Towards dual SPECT/optical bioimaging with a mitochondrial targeting, 99m Tc( I ) radiolabelled 1,8-naphthalimide conjugate

A series of six di ﬀ erent 1,8-naphthalimide conjugated dipicolylamine ligands ( L 1 – 6 ) have been synthesised and characterised. The ligands possess a range of di ﬀ erent linker units between the napthalimide ﬂ uoro-phore and dipcolylamine chelator which allow the overall lipophilicity to be tuned. A corresponding series of Re( I ) complexes have been synthesised of the form fac -[Re(CO) 3 ( L 1 – 6 )]BF 4 . The absorption and lumine-scence properties of the ligands and Re( I ) complexes were dominated by the intramolecular charge transfer character of the substituted ﬂ uorophore (typically absorption ca. 425 nm and emission ca. 520 nm). Photophysical assessments show that some of the variants are moderately bright. Radiolabelling experiments using a water soluble ligand variant ( L 5 ) were successfully undertaken and optimised with fac - [ 99m Tc(CO) 3 (H 2 O) 3 ] + . Confocal ﬂ uorescence microscopy showed that fac -[Re(CO) 3 ( L 5 )] + localises in the mitochondria of MCF-7 cells. SPECT/CT imaging experiments on naïve mice showed that fac -[ 99m Tc (CO) 3 ( L 5 )] + has a relatively high stability in vivo but did not show any cardiac uptake, demonstrating rapid clearance, predominantly via the biliary system along with a moderate amount cleared renally.


Pl e a s e n o t e:
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Thi s v e r sio n is b ei n g m a d e a v ail a bl e in a c c o r d a n c e wit h p u blis h e r p olici e s. S e e h t t p://o r c a .cf. a c. u k/ p olici e s. h t ml fo r u s a g e p olici e s.Co py ri g h t a n d m o r al ri g h t s fo r p u blic a tio n s m a d e a v ail a bl e in ORCA a r e r e t ai n e d by t h e c o py ri g h t h ol d e r s .

Introduction
The different bioimaging modalities each have intrinsic strengths and weaknesses, with no single clinically used technique providing comprehensive information.Prospective multimodal imaging agents are designed to combine the strengths of individual modalities, while offering complementary or additional clinically useful information not provided by a single technique. 1,2Single photon emission computed tomography (SPECT) is a high sensitivity nuclear imaging technique using short half-life gamma emitting radioisotopes that can image the whole body.In comparison, optical fluorescence imaging can utilise agents that emit visible or near-IR photons and can be applied to both pre-clinical in vivo and cellular microscopy studies.However, it lacks the excellent tissue penetration offered by nuclear imaging modalities.The combination of radioimaging and fluorescence imaging presents significant opportunities in the design of useful imaging agents. 3Combined single molecule SPECT/ fluorescence agents can provide early phase high sensitivity imaging of in vivo biodistribution using SPECT, followed by longitudinal optical imaging. 4][7] The clinical use of 99m Tc in SPECT imaging has been well established for many decades.99m Tc (t 1/2 = 6.01 h, γ = 142.7 keV) is a gamma emitting radioactive isotope which is used in >80% of clinical nuclear diagnostic imaging scans, with over 40 million SPECT scans carried out per year worldwide. 8What is less well understood is the behaviour of Tc agents at the sub-cellular level, since the image resolution of SPECT does not allow such determinations.Therefore intrinsically luminescent or fluorescently tagged 99m Tc SPECT agents 9 are potentially helpful in correlating in vitro and in vivo imaging properties, 10 and in determining their cellular uptake, trafficking and localisation. 11From an inorganic chemistry perspective, non-radioactive rhenium is the obvious choice for developing structurally analogous and chemically related complexes. 12Such an approach can be used to validate the development of appropriate chelates 13 for 99m Tc and develop experimental protocols for synthesis and purification. 14Indeed, rhenium complexes are now attracting considerable attention; in part for their potential as cancer therapy agents 15 as well as the existence of accessible radioisotopes appropriate for radionuclide therapies/theranostic applications. 16Binuclear Re(I) carbonyl complexes have shown accumulation in mitochondria leading to cell death. 17Indeed a bimetallic Re(I)/ 99m Tc(I) complex has also been described where the emissive nature of the Re(I) fragment can be exploited in optical studies. 18In the case of fluorescently tagged rhenium complexes, these species can be assessed with respect to cellular uptake using conventional confocal fluorescence microscopy techniques. 192][23] The relative ease of functionalisation together with advantageous absorption and emission properties in the visible region have led to a large number of reports on their application to cell imaging.For example, recent studies have shown that the 1,8-naphthalimide structure can dictate intracellular localisation, including effective mitochondrial targeting. 24ince mitochondria have essential roles in energy production, cell signalling, cell-cycle control and cell death, and mitochondrial function can be disrupted in both ischaemic heart cells and cancer cells, they are attractive targets.Thus the development of a SPECT/fluorescence multimodal imaging agents for targeting mitochondria would therefore allow for in vitro, in vivo and ex vivo examination of myocardial function and tumour growth. 25In this context, this current work describes the development of 1,8-naphthalimide functionalised chelates suitable for labelling with 99m Tc(I) for mitochondrial targeted multimodal imaging.During the course of these studies, work by Turnbull et al. has shown that a series of related 4-amino-1,8-naphthalimide fluorophores can be complexed with Re(I)/ 99m Tc(I).The overall charge of the complex was shown to strongly influence the biological attributes of the species. 26

Synthesis and characterisation of ligands and complexes
Scheme 1 shows the different chelator structures that were synthesised in this study.The compounds were designed to meet the coordination chemistry requirements of M(I) (where M = Re, Tc) with a face-capping tridentate donor set, fulfilled by a dipicolylamine (DPA) 27 chelating moiety.This is linked to the naphthalimide fluorophore for optical imaging via different length spacers of variable hydro-and lipophilicity.Thus, a series of six ligands were synthesised in three or four steps from 4-chloro-1,8-naphthalic anhydride (Scheme 2).Substitution at the 4-chloro position of the naphthalimide ring was achieved in DMSO at elevated temperature using an excess of diamine (often as the mono-Boc protected version).Following cleavage of the Boc group (trifluoroacetic acid in dichloromethane), treatment of N 1-6 with 2-pyridinecarboxalde-hyde in a one-pot reductive amination procedure gave the dipicolylamine derived target ligands, L [1][2][3][4][5][6] , in each case.All experimental details and spectroscopic characterisation data for the synthesis of intermediates and ligands are included in the Experimental section.
All isotopes of technetium are radioactive, and so natural rhenium is commonly used as a chemical analogue 28 to allow the synthetic and purification protocols to be optimised prior to the use of a radionuclide.The corresponding tricarbonyl Re(I) complexes were isolated by reacting 29 the precursor compounds with fac-[Re(CO) 3 (MeCN) 3 ]BF 4 to yield fac-[Re(CO) 3 (L)] BF 4 as air stable solids.Characterisation of the complexes was achieved using a range of spectroscopic and analytical techniques.The expected facial geometry with respect to the three carbonyl ligands was confirmed using IR spectroscopy.In all cases two CuO stretches were observed between 2040-1900 cm −1 which are attributed to the pseudo C 3v symmetry of the complexes.In addition to this, the naphthalimide carbonyl stretches were observed at 1700-1625 cm −1 and the [BF 4 ] − counter anion at ca. 1055 cm −1 .High resolution mass spectrometry data were obtained for each complex identifying the cationic fragment with the appropriate isotopic distribution for rhenium. 1 H NMR spectroscopy was able to clearly establish the formation of the complex in each case.Firstly, the methylene linkers of the dipicolylamine unit shift upon coordination and become diastereotopic with an observed coupling around 16 Hz. 28The aliphatic protons of the linker unit are also mildly shifted in the complex versus the ligand.The general pattern amongst the aromatic proton resonances is retained upon formation of the complex.Principally, data from 13 C NMR studies allowed identification, in all cases, of the rhenium-bound carbonyls (ca.195 ppm) and the naphthalimide carbonyls (ca.165 ppm).

Luminescence properties
Prior to radiolabelling and imaging studies, the electronic and optical properties of these species were investigated (Table 1).The UV-vis.spectra of all ligands were recorded in MeCN or chloroform solutions.Because of the relative hydrophilicity of the L 5 /L 6 systems, additional data in aqueous solution was also obtained on these ligands and their complexes.The absorption spectra showed some common features, with higher energy absorptions associated with the naphthalimide and pyridyl based π-π* transitions, and a lower energy, broad peak around 440 nm which was assigned to an intramolecular charge transfer (ICT) transition from the amine-functionalised naphthalimide chromophore.The position of the ICT peak showed a slight shift that was dependent upon the amine substituent and was shown to be sensitive to the polarity of solvent, as expected for a charge transfer transition. 30The absorption spectra of the corresponding Re(I) complexes (for example, Fig. 1) were comparable, with only minor shifts of the ICT band upon formation of the cationic Re(I) complex that are likely to be a result of the cationic charge perturbing the ICT transition. 31Typically the molar absorption coefficients (Table 1) of the ICT bands in the complexes were in the range of 7000-20 000 M −1 cm −1 .
Steady state emission spectra were obtained using an irradiation wavelength of 425 nm, corresponding to direct excitation of the ICT band of the ligand.All ligands and complexes showed an intense, featureless luminescence peak at 505-550 nm, giving Stokes' shift >3500 cm −1 , which is typical of this class of fluorophore (for example, Fig. 2).Again, the position of the peak was found to be moderately sensitive to the amine substituent (related to the relative electron donating power) and the solvent polarity.For example, measurements in aqueous media for L 5 /L 6 and their complexes resulted in a bathochromic shift of the emission maxima to ca. 540 nm, typical of positive solvatochromism (Fig. 2).Time-resolved experiments revealed lifetimes <15 ns in all cases, confirming the fluorescent nature of these ICT emitting species.Quantum yields were obtained for each species in the various solvents, and ranged 8-67%.For L 5 /L 6 and their rhenium(I) complexes

Radiolabelling with technetium-99m
Having established the Re(I) coordination chemistry and the optical properties of the complexes, radiolabelling of selected ligand systems was attempted with the SPECT imaging (gamma emitting) isotope technetium-99m.Radiolabelling studies were conducted using fac-[ 99m Tc(CO) 3 (H 2 O) 3 ] + , which was obtained from the reduction of [ 99m TcO 4 ] − using the method reported by Alberto et al. 32,33 Initial attempts to radiolabel L 1 (the ligand variant with an ethylene linker between the naphthalimide and the dipicolylamine chelate unit) led to the formation of a number of radioactive species indicative, perhaps, of the decomposition of the ligand.Certainly, the limited aqueous solubility of L 1 was likely to be a hindrance to efficient radiolabelling.Therefore, the focus of further radiochemistry experiments was focussed upon the more hydrophilic naphthalimide L 5 (the ligand variant incorporating the ethylenedioxodiamine linker) which demonstrated good water solubility.

Stability and mitochondrial uptake of fac-[ 99m Tc(CO) 3 (L 5 )] +
Lipophilicity is an important parameter in dictating the cellular localisation of organometallic complexes. 35Lipophilicity (log D 7.4 ) measurements were carried out on the radiolabelled species fac-[ 99m Tc(CO) 3 (L 5 )] + and showed a value of 0.67 ± 0.06.This suggests a species with suitable solubility for biological studies, but passive cell membrane permeability is likely to be less favourable. 36For drug-like species, such a value can also indicate that a renal clearance pathway will be observed. 37To mimic in vivo conditions, formulated fac-[ 99m Tc(CO) 3 (L 5 )] + was incubated with human serum at 37 °C.HPLC analysis did not reveal any significant instability up to 3 hours, indicating that the radiolabelled compound had sufficient stability to progress to preliminary in vivo imaging experiments.
To investigate fac-[ 99m Tc(CO) 3 (L 5 )] + behaviour towards mitochondria, experiments were carried out using mitochondria freshly isolated from rat heart tissue.fac-[ 99m Tc(CO) 3 (L 5 )] + accumulated in the mitochondria to a higher extent (55.68 ± 6.5%) than complexes containing the cationic triphenylpho-    sphonium mitochondria targeting group that has been frequently used in mitochondrial targeting. 38This uptake was highly dependent on mitochondrial membrane potential (MMP) as shown by a nearly complete abrogation (1.35 ± 0.14%) when the MMP was depolarised using carbonyl cyanide m-chlorophenylhydrazine (CCCP).
Having established the mitochondrial and cellular uptake properties, a scoping in vivo experiment was carried out with fac-[ 99m Tc(CO) 3 (L 5 )] + in a naïve mouse using SPECT/CT imaging (Fig. 6).Firstly, there were no signs of toxicity to the mouse during the in vivo studies.The tracer showed rapid clearance, predominantly via the biliary system along with a moderate amount cleared renally.This is consistent with the log D of fac-[ 99m Tc(CO) 3 (L 5 )] + , as some renal clearance was observed and more complex active transport mechanisms can influence hepatobiliary clearance. 39Urine analysis demonstrated that the tracer was of a relatively high stability (85.4 ± 1.4%) and suitable for further in vivo analysis.From these imaging studies, it was clear that cardiac uptake was not observed for fac-[ 99m Tc(CO) 3 (L 5 )] + .This suggests that in its current form the tracer will not be appropriate for assessing cardiac function in vivo and so structural modification would be required to ensure uptake and retention.

Conclusions
In conclusion, this study shows the design, synthesis and characterisation of a new class of radiotracer which is preferentially taken up into the mitochondria of cells.The tracer combines a naphthalimide fluorophore with a technetium(I) tricarbonyl unit to give a structure with excellent cellular and mitochondrial uptake characteristics.Cellular bioimaging shows good image quality in the MCF-7 cell line.The preliminary in vivo assessment of the aqueous soluble 99m Tc(I) compound shows rapid clearance, aligning with the requirements for nuclear imaging to ensure background signal is minimised.
These results now justify further investigation of the tumour uptake properties in vivo with appropriate tumour models and larger animal cohorts.
In its current form, fac-[ 99m Tc(CO) 3 (L 5 )] + does not demonstrate cardiac uptake in vivo.Therefore, our further studies will also consider structurally modified variants that can promote and optimise this characteristic.During the course of these studies, Luyt and co-workers reported a peptide-functionalised naphthalimide-based fluorophore that can be used to target CXCR4 expressing cells for potential dual mode imaging. 40his further suggests that the inherent fluorophore-chelate design presented herein holds great promise for future biological and bioimaging applications.

General considerations
All reagents and solvents were commercially available and were used without further purification if not stated otherwise.For the measurement of 1 H and 13 C NMR spectra a Bruker Fourier 300 (300 MHz), Bruker AVANCE HD III equipped with a BFFO SmartProbe™ (400 MHz) or Bruker AVANCE III HD with BBO Prodigy CryoProbe (500 MHz) was used.The obtained chemical shifts δ are reported in ppm and are referenced to the residual solvent signal.Spin-spin coupling constants J are given in Hz.
Low-resolution mass spectra were obtained by the staff at Cardiff University.High-resolution mass spectra were carried out at the EPSRC National Mass Spectrometry Facility at Swansea University.High resolution mass spectral (HRMS) data were obtained on a Waters MALDI-TOF mx at Cardiff University or on a Thermo Scientific LTQ Orbitrap XL by the EPSRC UK National Mass Spectrometry Facility at Swansea University.IR spectra were obtained from a Shimadzu IR-Affinity-1S FTIR.Reference to spectroscopic data are given for known compounds.UV-Vis studies were performed on a Shimadzu UV-1800 spectrophotometer as MeCN solutions (2.5 or 5 × 10 −5 M).Photophysical data were obtained on a JobinYvon-Horiba Fluorolog spectrometer fitted with a JY TBX picosecond photodetection module as MeCN solutions.Quantum yield measurements were obtained on aerated MeCN solutions of the complexes using [Ru(bpy) 3 ](PF 6 ) 2 in aerated MeCN as a standard (Φ = 0.016).Emission spectra were uncor-rected and excitation spectra were instrument corrected.The pulsed source was a nano-LED configured for 295 nm or 372 nm output operating at 1 MHz.Luminescence lifetime profiles were obtained using the JobinYvon-Horiba FluoroHub single photon counting module and the data fits yielded the lifetime values using the provided DAS6 deconvolution software.Product containing fraction (RCY = 47 ± 18%, n = 3, corrected for decay to the beginning of reaction) eluting at 12-14 min was collected, diluted twice with deionised water and loaded onto homemade SPE cartridge (Sep-Pak Light) containing 80-100 mg of Oasis C18 sorbent.The cartridge was washed with 2-3 mL of deionised water and dried by a current of inert gas.The product was eluted by 500 μL of ethanol, the solvent was evaporated under gentle heating aided by a current of inert gas and reformulated into PBS.For in vivo evaluation, the formulation was filtered using a 0.22 μm filter for sterility prior to animal administration.The decay corrected preparatory yield was 21 ± 11% (n = 3) and the full process requires less than 2 h from the start of synthesis.

Lipophilicity
Log P was measured using a standard shake-flask method.Briefly, an aliquot of the tracer solution in ethanol (∼1 MBq) was dried in a 1.5 mL Eppendorf tube and dissolved in 1 mL of 1 : 1 mixture of octanol/PBS.Solution was vigorously mixed in vortex shaker for 5 min at room temperature and phases separated by centrifugation at 14.5 krpm for 2 min.50 μLo fe a c h phase was diluted up to 1 mL and measured using an AGC.

Serum stability
An aliquot of the tracer in ethanol (∼3 MBq) was dried, dissolved in 0.5 mL of human blood serum in a HPLC vial and incubated while shaking at 600 rpm at 37 °C up to 3 hours.Samples were taken at time 0, 30 min, 60 min, 1 h, 2 h and 3 h; the protein was precipitated by addition of double volume of ice cold methanol and removed by centrifugation and the supernatant analysed by radio-HPLC.In vivo SPECT imaging and stability All animal procedures were approved by the University of Hull Animal Welfare Ethical Review Body (AWERB) and carried out in accordance with the Animals in Scientific Procedures Act 1986 and the UKCCCR Guideline 2010 41 by approved protocols following institutional guidelines (Home Office Project License number 60/4549 held by Dr Cawthorne).Female CD1 nude mice (25-30 g, Charles River, UK) were induced with 5% and maintained under anaesthesia with 3% isoflurane (oxygen at 1 L min −1 ).Temperature and respiration were monitored throughout the scan.SPECT-CT acquisition was carried out on a Mediso NanoSPECT/CTPLUS, equipped with multi-pinhole pyramid collimators.Animals were injected i.v. with ≈30 MBq (200 µL) of fac-[ 99m Tc(CO) 3 (L 5 )] + 30 min prior to a 25 min SPECT scan (energy window: 126.4-154.6 keV).To show anatomical co-registration, a CT scan (240 projections, 1 s exposure, 55 kVp X-rays) was acquired following the SPECT scan.SPECT and CT images were reconstructed with an iterative algorithm and with exact cone beam Filtered Back Projection, respectively (HiSPECT, Scivis GmbH, Germany).
After imaging experiments, mice urine was collected, proteins were precipitated by addition of double volume of ice cold methanol and removed by centrifugation at 14.5 krpm for 5 min and supernatant was analysed by radio-HPLC indicating 85.4 ± 1.4% stability (n = 1, triplicate analyses).

In vitro analysis
Cell culture.MCF-7 cells were purchased from the European Collection of Cell Cultures and grown in RPMI-1640 media.Media was supplemented with 10% fetal calf serum (FCS).Human glioblastoma cell line from (U87) from ATCC and it was cultured using DMDM media with 10% (v/v) heat inactivated fetal bovine serum (FBS).Media and FCS were purchased from Gibco/Life Technologies, UK.Cells were maintained in Nunc 75 cm 2 tissue culture flasks (Fischer Scientific, UK) inside a humidified 5% CO 2 incubator at 37 °C.Both cell lines were divided bi-weekly for maintenance of stock at a divided ratio of 1 : 8-1 : 10 for MCF-7 cells.For confocal and flow cytometry experiments cells were counted and seeding densities (below) were used.
Toxicity.Cells were seeded in 96 well plate with 1000 cells per well in 200 µl growth medium.The plates were incubated overnight at 37 °C allow cells to adhere.After 18 h media was removed from the wells and 100 μl of treated media with various concentrations of compounds was added.The plates were returned to the incubator.After 72 h, MTS reagent (Promega, UK) 20 µl was added to the wells.Plates were incubated for 3 h at 37 °C.Absorbance reading were taken at 490 nm using Synergy HT microplate reader (Biotek, USA).The absorbance value of each triplicate were averaged and the media only absorbance was subtracted.Compounds reading were normalised to the control reading.Empty plate and compound added background analysis was carried out to ensure low levels of non-specific fluorescence.
Flow cytometry.1×1 0 5 cells per well were seeded in six-well plates and cultured for 2-3 days until 70-80% confluent.Cell media was removed and 1 mL 0.1% FCS-containing media was added.Carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma Aldrich, UK) in DMSO (50 mM, 1 μL) was added into three wells (1 mL total volume) and 0.1% DMSO was added to the remaining three control wells (as a control).Plates were placed inside a CO 2 incubator for 30 min, followed by the addition of compound (500 nM, 50 µL media) to both groups.Plates were incubated inside the CO 2 incubator at 37 °C for 1 hour, subsequently, media was removed and the cells were washed twice with ice cold PBS buffer.Cells were harvested into PBS with cell scrapper and isolated by centrifugation at 200g for 5 min at 4 °C.The pellet was re-suspended in 300 μL of PBS and the cell suspension transferred into polypropylene FACS tubes (Falcon 2054) and analysed by a FACScan flow cytometer (BD Biosciences Europe, Erembodegem, Belgium).Dot plot was used to gate cells and the FL-1 channel was used to measure variation in fluorescence intensities.Mean fluorescence intensities were used to quantify for comparison.
Confocal microscopy.MCF-7 cells (2.5 × 10 3 ) and H9c2 cells (4 × 10 3 ) were seeded in Mattak confocal dishes (P35G-1.2-20-C,35 mm 2 ) and cultured for 2-3 days until 70-80% confluent.After reaching desired cell density, the growth media was replaced with 1 mL 0.1% FCS-containing media and cells were treated with fac-[Re(CO) 3 (L 5 )] + (500 nM, 50 µL) for 1 hour inside a CO 2 incubator under 5% CO 2 .Mitotracker deep red (MDR, 250 nM, 20 µL) was added 1 hour prior to compound to obtain double labelled cells for mitochondrial co-localisation study.After 30 min, the cells were washed twice with 1 mL PBS and media containing 15 mM HEPES was added.Unstained control samples of cells were used to check for auto fluorescence.Live cell images were obtained using ZEN software on a Zeiss LSM 710 inverted confocal microscope, equipped with an incubator chamber at 37 °C.Images were obtained using a Zeiss 63× water objective.
To measure the degree of overlap between subcellular localisation of fac-[Re(CO) 3 (L 5 )] + and MDR, a colocalisation coefficient was calculated following standard methods.Single-label control samples were prepared to eliminate fluorescence overlap between two fluorescence channels; in addition to fac-[Re (CO) 3 (L 5 )] + or MDR, untreated cells were assessed in order to eliminate background fluorescence.Single-label control samples were imaged under the same exposure settings as double-labelled samples.Exact coordinates were determined using crosshairs at X and Y coordinates from single-labelled controls and were identical for double-labelled samples.Once the exact coordinates were determined, the ZEN software gave tables containing values of co-localisation coefficient and overlap coefficient.
Mitochondrial uptake of [ 99m Tc(CO) 3 (L 5 )] + .Cardiac mitochondria were isolated following a modified method based on that reported by Boehm et al. 42 A male Sprague-Dawley rat was anaesthetised with Dolethal and the heart was excised, washed and trimmed in cold buffer.The trimmed heart was transferred into a centrifuge tube containing 5 ml of ice cold buffer and then minced.The minced cardiac tissue suspension was transferred to a Teflon glass homogeniser (Scientific Laboratory Supplies, UK) and a further 5 ml of ice cold buffer A was added (total 10 ml). 1 ml trypsin was added and gently homogenized using a tight fitting Teflon pestle (Scientific Laboratory Supplies, UK).After 15 min of trypsin digestion, 10 ml of trypsin inhibitor was added and mixed with three passes of the tight fitting Teflon pestle.The tissue suspension was then centrifuged at 600g for 10 min at 4 °C.The supernatant was transferred into another centrifuge tube and centrifuged again at 8000g for 10 min at 4 °C.The resultant pellet was resuspended in 10 ml of isolation buffer B and again centrifuged at 8000g for 10 min at 4 °C.The mitochondrial pellet was then resuspended in 100 µl of respiratory buffer containing 10 mM succinate.The total protein content of the isolated mitochondria was measured using Bio-Rad protein assay.10 µl of the isolated mitochondria suspension was added into 390 µl of water (40 times dilution).A standard solution was made with BSA to calibrate the protein amount.60 µl of the mitochondria suspension was added to 3 ml of Bio-Rad solution and 60 µl from each of the BSA stock solution dilutions were also taken and added to 3 ml of Bio-Rad solution.
Mitochondrial protein concentration was shown to be 1.23 µg µl −1 , as it was 40 times diluted, the original concentration was 49.46 µg µl −1 .Hence, the total mitochondrial protein amount was 4946 µg in 100 µl.150 µg protein was aliquoted into each Eppendorf.100 µl of respiratory buffer containing 10 mM succinate added to give the control (n = 3 for each tracer).10 µM CCCP was added into 100 µl of respiratory buffer containing 10 mM succinate and was then added in remaining Eppendorf tubes.All the Eppendorf tubes were transferred into the CO 2 incubator containing 5% CO 2 at 37 °C for 15 min and after that 0.5 MBq of tracer was added to the control and CCCP treated mitochondria.All of the Eppendorf tubes were transferred back into the CO 2 incubator for 30 min and after that the assay was terminated by centrifugation at 8000g for 5 min at 4 °C.The supernatant was collected and the mitochondrial pellet was washed three times with ice cold respiratory buffer containing 10 mM succinate.After the final wash, the mitochondrial pellet was resuspended into 1 ml of PBS.The amount of radioactivity was measured using the gamma counter (PerkinElmer Wallac wizard 3″).Percent accumulation was quantified by using the ratio of activity in pellet to that in the pellet plus supernatant.The experiment was carried out three times using internal triplicates.The data is presented as an average of each internal triplicate with the standard deviation among triplicates.

Ligands and precursors
Synthesis of compound C 1 . 434-Chloro-1,8-naphthalic anhydride (2 g, 8.6 mmol) was dissolved in ethanol (60 mL) and ethanolamine (0.79 mL, 12.9 mmol) was added.The solution was then heated at reflux for 12 h under a nitrogen atmosphere.Upon cooling to 0 °C precipitation occurred, and filtration yielded C 1 as a yellow solid (2.01 g, 85%). 1  Synthesis of compound C 2 . 44Prepared as for C 1 except using 2-methoxyethylamine (0.90 g, 12.9 mmol), yielding C 2 as a yellow solid (1.96 g, 79%). 1  Synthesis of N 1 .C 1 (0.3 g, 1.01 mmol) was dissolved in DMSO (4 mL) and N-Boc-ethylenediamine was added (0.47 g, 2.93 mmol) and the solution heated at reflux for 16 hours under a nitrogen atmosphere.The solution was flooded with 30 mL of water and neutralised with 0.1 M HCl.The product was then extracted with dichloromethane (3 × 15 mL) and washed with water (3 × 10 mL) and brine (3 × 10 mL).The solvent was then reduced to a minimum and precipitation induced by the dropwise addition of petroleum ether.Filtration of the precipitate followed by washing with diethyl ether (5 mL) yielded the Boc-protected intermediate product as a bright orange solid (0.27 g, 65%). 1  TFA in DCM for 24 hours under a nitrogen atmosphere.The solvents were then removed under vacuum, and the residue dissolved in methanol and the solvent removed again.This was repeated in triplicate to yield the final product as an oily yellow solid (0.178 g, 60%). 1    Synthesis of N 3 .C 1 (0.75 g, 2.73 mmol) was dissolved in DMSO (4 mL) and 1,6 diaminohexane (0.4 mL, 4.1 mmol) was added and the solution heated at reflux for 16 hours.The solution was flooded with 30 mL of water and neutralised with 0.1 M HCl.The product was then extracted with dichloromethane (3 × 15 mL) and washed with water (3 × 10 mL) and brine (3 × 10 mL).The solvent was then reduced to a minimum and precipitation induced by the dropwise addition of petroleum ether.Filtration of the precipitate followed by washing with diethyl ether (5 mL) yielded the product as an orange solid (0.422 g, 44%). 1    by stirring in 50% TFA in DCM (10 mL) for 24 hours under a nitrogen atmosphere.The solvents were then removed under vacuum, and the residue dissolved in methanol and the solvent removed again.This was repeated in triplicate to yield N 5 as a red oil (0.29 g, 91%). 1   Synthesis of L 1 .N 1 (0.11 g, 0.37 mmol) was dissolved in 1,2dichloroethane and 2-pyridinecarboxaldehyde (0.07 mL, 0.74 mmol) was added and the solution stirred for 2 hours under a nitrogen atmosphere.Sodium trisacetoxyborohydride (0.24 g, 1.11 mmol) was then added and the solution stirred at room temperature for 18 hours.The solution was then neutralised with saturated NaHCO 3 and the product extracted into chloroform, washed with water (3 × 20 mL) and brine (3 × 20 mL).The organic layer was collected then dried over MgSO 4 and filtered, and the solvent removed to yield the product as an orange oil (0.13 g, 73%). 1  necarboxaldehyde (0.07 mL, 0.74 mmol) was added and the solution stirred for 2 hours under a nitrogen atmosphere.Sodium trisacetoxyborohydride (0.24 g, 1.11 mmol) was then added and the solution stirred at room temperature for 18 hours.The solution was then neutralised with saturated NaHCO 3 and the product extracted into chloroform, washed with water (3 × 20 mL) and brine (3 × 20 mL).The organic layer was then dried over MgSO 4 and filtered, and the solvent removed yielding L 5 as an orange oil (0.13 g, 73%). 1  Synthesis of L 6 .Prepared as for L 5 but using N 6 (0.06 g, 0.15 mmol), 2-pyridine carboxaldehyde (0.03 g, 0.3 mmol) and sodium trisacetoxyborohydride (0.1 g, 0.45 mmol) and triethylamine (2 mL).L 2 was isolated as a red oil (0.063 g, 72%).

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Dalton Trans.,2020,49,511-523This journal is © The Royal Society of Chemistry 2020 Cellular cytotoxicity was assessed using U87 cells in vitro by the MTT assay, giving a CC 50 value of 72.7 μM (Fig.4), which indicates low cytotoxicity in vitro for the compound.Whole cell mitochondrial uptake of fac-[Re(CO) 3 (L 5 )] + was then studied using confocal fluorescence microscopy in human breast cancer cells (MCF-7) in culture.As a control, a benzyl chloride based mitochondrial stain (MitoTracker deep red, MDR) was used for dye co-localisation studies as its emission wavelength (665 nm) is sufficiently different from fac-[Re(CO) 3 (L 5 )] + (540 nm) thus allowing independent detection.Confocal fluorescence microscopy studies into the mitochondrial localisation for fac-[Re(CO) 3 (L 5

Fig. 6
Fig. 6 SPECT and CT overlaid images with the SPECT images acquired using the radiotracer fac-[ 99m Tc(CO) 3 (L 5 )] + .Sagittal and coronal views show the localisation of the tracer after 22-60 min.Red arrow: gall bladder; yellow arrow: biliary clearance into the biliary tract and small intestine; green arrow: bladder; blue arrow: kidney.
All work carried out with ionising radiations was carried out within the legal requirements of the Ionising Radiations Regulations 2017 and the Environmental Permitting Regulations 2010.The studies followed local rules and regulations to ensure safe and compliant handling of open source radioactive materials.The labelling precursor in a glass HPLC vial was dissolved in 50 μL of MeOH and was further diluted with 100 μL of 10 mM PBS solution.The solution was degassed by sonication and purged with argon for 10 min.Up to ∼400 MBq of freshly reduced fac-[ 99m Tc(CO) 3 (H 2 O) 3 ] + in a reduction medium (∼500 μL) was added, bringing the final precursor concentration to previously determined optimum 0.5-1.3mM range.The reaction mixture was incubated in a heating shaker at 70 °C mixing at 600 rpm for 30 min.After which it was cooled down to room temperature and injected onto semi-preparatory HPLC (ACE5 C18 5 Å 100 × 250).

Table 1
Solvent dependent intramolecular charge transfer absorption and emission data for the ligands and rhenium(I) complexes a Measurements obtained in aerated solutions; λ ex = 405 nm.b λ ex = 295 nm.c Using aerated MeCN solution of [Ru(bipy) 3 ](PF 6 ) 2 as a reference.d In acetonitrile.e In chloroform.f In water.
Synthesis of N6.Prepared as for N 5 but using C 2 (0.75 g, 2.59 mmol), first yielding the intermediate product as a red oil (0.46 g, 35%).