Search Results (25)

A novel, cost-effective, label-free biosensing strategy has been established for real-time quantification of alkaline phosphatase (ALP) activity, integrating the Tyndall effect with smartphone imaging technology. This method utilizes a handheld laser diode to probe the enzyme-triggered in situ assembly of Cu-guanosine monophosphate (Cu-GMP) coordination polymers, which exhibit tunable Tyndall scattering properties. In the absence of ALP, Cu²⁺ ions chelate with GMP to form Cu-GMP coordination polymers, generating an intense Tyndall effect. Conversely, ALP-mediated hydrolysis of GMP disrupts the formation of Cu-GMP coordination polymers, resulting in diminished light scattering. The intensity of the Tyndall effect is directly proportional to the concentration of Cu-GMP coordination polymers, which in turn correlates with ALP activity levels. A comprehensive investigation of experimental parameters was conducted, including pH, incubation temperature, GMP concentration, incubation time, synthesis duration, and CuSO₄ concentration. Under optimized conditions, the developed smartphone-assisted colorimetric assay enables the detection of ALP activity within the range of 0.375–3.75 U/mL, with a limit of detection of 0.184 U/mL. The application of this method to serum samples yielded recovery rates ranging from 102.6% to 109.0%. In summary, this smartphone-based colorimetric platform offers a portable and versatile approach for instrument-free detection of ALP activity, with potential applications in point-of-care diagnostics and resource-limited settings. Full article

Background: Fibroblast activation protein (FAP)-targeted theranostic radiopharmaceuticals have shown desired tumor-to-background organ selectivity due to the ubiquitous presence of FAP within the tumor microenvironment. However, suboptimal tumor retention and fast clearance have hindered their use to deliver effective cancer therapies. With well-documented FAP-targeting moieties and linkers appending them to optimal chelators, the development of copper radiopharmaceuticals has attracted considerable interest, given the fact that an ideal theranostic pair of copper radionuclides (⁶⁴Cu: t_1/2 = 12.7 h; 17.4% β⁺; E_β⁺_max = 653 keV and ⁶⁷Cu: t_1/2 = 2.58 d; 100% β⁻; E_β⁻_max = 562 keV) are available. Herein, we report our design, synthesis, and comparative evaluation of monovalent and divalent FAP-targeted theranostic conjugates constructed from our previously reported bifunctional chelator scaffold (BFS) based on 1,4,8,11-tetraaza-bicyclo [6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), which forms the most stable complex with Cu(II). Methods: After synthesis and characterization, the monovalent and divalent conjugates were radiolabeled with ⁶⁴Cu for in vitro cell assays, followed by in vivo positron emission tomography (PET) imaging evaluation in relevant mouse models. Results: Both ⁶⁴Cu-labeled conjugates showed high in vitro stability and anticipated FAP-mediated cell binding and internalization. The divalent one showed significantly higher FAP-specific tumor uptake than its monovalent counterpart. Conclusions: Our results demonstrate that the BFS-based multivalent approach can be practically used to generate FAP-targeted radiotheranostic agents for effective cancer diagnosis and treatment. Full article

Immunotherapy has transformed cancer treatment. Nevertheless, given the heterogeneity of clinical efficacy, the multiplicity of treatment options available and the possibility of serious adverse effects, selecting the most effective treatment has become the greatest challenge. Molecular imaging offers an attractive way for this purpose. ImmunoPET provides specific imaging with positron emission tomography (PET) using monoclonal antibodies (mAb) or its fragments as vector. By combining the high targeting specificity of mAb and the sensitivity of PET technique, immunoPET could noninvasively and dynamically reveal tumor antigens expression and provide theranostic tools of several types of malignancies. Because of their slow kinetics, mAbs require radioelements defined by a consistent half-life. Zirconium 89 (⁸⁹Zr) and Copper 64 (⁶⁴Cu) are radiometals with half-lives suitable for mAb labeling. Radiolabeling with a radiometal requires the prior use of a bifunctional chelate agent (BFCA) to functionalize mAb for radiometal chelation, in a second step. There are a number of BFCA available and much research is focused on antibody functionalization techniques or on developing the optimum chelating agent depending the selected radiometal. In this manuscript, we present a critical account of radiochemical techniques with radionuclides ⁸⁹Zr and ⁶⁴Cu and their applications in preclinical and clinical immuno-PET imaging. Full article

In this study, Cu²⁺ modulated silver nanoclusters were constructed for the turn-on, label-free detection of L-histidine. Six Ag NCs protected by oligonucleotides (DNA-Ag NCs) were tested in a series of experiments. Finally, A-DAN-Ag NCs were chosen as the best candidate due to their excellent fluorescent properties. The fluorescence of A-DAN-Ag NCs was quenched using Cu²⁺ through energy or electron transfer. However, quenched fluorescence could be restored dramatically in the presence of L-histidine due to Cu²⁺ liberation from A-DAN-Ag NCs and because of the chelation between the imidazole group of L-histidine and Cu²⁺. The proposed sensor exhibited high selectivity towards L-histidine over other amino acids, with a limit of detection (LOD) of 0.096 μM ranging from 0 to 8 μM. The proposed sensor succeeded in detecting L-histidine in diluted human urine. Therefore, the sensor has promising practical applications in biological systems. Full article

[⁶⁴Cu]Cu-diacetyl-bis(N⁴-methylthiosemicarbazone) ([⁶⁴Cu]Cu-ATSM) is a radioactive hypoxia-targeting therapeutic agent being investigated in clinical trials for malignant brain tumors. For the quality management of [⁶⁴Cu]Cu-ATSM, understanding trace metal impurities’ effects on the chelate formation of ⁶⁴Cu and ATSM is important. In this study, we conducted coordination chemistry studies on metal–ATSM complexes. First, the effects of nonradioactive metal ions (Cu²⁺, Ni²⁺, Zn²⁺, and Fe²⁺) on the formation of [⁶⁴Cu]Cu-ATSM were evaluated. When the amount of Cu²⁺ or Ni²⁺ added was 1.2 mol or 288 mol, equivalent to ATSM, the labeling yield of [⁶⁴Cu]Cu-ATSM fell below 90%. Little effect was observed even when excess amounts of Zn²⁺ or Fe²⁺ were added to the ATSM. Second, these metals were reacted with ATSM, and chelate formation was measured using ultraviolet–visible (UV-Vis) absorption spectra. UV-Vis spectra showed a rapid formation of Cu²⁺ and the ATSM complex upon mixing. The rate of chelate formation by Ni²⁺ and ATSM was lower than that by Cu-ATSM. Zn²⁺ and Fe²⁺ showed much slower reactions with the ATSM than Ni²⁺. Trace amounts of Ni²⁺, Zn²⁺, and Fe²⁺ showed little effect on [⁶⁴Cu]Cu-ATSM’ quality, while the concentration of impurity Cu²⁺ must be controlled. These results can provide process management tools for radiopharmaceuticals. Full article

Immune checkpoint inhibitor therapy targeting the PD-1/PD-L1 axis in cancer patients, is a promising oncological treatment. However, the number of non-responders remains high, causing a burden for the patient and the healthcare system. Consequently, a diagnostic tool to predict treatment outcomes would help with patient stratification. Molecular imaging provides said diagnostic tool by offering a whole-body quantitative assessment of PD-L1 expression, hence supporting therapy decisions. Four PD-L1 radioligand candidates containing a linker-chelator system for radiometalation, along with three hydrophilizing units—one sulfonic and two phosphonic acids—were synthesized. After labeling with ⁶⁴Cu, log D_7.4 values of less than −3.03 were determined and proteolytic stability confirmed over 94% intact compound after 48 h. Binding affinity was determined using two different assays, revealing high affinities up to 13 nM. µPET/CT imaging was performed in tumor-bearing mice to investigate PD-L1-specific tumor uptake and the pharmacokinetic profile of radioligands. These results yielded an unexpected in vivo distribution, such as low tumor uptake in PD-L1 positive tumors, high liver uptake, and accumulation in bone/bone marrow and potentially synovial spaces. These effects are likely caused by Ca²⁺-affinity and/or binding to macrophages. Despite phosphonic acids providing high water solubility, their incorporation must be carefully considered to avoid compromising the pharmacokinetic behavior of radioligands. Full article

Understanding the physicochemical properties of antibody–drug conjugates is critical to assess their quality at manufacturing and monitor them during subsequent storage. For radiometal–antibody complexes, it is important to control the properties of the antibody–chelator conjugate to maintain the quality of the final product. We have been developing ⁶⁴Cu-labeled anti-epidermal growth factor receptor antibody NCAB001 (⁶⁴Cu-NCAB001) for the early diagnosis and therapy of pancreatic cancer with positron-emission tomography. Here, we characterized the larger size variants contained in the antibody–chelator conjugate PCTA-NCAB001 by multi-angle light scattering coupled with size-exclusion chromatography. Secondly, we developed a chromatographic method to remove these size variants. Lastly, we demonstrated the stability of PCTA-NCAB001 after the removal of size variants. Dimer and oligomers were identified in PCTA-NCAB001. These larger size variants, together with some smaller size variants, could be removed by hydrophobic interaction chromatography. The PCTA-NCAB001 product, after the removal of these size variants, could be stored at 4 °C for six months. The methods developed here can be applied to assure the quality of PCTA-NCAB001 and other antibody–drug conjugates to facilitate the development of antibody–radiometal conjugates for positron-emission tomography and radioimmunotherapy of malignant cancers. Full article

Background: This study compared the effects of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as ⁶⁴Cu-chelating agents in newly developed prostate-specific membrane antigen (PSMA) target compounds, ⁶⁴Cu-cudotadipep and ⁶⁴Cu-cunotadipep, on pharmacokinetics. Methods: The in vitro stability of the chelators was evaluated using human and mouse serum. In vitro PSMA-binding affinity and cell uptake were compared using human 22Rv1 cells. To evaluate specific PSMA-expressing tumor-targeting efficiency, micro-positron emission tomography (mcroPET)/computed tomography (CT) and biodistribution analysis were performed using PSMA+ PC3-PIP and PSMA− PC3-flu tumor xenografts. Results: The serum stability of DOTA- or NOTA-conjugated ⁶⁴Cu-cudotadipep and ⁶⁴Cu-cunotadipep was >97%. The Ki value of the NOTA derivative, cunotadipep, in the in vitro affinity binding analysis was higher (2.17 ± 0.25 nM) than that of the DOTA derivative, cudotadipep (6.75 ± 0.42 nM). The cunotadipep exhibited a higher cellular uptake (6.02 ± 0.05%/1 × 10⁶ cells) compared with the cudotadipep (2.93 ± 0.06%/1 × 10⁶ cells). In the biodistribution analysis and microPET/CT imaging, the ⁶⁴Cu-labeled NOTA derivative, ⁶⁴Cu-cunotadipep, demonstrated a greater tumor uptake and lower liver uptake than the DOTA derivative. Conclusions: This study indicates that the PSMA-targeted ⁶⁴Cu-cunotadipep can be applied in clinical practice owing to its high diagnostic power for prostate cancer. Full article

Al(III) complexes have been recently investigated for their potential use in imaging with positron emission tomography (PET) by formation of ternary complexes with the radioisotope fluorine-18 (¹⁸F). Although the derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) are the most applied chelators for [Al¹⁸F]²⁺ labelling and (pre)clinical PET imaging, non-macrocyclic, semi-rigid pentadentate chelators having two N- and three O-donor atoms such as RESCA1 and AMPDA-HB have been proposed with the aim to allow room temperature labelling of temperature-sensitive biomolecules. The paucity of stability data on Al(III) complexes used for PET imaging instigated a complete thermodynamic and kinetic solution study on Al(III) complexes with aminomethylpiperidine (AMP) derivatives AMPTA and AMPDA-HB and the comparison with a RESCA1-like chelator CD3A-Bn (trans-1,2-diaminocyclohexane-N-benzyl-N,N′,N′-triacetic acid). The stability constant of [Al(AMPDA-HB)] is about four orders of magnitude higher than that of [Al(AMPTA)] and [Al(CD3A-Bn)], highlighting the greater affinity of phenolates with respect to acetate O-donors. On the other hand, the kinetic inertness of the complexes, determined by following the Cu²⁺-mediated transmetallation reactions in the 7.5–10.5 pH range, resulted in a spontaneous and hydroxide-assisted dissociation slightly faster for [Al(AMPTA)] than for the other two complexes (t_1/2 = 4.5 h for [Al(AMPTA)], 12.4 h for [Al(AMPDA-HB)], and 24.1 h for [Al(CD3A-Bn)] at pH 7.4 and 25 °C). Finally, the [AlF]²⁺ ternary complexes were prepared and their stability in reconstituted human serum was determined by ¹⁹F NMR experiments. Full article

Gold nanoparticles (AuNPs) are cutting-edge platforms for combined diagnostic and therapeutic approaches due to their exquisite physicochemical and optical properties. Using the AuNPs physically produced by femtosecond pulsed laser ablation of bulk Au in deionized water, with a capping agent-free surface, the conjugation of functional ligands onto the AuNPs can be tunable between 0% and 100% coverage. By taking advantage of this property, AuNPs functionalized by two different types of active targeting ligands with predetermined ratios were fabricated. The quantitatively controllable conjugation to construct a mixed monolayer of multiple biological molecules at a certain ratio onto the surface of AuNPs was achieved and a chelator-free ⁶⁴Cu-labeling method was developed. We report here the manufacture, radiosynthesis and bioevaluation of three different types of dual-ligand AuNPs functionalized with two distinct ligands selected from glucose, arginine–glycine–aspartate (RGD) peptide, and methotrexate (MTX) for tumor theragnosis. The preclinical evaluation demonstrated that tumor uptakes and retention of two components AuNP conjugates were higher than that of single-component AuNP conjugates. Notably, the glucose/MT- modified dual-ligand AuNP conjugates showed significant improvement in tumor uptake and retention. The novel nanoconjugates prepared in this study make it possible to integrate several modalities with a single AuNP for multimodality imaging and therapy, combining the power of chemo-, thermal- and radiation therapies together. Full article

A new Near InfraRed (NIR) fluorescent chemosensor for metal ions and anions is herein presented. The fluorophore is based on a styrylflavylium dye, a synthetic analogue of the natural anthocyanin family, with a di-(2-picolyl)amine (DPA) moiety as the metal chelating unit. The substitution pattern of the styrylflavylium core (with tertiary amines on positions 7 and 4′) shifts the optical properties of the dye towards the NIR region of the electronic spectra, due to a strong push-pull character over the π-conjugated system. The NIR chemosensor is highly sensitive to the presence of Zn²⁺, which induces a strong CHelation Enhanced Fluorescence (CHEF) effect upon binding to the DPA unit (2.7 fold increase). The strongest competing ion is Cu²⁺, with a complete fluorescence quenching, while other metals induce lower responses on the optical properties of the chemosensor. Subsequent anion screening of the Zn²⁺-chemosensor coordination compound has demonstrated a distinct selectivity towards adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP), with high association constants (K ~ 10⁶ M⁻¹) and a strong CHEF effect (2.4 and 2.9 fold fluorescence increase for ATP and ADP, respectively). Intracellular studies with the Zn²⁺-complexed sensor showed strong luminescence in the cellular membrane of Gram^– bacteria (E. coli) and mitochondrial membrane of mammalian cells (A659), which highlights its possible application for intracellular labelling. Full article

The development of ⁶⁴Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [⁶⁴Cu]Cu-15-5 complex prompted us to investigate its potential for the ⁶⁴Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with ⁶⁴Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [⁶⁴Cu]Cu-15-5- and [⁶⁴Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88–94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition. Full article

Copper radioisotopes are generally employed for cancer imaging and therapy when firmly coordinated via a chelating agent coupled to a tumor-seeking vector. However, the biologically triggered Cu²⁺-Cu⁺ redox switching may constrain the in vivo integrity of the resulting complex, leading to demetallation processes. This unsought pathway is expected to be hindered by chelators bearing N, O, and S donors which appropriately complements the borderline-hard and soft nature of Cu²⁺ and Cu⁺. In this work, the labelling performances of a series of S-rich polyazamacrocyclic chelators with [⁶⁴Cu]Cu²⁺ and the stability of the [⁶⁴Cu]Cu-complexes thereof were evaluated. Among the chelators considered, the best results were obtained with 1,7-bis [2-(methylsulfanyl)ethyl]-4,10,diacetic acid-1,4,7,10-tetraazacyclododecane (DO2A2S). DO2A2S was labelled at high molar activities in mild reaction conditions, and its [⁶⁴Cu]Cu²⁺ complex showed excellent integrity in human serum over 24 h. Biodistribution studies in BALB/c nude mice performed with [⁶⁴Cu][Cu(DO2A2S)] revealed a behavior similar to other [⁶⁴Cu]Cu-labelled cyclen derivatives characterized by high liver and kidney uptake, which could either be ascribed to transchelation phenomena or metabolic processing of the intact complex. Full article

This work suggested that Cu²⁺ ion coordinated by the peptide with a histidine (His or H) residue in the first position from the free N-terminal reveals oxidase-mimicking activity. A biotinylated polymer was prepared by modifying His residues on the side chain amino groups of lysine residues (denoted as K_H) to chelate multiple Cu²⁺ ions. The resulting biotin-poly-(K_H-Cu)₂₀ polymer with multiple catalytic sites was employed as the signal label for immunoassay. Prostate specific antigen (PSA) was determined as the model target. The captured biotin-poly-(K_H-Cu)₂₀ polymer could catalyze the oxidation of o-phenylenediamine (OPD) to produce fluorescent 2,3-diaminophenazine (OPDox). The signal was proportional to PSA concentration from 0.01 to 2 ng/mL, and the detection limit was found to be eight pg/mL. The high sensitivity of the method enabled the assays of PSA in real serum samples. The work should be valuable for the design of novel biosensors for clinical diagnosis. Full article

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as a target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic positron emission tomography (PET) imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with ⁶⁴Cu²⁺, the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with ²²⁵Ac³⁺ and applied for targeted alpha therapy. A single injection of the ²²⁵Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy. Full article