Hydrophilic cyanine dyes as contrast agents for near-infrared tumor imaging: synthesis, photophysical properties and spectroscopic in vivo characterization

We have synthesized a group of glucamine and gluosamine-substituted cyanine dyes structurally related to indocyanine green (ICG) and have characterized these compounds with regard to their potential as contrast agents for biomedical optical imaging. The compounds reported herein exhibit increased hydrophilicity and less plasma protein binding (< 50%), and are thus expected to have different pharmacokinetic properties compared with ICG. Furthermore, we measured enhanced fluorescence quantum yields (7-15%) in a physiological environment with respect to ICG. For the derivative with the highest hydrophilicity (5a) the efflux from tumor and normal tissue was monitored by intensity-modulated diffuse optical spectroscopy after intravenous injection into tumor-bearing rats. In comparison with ICG, 5a exhibited a considerably enhanced tissue-efflux half-life (73 min versus less than 10 min for ICG in tumor tissue), a two-fold higher initial tissue absorption coefficient compared to ICG, and finally, it generated an elevated tumor-to-tissue concentration gradient up to 1 h after injection. In conclusion, compounds such as 5a are promising contrast agents for optical imaging, and could facilitate highly sensitive and specific detection of breast cancer or other malignancies by utilizing mechanisms similar to contrast-enhanced magnetic resonance imaging or computerized tomography.     

Silica-coated quantum dots for optical evaluation of perfluorocarbon droplet interactions with cells

There has been recent interest in developing new, targeted, perfluorocarbon (PFC) droplet-based contrast agents for medical imaging (e.g., magnetic resonance imaging, X-ray/computed tomography, and ultrasound imaging). However, due to the large number of potential PFCs and droplet stabilization strategies available, it is challenging to determine in advance the PFC droplet formulation that will result in the optimal in vivo behavior and imaging performance required for clinical success. We propose that the integration of fluorescent quantum dots (QDs) into new PFC droplet agents can help to rapidly screen new PFC-based candidate agents for biological compatibility early in their development. QD labels can allow the interaction of PFC droplets with single cells to be assessed at high sensitivity and resolution using optical methods in vitro, complementing the deeper depth penetration but lower resolution provided by PFC droplet imaging using in vivo medical imaging systems. In this work, we introduce a simple and robust method to miscibilize silica-coated nanoparticles into hydrophobic and lipophobic PFCs through fluorination of the silica surface via a hydrolysis-condensation reaction with 1H,1H,2H,2H-perfluorodecyltriethoxysilane. Using CdSe/ZnS core/shell QDs, we show that nanoscale, QD-labeled PFC droplets can be easily formed, with similar sizes and surface charges as unlabeled PFC droplets. The QD label can be used to determine the PFC droplet uptake into cells in vitro by fluorescence microscopy and flow cytometry, and can be used to validate the fate of PFC droplets in vivo in small animals via fluorescence microscopy of histological tissue sections. This is demonstrated in macrophage and cancer cells, and in rabbits, respectively. This work reveals the potential of using QD labels for rapid, preclinical, optical assessment of different PFC droplet formulations for their future use in patients.     

An enzymatically activated fluorescence probe for targeted tumor imaging

Beta-galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-betaGal (J. Am. Chem. Soc. 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-betaGal, provides a dramatic fluorescence enhancement upon reaction with beta-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-betaGal, after confirming that tumors in the model could be labeled with an avidin-beta-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-betaGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 microm. This strategy has potential clinical application, for example, in video-assisted laparoscopic tumor resection.     

In vivo measurement of vascular modulation in experimental tumors using a fluorescent contrast agent

Abstract We compared the effectiveness of three optical techniques based on fluorescence imaging and spectroscopy with indocyanine green (ICG) contrast agent to evaluate in vivo the disruption of the active vasculature induced by a vascular targeting agent. The blood perfusion of the MDA-MB-435 tumor model transplanted in nude mice was estimated from the signal of the contrast agent measured immediately after its systemic injection in mice. Optical measurements were performed using a fluorescence imaging setup and a fiber-based time correlated single photon counting (TCSPC) apparatus. This latter apparatus was used to measure the tumor fluorescence in transmittance geometry and the change in the basal optical absorption induced by the contrast agent, thus providing an alternative estimation of the blood content in the tumor. Mice were divided into four groups. Three groups were treated with different doses of the vascular disrupting agent ZD6126, the fourth group (control group) received the drug vehicle only. Optical measurements were carried out 3 h after pharmacologic treatment. After 24 h, mice were killed, tumors were excised and the extent of necrosis was evaluated with standard histologic analysis. On fluorescence imaging ICG emission from tumors of mice treated with ZD6126 significantly was lower compared with the emission from control mice. The histologic sections also showed a significantly higher amount of necrosis in tumors of treated mice. Both these findings, which correlate with each other, indicate an effective vascular shutdown induced by the drug. However, ICG fluorescence measured with the TCSPC apparatus in transmittance geometry and the estimate of the change in optical absorption did not allow a statistically significant differentiation between treated and control groups.     

Synthesis of 7alpha-(fluoromethyl)dihydrotestosterone and 7alpha-(fluoromethyl)nortestosterone, structurally paired androgens designed to probe the role of sex hormone binding globulin in imaging androgen receptors in prostate tumors by positron emission tomography

Although prostate cancer growth is regulated by androgens through the androgen receptor (AR), in vitro assays of AR levels in prostate tumors have limited prognostic value. This might be improved by direct measurement of tumor AR in vivo using positron emission tomography (PET) imaging with fluorine-18-labeled androgens. Most AR PET imaging agents have been designed to limit steroid binding to serum proteins, but there is evidence that binding to sex hormone binding globulin (SHBG) might enhance tumor uptake. To probe the role of SHBG in prostate tumor uptake of PET imaging agents, we have synthesized two fluoro steroids, 7alpha-(fluoromethyl)dihydrotestosterone (7alpha-FM-DHT) and 7alpha-(fluoromethyl)nortestosterone (7alpha-FM-norT), by a route amenable to their labeling with [18F]fluoride ion. Both compounds have high affinity for AR, but 7alpha-FM-norT has much lower affinity for SHBG. Thus, these two fluoro steroids are well matched in terms of their site of fluorine labeling, similarity of structure, and equivalent AR binding affinity-but contrasting SHBG binding-and therefore can be used as agents for evaluating the role of SHBG binding in the target tissue uptake of AR PET imaging agents in humans.     

Near-infrared quantum dots for deep tissue imaging

Developments in nanotechnology have paved the way for the early detection, treatment, and prevention of several tumors which affect mankind. In the past few years, near-infrared (NIR) fluorescence imaging techniques have emerged that enable the in vivo imaging of physiological, metabolic, and molecular function. The NIR window, also known as the diagnostic window (700–900 nm), can be explored for sensitive detection techniques. Nanoparticles, particularly semiconductor quantum dots (QDs), can be utilized for the purpose of optical imaging. These semiconductor QDs possess novel electronic, optical, magnetic, and structural properties which are quite different from those of bulk materials. NIR QDs with these unique properties can be utilized as contrast agents for optical imaging, particularly for deep tissue imaging. Deep tissue imaging provides more information about the pathological status of the disease, which makes the treatment more effective and efficient. In this review we highlight the importance of NIR QDs as probes for optical imaging. We describe the different types of NIR QDs, their synthesis, and their application for deep tissue imaging along with recently developed self-illuminating NIR QDs.     

Tumor targeting by surface-modified protein microspheres

Protein microspheres have been used in the fields of biomedical imaging and drug delivery, but surface modification for cell targeting has been problematic. We have for the first time used an electrostatic adhesion approach to adhere arginine-glutamic acid-aspartic acid (RGD) containing peptides to the surface of protein microspheres for the purpose of targeting these vesicles to tumor cells. RGD sequences are recognized by integrin membrane receptors, which are overexpressed in various tumors. We have succeeded in modifying the surface of serum albumin core-shell microspheres, which have a fluorescent nonaqueous core by using several polylysine peptides containing the RGD sequence. Fluorescence microscopy reveals that these modified microspheres are selectively bound and taken up by HT29 human colon cancer cells in vitro.     

A far-red fluorescent contrast agent to image epidermal growth factor receptor expression

Recent developments in optical technologies have the potential to improve the speed and accuracy of screening and diagnosis of curable precancerous lesions and early cancer, thereby decreasing the costs of detection and management of epithelial malignancies. The development of molecular-specific contrast agents for markers of early neoplastic transformation could improve the detection and molecular characterization of premalignant lesions. In the oral cavity, epidermal growth factor receptor (EGFR) overexpression has been identified in early stages of premalignant lesions of the oral squamous cell carcinoma; therefore, real-time assessment of EGFR expression could serve as a biomarker for oral neoplasia. The purpose of our study was to develop a molecular-specific optical contrast agent targeted against EGFR for in vivo assessment of epithelial neoplasia using a monoclonal antibody and the far-red fluorescent dye, Alexa Fluor 660 streptavidin. In addition to demonstrating the specificity of the contrast agent for EGFR in cell lines, we document the ability to achieve penetration through 500 microm thick epithelial layers using multilayer tissue constructs and permeability-enhancing agents. Finally, using the fluorescence intensity of the contrast agent on fresh oral cavity tissue sections, we were able to distinguish abnormal from normal oral tissue. This contrast agent should have important clinical applications for use in conjunction with fluorescence spectroscopy or imaging (or both) to facilitate tumor detection and demarcation.     

Sensitive and selective tumor imaging with novel and highly activatable fluorescence probes.

Selective and sensitive tumor imaging in vivo is one of the most requested methodologies in medical sciences. Although several imaging modalities have been developed including positron emission tomography (PET) and magnetic resonance (MR) imaging for the detection of tumors, none of these modalities can activate the signals upon being accumulated or uptaken to tumor sites. Among these modalities, only optical fluorescence imaging has a marked advantage, that is, their signals can be dramatically increased upon detecting some biological features. In this short review, I will introduce some recent strategies for activatable optical fluorescence imaging of tumors, and discuss their advantages over other modalities.     

In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.     

A Preliminary Study of PSMA Fluorescent Probe for Targeted Fluorescence Imaging of Prostate Cancer

Purpose: With the increasing detection rate of early prostate cancer (PCa), the proportion of surgical treatment is increasing. Surgery is the most effective treatment for PCa. Precise targeting of tumors during surgery can reduce the incidence of positive surgical margins (PSMs) and preserve the neurovascular bundles (NVBs) as much as possible. The objective of this study was to synthesize a PSMA fluorescent probe (PSMA-Cy5) and verify the targeting specificity of the probe for prostate cancer, thereby providing a theoretical basis for the development of PSMA fluorescent probes for clinical application in the future. Methods: In this study, a novel water-soluble 3H-indocyanine-type bioluminescent dye-Cy5-labeled prostate-specific membrane antigen (PSMA) ligand (PSMA-Cy5) was synthesized by liquid phase synthesis. The PSMA ligand was developed based on the glutamine-urea-lysine (Glu-urea-Lys) structure. The new fluorescent probe was evaluated in vitro and in vivo, and its safety was evaluated. Confocal microscopy was used to observe the binding uptake of PSMA-Cy5 with PSMA (+) LNCaP cells, PSMA (-) PC3 cells and blocked LNCaP cells. In in vivo optical imaging studies, the targeting specificity of PSMA (+) 22Rv1 tumors to probe binding was validated by tail vein injection of PSMA-Cy5. The safety of the PSMA-Cy5 probe was evaluated by histopathological analysis of mouse organs by a single high-dose tail vein injection of PSMA-Cy5. Results: In vitro fluorescence cell uptake experiments showed that the binding of PSMA-Cy5 to LNCaP cells has targeting specificity. PC3 cells and blocked LNCaP cells showed almost no uptake. The results of in vivo optical imaging studies showed that the tumor-to-background ratio in the 22Rv1 group was 3.39 ± 0.47; in the 22Rv1 blocking group it was 0.78 ± 0.15, and in the PC3 group it was 0.94 ± 0.09, consistent with the in vitro results. After a high-dose injection of PSMA-Cy5, there were no abnormalities in the tissues or organs of the mice. The probe showed good safety. Conclusions: PSMA-Cy5 is a probe with good targeting specificity and low toxicity that can accurately visualize tumors in vivo. This study has an important reference value for the development of PSMA fluorescent probes. In the future, it can be applied to precise tumor imaging during radical prostatectomy to reduce the incidence of postoperative PSM.     

Biofunctional magnetic nanoparticles as contrast agents for magnetic resonance imaging of pancreas cancer

We report on the fabrication and characterization of biofunctional magnetic nanoparticles as contrast agents for magnetic resonance imaging. The anti-cancer antigen 19-9 monoclonal antibody (a cancer-targeting antibody) was conjugated onto the magnetic contrast agents in an effort to detect pancreatic tumor. The structure, size, morphology and magnetic property of the biofunctional magnetic nanoparticles are characterized systematically by means of transmission electron microscopy and X-ray diffractometry. Furthermore, the interaction between the nanoparticles and pancreas cancers cells are investigated by atomic force microscope and transmission electron microscopy. Magnetic resonance imaging demonstrates that the conjugated nanoparticles can effectively target cancer cells both in vitro and in vivo, suggesting that they potentially can be used as contrast agents for magnetic resonance imaging of pancreas cancer.     

A comparative study of the cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl) chlorin encapsulated in surface-modified submicronic oil/water carriers in HT29 tumor cells

The poor selectivity of photosensitizers for tumor tissue remains a drawback in photodynamic therapy (PDT) and could be improved by adapted formulations. The cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl)chlorin (mTHPC) encapsulated in submicronic colloidal carriers have been studied in macrophage-like J774 cells and HT 29 human adenocarcinoma cells. Nanocapsules with an external layer made of poly(D,L lactic acid) (PLA NCs), PLA grafted with polyethylene glycol (PLA-PEG NCs), PLA coated with poloxamer 188 (polox PLA NCs) and oil/water nanoemulsion (NE) have been examined. The cellular uptake by J774, as determined by microspectroflorimetry, is reduced with mTHPC encapsulated into surface-modified NCs--PLA-PEG and polox PLA--compared with naked PLA, indicating a possible limitation of the clearance of such carriers by the reticuloendothelial system. Encapsulation also modifies the interaction between mTHPC and HT29 cells. Compared with the manufacturer's solution (PEG, ethanol, water), the cellular uptake is strongly reduced. However, the HT29 phototoxicity is much less affected and a protecting effect against plasma proteins is observed. Fluorescence microscopy reveals a specific punctate fluorescence pattern with PLA-PEG and polox PLA NCs in contrast to a more diffuse distribution with NE and solution, indicating that photodamage targeting could be different. These findings suggest that photosensitizers encapsulated into surface-modified nanocapsules could be a promising approach for improving PDT efficacy and this has to be confirmed in vivo.     

Quantitative DNA imaging in breast tumor cells by a Hadamard transform fluorescence imaging microscope.

This paper presents a novel Hadamard transform (HT) fluorescence imaging microscope by combining multiplexed imaging technique with a conventional upright fluorescence microscope for single-cell imaging and quantitative cellular analysis. The HT imaging microscope can provide 511 x 512-pixel single-cell image with high sensitivity within 21 s. In this study, the high potential value of the microscope in biomedical analysis has been demonstrated by using it to evaluate the malignancy degree of thirty cases of human breast tumors based on the measurements of cellular DNA contents, with conclusions highly accordant with pathological diagnosis. The results show that the HT microscope has the ability to analyze very small specimens and the capability of detecting very high ploidy cells, which are advantages over flow cytometry. The microscope was also successfully applied to cellular morphological analysis, and it was demonstrated that a significant linear relationship exists between tumor nuclear DNA contents and the nuclear area, and malignant and benign tumors are significantly different in both DNA contents and nuclear area. The reliability of the HT microscope in cellular DNA measurements was also investigated.     

Development of a hypoxia-selective near-infrared fluorescent probe for non-invasive tumor imaging

A near-infrared fluorochrome, GPU-311, was designed, synthesized and evaluated for its application in non-invasive imaging of tumor hypoxia. Efficient synthesis was achieved by nucleophilic substitution and click chemistry ring using the bifunctional tetraethylene glycol linker 2 containing thiol and azide groups for the conjugation of the propargylated nitroimidazole 1 and the heptamethine cyanine dye 3 bearing a 2-chloro-1-cyclohexenyl ring. GPU-311 exhibited long excitation and emission wavelength (Ex/Em=785/802?nm) and a decent quantum yield (0.05). The water solubility and hydrophilicity of GPU-311 increased. After in vitro treatment of SUIT-2/HRE-Luc pancreatic cancer cells with GPU-311, a higher level of fluorescence was observed selectively in hypoxia than in normoxia. However, in vivo fluorescence imaging of a mouse xenograft model after GPU-311 administration revealed inadequate accumulation of GPU-311 in tumors due to its rapid elimination through the liver.     

The serum protein binding of pharmacologically active gallium(III) compounds assessed by hyphenated CE‐MS techniques

Transition metal‐based drugs exhibit high affinity to the soft donors of human serum proteins, especially of the high‐abundance protein HSA and of transferrin (Tf), whereas Ga(III) salts are known to bind to Tf and other iron‐containing metalloproteins, thereby interfering with the iron metabolism. Herein, the utilization of CE‐MS methods for studying the binding behavior of a therapeutic gallium nitrate formulation and the anticancer drug candidate Tris(8‐oxyquinolinato)gallium(III) to Tf and HSA under simulated physiological conditions is described. Both the Ga(III) salt and the complex were found to bind to Tf exclusively in the presence of carbonate, however, at different kinetics and to a different extent. Fe(III) induces the release of the Ga ions due to the higher affinity constant and also prevents the Ga(III) species from accessing the iron‐binding pockets of Tf. In contrast, only low affinity to HSA was observed and even when present at ca. 20‐fold excess, the majority of the Ga was attached to Tf.     

In vivo fluorescence imaging in the second near-infrared window with long circulating carbon nanotubes capable of ultrahigh tumor uptake

Cancer imaging requires selective high accumulation of contrast agents in the tumor region and correspondingly low uptake in healthy tissues. Here, by making use of a novel synthetic polymer to solubilize single-walled carbon nanotubes (SWNTs), we prepared a well-functionalized SWNT formulation with long blood circulation (half-life of ～30 h) in vivo to achieve ultrahigh accumulation of ～30% injected dose (ID)/g in 4T1 murine breast tumors in Balb/c mice. Functionalization dependent blood circulation and tumor uptake were investigated through comparisons with phospholipid-PEG solubilized SWNTs. For the first time, we performed video-rate imaging of tumors based on the intrinsic fluorescence of SWNTs in the second near-infrared (NIR-II, 1.1-1.4 μm) window. We carried out dynamic contrast imaging through principal component analysis (PCA) to immediately pinpoint the tumor within ～20 s after injection. Imaging over time revealed increasing tumor contrast up to 72 h after injection, allowing for its unambiguous identification. The 3D reconstruction of the SWNTs distribution based on their stable photoluminescence inside the tumor revealed a high degree of colocalization of SWNTs and blood vessels, suggesting enhanced permeability and retention (EPR) effect as the main cause of high passive tumor uptake of the nanotubes.     

二{[5,5'-10,20-二苯基-15-(4-吡啶基)卟啉]Zn(Ⅱ)]}丁二炔近红外荧光化合物的合成及光学性质

合成了具有近红外荧光特性的卟啉化合物二{[5,5’-10,20-二苯基-15-(4-吡啶基)卟啉]Zn(Ⅱ)]}丁二炔,通过核磁共振波谱和质谱对其结构进行了确认.通过紫外吸收光谱法测定了其光学性质,利用HWHR光学平台测定了化合物在近红外区激发产生的荧光,并通过动物组织模型评价其荧光深层次成像效果.结果表明,该化合物具...     

In vivo fluorescence spectroscopy of nonmelanoma skin cancer

In vivo and ex vivo tissue autofluorescence (endogenous fluorescence) have been employed to investigate the presence of markers that could be used to detect tissue abnormalities and/or malignancies. We present a study of the autofluorescence of normal skin and tumor in vivo, conducted on 18 patients diagnosed with nonmelanoma skin cancers (NMSC). We observed that both in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) the endogenous fluorescence due to tryptophan residues was more intense in tumor than in normal tissue, probably due to epidermal thickening and/or hyperproliferation. Conversely, the fluorescence intensity associated with dermal collagen crosslinks was generally lower in tumors than in the surrounding normal tissue, probably because of degradation or erosion of the connective tissue due to enzymes released by the tumor. The decrease of collagen fluorescence in the connective tissue adjacent to the tumor loci was validated by fluorescence imaging on fresh-frozen tissue sections obtained from 33 NMSC excised specimens. Our results suggest that endogenous fluorescence of NMSC, excited in the UV region of the spectrum, has characteristic features that are different from normal tissue and may be exploited for noninvasive diagnostics and for the detection of tumor margins.     

A Dendron-Based Fluorescence Turn-On Probe for Tumor Detection

Specifically amplifying the emission signals of optical probes in tumors is an effective way to improve the tumor-imaging sensitivity and contrast. In this paper, the first case of dendron-based fluorescence turn-on probes mediated by a Förster resonance energy transfer (FRET) mechanism is reported. Dendrons up to the fourth generation with a hydrophilic oligo(ethylene glycol) scaffold are synthesized by a solid-phase synthesis strategy, and show precise and defect-free chemical structures. To construct the fluorescence turn-on probe, one Cy5.5 molecule is conjugated to the focal of a G3 dendron through a robust linkage and eight Black Hole Quencher 3 (BHQ-3) molecules are conjugated to its periphery through a PEG chain bearing a reductively cleavable disulfide linkage. By in vitro and in vivo experiments, it is demonstrated that the fluorescence of the dendron-based probe can be activated effectively and rapidly in the reductive environments of tumor cells and tissues, and the probe thus exhibits amplified tumor signals and weak normal tissue signals. Compared with the reported nanoscale turn-on probes, the dendron-based probe has several significant advantages, such as well-defined chemical structure, precisely controllable fluorophore/quencher conjugation sites and ratio, desirable chemical stability, and reproducible pharmacokinetic and pharmacological profiles, and is very promising in tumor detection.