Celeste Regino

Toxicity of organic fluorophores used in molecular imaging: literature review.

Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of 19 widely used fluorophores was conducted by searching 26 comprehensive biomedical and chemical literature databases and analyzing the retrieved material. These fluorophores included Alexa Fluor 488 and 514, BODIPY FL, BODIPY R6G, Cy 5.5, Cy 7, cypate, fluorescein, indocyanine green, Oregon green, 8-phenyl BODIPY, rhodamine 110, rhodamine 6G, rhodamine X, rhodol, TAMRA, Texas red, and Tokyo green. Information regarding cytotoxicity, tissue toxicity, in vivo toxicity, and mutagenicity was included. Considerable toxicity-related information was available for the Food and Drug Administration (FDA)-approved compounds indocyanine green and fluorescein, but published information on many of the non-FDA-approved fluorophores was limited. The information located was encouraging because the amounts of fluorophore used in molecular imaging probes are typically much lower than the toxic doses described in the literature. Ultimately, the most effective and appropriate probes for use in patients will be determined by their fluorescent characteristics and the safety of the conjugates.

Multimodal Nanoprobes for Radionuclide and Five-Color Near-Infrared Optical Lymphatic Imaging

Current contrast agents generally have one function and can only be imaged in monochrome; therefore, the majority of imaging methods can only impart uniparametric information. A single nanoparticle has the potential to be loaded with multiple payloads. Such multimodality probes have the ability to be imaged by more than one imaging technique, which could compensate for the weakness or even combine the advantages of each individual modality. Furthermore, optical imaging using different optical probes enables us to achieve multicolor in vivo imaging, wherein multiple parameters can be read from a single image. To allow differentiation of multiple optical signals in vivo, each probe should have a close but different near-infrared emission. To this end, we synthesized nanoprobes with multimodal and multicolor potential, which employed a polyamidoamine dendrimer platform linked to both radionuclides and optical probes, permitting dual-modality scintigraphic and five-color near-infrared optical lymphatic imaging using a multiple-excitation spectrally resolved fluorescence imaging technique.

A dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fluorescence imaging to localize the sentinel lymph node in mice

To preoperatively and intraoperatively localize the sentinel lymph node (SLN), a single hybrid probe for MR and near infrared (NIR) optical imaging was synthesized and tested.

Synthesis, Characterization, and Biological Evaluation of Integrin αvβ3-Targeted PAMAM Dendrimers

Ligand size and valency strongly influence the receptor uptake and clearance of tumor angiogenesis imaging agents. The structures of successful imaging agents exhibit a high degree of variability, encompassing small monovalent arginine-glycine-aspartic acid (RGD)-containing peptides, multivalent RGD-oligomers, and a monoclonal antibody against integrin alpha-v-beta-3 (alpha-v-beta-3). We have pursued a nanoscale approach to imaging of angiogenesis using rationally designed polyamidoamine (PAMAM) dendrimers covalently adorned with RGD-cyclopeptides. An orthogonal oxime-ligation strategy was applied to chemoselectively effect conjugation of the PAMAM dendrimers with RGD-cyclopeptides for targeting alpha vbeta 3. Fluorescent dyes for optical imaging and chelates for gadolinium-based magnetic resonance (MR) imaging were subsequently appended to create robust multimodal macromolecular imaging agents. Fluorescence microscopy revealed selective binding of the resulting RGD peptide-bearing dendrimer with empty chelates to alpha-v-beta-3-expressing cells, but somewhat reduced selectivity was observed following Gd(III) complexation. The expected incomplete saturation of chelates with Gd(III) ions permitted radiometal complexation, and an in vivo tissue distribution of the resulting agent in M21 melanoma tumor-bearing mice showed mostly renal and reticuloendothelial accumulation, with the tumor:blood ratio peaking (3.30+/-0.03) at 2 h postinjection.

Influence of dendrimer generation and polyethylene glycol length on the biodistribution of PEGylated dendrimers.

Dendrimers are a potential drug carrier. Because modification with polyethylene glycol (PEG) is known to improve the blood retention, PEGylated dendrimers have been studied as a useful drug carrier. In this study, three types of PEGylated L-lysine-bearing polyamidoamine dendrimers (PEG2k-Lys-PAMAM (G4), PEG5k-Lys-PAMAM (G4), PEG2k-Lys-PAMAM (G5)) were synthesized, which are composed of a dendrimer of different generations (generations 4 and 5) and PEG chains with different molecular weights (2k and 5k). An acetylated L-lysine-bearing dendrimer was also synthesized as a non-PEGylated dendrimer. Bifunctional diethylenetriaminepentaacetic acid (pSCN-benzyl-DTPA) was bound to the epsilon -amino group of lysine in a dendrimer, to be labeled with radioactive indium-111. These PEGylayed dendrimers showed longer blood retention and lower accumulation in other normal organs such as the kidneys than the non-PEGylated dendrimer. The PEGylated dendrimers with the higher generation and the longer PEG led the greater blood retention.

In vivo target-specific activatable near-infrared optical labeling of humanized monoclonal antibodies

Imaging with labeled monoclonal antibodies may be useful in detecting, staging, and monitoring tumors. Despite their high affinity and specificity, a critical limitation of antibody imaging is the high background signal due to prolonged clearance from the blood, which reduces the tumor-to-background ratio. To address this problem, we developed a molecular imaging probe consisting of multiple self-quenching fluorophores [Cy5.5 or Alexa Fluor 680 (Alexa680)] conjugated to a monoclonal antibody (trastuzumab) to synthesize Tra-Cy5.5(SQ) or Tra-Alexa680(SQ), respectively. This agent only becomes fluorescently “active” after cellular internalization but is quenched in the unbound state leading to high tumor-to-background ratios. The in vitro quenching capacity for both conjugates was ∼9-fold. In vivo imaging experiments were done in mice bearing both 3T3/HER-2+ and BALB/3T3/ZsGreen/HER-2− xenografts. Tra-Alexa680(SQ) produced specific enhancement in the 3T3/HER-2+ tumors but not in the HER-2− control tumors. However, Tra-Cy5.5(SQ) produced nonspecific enhancement in both 3T3/HER-2+ and control tumors. In conclusion, whereas Cy5.5-conjugates produced nonspecific results as well as rapid liver accumulation, conjugating multiple Alexa680 molecules to a single monoclonal antibody resulted in a near-infrared optical agent that activated within specific target tumors with high tumor-to-background ratio with considerable potential for clinical translation. [Mol Cancer Ther 2009;8(1):232–9]

Dual-modality molecular imaging using antibodies labeled with activatable fluorescence and a radionuclide for specific and quantitative targeted cancer detection

Multimodality molecular imaging should have potential for compensating the disadvantages and enhancing the advantages of each modality. Nuclear imaging is superior to optical imaging in whole body imaging and in quantification due to good tissue penetration of gamma rays. However, target specificity can be compromised by high background signal due to the always signal ON feature of nuclear probes. In contrast, optical imaging can be superior in target-specific imaging by employing target-specific signal activation systems, although it is not quantitative because of signal attenuation. In this study, to take advantage of the mutual cooperation of each modality, multimodality imaging was performed by a combination of quantitative radiolabeled probe and an activatable optical probe. The monoclonal antibodies, panitumumab (anti-HER1) and trastuzumab (anti-HER2), were labeled with 111In and ICG and tested in both HER1 and HER2 tumor bearing mice by the cocktail injection of radiolabeled and optical probes and by the single injection of a dual-labeled probe. The optical and nuclear images were obtained over 6 days after the conjugates injection. The fluorescence activation properties of ICG labeled antibodies were also investigated by in vitro microscopy. In vitro microscopy demonstrated that there was no fluorescence signal with either panitumumab-ICG or trastuzumab-ICG, when the probes were bound to cell surface antigens but were not yet internalized. After the conjugates were internalized into the cells, both conjugates showed bright fluorescence signal only in the target cells. These results show that both conjugates work as activatable probes. In in vivo multimodality imaging by injection of a cocktail of radio-optical probes, only the target specific tumor was visualized by optical imaging. Meanwhile, the biodistribution profile of the injected antibody was provided by nuclear imaging. Similar results were obtained with radio and optical dual-labeled probes, and it is confirmed that pharmacokinetic properties did not affect the results above. Here, we could characterize the molecular targets by activatable optical probes and visualize the delivery of targeting molecules quantitatively by radioactive probes. Multimodality molecular imaging combining activatable optical and radioactive probes has great potential for simultaneous visualization, characterization, and measurement of biological processes.

Dendrimer-based MRI contrast agents: the effects of PEGylation on relaxivity and pharmacokinetics

UNLABELLED Polyethylene glycol (PEG) surface modification can make nanomaterials highly hydrophilic, reducing their sequestration in the reticuloendothelial system. In this study, polyamidoamine (PAMAM) dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated. Specifically, Gd chelate-bearing PAMAM dendrimers (generations 4 and 5; G4 and G5) were conjugated with two different PEG chains (2 kDa and 5 kDa; 2k and 5k). Long PEG chains (5k) on the smaller (G4) dendrimer resulted in reduced relaxivity compared to non-PEGylated dendrimers, whereas short PEG chains (2k) on a larger (G5) dendrimer produced relaxivities comparable to non-PEGylated G4 dendrimers. The relaxivity of all PEGylated or lysine-conjugated dendrimers increased at higher temperature, whereas that of intact G4 Gd-PAMAM dendrimer decreased. All PEGylated dendrimers had minimal liver and kidney uptake and remained in circulation for at least 1 hour. Thus, surface-PEGylated Gd-PAMAM dendrimers showed decreased plasma clearance and prolonged retention in the blood pool. Shorter PEG, higher generation conjugates led to higher relaxivity. FROM THE CLINICAL EDITOR In this study, polyamidoamine dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated.

Determination of Optimal Rhodamine Fluorophore for in Vivo Optical Imaging

Optical imaging has the potential to improve the efficacy of surgical and endoscopic approaches to cancer treatment; however, the optimal type of fluorescent probe has not yet been established. It is well-known that rhodamine-core-derived fluorophores offer a combination of desirable properties such as good photostability, high extinction coefficient, and high fluorescence quantum yield. However, despite the ubiquitous use of rhodamine fluorophores for in vivo optical imaging, it remains to be determined if unique chemical properties among individual rhodamine core family members affect fluorophore parameters critical to in vivo optical imaging applications. These parameters include preserved fluorescence intensity in low pH environments, similar to that of the endolysosome; efficient fluorescence signal despite conformational changes to targeting proteins as may occur in harsh subcellular environments; persistence of fluorescence after cellular internalization; and sufficient signal-to-background ratios to permit the identification of fluorophore-targeted tumors. In the present study, we conjugated 4 common rhodamine-core based fluorescent dyes to a clinically feasible and quickly internalizing D-galactose receptor targeting reagent, galactosamine serum albumin (GmSA), and conducted a series of in vitro and in vivo experiments using a metastatic ovarian cancer mouse model to determine if differences in optical imaging properties exist among rhodamine fluorophores and if so, which rhodamine core possesses optimal characteristics for in vivo imaging applications. Herein, we demonstrate that the rhodamine-fluorophore, TAMRA, is the most robust of the 4 common rhodamine fluorophores for in vivo optical imaging of ovarian cancer metastases to the peritoneum.

A dual CT-MR dendrimer contrast agent as a surrogate marker for convection-enhanced delivery of intracerebral macromolecular therapeutic agents.

The feasibility of using Gd dendrimer-based macromolecules (Gd-G8 dendrimer) as a dual CT and MR contrast agent for monitoring convection-enhanced delivery of therapy in the brain is evaluated both in vitro and in vivo with optimal dosing established. In vitro CT attenuation values of the Gd-based agents ( approximately 6.0 HU mM(-1)) were approximately 1.6 times greater than iodine-based agents and the attenuation of the Gd-DTPA was comparable to Gd-G8 dendrimer. Visible enhancement was observed on both CT and MR using Gd-G8 dendrimer over a range of 23-78 mM; however, a concentration of at least 47 mM in Gd was required for adequate delineation of the injection site on both CT and MR. MR offers greater sensitivity than CT in estimating the volume of distribution (V(d)) and effectively quantified the agents concentration and diffusion using T(1) mapping at much lower concentrations of Gd (<10 mM in [Gd]).