Stephen James Paisey

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.

The advent of total-body Positron Emission Tomography (PET) has vastly broadened the range of research and clinical applications of this powerful molecular imaging technology¹. Such possibilities have accelerated progress in ¹⁸F-radiochemistry with numerous methods available to ¹⁸F-label (hetero)arenes and alkanes². However, access to ¹⁸F-difluoromethylated molecules in high molar activity (A_m) is largely an unsolved problem, despite the indispensability of the difluoromethyl group for pharmaceutical drug discovery³. We report herein a general solution by introducing carbene chemistry to the field of nuclear imaging with a [¹⁸F]difluorocarbene reagent capable of a myriad of ¹⁸F-difluoromethylation processes. In contrast to the tens of known difluorocarbene reagents, this ¹⁸F-reagent is carefully designed for facile accessibility, high molar activity and versatility. The issue of A_m is solved using an assay examining the likelihood of isotopic dilution upon variation of the electronics of the difluorocarbene precursor. Versatility is demonstrated with multiple [¹⁸F]difluorocarbene based reactions including O–H, S–H and N–H insertions, and cross-couplings that harness the reactivity of ubiquitous functional groups such as (thio)phenols, N-heteroarenes, and aryl boronic acids that are easy to install. Impact is illustrated with the labelling of highly complex and functionalised biologically relevant molecules and radiotracers.

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with O⁶-methylguanine-DNA-methyltransferase (MGMT) expression and mismatch repair deficiency. Hence, a potent N(3)-propargyl analogue (N3P) was derived, which not only evades MGMT but also remains effective in mismatch repair deficient cells. Due to the poor pharmacokinetic profile of N3P (t_1/2 < 1 h) and to bypass the blood–brain barrier, we proposed convection enhanced delivery (CED) as a method of administration to decrease dose and systemic toxicity. Moreover, to enhance N3P solubility, stability, and sustained distribution in vivo, either it was incorporated into an apoferritin (AFt) nanocage or its sulfobutyl ether β-cyclodextrin complex was loaded into nanoliposomes (Lip). The resultant AFt-N3P and Lip-N3P nanoparticles (NPs) had hydrodynamic diameters of 14 vs 93 nm, icosahedral vs spherical morphology, negative surface charge (−17 vs −34 mV), and encapsulating ∼630 vs ∼21000 N3P molecules per NP, respectively. Both NPs showed a sustained release profile and instant uptake within 1 h incubation in vitro. In comparison to the naked drug, N3P NPs demonstrated stronger anticancer efficacy against 2D TMZ-resistant DIPG cell cultures [IC₅₀ = 14.6 (Lip-N3P) vs 32.8 μM (N3P); DIPG-IV) and (IC₅₀ = 101.8 (AFt-N3P) vs 111.9 μM (N3P); DIPG-VI)]. Likewise, both N3P-NPs significantly (P < 0.01) inhibited 3D spheroid growth compared to the native N3P in MGMT⁺ DIPG-VI (100 μM) and mismatch repair deficient DIPG-XIX (50 μM) cultures. Interestingly, the potency of TMZ was remarkably enhanced when encapsulated in AFt NPs against DIPG-IV, -VI, and -XIX spheroid cultures. Dynamic PET scans of CED-administered zirconium-89 (⁸⁹Zr)-labeled AFt-NPs in rats also demonstrated substantial enhancement over free ⁸⁹Zr radionuclide in terms of localized distribution kinetics and retention within the brain parenchyma. Overall, both NP formulations of N3P represent promising approaches for treatment of TMZ-resistant DIPG and merit the next phase of preclinical evaluation.

Texture analysis for quantification of intratumor uptake heterogeneity in PET/CT images has received increasing attention. This allows the extraction of a large number of ‘radiomic’ features to be correlated with end point information such as tumor type, therapy response, prognosis. The conventional complex workflow for calculation of texture features introduces numerous confounding variables. This non exhaustively includes, imaging time post administration of radiopharmaceutical and the method and extent of functional volume segmentation. A lack of understanding on the dependency of texture features with these variables serves as a detriment to the urgent need to standardize texture measurements to pool results from different imaging centers. The utilization of machine learning techniques for feature (and their combinations) selection serves as a promising method to alleviate redundancy in radiomics. To this avail, we introduce for the first time the application of a Kohonen self-organizing feature map to identify the emergent properties present when performing texture analysis. The application of the self-organizing map to radiomic analysis serves as a powerful general-purpose exploratory instrument to reveal the statistical indicators of texture distributions. For this purpose, texture features from PET-CT images of 8 pre-clinical mice with mammary carcinoma xenografts were analyzed with varying post injection imaging time and tumor segmentation contour size. This varying distribution of texture parameters were interpreted by the self-organizing map to reveal two distinct clusters of texture features which are dependent on contour size, providing additional evidence that contour size and hence segmentation method is a confounding variable when performing texture analysis. Furthermore, the self-organizing map can be utilized as a method to incorporate this revealed dependency in a prediction model in the presence of end point information, which will be an area of future work.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time ⁸⁹Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of ⁸⁹Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-⁸⁹Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

The non-covalent radiolabelling of new graphene oxide-bis(thiosemicarbazonato) nanohybrids with gallium-68 and zirconium-89 ions is reported herein for the first time.