Structural Investigations, Cellular Imaging, and Radiolabeling of Neutral, Polycationic, and Polyanionic Functional Metalloporphyrin Conjugates.

Over the past decade, porphyrin derivatives have emerged as invaluable synthetic building blocks and theranostic kits for the delivery of cellular fluorescence imaging and photodynamic therapy. Tetraphenylporphyrin (TPP), its metal complexes, and related derivatives have been investigated for their use as dyes in histology and as components of multimodal imaging probes. The photophysical properties of porphyrin-metal complexes featuring radiometals have been a focus of our attention for the realization of fluorescence imaging probes coupled with radioimaging capabilities and therapeutic potential having "true" theranostic promise. We report hereby on the synthesis, radiochemistry, structural investigations, and preliminary in vitro and in vivo uptake studies on a range of functionalized porphyrin-based derivatives. In pursuit of developing new porphyrin-based probes for multimodality imaging applications, we report new functionalized neutral, polycationic, and polyanionic porphyrins incorporating nitroimidazole and sulfonamide moieties, which were used as targeting groups to improve the notoriously poor pharmacokinetics of porphyrin tags. The resulting functional metalloporphyrin species were stable under serum challenges and the nitroimidazole and sulfonamide derivatives remained fluorescent, allowing in vitro confocal studies and visualization of the lysosomal uptake in a gallium(III) sulfonamide derivative. The molecular structures of selected porphyrin derivatives were determined by single crystal X-ray diffraction using synchrotron radiation. We also investigated the nature of the emission/excitation behavior of model functional porphyrins using in silico approaches such as TD DFT in simple solvation models. The conjugation of porphyrins with the [7-13] and [7-14] fragments of bombesin was also achieved, to provide targeting of the gastrin releasing peptide receptor (GRPR). Depending on the metal, probe conjugates of relevance for single photon emission computed tomography (SPECT) or positron emission tomography (PET) probes have been designed and tested hereby, using TPP and related functional free base porphyrins as the bifunctional chelator synthetic scaffold and 111In[In] or 68Ga[Ga], respectively, as the central metal ions. Interestingly, for simple porphyrin conjugates good radiochemical incorporation was obtained for both radiometals, but the presence of peptides significantly diminished the radio-incorporation yields. Although the gallium-68 radiochemistry of the bombesin conjugates did not show radiochemical incorporation suitable for in vivo studies, likely because the presence of the peptide changed the behavior of the TPP-NH2 synthon taken alone, the optical imaging assays indicated that the conjugated peptide tags do mediate uptake of the porphyrin units into cells.

Preliminary Study of a 1,5-Benzodiazepine-Derivative Labelled with Indium-111 for CCK-2 Receptor Targeting.

The cholecystokinin-2 receptor (CCK-2R) is overexpressed in several human cancers but displays limited expression in normal tissues. For this reason, it is a suitable target for developing specific radiotracers. In this study, a nastorazepide-based ligand functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator (IP-001) was synthesized and labelled with indium-111. The radiolabeling process yielded >95% with a molar activity of 10 MBq/nmol and a radiochemical purity of >98%. Stability studies have shown a remarkable resistance to degradation (>93%) within 120 h of incubation in human blood. The in vitro uptake of [111In]In-IP-001 was assessed for up to 24 h on a high CCK-2R-expressing tumor cell line (A549) showing maximal accumulation after 4 h of incubation. Biodistribution and single photon emission tomography (SPECT)/CT imaging were evaluated on BALB/c nude mice bearing A549 xenograft tumors. Implanted tumors could be clearly visualized after only 4 h post injection (2.36 ± 0.26% ID/cc), although a high amount of radiotracer was also found in the liver, kidneys, and spleen (8.25 ± 2.21%, 6.99 ± 0.97%, and 3.88 ± 0.36% ID/cc, respectively). Clearance was slow by both hepatobiliary and renal excretion. Tumor retention persisted for up to 24 h, with the tumor to organs ratio increasing over-time and ending with a tumor uptake (1.52 ± 0.71% ID/cc) comparable to liver and kidneys.

Applications of "Hot" and "Cold" Bis(thiosemicarbazonato) Metal Complexes in Multimodal Imaging.

The applications of coordination chemistry to molecular imaging has become a matter of intense research over the past 10 years. In particular, the applications of bis(thiosemicarbazonato) metal complexes in molecular imaging have mainly been focused on compounds with aliphatic backbones due to the in vivo imaging success of hypoxic tumors with PET (positron emission tomography) using (64) CuATSM [copper (diacetyl-bis(N4-methylthiosemicarbazone))]. This compound entered clinical trials in the US and the UK during the first decade of the 21(st) century for imaging hypoxia in head and neck tumors. The replacement of the ligand backbone to aromatic groups, coupled with the exocyclic N's functionalization during the synthesis of bis(thiosemicarbazones) opens the possibility to use the corresponding metal complexes as multimodal imaging agents of use, both in vitro for optical detection, and in vivo when radiolabeled with several different metallic species. The greater kinetic stability of acenaphthenequinone bis(thiosemicarbazonato) metal complexes, with respect to that of the corresponding aliphatic ATSM complexes, allows the stabilization of a number of imaging probes, with special interest in "cold" and "hot" Cu(II) and Ga(III) derivatives for PET applications and (111) In(III) derivatives for SPECT (single-photon emission computed tomography) applications, whilst Zn(II) derivatives display optical imaging properties in cells, with enhanced fluorescence emission and lifetime with respect to the free ligands. Preliminary studies have shown that gallium-based acenaphthenequinone bis(thiosemicarbazonato) complexes are also hypoxia selective in vitro, thus increasing the interest in them as new generation imaging agents for in vitro and in vivo applications.

Shedding light on the use of graphene oxide-thiosemicarbazone hybrids towards the rapid immobilisation of methylene blue and functional coumarins.

Coumarins, methylene blue derivatives, as well as related functional organic dyes have become prevalent tools in life sciences and biomedicine. Their intense blue fluorescence emission makes them ideal agents for a range of applications, yet an unwanted facet of the interesting biological properties of such probes presents a simultaneous environmental threat due to inherent toxicity and persistence in aqueous media. As such, significant research efforts now ought to focus on their removal from the environment, and the sustainable trapping onto widely available, water dispersible and processable adsorbent structures such as graphene oxides could be advantageous. Additionally, flat and aromatic bis(thiosemicarbazones) (BTSCs) have shown biocompatibility and chemotherapeutic potential, as well as intrinsic fluorescence, hence traceability in the environment and in living systems. A new palette of graphene oxide-based hierarchical supramolecular materials incorporating BTSCs were prepared, characterised, and reported hereby. We report on the supramolecular entrapping of several flat, aromatic fluorogenic molecules onto graphene oxide on basis of non-covalent interactions, by virtue of their structural features with potential to form aromatic stacks and H-bonds. The evaluations of the binding interactions in solution by between organic dyes (methylene blue and functional coumarins) and new graphene oxide-anchored Zn(ii) derivatised bis(thiosemicarbazones) nanohybrids were carried out by UV-Vis and fluorescence spectroscopies.

Α Humanized RANKL Transgenic Mouse Model of Progestin-Induced Mammary Carcinogenesis for Evaluation of Novel Therapeutics.

Receptor activator of nuclear factor-κB ligand (RANKL) is critically involved in mammary gland pathophysiology, while its pharmaceutical inhibition is being currently investigated in breast cancer. Herein, we investigated whether the overexpression of human RANKL in transgenic mice affects hormone-induced mammary carcinogenesis, and evaluated the efficacy of anti-RANKL treatments, such as OPG-Fc targeting both human and mouse RANKL or Denosumab against human RANKL. We established novel MPA/DMBA-driven mammary carcinogenesis models in TgRANKL mice that express both human and mouse RANKL, as well as in humanized humTgRANKL mice expressing only human RANKL, and compared them to MPA/DMBA-treated wild-type (WT) mice. Our results show that TgRANKL and WT mice have similar levels of susceptibility to mammary carcinogenesis, while OPG-Fc treatment restored mammary ductal density, and prevented ductal branching and the formation of neoplastic foci in both genotypes. humTgRANKL mice also developed MPA/DMBA-induced tumors with similar incidence and burden to those of WT and TgRANKL mice. The prophylactic treatment of humTgRANKL mice with Denosumab significantly prevented the rate of appearance of mammary tumors from 86.7% to 15.4% and the early stages of carcinogenesis, whereas therapeutic treatment did not lead to any significant attenuation of tumor incidence or tumor burden compared to control mice, suggesting the importance of RANKL primarily in the initial stages of tumorigenesis. Overall, we provide unique genetic tools for investigating the involvement of RANKL in breast carcinogenesis, and allow the preclinical evaluation of novel therapeutics that target hormone-related breast cancers.

Functional, Aromatic, and Fluorinated Monothiosemicarbazones: Investigations into Their Structures and Activity toward the Gallium-68 Incorporation by Microwave Irradiation.

We report on the synthesis and spectroscopic characterization of a new series of coordinating monothiosemicarbazones incorporating aromatic backbones, featuring O/N/S donor centers monosubstituted with different aliphatic, aromatic, fluorinated, and amine-functionalized groups at their N centers. Their ability to bind metal ions such as Zn(II) and Ga(III) was explored, and the formation of two different coordination isomers of the Zn(II) complex was demonstrated by X-ray diffraction studies using synchrotron radiation. These studies showed the planar geometry for the coordinated mono(thiosemicarbazone) ligand and that the metal center can adopt either a heavily distorted tetrahedral Zn center (placed in an N/S/S/N environment, with CN = 4) or a pseudo-octahedral geometry, where the Zn(II) center is in the O/N/S/S/N/O environment, and CN = 6. Furthermore, 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) assays and cellular imaging in living cells were subsequently performed in two different cancer cell lines: PC-3 (a standard cell line derived from a bone metastasis of a stage IV prostate cancer) and EMT6 (a commercial murine mammary carcinoma cell line). The radiolabeling of new functional and aromatic monothiosemicarbazones with either gallium-68 (under pH control) or fluorine-18 is discussed. The potential of this class of compounds to act as synthetic scaffolds for molecular imaging agents of relevance to positron emission tomography was evaluated in vitro, and the cellular uptake of a simultaneously fluorinated and [68Ga]-labeled mono(thiosemicarbazone) was investigated and is reported here.

Transforming the Chemical Structure and Bio-Nano Activity of Doxorubicin by Ultrasound for Selective Killing of Cancer Cells.

Reconfiguring the structure and selectivity of existing chemotherapeutics represents an opportunity for developing novel tumor-selective drugs. Here, as a proof-of-concept, the use of high-frequency sound waves is demonstrated to transform the nonselective anthracycline doxorubicin into a tumor selective drug molecule. The transformed drug self-aggregates in water to form ≈200 nm nanodrugs without requiring organic solvents, chemical agents, or surfactants. The nanodrugs preferentially interact with lipid rafts in the mitochondria of cancer cells. The mitochondrial localization of the nanodrugs plays a key role in inducing reactive oxygen species mediated selective death of breast cancer, colorectal carcinoma, ovarian carcinoma, and drug-resistant cell lines. Only marginal cytotoxicity (80-100% cell viability) toward fibroblasts and cardiomyocytes is observed, even after administration of high doses of the nanodrug (25-40 µg mL-1 ). Penetration, cytotoxicity, and selectivity of the nanodrugs in tumor-mimicking tissues are validated by using a 3D coculture of cancer and healthy cells and 3D cell-collagen constructs in a perfusion bioreactor. The nanodrugs exhibit tropism for lung and limited accumulation in the liver and spleen, as suggested by in vivo biodistribution studies. The results highlight the potential of this approach to transform the structure and bioactivity of anticancer drugs and antibiotics bearing sono-active moieties.

Optical to Planar X-ray Mouse Image Mapping in Preclinical Nuclear Medicine Using Conditional Adversarial Networks.

In the current work, a pix2pix conditional generative adversarial network has been evaluated as a potential solution for generating adequately accurate synthesized morphological X-ray images by translating standard photographic images of mice. Such an approach will benefit 2D functional molecular imaging techniques, such as planar radioisotope and/or fluorescence/bioluminescence imaging, by providing high-resolution information for anatomical mapping, but not for diagnosis, using conventional photographic sensors. Planar functional imaging offers an efficient alternative to biodistribution ex vivo studies and/or 3D high-end molecular imaging systems since it can be effectively used to track new tracers and study the accumulation from zero point in time post-injection. The superimposition of functional information with an artificially produced X-ray image may enhance overall image information in such systems without added complexity and cost. The network has been trained in 700 input (photography)/ground truth (X-ray) paired mouse images and evaluated using a test dataset composed of 80 photographic images and 80 ground truth X-ray images. Performance metrics such as peak signal-to-noise ratio (PSNR), structural similarity index measure (SSIM) and Fréchet inception distance (FID) were used to quantitatively evaluate the proposed approach in the acquired dataset.

Conjugated Photosensitizers for Imaging and PDT in Cancer Research.

Early cancer detection and perfect understanding of the disease are imperative toward efficient treatments. It is straightforward that, for choosing a specific cancer treatment methodology, diagnostic agents undertake a critical role. Imaging is an extremely intriguing tool since it assumes a follow up to treatments to survey the accomplishment of the treatment and to recognize any conceivable repeating injuries. It also permits analysis of the disease, as well as to pursue treatment and monitor the possible changes that happen on the tumor. Likewise, it allows screening the adequacy of treatment and visualizing the state of the tumor. Additionally, when the treatment is finished, observing the patient is imperative to evaluate the treatment methodology and adjust the treatment if necessary. The goal of this review is to present an overview of conjugated photosensitizers for imaging and therapy.

Synthesis, Radiolabelling and In Vitro Imaging of Multifunctional Nanoceramics.

Molecular imaging has become a powerful technique in preclinical and clinical research aiming towards the diagnosis of many diseases. In this work, we address the synthetic challenges in achieving lab-scale, batch-to-batch reproducible copper-64- and gallium-68-radiolabelled metal nanoparticles (MNPs) for cellular imaging purposes. Composite NPs incorporating magnetic iron oxide cores with luminescent quantum dots were simultaneously encapsulated within a thin silica shell, yielding water-dispersible, biocompatible and luminescent NPs. Scalable surface modification protocols to attach the radioisotopes 64Cu (t1/2=12.7 h) and 68Ga (t1/2=68 min) in high yields are reported, and are compatible with the time frame of radiolabelling. Confocal and fluorescence lifetime imaging studies confirm the uptake of the encapsulated imaging agents and their cytoplasmic localisation in prostate cancer (PC-3) cells. Cellular viability assays show that the biocompatibility of the system is improved when the fluorophores are encapsulated within a silica shell. The functional and biocompatible SiO2 matrix represents an ideal platform for the incorporation of 64Cu and 68Ga radioisotopes with high radiolabelling incorporation.