Lead sulfide near-infrared quantum dot bioconjugates for targeted molecular imaging

In this paper, we report the use of lead sulfide quantum dot (PbS QD) bioconjugates as near infrared (NIR) contrast agents for targeted molecular imaging with expanded emission wavelengths beyond 1000 nm. The red-shifted emission band, coupled with the small particle size, which will facilitate clearance, both afford PbS QDs unique properties for noninvasive, high resolution in vivo NIR imaging applications. We have performed imaging experiments at the molecular level using surface-modified PbS NIR QDs, together with our lab-built NIR imaging system. This novel instrumentation and fluorescent contrast agent have enabled us to study the relatively unexplored NIR biomedical imaging spectral region of 900-1200 nm. Preliminary experimental results indicate that PbS-QD/antibody bioconjugates are promising candidates for targeted NIR molecular imaging and future in vivo NIR tissue imaging applications.     

IRDye78 conjugates for near-infrared fluorescence imaging

The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.     

Radiolabeling of folic acid-modified chitosan with (99m)Tc as potential agents for folate-receptor-mediated targeting

The feasibility of chitosan (CS) as a backbone for the design of (99m)Tc-labeled targeting agent was evaluated in this study. Chitosan-folate conjugate (CSFA) and chitosan-folate dithiocarbamate (CSFADTC) were synthesized, characterized and radiolabeled with (99m)Tc as model compounds for folate-receptor (FR) targeting. (99m)Tc-complexes were prepared with high radiochemical purity and high stability. The hydrophilicities of these (99m)Tc-complexes were determined by partition coefficient experiments. The results of biodistribution in normal mice showed that the folic-acid modified agents ((99m)Tc-CSFA and (99m)TcN-CSFADTC) had obviously higher uptake in FR-positive kidney and much lower liver and spleen uptakes than that of non-folic-acid modified (99m)Tc-agent, and the kidney uptakes of FA-modified agents could be blocked significantly by the corresponding cold ligand. Furthermore in vitro and in vivo specific studies will be done in cell line and tumor bearing mice to confirm the usefulness of this chitosan backbone for FR targeting agent design.     

Fast multispectral optoacoustic tomography (MSOT) for dynamic imaging of pharmacokinetics and biodistribution in multiple organs

The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 μm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

Controlled modulation of serum protein binding and biodistribution of asymmetric cyanine dyes by variation of the number of sulfonate groups

To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n = 1-4; DY-675, DY-676, DY-677, DY-678, respectively), were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n = 12; up to 180 minutes postinjection) using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the basis for the rationalization of future contrast agent developments.     

Quantitative imaging of lymph function

Functional lymphatic imaging was demonstrated in the abdomen and anterior hindlimb of anesthetized, intact Yorkshire swine by using near-infrared (NIR) fluorescence imaging following intradermal administration of 100-200 microl of 32 microM indocyanine green (ICG) and 64 microM hyaluronan NIR imaging conjugate to target the lymph vascular endothelial receptor (LYVE)-1 on the lymph endothelium. NIR fluorescence imaging employed illumination of 780 nm excitation light ( approximately 2 mW/cm(2)) and collection of 830 nm fluorescence generated from the imaging agents. Our results show the ability to image the immediate trafficking of ICG from the plexus, through the vessels and lymphangions, and to the superficial mammary, subiliac, and middle iliac lymph nodes, which were located as deep as 3 cm beneath the tissue surface. "Packets" of ICG-transited lymph vessels of 2-16 cm length propelled at frequencies of 0.5-3.3 pulses/min and velocities of 0.23-0.75 cm/s. Lymph propulsion was independent of respiration rate. In the case of the hyaluronan imaging agent, lymph propulsion was absent as the dye progressed immediately through the plexus and stained the lymph vessels and nodes. Lymph imaging required 5.0 and 11.9 microg of ICG and hyaluronan conjugate, respectively. Our results suggest that microgram quantities of NIR optical imaging agents and their conjugates have a potential to image lymph function in patients suffering from lymph-related disorders.     

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.     

新型近红外荧光探针MHI85在胆道系统成像的实验研究

目的:观察一种新型近红外荧光探针MHI85在器官中的成像特点,寻找特异性的器官成像荧光探针,为手术提供帮助。方法:用海洋光学测量系统检测近红外荧光探针MHI85的吸光度和荧光强度,分析其光学特点。随后将近红外荧光探针MHI85注射到CD-1小鼠体内,4小时后观察小鼠体内腹腔、胆囊和胆管、离体小鼠腹部脏器的近红外荧光成像情况。并测量离体脏器的信号背景比(SBR)。结果:近红外荧光探针MHI85最大吸收峰值和荧光峰值分别在690 nm和713 nm,说明其发光谱在700 nm左右,且成像稳定。利用小动物活体成像系统发现,近红外荧光探针MHI85在小鼠胆囊、胆囊管、左右肝管、肝总管可见明显荧光信号。心、肺、肝、胰、脾、肾、十二指肠、小肠均无荧光信号,而胆囊中可见明显的荧光信号。离体脏器SBR结果显示,胆囊的SBR明显高于其他脏器。结论:近红外荧光分子探针MHI85对胆囊及胆道系统具有良好的靶向性,且成像清晰、定位准确。     

Galactosyl Human Serum Albumin-NMP1 Conjugate: A Near Infrared (NIR)-Activatable Fluorescence Imaging Agent to Detect Peritoneal Ovarian Cancer Metastases

Patient survival depends on the completeness of resection of peritoneal ovarian cancer metastases (POCM), and therefore, it is important to develop methods to enhance detection. Previous probe designs based on activatable galactosyl human serum albumin (hGSA)-fluorophore pairs, which target lectin receptors expressed on POCM, have used only visible range dyes conjugated to hGSA. However, imaging probes emitting fluorescence in the NIR range are advantageous because NIR photons have deeper in vivo tissue penetration and result in lower background autofluorescence than those emitting in the visible range. A NIR-activatable hGSA fluorophore was synthesized using a bacteriochlorin-based dye, NMP1. NMP1 has two unique absorption peaks, one in the green range and the other in the NIR range, but emits at a NIR peak of 780 nm. NMP1, thus, has two different Stokes shifts that have the potential to allow imaging of POCM both at the peritoneal surface and just below it. hGSA was conjugated with 2 NMP1 molecules to create a self-quenching complex (hGSA-NMP1). The activation ratio of hGSA-NMP1 was measured by the fluorescence intensity before and after exposure to 10% SDS. The activation ratio of hGSA-NMP1 was ～100-fold in vitro. Flow cytometry, fluorescence microscopy, and in vivo spectral fluorescence imaging were carried out to compare hGSA-NMP1 with hGSA-IR800 and hGSA-ICG (two always-on control agents with similar emission to NMP1) in terms of comparative fluorescence signal and the ability to detect POCM in mice models. The sensitivity and specificity of hGSA-NMP1 for POCM implant detection were determined by colocalizing NMP1 emission spectra with red fluorescent protein (RFP) expressed constitutively in SHIN3 tumor implants at different depths below the peritoneal surface. In vitro, SHIN3 cells were easily detectable after 3 h of incubation with hGSA-NMP1. In vivo submillimeter POCM foci were clearly detectable with spectral fluorescence imaging using hGSA-NMP1. Among 555 peritoneal lesions, hGSA-NMP, using NIR and green excitation light, respectively, detect 75% of all lesions and 91% of lesions ～0.8 mm or greater in diameter. Few false positives were encountered. Nodules located at a depth below the small bowel surface were only depicted with hGSA-NMP1. We conclude that hGSA-NMP1 is useful in imaging peritoneal ovarian cancer metastases, located both superficially and deep in the abdominal cavity.     

Structure-activity relationships of 111In- and 99mTc-labeled quinolin-4-one peptidomimetics as ligands for the vitronectin receptor: potential tumor imaging agents

The integrin receptor alpha(v)beta(3) is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. Radiolabeled alpha(v)beta(3) antagonists have demonstrated potential application as tumor imaging agents and as radiotherapeutic agents. This report describes the total synthesis of eight new HYNIC and DOTA conjugates of receptor alpha(v)beta(3) antagonists belonging to the quinolin-4-one class of peptidomimetics, and their radiolabeling with (99m)Tc (for HYNIC) and (111)In (for DOTA). Tethering of the radionuclide-chelator complexes was achieved at two different sites on the quinolin-4-one molecule. All such derivatives maintained high affinity for receptor alpha(v)beta(3) and high selectivity versus receptors alpha(IIb)beta(3), alpha(v)beta(5), alpha(5)beta(1). Biodistribution of the radiolabeled compounds was evaluated in the c-neu Oncomouse mammary adenocarcinoma model. DOTA conjugate (111)In-TA138 presented the best biodistribution profile. Tumor uptake at 2 h postinjection was 9.39% of injected dose/g of tissue (%ID/g). Activity levels in selected organs was as follows: blood, 0.54% ID/g; liver, 1.94% ID/g; kidney, 2.33% ID/g; lung, 2.74% ID/g; bone, 1.56% ID/g. A complete biodistribution analysis of (111)In-TA138 and the other radiolabeled compounds of this study are presented and discussed. A scintigraphic imaging study with (111)In-TA138 showed a clear delineation of the tumors and rapid clearance of activity from nontarget tissues.     

Fluorescence molecular imaging of small animal tumor models

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.     

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

Non-Invasive In Vivo Imaging of Near Infrared-labeled Transferrin in Breast Cancer Cells and Tumors Using Fluorescence Lifetime FRET

The conjugation of anti-cancer drugs to endogenous ligands has proven to be an effective strategy to enhance their pharmacological selectivity and delivery towards neoplasic tissues. Since cell proliferation has a strong requirement for iron, cancer cells express high levels of transferrin receptors (TfnR), making its ligand, transferrin (Tfn), of great interest as a delivery agent for therapeutics. However, a critical gap exists in the ability to non-invasively determine whether drugs conjugated to Tfn are internalized into target cells in vivo. Due to the enhanced permeability and retention (EPR) effect, it remains unknown whether these Tfn-conjugated drugs are specifically internalized into cancer cells or are localized non-specifically as a result of a generalized accumulation of macromolecules near tumors. By exploiting the dimeric nature of the TfnR that binds two molecules of Tfn in close proximity, we utilized a Förster Resonance Energy Transfer (FRET) based technique that can discriminate bound and internalized Tfn from free, soluble Tfn. In order to non-invasively visualize intracellular amounts of Tfn in tumors through live animal tissues, we developed a novel near infrared (NIR) fluorescence lifetime FRET imaging technique that uses an active wide-field time gated illumination platform. In summary, we report that the NIR fluorescence lifetime FRET technique is capable of non-invasively detecting bound and internalized forms of Tfn in cancer cells and tumors within a live small animal model, and that our results are quantitatively consistent when compared to well-established intensity-based FRET microscopy methods used in in vitro experiments.     

Polyethylene Glycol (PEG) Linked to Near Infrared (NIR) Dyes Conjugated to Chimeric Anti-Carcinoembryonic Antigen (CEA) Antibody Enhances Imaging of Liver Metastases in a Nude-Mouse Model of Human Colon Cancer

We report here that polyethylene glycol (PEG) linked to near infrared dyes conjugated to chimeric mouse-human anti-carcinoembryonic antigen (CEA) antibody greatly improves imaging of liver metastases in a nude mouse model of colon-cancer experimental metastases. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to the chimeric anti-CEA antibody. The dyes were initially injected intravenously into nude mice without tumors. Tissue biodistribution was determined by tissue sonication and analyzing tissue dye concentration profiles over time. PEGylated dyes had significantly lower accumulation in the liver (p = 0.03 for the 650 dyes; p = 0.002 for the 750 dyes) compared to non-PEGylated dyes. In an experimental liver metastasis model of HT-29 colon cancer, PEGylated dyes conjugated to the anti-CEA antibody showed good labeling of metastatic tumors with high contrast between normal and malignant tissue which was not possible with the non-PEGylated dyes since there was so much non-specific accumulation in the liver. PEGylation of the DyLight 650 and 750 NIR dyes significantly altered tissue biodistribution, allowing brighter tissue labeling, decreased accumulation in normal organs, particularly the liver. This enabled high fidelity and high contrast imaging of liver metastases.     

High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents

For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena—such as the presence of immune system cells, tumor angiogenesis, and metastasis—may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 μm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging.     

Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide

Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M(2)) that targets MMP-2/9 and is dual-labeled with IRDye 800 CW and (68)Ga. Since (68)Ga chelation typically requires low pH (3.5-4) and elevated heating temperatures (95 °C), we sought to evaluate the impact of (68)Ga labeling on the optical properties of M(2). An efficient method for preparation of (68)Ga-M(2) was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that (68)Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although (68)Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using (68)Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.     

Fluorescence-enhanced,near infrared diagnostic imaging with contrast agents

The deep tissue propagation of near-infrared (NIR) light between 700-900 nm offers new opportunities for diagnostic imaging when employing sensitive detection techniques and NIR excitable fluorescent agents that target and report disease and metabolism. Herein, we highlight approaches for illuminating tissues and monitoring the re-emitted fluorescence for tomographic reconstruction, strategies for developing fluorescent dye constructs, and clinical opportunities for fluorescence-enhanced NIR optical imaging.     

In vivo resolution of multiexponential decays of multiple near-infrared molecular probes by fluorescence lifetime-gated whole-body time-resolved diffuse optical imaging

The biodistribution of two near-infrared fluorescent agents was assessed in vivo by time-resolved diffuse optical imaging. Bacteriochlorophyll a (BC) and cypate-glysine-arginine-aspartic acid-serine-proline-lysine-OH (Cyp-GRD) were administered separately or combined to mice with subcutaneous xenografts of human breast adenocarcinoma and slow-release estradiol pellets for improved tumor growth. The same excitation (780 nm) and emission (830 nm) wavelengths were used to image the distinct fluorescence lifetime distribution of the fluorescent molecular probes in the mouse cancer model. Fluorescence intensity and lifetime maps were reconstructed after raster-scanning whole-body regions of interest by time-correlated single-photon counting. Each captured temporal point-spread function (TPSF) was deconvolved using both a single and a multiexponental decay model to best determine the measured fluorescence lifetimes. The relative signal from each fluorophore was estimated for any region of interest included in the scanned area. Deconvolution of the individual TPSFs from whole-body fluorescence intensity scans provided corresponding lifetime images for comparing individual component biodistribution. In vivo fluorescence lifetimes were determined to be 0.8 ns (Cyp-GRD) and 2 ns (BC). This study demonstrates that the relative biodistribution of individual fluorophores with similar spectral characteristics can be compartmentalized by using the time-domain fluorescence lifetime gating method.     

Secreted Luciferase for In Vivo Evaluation of Systemic Protein Delivery in Mice

A naturally secreted Gaussia luciferase (Gluc) has been utilized as a reporter for bioluminescence imaging (BLI) evaluation. However, the potential application of Gluc for in vivo monitoring of systemic protein delivery, as well as its natural biodistribution, has not been studied. To examine Gluc secretion and uptake profile, we injected Gluc-encoding plasmids into mice by hydrodynamic tail-vein injection. Whole-body BLI showed that imaging quantification obtained at pawpad was directly correlated to blood Gluc activities. When gene expression was restricted to the liver by the use of a hepatic promoter, in vivo Gluc biodistribution analysis revealed the kidney/bladder, stomach/intestine, and lung as the major uptake organs. Three-dimensional BLI identified liver/stomach and lung as the main internal luminescent sources, demonstrating the feasibility of detecting major uptake organs in live animals by 3D BLI with high-background signals in circulation. Notably, Gluc levels in capillary-depleted brain samples from Gluc-injected mice were comparable to controls, suggesting that Gluc may not cross the blood?Cbrain barrier. Gluc uptake kinetics and intracellular half-life were assessed in various types of cell lines, implicating the involvement of non-specific pinocytosis. These results suggest that Gluc-based system may provide a useful tool for in vivo evaluation of protein/agent biodistribution following systemic delivery.