Multimodal Prussian blue analogs as contrast agents for X-ray computed tomography.

Prussian blue analogs (PBAs) are versatile materials with a wide range of applications. Due to their tunability, intrinsic biocompatibility, as well as low toxicity, these nanoscale coordination polymers have been successfully studied as multimodal contrast agents for multiple imaging techniques. Herein, we report the expanded biomedical application of PBAs to X-ray computed tomography (CT). In our systematic study of the series A{MnII[FeIII(CN)6]} (A = K+, Rb+, Cs+), we showed that derivatives incorporating Rb+ and Cs+ ions in the tetrahedral sites of the parent face-centered cubic cyano-bridged networks exhibited substantially increased X-ray attenuation coefficients, thus yielding significant contrast compared to the clinically approved X-ray contrast agent iohexol at the same concentrations. Additionally, our µ-CT studies revealed that these PBAs could be useful as dual-energy CT contrast agents for different biological specimens by using the lower varying scanning X-ray tube voltages. Finally, in vitro studies using U87-Luc cells treated with PBAs, including cellular CT imaging and bioluminescence cell viability assays, revealed that PBAs were taken up by the glioblastoma cells, with moderate biocompatibility at concentrations below the mM range.

A corrole nanobiologic elicits tissue-activated MRI contrast enhancement and tumor-targeted toxicity.

Water-soluble corroles with inherent fluorescence can form stable self-assemblies with tumor-targeted cell penetration proteins, and have been explored as agents for optical imaging and photosensitization of tumors in pre-clinical studies. However, the limited tissue-depth of excitation wavelengths limits their clinical applicability. To examine their utility in more clinically-relevant imaging and therapeutic modalities, here we have explored the use of corroles as contrast enhancing agents for magnetic resonance imaging (MRI), and evaluated their potential for tumor-selective delivery when encapsulated by a tumor-targeted polypeptide. We have found that a manganese-metallated corrole exhibits significant T1 relaxation shortening and MRI contrast enhancement that is blocked by particle formation in solution but yields considerable MRI contrast after tissue uptake. Cell entry but not low pH enables this. Additionally, the corrole elicited tumor-toxicity through the loss of mitochondrial membrane potential and cytoskeletal breakdown when delivered by the targeted polypeptide. The protein-corrole particle (which we call HerMn) exhibited improved therapeutic efficacy compared to current targeted therapies used in the clinic. Taken together with its tumor-preferential biodistribution, our findings indicate that HerMn can facilitate tumor-targeted toxicity after systemic delivery and tumor-selective MR imaging activatable by internalization.

Cellular uptake and cytotoxicity of a near-IR fluorescent corrole-TiO2 nanoconjugate.

We are investigating the biological and biomedical imaging roles and impacts of fluorescent metallocorrole-TiO2 nanoconjugates as potential near-infrared optical contrast agents in vitro in cancer and normal cell lines. The TiO2 nanoconjugate labeled with the small molecule 2,17-bis(chlorosulfonyl)-5,10,15-tris(pentafluorophenyl)corrolato aluminum(III) (1-Al-TiO2) was prepared. The nanoparticle 1-Al-TiO2 was characterized by transmission electron microscopy (TEM) and integrating-sphere electronic absorption spectroscopy. TEM images of three different samples of TiO2 nanoparticles (bare, H2O2 etched, and 1-Al functionalized) showed similarity in shapes and sizes with an average diameter of 29nm for 1-Al-TiO2. Loading of 1-Al on the TiO2 surfaces was determined to be ca. 20-40mg 1-Al/g TiO2. Confocal fluorescence microscopy (CFM) studies of luciferase-transfected primary human glioblastoma U87-Luc cells treated with the nanoconjugate 1-Al-TiO2 as the contrast agent in various concentrations were performed. The CFM images revealed that 1-Al-TiO2 was found inside the cancer cells even at low doses (0.02-2µg/mL) and localized in the cytosol. Bioluminescence studies of the U87-Luc cells exposed to various amounts of 1-Al-TiO2 showed minimal cytotoxic effects even at higher doses (2-2000µg/mL) after 24h. A similar observation was made using primary mouse hepatocytes (PMH) treated with 1-Al-TiO2 at low doses (0.0003-3µg/mL). Longer incubation times (after 48 and 72h for U87-Luc) and higher doses (>20µg/mL 1-Al-TiO2 for U87-Luc and >3µg/mL 1-Al-TiO2 for PMH) showed decreased cell viability.

Decorating metal oxide surfaces with fluorescent chlorosulfonated corroles.

We have prepared 2,17-bis(chlorosulfonyl)-5,10,15-tris(pentafluorophenyl)corrole (1), 2,17-bis(chlorosulfonyl)-5,10,15-tris(pentafluorophenyl)corrolatoaluminum(III) (1-Al), and 2,17-bis(chlorosulfonyl)-5,10,15-tris(pentafluorophenyl)corrolatogallium(III) (1-Ga). The metal complexes 1-Al and 1-Ga were isolated and characterized by electronic absorption and NMR spectroscopies, as well as by mass spectrometry. Relative emission quantum yields for 1, 1-Al, and 1-Ga, determined in toluene, are 0.094, 0.127, and 0.099, respectively. Reactions between 1, 1-Al, and 1-Ga and TiO2 nanoparticles (NPs) result in corrole-TiO2 NP conjugates. The functionalized NP surfaces were investigated by solid-state Fourier transform infrared and X-ray photoelectron spectroscopies and by confocal fluorescence imaging. The fluorescence images for 1-Al-TiO2 and 1-Ga-TiO2 suggest a promising application of these NP conjugates as contrast agents for noninvasive optical imaging.

Large Field of View Scanning Fluorescence Lifetime Imaging System for Multimode Optical Imaging of Small Animals.

We describe a scanning fluorescence lifetime imaging (SFLIM) system that provides a large field of view (LFOV), using a femtosecond (fs) pulsed laser, for multi-mode optical imaging of small animals. Fluorescence lifetime imaging (FLIM) can be a useful optical method to distinguish between fluorophores inside small animals. However, difficulty arises when LFOV is required in FLIM using a fs pulsed laser for the excitation of the fluorophores at low wavelengths (<500nm), primarily because the field of view of the pulsed blue excitation light generated from the second harmonic of the fs pulsed light is limited to about a centimeter in diameter due to the severe scattering and absorption of the light inside tissues. Here, we choose a scanning method in order to acquire a FLIM image with LFOV as one alternative. In the SFLIM system, we used a conventional cooled CCD camera coupled to an ultra-fast time-gated intensifier, a tunable femtosecond laser for the excitation of fluorophores, and an x-y moving stage for scanning. Images acquired through scanning were combined into a single image and then this reconstructed image was compared with images obtained by spectral imaging. The resulting SFLIM system is promising as an alternative method for the FLIM imaging of small animals, containing fluorophores exited by blue light, for LFOV applications such as whole animal imaging.

Amphiphilic corroles bind tightly to human serum albumin.

Amphiphilic 2,17-bis-sulfonato-5,10,15(trispentafluorophenyl)corrole (2) and its Ga and Mn complexes (2-Ga and 2-Mn) form tightly bound noncovalent conjugates with human serum albumin (HSA). Protein-induced changes in the electronic absorption, emission, and circular dichroism spectra of these corroles, as well as results obtained from HPLC profiles of the conjugates and selective fluorescence quenching of the single HSA tryptophan, are interpreted in terms of multiple corrole:HSA binding sites. High-affinity binding sites, close to the unique tryptophan, are fully occupied at very low concentrations. At biologically relevant HSA concentrations (2-3 orders of magnitude larger than those employed in our studies), all corroles (2, 2-Ga, and 2-Mn) may be considered as fully conjugated.

Isolation of the novel dirhodium(II/II) thiolate compound Rh(2)(eta(1)-C(6)H(5)S)(2)(mu-C(6)H(5)S)(2)(bpy)(2).

The reaction of the anticancer active compound [Rh(2)(mu-O(2)CCH(3))(2)(bpy)(2)(CH(3)CN)(2)][BF(4)](2) (1) (bpy = 2,2'-bipyridine) with NaC(6)H(5)S under anaerobic conditions yields Rh(2)(eta(1)-C(6)H(5)S)(2)(mu-C(6)H(5)S)(2)(bpy)(2).CH(3)OH (2), which was characterized by UV-visible, IR, and (1)H NMR spectroscopies as well as single-crystal X-ray crystallography. Compound 2 crystallizes as dark red platelets in the monoclinic space group C2/c with cell parameters a = 20.398(4) A, b = 11.861(2) A, c = 17.417(4) A, beta = 108.98 degrees, V = 3984.9(14) A(3), Z = 4. The main structural features are the presence of a [Rh(2)](4+) core with a Rh-Rh distance of 2.549(2) A bridged by two benzene thiolate ligands in a butterfly-type arrangement. The axial positions of the [Rh(2)](4+) core are occupied by two terminal benzene thiolates. Cyclic voltammetric studies of 2 reveal that the compound exhibits an irreversible oxidation at +0.046 V in CH(3)CN, which is in accord with the fact that the compound readily oxidizes in the presence of O(2). The fact that this unusual dirhodium(II/II) thiolate compound is formed under these conditions is an important first step in understanding the metabolism of dirhodium anticancer active compounds with thiol-containing peptides and proteins.

Inhibition of transcription in vitro by anticancer active dirhodium(II) complexes.

The DNA binding and inhibition of transcription in vitro by neutral Rh(2)(mu-O(2)CCH(3))(4) and cationic cis-[Rh(2)(mu-O(2)CCH(3))(2)(phen)(2)](2+) complexes were investigated. The binding constants of the two complexes to calf-thymus DNA were estimated from absorption titrations to be 4.6 x 10(2) M(-)(1) and 1.7 x 10(4) M(-)(1) for Rh(2)(mu-O(2)CCH(3))(4) and cis-[Rh(2)(mu-O(2)CCH(3))(2)(phen)(2)](2+), respectively. The shift to higher energies of the low-energy absorption of the complexes upon addition of DNA is consistent with axial binding of the complexes to duplex DNA. The relative concentrations, [complex]/[DNA], of Rh(2)(mu-O(2)CCH(3))(4) and cis-[Rh(2)(mu-O(2)CCH(3))(2)(phen)(2)](2+) at which 50% of the transcription is inhibited (R(inh)(50)), are 0.0031 and 0.0011, respectively. These concentrations are significantly lower than that required for activated cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), with R(inh)(50) = 0.0085 under similar experimental conditions. Upon incubation of cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) with the template DNA prior to the addition of the enzyme and nucleobases necessary for the transcription reaction for 30 min at 37 degrees C, significantly lower concentrations of the complex were required to attain 50% inhibition. In contrast, similar incubation of the DNA with the dirhodium complexes did not result in better transcription inhibition. Experiments designed to elucidate the mechanism of the observed inhibition indicate that, unlike cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), Rh(2)(mu-O(2)CCH(3))(4) and cis-[Rh(2)(mu-O(2)CCH(3))(2)(phen)(2)](2+) appear to interact directly with the enzyme T7-RNA polymerase as their mode of inhibition.