN38 tumor cells expressing the SST2 receptor or a GFP- labeled VIP (VPAC1) receptor. Furthermore, biodistribution of the conjugates was examined by laser-induced fluorescence imaging in nude mice bearing SST2 or VPAC1 receptor- expressing tumors. After incubation of RIN38 SSTR2 cells in the presence of 100 nM indotricarbocyanine-SST analogue, cell-associated fluorescence increased, whereas no increase was observed when receptor-medicated endocytosis was inhibited. Indodicarbocyanine-VIP accumulated in RIN38 VPAC1 cells and co-localization with the GFP-labeled VPAC1 receptor was observed. After injection of indotricarbocyanine-SST analogue into tumor-bearing nude mice, SST2 receptor-positive tumors could be visualized for a time period from 10 min to at least 48 h. After application of indodicarbocyanine-VIP, a fluorescence signal in VIP1 receptor-expressing tumors was only detected during the first hour. We conclude that cyanine dye-labeled VIP and SST analogue are novel, targeted contrast agents for the optical imaging of tumors expressing the relevant receptor.
We describe the design and properties of cyanine dye-peptide conjugates synthesized on solid supports for screening assays. With this approach, we demonstrate the feasibility of including a diagnostic molecule into a spot synthesis of membrane-bound peptide libraries in order to permit screening of complete dye-peptide conjugates for their potential as fluorescent contrast agents in biomedical optical imaging. A cellulose support, which is modified with a linker permitting cleavage of the dye-peptide conjugates from the support, was prepared. The attachment of dyes to the peptides is exemplified with carboxy-substituted indotricarbocyanines, which can be covalently linked to the N-terminal amino group or a lysin in the course of the synthesis. Several dye-peptide conjugates were obtained by automated peptide synthesis on resins. Furthermore, model sequences consisting of up to 11 amino acids were synthesized on cellulose in sufficient amounts and purity, thus permitting direct testing of these compounds in cell- based assays and fluorescence microscopy. The dyes show only negligible alterations in their absorption and fluorescence properties when attached to low molecular weight peptides. In conclusion, membrane-bound peptide libraries provide a powerful tool to generate large diversities of dye-labeled peptides and to make these compounds available for biological screening assays.
The investigation of cyanine dyes as contrast agents in optical tumor imagin has been a focus of our recent work. We have shown that i.v. injected hydrophilic indotricarbocyanine derivatives enable tumor detection by fluorescence imagin and by frequency-domain absorption spectroscopy. Our current objective is to extend this approach by conjugating these dyes with specific biomolecules in order to enhance targetability and to introduce acid-cleavable links that enable dye release in acidic cell compartments. Accordingly, we have synthesized cyanine dyes which contain different acid-cleavable hydrazone links and which were coupled to peptides, proteins and antibodies. We have studied the release of the dyes under various pH conditions. Our results show that dye release from transferrin increased under acidic conditions, while at neutral pH the stability was higher. Additionally, we observed pH-dependent fluorescence enhancement during cleavage. Cellular fluorescence microscopy experiments indicated that intracellular trapping is possible. In conclusion, cyanine dyes bound to biomolecules by acid- cleavable bonds could act as promising optical contrast agents. Further work will include optimization of release rates by chemical modification and in vivo imaging studies.
Many tumor cells are characterized by the overexpression of highly specific transferrin receptors. Transferrin is bound to its receptor, which is followed by rapid internalization into intracellular compartments; it can thus be used for the specific delivery of coupled contrast agents in to tumor cells. The aim of our study was to investigate the tumor- specificity of transferrin-modified contrast agents for optical imaging and magnetic resonance imaging (MRI). Indotricarbocyanine, a near-IR absorbing dye, and ultrasmall superparamagnetic iron oxide particles, a contrast material for MRI, were covalently coupled to transferrin and examined in vitro and in vivo for their potential as tumor-specific contrast agents. CEll culture experiments using HT29 and A431 tumor cells showed specific uptake of transferrin- modified contrast agents by the cells at 37 degrees C, while uptake was inhibited at 4 degrees C or in the presence of an excess of unlabeled transferrin. Optical imaging of tumor- bearing nude mice after intravenous injection of transferrin-ICC resulted in pronounced tumor fluorescence. Tumor specificity was also observe in MRI, where transferrin-USPIO accumulated in SMT2A tumor-bearing rats. We conclude that transferrin-modified contrast agents for optical imaging and MRI can be specifically delivered to tumor tissue by use of transferrin receptor pathways.
KEYWORDS: Tumors, Tissues, Luminescence, Microscopy, Signal detection, In vivo imaging, Indocyanine green, Plasma, Near infrared, Fluorescence spectroscopy
In this contribution we use intravital microscopy to study the dynamics of extravasation into normal and tumor tissue of several hydrophilic cyanine dyes used as near-infrared (NIR) contrast agents. The technique provides information about the angiographic properties of the dyes and about their interaction with tumor tissue under dynamic conditions in vivo. In our previous work we demonstrated that several NIR- absorbing fluorescent dyes enable in vivo fluorescence detection of tumors in mice and rats. However, the mechanism leading to dye accumulation and enhanced fluorescence in tumors is not fully understood. Increased extravasation of dyes into tumor tissue due to pathologically altered tumor vessels may be an important factor in this process. Indocyanine green (ICG) displayed predominantly intravascular distribution and rapid elimination resulting in enhanced fluorescence signal of vessels during the first 15 min after administration only. No elevated extravasation into tumor tissue was observed with ICG. A hydrophilic indotricarbocyanine derivative with a high molecular weight displayed prolonged intravascular distribution and increased fluorescence signal of the vasculature compared to surrounding tissue for up to five hours. Rapid extravasation and accumulation in tumor areas, yielding elevated contrast of tumors up to 15 min after administration, was observed with hydrophilic, low molecular weight indotricarbocyanine derivatives.
Optical mammography is a new imaging modality currently under development. A contrast agent capable of adding malignancy differentiation signatures is expected to enhance the sensitivity and specificity characteristics of the technique. We report the in vivo characterization of hydrophilic analogs of indocyanine green (ICG) that lead to enhanced tumor absorbance compared to surrounding tissue. The tumor efflux of these dyes was quantitatively studied in tumor-bearing rats by frequency-modulated near-infrared spectroscopy. We demonstrate that the half-lives of hydrophilic cyanine dyes in tumor tissue are considerably enhanced compared to ICG, thus providing a prolonged time window for diagnosis. Moreover, absorption differences between the tumor and normal tissue were observed for 3 hours after injection of the hydrophilic glucamine derivative NIR96010. In conclusion, pharmacokinetic properties as present with NIR96010 may facilitate contrast- agent-aided optical mammography.
The influence of the molecular weight of cyanine dye- poly(ethylene glycol) (PEG) conjugates on their pharmacokinetic behavior and on the contrast between malignant and normal tissue in fluorescence images was studied. PEG conjugates with a molecular weight ranging from 1800 to 40000 g mol-1 were investigated in a rat model. A tunable, pulsed, solid-state laser system and an intensified CCD camera served to record fluorescence images of different tumor-bearing mice and rats. The time window of increased contrast between tumor and normal tissue in fluorescence images can be adjusted by the molecular weigh of PEG residues. Furthermore, we were able to demonstrate the visualization of subcutaneous blood vessels.
Cyanine dyes are promising near-IR contrast agents because of their high molar absorption between 700 and 1000 nm, minimal phototoxicity and convenient synthetic availability. It is known that the derivatization of drugs or contrast agents with polyethylene glycol residues leads to enhanced retention in tumor tissue. The purpose of this study was to generate indotricarbocyanines with improved pharmacological properties enabling in vivo fluorescence detection of tumors. Several hydrophilic indotricarbocyanine- poly(ethylene glycol) conjugates of different molecular weight were synthesized and characterized physicochemically and photophysically in order to test their applicability as near IR contrast media.
In this study indotricarbocyanines were investigated in vivo as near-infrared contrast agents. The known dye indocyanine green (ICG) has several disadvantages regarding its use in near-infrared imaging. ICG has a very short plasma half- life, limited tolerability and is unstable in aqueous solutions. Therefore, several indotricarbocyanine dyes, structurally related to ICG but with different hydrophilicities and physicochemical properties, were synthesized. The tolerability of synthesized dyes was tested in mice. The pharmacokinetic behavior and elimination characteristics were studied in a rat model. The in vivo imaging properties of synthesized dyes were investigated using a tunable, pulsed, solid state laser system for excitation and an intensified CCD camera for fluorescence imaging of different tumor-bearing nude mice models and mamma-carcinoma-bearing rat models. The dye-specific fluorescence exitance was followed at different times after dye administration. The results are demonstrated in comparison to indocyanine green. Synthesized hydrophilic indotricarbocyanine dyes had longer plasma half-lives and increasing renal elimination, corresponding to higher hydrophilicity. Tolerability in mice was increased up to 60- fold compared to ICG. Increased fluorescence exitance in tumors was observed for several dyes 24 h p.i. in the tumor models studied, whereas ICG showed no tumor fluorescence signal under the same conditions.
The diagnostic potential of the near-infrared imaging method can be enhanced using fluorescent dyes as contrast agents. In the pool of fluorescent dyes (e.g. tetrapyrroles, polymethines) the cyanine dyes are the most promising ones because of their high molar absorption between 700 and 1000 nm, minimal phototoxicity and convenient synthetic availability. The potential of indocyanine green (ICG) as a contrast agent for optical mammography has been investigated. The major drawbacks of this compound are a rapid liver uptake after intravenous injection, a low fluorescence quantum yield and low stability in aqueous solutions. Our purpose was to generate indotricarbocyanine derivatives structurally related to ICG with improved photophysical and pharmacological properties. Several dyes more hydrophilic than ICG and covering a wide polarity range were synthesized. The dyes were characterized photophysically (absorption and fluorescence spectra, molar absorptivities and fluorescence quantum yields in different physiological media) and physicochemically (partition coefficients, plasma protein binding).
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