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. 2025 Jun 24;3(10):653–662. doi: 10.1021/cbmi.5c00024

Imaging Copper Levels during Life in the Brain and beyond Using a Fluorescent Copper Sensor with Multimodal Capacity

Liam D Adair †,§, Benjamin G Trist ∥,⊥, Marcus E Graziotto †, Tom J Hawtrey †,‡,§, Benjamin D Rowlands ∥,⊥, Sarah A Rosolen ∥,⊥, Veronica Cottam ∥,⊥, Michael Kuligowski #, Jessica L Chitty ∇,○, Ellie T Y Mok ∇,○, Thomas R Cox ∇,○, Michael P Gotsbacher ⊥, Kay L Double ∥,⊥, Elizabeth J New †,§,*
PMCID: PMC12569956  PMID: 41169453

Abstract

Copper is an essential trace element for normal development and function throughout the body, including the central nervous system (CNS). Alterations to cellular copper levels result in severe neurological consequences and are linked to a range of CNS disorders, positioning treatments that restore copper balance as promising therapies for these disorders. However, despite the clear relationship between copper balance and CNS health, there are limited tools to measure copper levels in vivo in humans. This constitutes a significant challenge for both diagnosing disorders of copper imbalance and monitoring the efficacy of copper-altering treatments for these disorders. Here we report the synthesis and characterization of Fluorine-labeled Naphthalimide Copper sensor 1 (F-NpCu1), a fluorescent sensor for copper that contains a fluorine atom for future radiolabeling for clinical application. We demonstrate that the probe exhibits good stability and is highly selective for copper above other transition metals present in biological tissues. Copper binding promotes covalent bond formation between the sensor and proximal cellular proteins. F-NpCu1 is nontoxic and can be measured using fluorescence microscopy in living cells and fixed tissue sections from both mouse brain and pancreas. Furthermore, F-NpCu1 exhibits good blood-brain-barrier permeability and can report differences in brain copper levels induced by copper modulating therapies in living mice using intravital fluorescence microscopy. This study represents a promising advance toward the development of the first clinical tool for measuring copper in living humans, including in the CNS, with radiolabeling studies underway to develop 18F-NpCu1 for PET imaging of copper in vivo.

Keywords: copper, sensor, intravital microscopy, in vivo imaging, brain, neurological

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Copper is the second-most abundant transition metal in the body, after iron, and is an essential cofactor in many key enzymes, including mitochondrial cytochrome C oxidase, copper/zinc superoxide dismutase (SOD1) and lysyl oxidase. The redox activity of copper that underpins its crucial roles in health can also result in detrimental effects in disease, particularly when copper homeostasis is disrupted through perturbations to copper regulatory pathways. For example, excess copper can cause oxidative stress through Fenton-like chemistry, and is involved in a recently identified cell death pathway, cuproptosis.

Copper levels in the central nervous system (CNS) are second only to those in the liver, and maintenance of physiological copper levels within the brain is crucial. The severe consequences of CNS copper imbalance are seen in the rare genetic disorders Menkes and Wilson’s diseases, which cause copper deficiency and copper overload in the brain, respectively. Treatments that restore copper levels in Menkes disease have clinical benefits and can extend life, with the copper delivery drug elesclomol approved by the FDA in 2023 for the treatment of this disorder. Similarly, early normalization of CNS copper levels in Wilson’s disease patients using the FDA-approved copper chelators penicillamine, tetrathiomolybdate or trientine tetrahydrochloride, or the copper absorption inhibitor zinc acetate, can mitigate hepatic, neurologic, and psychiatric symptoms. In addition to rare genetic disorders, we have identified reduced copper levels in degenerating regions of the Parkinson’s disease (PD) brain, ,, which are present in early stages of the disease and are absent in regions which do not degenerate. Further, alterations to the subcellular distribution of copper have been detected in the degenerating spinal cords of amyotrophic lateral sclerosis cases. These findings have led to preclinical − and clinical trials (clinicaltrials.gov: NCT04082832, NCT03204929) of copper supplementation in both diseases. In addition to the alterations in CNS copper levels observed in these disorders, ,, elevated levels of bioavailable copper have been linked to Alzheimer’s disease, and the requirement of proliferating cancer cells for high levels of copper has sparked promising trials of copper chelation agents as therapies for a range of cancers, such as neuroblastoma, breast cancer and pancreatic cancer. −

Given the clear importance of copper in a range of CNS disorders, there have been diverse research efforts to measure and monitor brain copper levels. Advanced analytical techniques such as laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron X-ray fluorescence microscopy (XFM) enable high-sensitivity and high-resolution mapping of copper levels in postmortem brain tissues. However, they cannot be performed on living samples nor can they distinguish bioavailable copper from within the total copper pool. There have been some elegant tools developed to image copper in living mice, including near-IR fluorescent sensors applied to mouse liver, and mouse brain with craniotomy, bioluminescent probes used to image mouse liver, and photoacoustic sensors applied in mouse liver and mouse brain. , However, such approaches have not been translated to in vivo imaging in humans, particularly in brain imaging, primarily because of tissue penetration limitations, although some progress has been made to apply photoacoustic imaging in living human brain.

At present, no tool exists to measure copper levels in the living brain and spinal cord in humans, constituting a significant challenge for supporting the diagnosis or monitoring the efficacy of copper-altering treatments in these disorders. It is not possible to measure copper levels in urine or blood as a proxy; for example, we have reported that copper levels in PD patient biofluids do not correlate with those measured in post mortem CNS tissues from PD patients. Given that the primary aim of in vivo studies is to better understand copper perturbations in the living mammalian brain, with the goal of translating findings into improved disease diagnoses and treatments in humans, there is a clear and urgent need to develop new molecular imaging tools that enable in vivo measurement of tissue copper levels.

Positron emission tomography (PET) and fluorescence imaging are key modalities for in vivo imaging. ,,, PET enables the transient estimation of physiological changes deep within tissues using specific radiotracers, whereas fluorescence imaging techniques use fluorescent probes to label targets within superficial tissues at high resolution. Both play pivotal roles in clinical biomedical research, particularly oncology, given their capacity to improve disease diagnosis and monitoring, and guide drug development through in vivo visualization of target location and engagement. , Importantly, both modalities are highly compatible, employ imaging agents at trace quantities minimizing toxicity concerns, and together enable imaging of molecular markers of interest at high spatial and long temporal resolutions. Recognizing the potential for these compatible imaging modalities to enable measurement of copper levels in mammalian tissues in vivo, we developed Fluorine-labeled Naphthalimide Copper sensor 1 (F-NpCu1), a fluorescent sensor for copper that contains a fluorine atom for future radiolabeling (Figure A). In this study, we investigate the activity of the cold compound prior to radiolabeling and PET imaging. Importantly, there is currently no PET probe available for measurement of endogenous copper, with current methods relying only on the application of exogenous 64Cu radionuclide, − which does not enable monitoring of native copper pools.

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F-NpCu1 mechanism of action and photophysical properties. (A) Structure of F-NpCu1 and mechanism for proximal labeling of proteins in the presence of copper. (B) Excitation (dashed line) and emission (solid line) spectra for F-NpCu1 (5 μM), collected in HEPES buffer (20 mM, pH 7.4). (C) HPLC chromatograms of the reaction between 50 μM F-NpCu1 and metal ions (5 equiv of Cu2+, Cu+, Cu2+ + 25 equiv BCS) in PBS in the presence of 10 equiv of L-serine methyl ester and 2.0 mM GSH.

To ensure accumulation at sites of high copper levels, we employed a strategy where the probe becomes immobilized upon copper binding through proximal protein labeling, with the signal from the probe (either fluorescence or PET) proportional to copper concentration. This strategy has been used for identification of proteins associated with zinc homeostasis, and more recently Chang and colleagues have reported a set of dyes that label proximal proteins once activated by copper. , We therefore chose to use the acyl imidazole featuring thioether groups used by Chang. To facilitate radiolabeling in future studies, we required a fluorine-containing group that could be readily incorporated at a final synthetic step, and therefore chose N-succinimidyl-4-[18F]­fluorobenzoate ([18F]­SFB) as a well-established prosthetic agent, which can be installed under mild conditions. − Finally, we required our molecule to exhibit good blood-brain-barrier (BBB) permeability. With the copper-sensing and fluorinated groups already chosen, we computationally screened a range of potential fluorophores to determine which exhibited the best properties (Table S1). The QikProp tool in Schrödinger was chosen for this, and the QPPCaco output was chosen to assess general cell permeability, while QPPMDCK was used to evaluate the predicted BBB permeability, with both of these metrics being suitable for assessing nonorally available drugs. One fluorophore-copper sensing conjugate that showed both improved predicted cell permeability and BBB permeability compared with the previously published probes was 4-amino-1,8-naphthalimide (Table S1, Entry 3). Incorporation of a fluorine atom into the structure, as well as being essential for our future plans to radiolabel this compound, is also predicted to further enhance these properties (Table S1, Entries 4–5). These favorable properties, coupled with the established chemistry for functionalizing this fluorophore at multiple positions, led us to the design of our target probe, F-NpCu1 (Figure A).

F-NpCu1 was synthesized in six steps from commercially available 4-bromo-1,8-naphthalic anhydride (Scheme S1). Synthetic handles were introduced at both the imide and 4-positions of the naphthalimide core, followed by subsequent functionalization with the copper sensing group and the fluorine containing SFB moiety. Crucially, installation of the SFB group was achieved by amide bond formation in the final synthetic step, with this proceeding under mild conditions over 4 h. This gives us good confidence that this approach will be able to be adapted successfully for the planned radiolabeling. Typically, the efficiency of these reactions with [18F]­SFB are enhanced by using an excess of the nonradiolabeled coupling partner and optimizing the reaction temperature to decrease the reaction time, minimizing lost yield due to radioactive decay (18F half-life = 109.8 min). This chemistry would be performed in an automated synthesis module with prep-HPLC employed to purify the radiolabeled probe.

The photophysical properties of F-NpCu1 were investigated in HEPES buffer (20 mM, pH 7.4) (Figure B): the compound has an excitation maximum at 448 nm and an emission maximum at 553 nm (Figure S1). We then measured the absorption and emission of the probe and hydrolyzed product (without the Cu sensing group) in the presence of Cu­(I) and Cu­(II) to demonstrate the emission and absorption spectra were unaffected (Figure S2). To demonstrate the response of the probe to copper, HPLC studies were used to monitor the stability of F-NpCu1 (50 μM) in PBS buffer with 5 equiv of L-serine methyl ester and glutathione (2.0 mM) with or without the addition of metal ions (Figure C; Figure S3). The probe alone in these aqueous conditions showed good stability over a period of 8 h, while addition of Cu­(I) or Cu­(II) led to a greatly accelerated reaction corresponding to cleavage of the copper responsive acyl imidazole moiety. After 1 h, 93% of the probe had reacted in the presence of 5 equiv of Cu­(II) compared to only 4% of the probe without addition of the metal. Addition of 25 equiv of the copper chelator, bathocuproinedisulfonic acid disodium salt (BCS) (1.25 mM) along with 5 equiv of Cu­(II) suppressed the reaction, leaving F-NpCu1 intact after 1 h.

It is important to note that Cu­(I) and Cu­(II) have different physiological and pathological roles, with the potent and redox-active Cu­(I) predominating within cells, and the more common extracellular form, Cu­(II), now reported to be present under conditions of oxidative stress. Therefore, while F-NpCu1 is a useful sensor for the total labile copper pool, which is known to be perturbed in disease, further development could install a more oxidation-state-specific receptor to specifically probe either Cu­(I) or Cu­(II).

To determine the selectivity of F-NpCu1 for copper, the HPLC experiments were repeated against a panel of similar and biologically relevant metals, with incubation for 1 h with 5 equiv (250 μM) of Fe­(II), Fe­(III), Co­(II), Ni­(II) or Zn­(II) or with 50 equiv (2.5 mM) of Mg­(II) or Ca­(II), all exhibiting a significantly lower extent of reaction than that induced by copper (Figure S2; Table S2).

Having successfully characterized the specificity of F-NpCu1 for copper in vitro, we next sought to assess its potential to report differences in tissue copper content when it is applied exogenously using microscopy. Application of F-NpCu1 to fixed brain tissue sections from the cortices of wild-type C57BL/6 mice revealed concentration-dependent increases in whole-tissue fluorescence at 540 nm following excitation with a 445 nm laser, which was absent at the same acquisition settings in F-NpCu1-naive tissues (Figure S4). These data suggest that F-NpCu1 may be a suitable copper sensor for fixed postmortem tissues from the mammalian central nervous system.

To assess the application of F-NpCu1 in the pathological diagnosis of neurological disorders of copper imbalance, we applied F-NpCu1 to paraffin-embedded fixed postmortem brain tissue sections from Dementia with Lewy bodies (DLB) and Incidental Lewy Body Disease patients (ILBD) (Table S3), both of which exhibit significant copper deficiency in the substantia nigra (SN) but not the occipital cortex (OCx). , Consistent with these data, we observed a 21% decrease in fluorescence intensity in the SN of DLB and ILBD cases compared with age-matched controls and no change in fluorescence intensity within gray or white matter of the OCx between groups (Figure A,B; Figure S5). These data closely align with quantification of copper in these same brain tissues using inductively coupled plasma mass spectrometry (ICP-MS)­(Figure C). Similar results were obtained using F-NpCu1 and ICP-MS on fixed brain tissue sections from copper-deficient Ctr1 ± mice. Fluorescence intensity and copper levels were proportionally lower in the cortex of these mice compared with those of wild-type mice (Figure D,E; Figure S6). Collectively our data indicate that F-NpCu1 could potentially quantify differences in brain copper in postmortem tissues from patients with neurological disorders of copper deficiency, as well as preclinical models of these diseases.

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Exogenous application of F-NpCu1 to human and mouse brain and pancreatic cancer tissues exhibiting distinct copper contents. Images of F-NpCu1 (green) and Hoechst (cell nuclei, blue) staining in (A) were taken using the same acquisition settings in the substantia nigra pars compacta (SNc) and in the white and gray matter of the occipital cortex (OCx) of Lewy body disease (LBD) patients and age-matched controls (n = 3–4/group). Quantification of tissue copper content was performed according to mean F-NpCu1 fluorescence intensity (B) and ICP-MS (C), with the latter conducted using adjacent tissue sections. Matching experiments were performed using cortical tissues from wild-type (WT) and copper deficient (Ctr1 ± ) mice treated with either 15 mg/kg diacetylbis­(N(4)-methylthiosemicarbazonato) copper II (CuATSM) or vehicle (SSV) (D–F), as well as healthy pancreatic tissues and pancreatic tumors excised from female NOD. Cg-PrkdcscidIL2rgtm1Wjl/SzAusb mice orthotopically implanted with KPC cancer cells and CAFs, compared to aged-matched healthy control pancreas tissue (G–I). Scale bars in panels A and D represent 70 μm, while those in panel G represent 50 μm. Full histochemical staining panels for A, D and G is presented in Figures S4–S6. Copper levels (63Cu; ppb) measured using ICP-MS were normalized to the phosphorus content (31P; ppm) to account for differences in tissue input. Data points in B, C, E, F, H, I represent individual mice/postmortem tissue cases, each averaged from triplicate measurements, while brackets represent mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Pharmacological restoration of brain copper is a key treatment approach for neurological disorders of copper deficiency. , To determine whether F-NpCu1 is sensitive to alterations in brain copper induced by copper delivery compounds, we applied it to brain tissues from Ctr1 ± and wild-type mice that had undergone treatment with the copper delivery drug diacetylbis­(N(4)-methylthiosemicarbazonato) copper II (CuATSM). We observed a significant increase in fluorescence intensity in CuATSM-treated mice compared with mice administered the vehicle alone (Figure D,E; Figure S6), which aligned with tissue copper levels measured using ICP-MS (Figure C). These data suggest that F-NpCu1 is responsive to changes in brain tissue copper content elicited by copper-modifying therapies.

While the F-NpCu1 fluorescence intensity was well-aligned with ICP-MS results when comparing copper distribution across different brain regions, we observed differences between the two measurements when examining copper distribution at a higher resolution. Fluorescence intensity in the human OCx, for example, was significantly higher in white matter (cell projections) compared with gray matter (cell bodies; Figure A-C; Figure S5), while in the SN it was greater in neuronal projections compared with neuronal soma or surrounding neuropil (Figure S5). This is in stark contrast to previous data obtained using several analytical techniques, which demonstrate that cerebral gray matter contains 1.8-to-2.6-fold more copper than cerebral white matter. As complexation of copper to BCS suppressed activation of F-NpCu1 (Figure C; Figure S3), this is consistent with F-NpCu1 activation being only mediated by the bioavailable copper pool.

To investigate the applicability of F-NpCu1 to other tissue types and disease contexts, we applied F-NpCu1 to paraffin-embedded mouse tissue sections of healthy pancreas and pancreatic tumor. The fluorescence intensity of F-NpCu1 in pancreatic tumor tissue decreased by 16% compared to healthy pancreas tissue (Figure G,H; Figure S7), despite the latter exhibiting a 66% increase in tissue total copper levels as determined by ICP-MS (Figure I). Due to the high metabolic and tissue remodelling nature of pancreatic cancer, tumors exhibit significantly greater expression of cytochrome c oxidase (CcO) and Lysyl Oxidase (LOX) cuproenzymes. , Thus, these results reinforce the hypothesis that F-NpCu1 is selective for bioavailable copper and thus provides valuable complementary information to techniques such as ICP-MS that profile the total copper pool. The decreased F-NpCu1 fluorescence indicates a lower bioavailable copper level in pancreatic tumor tissue, which may have implications for the role of copper in both tumor progression and treatment.

The high specificity and sensitivity of F-NpCu1 for copper in vitro and ex vivo tissue sections prompted us to evaluate its potential to report on copper levels in living cells. Cell viability of neuron-like SH-SY5Y cells after extended incubation (24 h) with high concentrations of F-NpCu1 was assessed by the alamarBlue assay (Figure S8). These results demonstrated that F-NpCu1 showed negligible cytotoxicity at higher concentrations and a longer incubation time than we used in subsequent imaging experiments. Following the confirmation of low cytotoxicity, we next evaluated the performance of the probe in reporting on the cellular copper content in SH-SY5Y cells in situ (Figure A). As expected, SH-SY5Y cells exhibited weak fluorescence following incubation with F-NpCu1 (10 μM) which increased significantly in cells pretreated with CuATSM (5 – 10 μM) and decreased in cells pretreated with the copper chelator ATTM (200 μM) (Figure B). These results combined with HPLC selectivity and ex vivo tissue staining data suggest that F-NpCu1 could be used for semiquantitative analysis of copper content with high selectivity in living cells.

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Results of confocal microscope imaging of live SH-SY5Y cells treated with F-NpCu1 (10 μM, 30 min). (A) Representative images of control cells, cells pretreated with CuATSM (5 or 10 μM, 5 h), or cells pretreated with ATTM (200 μM, 5 h). (B) Quantification of the fluorescence intensity within cells. Scale bars: 50 μm. Calibration bar = fluorescence intensity. **p < 0.001, ****p < 0.0001 (n = 4, average of 6 representative ROIs).

Following successful semiquantitative measurement of copper by F-NpCu1 in living cells, we next sought to assess the ability of F-NpCu1 to detect copper in living mice. Toxicology screening reported no morbidity or mortality nor treatment-related changes in mouse health (Table S4), over an eight day period following intravenous administration of a single bolus dose of 4 – 12 mg/kg F-NpCu1 to C57BL/6 mice (Agilex Biolabs, study number UOS-015). There were also no findings of dose-related changes in body weight or macroscopic pathology, which were considered related to treatment (Figure S9), establishing a high safety and tolerability of F-NpCu1 at doses up to 12 mg/kg. To assess in vivo copper detection by F-NpCu1, anaesthetized C57BL/6 mice were administered 12 mg/kg F-NpCu1 intravenously and whole-body fluorescence emission (measured as radiant efficiency) quantified in real time for 90 min postinjection using an IVIS Spectrum CT in vivo imaging system (Figure A; Figure S10). Dimethyl sulfoxide (DMSO) solutions with and without polyethylene glycol (PEG)­400 were evaluated as vehicles for probe delivery, as both are safe for human injection at concentrations found to completely solubilize F-NpCu1 (140 μL injection vol; 0.83 mL/kg DMSO, 3.33 mL/kg PEG400). Whole-body fluorescence was successfully detected above baseline following administration of F-NpCu1 solubilized in both vehicles, with radiant efficiency peaking slightly later in the PEG400 vehicle (7 min) compared with DMSO (5 min; Figure A). Radiant efficiency was also more sustained over the 90 min time course using PEG400 vehicle compared with DMSO, which exhibited negligible fluorescence beyond 70 min postinjection.

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Whole-body fluorescence imaging of F-NpCu1 in living mice and ex vivo fluorescence imaging of organs harvested from these mice following in vivo imaging. (A) Real-time, whole body fluorescence emission (measured as radiant efficiency) was measured in C57BL/6 mice administered 12 mg/kg F-NpCu1 solubilized in PBS containing 12.5% DMSO ± 52.5% polyethylene glycol (PEG)­400 via tail vein injection (n = 1/vehicle formulation). Total radiant efficiency was calculated for each minute by subtracting baseline fluorescence measurements made prior to probe administration from F-NpCu1 measurements (A). The gastrointestinal tract (GIT), liver, brain, kidneys, lungs, heart and spleen were excised following in vivo fluorescence imaging for ex vivo fluorescence measurements to be made using the same acquisition parameters (B,C). Metal levels in these organs were then measured using ICP-MS (D). Data in parts A and C represent mean values, while data in part d represent mean ± SEM.

We next assessed organ-specific fluorescence as a measure of the ability of F-NpCu1 to profile the natural biodistribution of copper throughout the body. Obtaining these measurements in vivo was initially challenging due to the variable anatomical positions of organs, which affected the fluorescence emission externally detected. We intend to address this issue in the future by radiolabeling F-NpCu1. Nonetheless, we were still able to assess probe biodistribution in this study by euthanizing mice following real-time, whole-body fluorescence measurements and removing target organs to quantify organ-specific fluorescence emission ex vivo. Irrespective of the F-NpCu1 vehicle formulation, the gastrointestinal tract consistently exhibited the highest fluorescence emission, followed by the liver, brain, and other peripheral organs (Figure B,C). Ex vivo organ fluorescence was consistently higher using the PEG400 vehicle compared with DMSO (Figure C), resulting in the adoption of this vehicle for all subsequent experiments. These data closely matched organ-specific copper levels quantified using ICP-MS following IVIS analyses (Figure D), indicating that the bioaccumulation of F-NpCu1 is indeed proportional to the tissue copper content. These results provide strong confidence that the labeling reaction occurs rapidly enough in vivo to prevent free diffusion of the probe after interaction with copper.

BBB permeability constitutes one of the greatest challenges in developing any in vivo imaging tool for CNS disorders. While fluorescence emission was observed in brains harvested from mice injected with F-NpCu1, it was unclear whether this observation signified probe activation within brain vasculature or in surrounding tissues following its diffusion across the blood brain barrier. Intravital microscopy (IMV) could be used to address this question, as it enables real-time observation of fluorescent sensors within the living mouse brain. This technique would allow visualization of F-NpCu1 blood brain barrier infiltration and diffusion into surrounding brain tissues and provide insight into the potential for this tool to be translated from preclinical models to human patients for clinical use.

To facilitate high-resolution deep in vivo fluorescence imaging, we performed craniotomy surgeries on anaesthetized mice to create cranial windows over the parietal cortex (Figure S11). F-NpCu1 (4 mg/kg) and the vascular dye TRITC-Dextran (100 μL, 1 mg/mL) were then injected intravenously via the tail vein to mimic clinical administration of F-NpCu1 in a human, and cortical tissue and vasculature were imaged through the cranial window over 60 min and each compartment quantified separately (Figure A; Figure S12). Fluorescence emission from F-NpCu1 was clearly visible in brain vasculature within 1 min of tail vein injection. It rapidly diffused into surrounding brain tissue, and the emission peaked 3 min postinjection, remaining detectable until 45 min postinjection (Figure A). These data indicate suitable BBB permeability of F-NpCu1 for the in vivo detection of CNS copper levels.

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Intravital fluorescence imaging of F-NpCu1 in the living mouse brain. (A) Anesthetized SWISS mice underwent a craniotomy surgery to create a cranial window exposing parietal cortex, which was then imaged using a Leica SP8 Deep In Vivo Explorer (DIVE) multiphoton microscope with a water dipping objective lens. Three minutes after imaging commenced, dextran was intravenously injected to visualize vasculature (blue) and 4.0 mg/kg F-NpCu1 (green) administered intravenously through the tail vein. Dextran and F-NpCu1 were excited at 920 nm, with emissions measured at 616–684 and 504–577 nm, respectively. Images represent summative depth projections of 130 μm. Scale bars = 100 μm. Stars indicate vascular staining, and hashtags indicate signal in brain tissue. Individual fluorescence microscopy panels are displayed in Figure S11. Untreated mice (n = 3) received no treatment and thus represent physiological brain copper levels, while CuATSM-treated mice (n = 3) were orally administered 15 mg/kg CuATSM daily for 8 days prior to imaging. Average fluorescence emission per unit area (arbitrary units) was calculated within brain tissue (B) and vasculature (C) every 75 s in five representative ROIs within images by subtracting baseline fluorescence from that ROI acquired prior to probe injection from all subsequent fluorescence measurements. Fluorescence intensity was significantly higher in CuATSM treated mice compared with untreated mice (Paired t-test: α < 0.05, t = 19.03, df = 64, n = 65, p < 0.0001), but was unchanged in vasculature between these groups (Paired t-test: α < 0.05, t = 0.84, df = 64, n = 65, p = 0.41). Data represent the mean ± SEM.

The ability to identify differences in brain copper levels in the clinic constitutes a critical unmet need for neurological disorders exhibiting copper dysregulation, promising unparalleled advances in the diagnosis, monitoring, and treatment of these disorders. Having demonstrated that F-NpCu1 crosses the blood brain barrier and diffuses into tissue, we next investigated the ability of F-NpCu1 to differentiate brain copper content in living mice treated with the copper delivery drug, CuATSM, compared with untreated control mice. CuATSM-treated mice exhibited >3-fold higher fluorescent emission in their brain tissue compared to untreated mice at all time points postinjection (Figure B), which was not observed in vasculature at any time point up to 57 min postinjection (Figure C). Elevated copper levels in these same tissues were confirmed using ICP-MS following deep in vivo imaging (Figure S13), cross-validating the ability of F-NpCu1 to identify differences in brain copper in vivo. The rapid peak in fluorescent intensity and subsequent clearance within the 60 min scanning period gives us confidence that this probe will be compatible with the planned PET imaging as 18F has a half-life of 109.8 min and routine [18F]­FDG scans are conducted within 60–90 min of injection.

In summary, we report the synthesis of copper sensor F-NpCu1, which accumulates in the brain and periphery following intravenous injection through proximal protein labeling, in a manner that is proportionate to tissue copper content. Crucially, this sensor exhibits good BBB permeability and can be imaged by intravital microscopy deep within the brains of living mice, reporting on changes in copper levels induced by the copper delivery drug CuATSM. The fluorescence output of F-NpCu1 has not only enabled rigorous in vitro and tissue studies to validate the performance of the probe, which would be extremely difficult without fluorescence emission, but also demonstrated the utility of the probe as a useful fluorescent tool. However, to move into further in vivo studies, and toward development of a diagnostic tool, we will harness the fluorine-containing SFB group to include radionuclide 18F for PET imaging. We are now proceeding with radiolabeling studies to develop 18 F-NpCu1 for PET imaging of copper in living mice and humans. While we have focused here on studies of neurodegenerative disorders and pancreatic cancer, the many other indications in which copper imbalances are implicated highlights the translational potential of 18 F-NpCu1, including genetic copper disorders (e.g., Wilson’s disease) and other cancers (e.g., neuroblastoma) and neurodegenerative conditions (e.g., amyotrophic lateral sclerosis).

Supplementary Material

im5c00024_si_001.pdf (4.1MB, pdf)

Acknowledgments

The authors acknowledge the National Health and Medical Research Council of Australia (APP2019931, GNT2033065, GNT2013881), the Australian Research Council (CE200100012, DP210102148), the Human Frontier Science Program (RGP0060/2021) and Cancer Council NSW (RG21-11) for funding. ETYM is supported by an Australian Government Research Training Program Scholarship. We acknowledge the scientific and technical assistance of Sydney Analytical Core Research Facility at the University of Sydney, the Australian Microscopy and Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis (ACMM), and the Preclinical Imaging Facility and Laboratory Animal Services, both at the University of Sydney. The authors would like to acknowledge the use of the In Silico Drug Design Facility funded through the Centre for Drug Discovery Innovation at the University of Sydney. Human postmortem brain tissues were obtained from the Sydney Brain Bank (Sydney, Australia) and The London Neurodegenerative Diseases Brain Bank (London, UK), which receives funding from the Medical Research Council and the Brains for Dementia Research programme, jointly funded by Alzheimer’s Research UK and Alzheimer’s Society. We acknowledge the Garvan Institute histopathology and biological testing facilities for their services. We thank Agilex Biolabs Pty Ltd, Queensland, Australia for the toxicology studies.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00024.

  • Details about materials, synthesis and characterization, experimental methods, additional figures including fluorescence spectra, HPLC data, chemical property predictions and cell, tissue and animal imaging (PDF)

¶.

L.D.A., B.G.T., and M.E.G. contributed equally.

The authors declare no competing financial interest.

Published as part of Chemical & Biomedical Imaging special issue “Bioimaging of Metals”.

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