Development of a suite of gadolinium-free OATP1-targeted paramagnetic probes for liver MRI

Abstract

Five amphiphilic, anionic Mn(II) complexes were synthesized as contrast agents targeted to organic anion transporting peptide transporters (OATP) for liver magnetic resonance imaging (MRI). The Mn(II) complexes are synthesized in 3 steps, each from the commercially available trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA) chelator, with T₁-relaxivity of complexes ranging between 2.3 and 3.0 mM⁻¹s⁻¹ in phosphate buffered saline at applied field strength of 3.0 T. Pharmacokinetics were assessed in female BALB/c mice by acquiring T₁-weighted images dynamically for 70 minutes after agent administration and determining contrast enhancement and washout in various organs. Uptake of Mn(II) complexes in human OATPs was investigated through in vitro assays using MDA-MB-231 cells engineered to express either OATP1B1 or OATP1B3 isoforms. Our study introduces a new class of Mn-based OATP-targeted contrast that can be broadly tuned via simple synthetic protocols.

INTRODUCTION

Gadolinium-based contrast agents (GBCAs) have long been implemented into magnetic resonance imaging (MRI) scans to better elucidate pathologies that do not present with vastly different longitudinal (T₁) and transverse relaxation times (T₂). Many Gd-based agents have been used for clinical MRI, due to the favourable properties of the Gd(III) ion for generating T₁ contrast (high spin quantum number (7/2), fast water exchange, and long electronic relaxation times)¹ and can be broadly categorized into either (i) non-specific or (ii) targeted agents, dependent on their transport and localization in the body upon intravenous delivery. Targeted GBCAs that have been tested or used routinely in humans include blood pool agents that bind to albumin, and liver targeted agents.

Gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA), known commercially as Primovist^® in Europe and Canada, and Eovist^® in the USA (Bayer Healthcare) is a liver-targeted MR contrast agent, that is clinically administered to better delineate focal lesions present in the liver, or metastatic disease arising from primary tumour sites other than the liver^2,3. Targeted agents such as Gd-EOB-DTPA have an initial venous transport phase where their distribution is akin to non-specific MR agents, before being taken up into hepatocytes through one or two bi-directional transmembrane transporters, called organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1, OATP1B3)^4–6. These transporters share 80% sequence homology⁷, and possess some of the same natural substrates including bilirubin, bile acids, conjugated steroids, eicosanoids, and thyroid hormones^8–11. Liver targeting by Gd-EOB-DTPA is modulated through the addition of the ethoxy-benzyl side chain that helps give the molecule an amphiphilic character to promote uptake through these OATP1 transporters. Upon residency in the hepatocyte, surrounding water protons will experience enhanced T₁ relaxation, leading to hyperintensities on T₁-weighted images. The molecule is then pumped out of the cell through one of several unidirectional ATP-dependent multidrug resistance proteins (MRP) that either pump the agent back into the bloodstream or into bile canaliculi and eventually clear the subject through the gastrointestinal tract.

In the last decade, Gd-EOB-DTPA has also been used in non-clinical studies as a reporter probe for tracking cells engineered to ectopically express OATP transporters, motivated by pioneering work on the rat-derived OATP1A1¹². Most recently human OATPs (OATP1B3 and OATP1B1) have been expressed in cancer cells to perform longitudinal, 3D tracking of engineered cells in vivo (Figure 1)^12–18.

Figure 1.

Simplified schematic of the OATP1 reporter present at the cell surface, and the bidirectional transport mechanism of OATP1-targeted contrast agents

GBCAs possess either a linear or macrocyclic chelator, which can have implications in their safety profiles. Unlike macrocyclic chelators which are considered sufficiently stable, Gd(III) agents with linear chelators, such as Gd-EOB-DTPA, are less stable¹⁹. In patients with renal impairment, the resulting prolonged exposure to GBCAs can lead to increased likelihood of demetallation of Gd(III) from the chelator, particularly when the less-stable linear agents are used which can be a risk factor for nephrogenic systemic fibrosis (NSF), a non-specific fibrotic disorder that presents as hardening of the skin and nonspecific scleroderma-like skin lesions^20–26. The amount of circulating free Gd(III) is the main determinant of tissue toxicity²⁷ and is problematic as Gd(III) has an ionic radius similar to Ca(II), and can act as a calcium antagonist in essential biological processes^28–30. Additionally, vacancies in chelators upon Gd(III) demetallation can lead to transmetallation whereby endogenously present metals can fill the chelator’s vacancy^31,32. Reported cases of Gd(III) deposition in the brain led the European Medicines Agency to suspend the use of three linear GBCAs in 2017, and the U.S. Food & Drug Administration also placed a warning on the use of all linear GBCAs in 2017^33,34. As there is no clinical alternative to Gd-EOB-DTPA, its use has continued due to the important clinical information it provides. However, an OATP1-targeted gadolinium-free probe that provides similar information but lacks the safety issues associated with linear GBCAs would be beneficial.

Several groups have investigated alternative paramagnetic ions to generate T₁ contrast, including Fe(III), and Mn(II)^35,36,37. Both ions present attractive alternatives as they are essential nutritional elements, where low-level exposures generated through complex dissociation can be incorporated into labile endogenous pools or excreted in the case of Mn(II)³⁸. There is a clinical precedence of Mn-based MRI contrast agents. The complex Manganese-dipyridoxal diphosphate (Mn-DPDP, mangafodipir) was marked for liver scans but its use was discontinued due to poor clinical performance and low sales.^39,40 Mn-DPDP underwent spontaneous dissociation in the bloodstream, and liver enhancement following Mn-DPDP injection is predominantly due to hepatocellular accumulation of de-chelated Mn(II) ions. More recently, a novel manganese porphyrin agent termed Mn-TriCP-PhOEt has been developed to target OATP1, while displaying high relaxivity due to the presence of a water co-ligand in either axial position⁴¹. Another promising Mn-based probe is Mn-PyC3A^38,42,43,44, which is being developed as an extracellular fluid contrast agent and which recently entered first in human clinical trials (NCT05413668). Amphiphilic derivatives of Mn-PyC3A have been demonstrated as effective liver MRI agents in mice, but affinities for human OATPs were not evaluated. One such derivative is Mn-PyC3A-3-OBn, which was prepared by a multistep synthesis, requiring chromatographic purification of intermediates, representing a substantial challenge to scale up required for future development work⁴⁵. Here, we seek to develop liver-targeted manganese-based contrast agents through a simplified synthesis pathway, specifically designed for liver uptake in rodents, as well as through the human OATP1B1 and OATP1B3 transporters. Five novel agents were developed using trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid as a starting reagent, by conjugating varied lipophilic side chains through mono-amide conjugation. We then characterized these agents through relaxivity measurements, in vivo liver imaging in mice, and through quantitation of in vitro cell uptake through ectopically expressed human OATP1 transporters.

RESULTS

Synthesis of Five Novel Mn(II) Complexes

Because free, bioavailable Mn accumulates in tissues such as the liver, pancreas, and heart, and can exhibit acute cardiotoxicity,⁴⁶, we sought to develop OATP-targeted Mn-complexes that were both thermodynamically stable and kinetically inert to Mn release. Based on prior accounts demonstrating efficient whole-body elimination of Mn administered as the complex Mn-PyC3A^42–44, we reasoned that structurally related acyclic, hexadentate ligands built from the rigidifying trans-1,2-cyclohexylenediamine backbone would provide complexes of comparable stability. We also sought to develop complexes that could be easily synthesized, modified, and scaled for potential future development work. In this regard, we reasoned that reacting lipophilic amines with the common synthon 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid monoanhydride (CDTA-mA), with would enable facile access to suitable Mn chelators.

The complexes were prepared in 3 steps. CDTA-mA was generated by stirring commercially available CDTA in an excess of acetic anhydride with pyridine added. Next, the ligands were assembled by reacting CDTA-mA with the corresponding either p-chloraniline, 3,5-(bis)trifluoromethylaniline, 1-phenylpiperazine, benzylamine, or 4-ethoxybenzylame. The corresponding Mn complexes Mn-1 through Mn-5 were then synthesized by reacting ligands 1–5 with 1 molar equiv. MnCl₂ and raising the pH to > 6.5 (Figure 2). Detailed ligand synesis schematics shown in Supplementary Figure S1–S5, and Liquid Chromatography-Mass Spectrometer data the compounds used in vivo is shown in Supplementary Figure S6.

Figure 2.

Synthesis of Mn 1-5. Reagents and conditions: (i) acetic anhydride, pyridine, RT, 38h; (ii) benzylamine, p-chloroaniline, 3,5-(bis)trifluoromethylaniline, 1-phenylpiperazine, or 4-ethoxybenzylanmine, DMF, RT, 12h; (iii) MnCl₂, pH 6.5–7.5, RT.

Functional Assay for Kinetic Inertness

We recorded Mn(II) dissociation kinetics for representative complex Mn-2 under conditions designed for force Zn²⁺ trans-chelation (pH 6.0 50 mM MES buffer, 25 °C, 0.7 mM Mn(II) complex, 30 mM Zn(OTf)₂) (Supplemental Figure S7). For reference, the dissociation kinetics of the well-characterized kinetically inert complex Mn-CDTA was also measured under identical conditions⁴⁷. Dissociation of Mn(II) was monitored by measuring r₂ change, as previously described. Under these reaction conditions, the half-lives for dissociation of Mn(II) from Mn-2 and Mn-CDTA are 171 min and 42 min, respectively. We note that Mn-PyC3A, which is very robust against Mn dissociation in vivo^42–44, dissociates Mn(II) roughly 2-fold more slowly than Mn-CDTA under comparable reaction conditions³⁸. Given the comparable kinetic inertness of representative complex Mn-2 to Mn-PyC3A under this set of dissociation forcing conditions, we posit that complexes Mn-1 through Mn-5 are unlikely to release any substantial quantity of Mn(II) in vivo.

Relaxivity Measurements Reveal Moderate Relaxivity of Novel Probes at 3 T relative to Gd-EOB-DTPA

At 3 T, the r₁ relaxivities ranged from 2.3 to 3.0 s⁻¹ mM⁻¹ (Figure 3). For all the Mn(II) complexes, the r₁ values are consistent with the presence of an inner sphere water co-ligand. Although a detailed investigation of the physical mechanisms underpinning Mn(II) complex relaxivity are beyond the scope of this study, we tentatively posit that the small differences in relaxivity likely reflect differences in rotation correlation time (τ_R). The molecular weights of the complexes shown in Figure 2 range between 489 and 612 Da. Prior studies to evaluate Mn-PyC3A derived complexes appended with OATP-targeting groups demonstrated how relaxivity at 1.4T varied comparably across a series of compounds comprising a comparable range of molecular weights. Here, relaxivity may reflect differences in molecular weight in a similar fashion. We also note for that the two complexes with greatest relaxivity, Mn-2 and Mn-3, the OATP-targeting moieties are appended via rigid linkages so that the rotational motions of the paramagnetic Mn(II) ion are strongly coupled to the global motions of the entirely molecule. Conversely, the rotational motions of Mn-5 - the complex with the lowest relaxivity – can partially decouple from its OATP-targeting moiety enabling the paramagnetic ion to experience an effective τ_R that is somewhat less than that of the global molecular τ_R. Smaller variations in relaxivity are potentially attributable to changes in water co-ligand mean residency time (τ_m). For example, if we assume τ_R ~120 at 37 °C based on prior accounts of complexes with comparable molecular weight, τ_m change between 1 ns and 50 ns can cause fluctuations in relaxivity of 0.4 mM⁻¹s⁻¹ at 3.0T. Finally, small changes in the effective number of water co-ligand, caused by fractional equilibrium composition comprising species of q = 1 and q = 0, or even very small variations in effective distance between the paramagnetic ion and ¹H nuclei of coordinated water co-ligands could also represent potential sources of variation in relaxivity. Ultimately, in vivo efficacy for liver imaging and other applications regarding OATP-mediated cell uptake will reflect OATP-avidity and pharmacokinetics as well as relaxivity. In this regard, we reserve detailed characterization of molecular mechanisms underpinning relaxivity until a candidate for further development emerges.

Figure 3.

Relaxivity characterization of five novel manganese MR probes compared to Gd-EOB-DTPA for reference, measured at the imaging field strength of 3T.

In vivo MRI Demonstrates Novel Mn(II) Probes Generate Positive Contrast in Mouse Livers

The pharmacokinetics and biodistribution of three representative complexes, Mn-2, Mn-3, and Mn-5, were evaluated in mice using dynamic whole-body MRI at 3 T. These were selected for in vivo analysis based on the superior relaxivity of Mn-2 and Mn-3 at our imaging field strength, and to compare the effect of the ethoxybenzyl compound possessed by Mn-5 on transport between analogous Mn(II) and Gd(III) compounds. Representative coronal T₁-weighted images of BALB/c post 0.1 mmol/kg Mn(II) probe delivery are shown in Figure 4, with three out of the thirty total timepoints post-contrast shown at linearly spaced time intervals. Slices displaying liver and bladder enhancement are displayed in Figure 4A–C, while coronal slices displaying kidney enhancement are shown in Figure 4G–I. Percentage contrast enhancement (% CE) maps were generated by segmenting whole-liver (Figure 4D–F) and whole-kidney (Figure 4J–L) slices and mapping percentage contrast enhancement (following Equation 1 in Materials and Methods) on a pixel-by-pixel basis for the entire imaging time course. A representative animal who received a 0.1 mmol/kg injection of Gd-EOB-DTPA is shown in Supplemental Figure S9 to serve as a positive control. The images demonstrate strong blood pool enhancement in the first scans acquired after injection, which decreased rapidly via mixed renal and hepatobiliary clearance mechanisms.

Figure 4.

T₁-weighted images and regional contrast enhancement maps for liver imaging in mice at 3 T. Coronal slices through the liver are shown at representative timepoints pre- and post- administration of 0.1 mmol/kg (A) Mn-2, (B) Mn-3, (C) Mn-5. Voxel-by-voxel contrast enhancement map of whole liver sections post Mn-2 (D), Mn-3 (E), or Mn-5 (F), colour bar represents percentage contrast enhancement of a voxel. Coronal slices through the kidney are shown at representative timepoints pre- and post- administration of 0.1 mmol/kg (G) Mn-2, (H) Mn-3, (I) Mn-5. Voxel-by-voxel contrast enhancement map of whole kidney sections post Mn-2 (J), Mn-3 (K), or Mn-5 (L), colour bar represents percentage contrast enhancement of a voxel.

The mean signal intensity in the liver was significantly higher than the mean signal intensity in the ROI pre-contrast continuously up until 49.3 minutes post Mn-2 (Figure 5A; p < 0.0001 from 3.3 minutes to 30.9 minutes, p < 0.0065 from 33.2 minutes to 49.3 minutes), continuously for 47.0 minutes post Mn-3 (Figure 5B; p < 0.0001 from 3.3 minutes to 37.8 minutes, p < 0.04 from 40.1 minutes to 47.0 minutes), and continuously for 33.2 minutes post Mn-5 delivery (Figure 5C; p < 0.0001 from 3.3 minutes to 30.9 minutes and p < 0.0003 at 33.2 minutes). The highest % CE values (referred to here as peak % CE values) for Mn-2, Mn-3, and Mn-5 were 212 ± 43 %, 226 ± 50 %, 195 ± 31 %, respectively, and not significant from each other (Figure 5G). Peak %CE values occurred at 5.6 minutes for Mn-2 and Mn-3, and at 7.9 minutes for Mn-5 (Figure 5A,B,C). The period of strongest enhancement in the liver for each of the agents are defined here as the interval for which there was no significant difference between the given % CE value and the peak % CE value. The period of enhancement was 11.5 minutes, 6.9 minutes and 13.8 minutes in length for Mn-2, Mn-3, and Mn-5, respectively (Figure 5D,E,F).

Figure 5.

Mean signal intensity curves for liver post Mn-2 (A), Mn-3 (B), Mn-5 (C) indicating where increase relative to pre-contrast was significant, error bars indicated standard deviation between animals. Percentage contrast enhancement curves for liver post Mn-2 (D), Mn-3 (E), Mn-5 (F), indicating for which time interval the %CE was not significantly different from the peak %CE value. (G) Peak percentage contrast enhancement in the liver of mice post Mn-2, Mn-3, or Mn-5 probe injection. Error bars indicate standard deviation between animals. (n=4 for Mn-2 and Mn-3, n=3 for Mn-5)

Washout curves for liver, kidney, and blood are shown in Figure 6A–C. Washout kinetics analysis determined blood half-lives of 7.5 ± 0.5 min, 5.0 ± 0.6 min, and 4.9 ± 0.4 min for Mn-2, Mn-3, and Mn-5, respectively (Figure 6D). Half-lives of 44 ± 27 minutes, 39 ± 30 minutes, and 36 ± 6.5 minutes in the liver (Figure 6E), 11 ± 1.8 minutes, 8.7 ± 0.5 minutes and, 7.8 ± 1.4 minutes in the kidney (Figure 6F), for Mn-2, Mn-3, and Mn-5, respectively. Washout times in the liver were not significantly different from each other. The washout half-life of Mn-2 in the kidney was not significantly different from that of Mn-3 and was significantly longer than Mn-5 (p < 0.036). The washout half-life of Mn-2 was significantly longer than both Mn-3 and Mn-5 (p < 0.0001). Area under the curve (AUC) analysis was performed for the liver, kidney and blood washout curves and revealed that there was no significant difference in the AUC for the liver and kidney curves for Mn-2, and that the AUC for the liver and kidney curves were significantly higher than the AUC for the blood washout curve (p < 0.0013 and p < 0.0024) (Figure 6G). There was once again no significant difference in the AUC for the liver and kidney curves for Mn-3, and significantly greater AUC for the liver curve compared to the blood curve (p < 0.0003), but different from Mn-2, for Mn-3 there was no significant difference in the AUC for the blood and kidney curves. Mn-5 displayed no significant difference in the AUC for the liver and kidney curves, as well as the kidney and blood curves but there was a significant difference in the AUC for the liver and blood curves (p < 0.031).

Figure 6.

Percentage contrast enhancement curves for dynamic imaging time course post Mn-2, Mn-3, or Mn-5 administration in the (A) liver, (B) kidney, (C) blood. Half-life washout of (D) blood, (E) liver, (F) kidney. (G) Area under the curve (AUC) analysis for liver, kidney, and blood curves. (n=4 for Mn-2 and Mn-3, n=3 for Mn-5)

Finally, the peak % CE in the liver was compared to the % CE generated in the liver of mice who received an equivalent dose of Gd-EOB-DTPA. The peak % CE relative to Gd-EOB-DTPA was 72 ± 15%, 77 ± 17%, 67 ± 11% for Mn-2, Mn-3, and Mn-5, respectively. Supplemental Figure S8 shows the enhancement of the gallbladder and the bladder, further confirming the mixed renal/hepatobiliary clearance mechanism, with longer retention times than we could elucidate in the 70-minute imaging session. Percentage contrast enhancement curves in the brain and muscle show initial peaks in enhancement followed by %CE that approaches zero. Supplemental Figure S9 shows a representative animal post 0.1 mmol/kg injection of Gd-EOB-DTPA at six representative timepoints, as well as kidney and liver washout curves for reference.

In vitro Human OATP1B1/OATP1B3 Uptake Assays Display Preferential Transporter Uptake

MDA-MB-231 human triple negative breast cancer cells were engineered to stably co-express a null plasmid (control cell group), then re-engineered to express zsGreen and either OATP1B1 or OATP1B3 (Figure 7A). Cells were analyzed using flow cytometry post-transduction and confirmed that control cells were zsGreen negative and revealed a transduction efficiency of 83.6% and 80.6% for the OATP1B1 and OATP1B3-engineered cells, respectively (Figure 7B). A Western Blot was performed to evaluate OATP1B3 expression in each of the three engineered cell populations (Supplementary Figure S11). Relaxation rate analysis measured the pixel-by-pixel relaxation rates in Hz, from which an average cell pellet relaxation rate value was used for statistical agent uptake analysis (Figure 7C). The average relaxation rates between Mn-treated control cells and Mn-treated engineered cells were compared using a paired one-tailed t-test. The results indicate that Mn-1 and Mn-3 displayed significant uptake through only the OATP1B1 transporter (p < 0.0277 and p < 0.02, respectively), while none of the five agents displayed significant uptake through the OATP1B3 transporter. Supplementary Figure S10 shows uptake assay data after cell incubation in a molar equivalent dose of Gd-EOB-DTPA.

Figure 7:

In vitro analysis of MDA-MB-231 cells engineered with either human OATP1B1 or OATP1B3. (A) Plasmid constructs and (B) associated fluorescence associated cell sorting (FACS) histograms showing zsGreen expression. Engineered cells were incubated in 1.6 mM of one of the five novel Mn(II) probes for 90 minutes before being washed and collected for analysis. (C) Representative R₁ map showing relaxation rates of cell pellets incubated in 1.6 mM of Mn-3. Relaxation rate changes due to Mn(II) incubation compared to treated control cells for the (D) OATP1B1 group and the (E) OATP1B3 group (n=3, p < 0.0277 for Mn-1 and p < 0.02 for Mn-3, respectively).

DISCUSSION & CONCLUSIONS

Here we present a novel approach to develop a library of compounds born from a common chelator. Our findings indicate that despite the relaxivity of the novel manganese agents at 3 T investigated being nearly half that of the relaxivity of Gd-EOB-DTPA, we observed significant increases in signal intensities in the liver upon 0.1 mmol/kg dose of all the manganese probes evaluated. Peak contrast enhancement in the liver relative to that of Gd-EOB-DTPA reveals that Mn-2, Mn-3, and Mn-5 produced peak contrast enhancement that was 72 ± 15 %, 77 ± 17 %, and 67 ± 11 % that of the contrast enhancement generated in the liver when Gd-EOB-DTPA when given in equimolar quantities. This finding indicates that despite the ~50% lower agent relaxivity for the Mn(II) probe, the peak contrast enhancement relative to an equimolar dose of Gd(III) was > 50%. It is at present unclear exactly why the Mn complexes reported here provide greater liver enhancement than Gd-EOB-DTPA. It is possible that our Mn complexes are more avidly accumulated by hepatocytes than Gd-EOB-DTPA. Alternately, differences in delayed liver enhancement may reflect differences in the kinetics of hepatocellular clearance. We also did not determine the Mn complex speciation in hepatocytes. For example, it is not presently known whether the complexes could get a relaxivity boost due to interactions with hepatocellular proteins, or whether metabolism in the liver results in liberation of free Mn. This information may be ascertained through carefully designed ADME (absorption, distribution, metabolism, and excretion) experiments, but such studies are beyond the scope of this paper.

Dynamic whole-body imaging of mice post injection revealed that half-life washout times of were not significantly different in the liver, suggesting that the three probes explored in vivo have similar hepatobiliary clearance properties. The AUC Liver/Kidney ratio revealed that Mn-2 cleared in approximately equal liver/kidney proportions, whereas Mn-3 and Mn-5 cleared predominately through the liver. For the purposes of probes for cell tracking, the main parameter of highest importance continues to be peak contrast enhancement, so we report that given the lack of significant difference in the peak liver enhancement between the three agents, any one of the compounds would be a worthy candidate for Gd(III)-free imaging of the liver or cellular imaging of OATP-engineered cells. Though, it is important to note that rodent OATP studies do not necessarily predict human behavior – for example, the Gd(III) contrast agent Gd-BOPTA is taken up nearly 50% in the liver in rats, but < 5% accumulates in human hepatocytes^48,49.

Extending this study to the investigation of agent uptake through human OATPs revealed that some agents show preferential uptake through the OATP1B1-engineered cells, while no significant uptake was seen in OATP1B3 cells, relative to treated control cells. Also interestingly, the treated control cells displayed significantly different relaxation rates post-incubation compared to untreated control cells for all five agents. The uptake experiment was also completed using Gd-EOB-DTPA, following the same 90-minute incubation protocol as with the Mn(II) agents, and a ~25% increase in relaxation rates resulted from incubating control cells with Gd-EOB-DTPA (Supplemental Figure S10). In previous studies, cells were incubated for just 60 minutes before assessing relaxation rate changes at 3.0 T. In these past experiments, we saw no significant uptake of agent into control cells^14,15,50. We propose that the additional 30-minute incubation may have facilitated the large concentration gradient between the intra- and extra-cellular compartments to drive Mn(II) into the control cells, providing an inaccurate picture of the OATP-mediated transport of the novel Mn(II) agents. This could also be the result of probes interacting with the outer cell membrane through electrostatic or lipophilic interactions, though this work did not explore this theory with measurements. There is also the possibility of some non-specific uptake occurring in cells lacking the OATP transporter, or uptake of dissociated Mn(II). Notably, besides predicted clearance organs (e.g., kidney), we did not see any significant enhancement of tissues lacking the OATP1 transporter in vivo (e.g., muscle).

Mn-based probes offer an attractive alternative to Gd(III) for generating strong positive MR contrast. That being said, the measured r₁ relaxivity of the novel Mn(II) probes described here is lower than of Gd-EOB-DTPA by a factor of ~2 at 3 T. This relaxivity disparity is especially relevant for cell tracking, where the goal is detection of small numbers of engineered cells that reside in tissues with relatively high background signal. From a cellular sensitivity perspective, one could compensate for this lower r₁ relaxivity by administering OATP-targeted probes at higher concentrations to provide a greater relaxation enhancement effect in engineered cells. Even when Gd(III) based agents are used, high doses can be necessary to achieve highly sensitive detection of OATP-engineered cells¹⁸. With both the real and perceived risks of Gd(III), such high doses are not feasible as a standard imaging tool. We propose that an alternative Mn(II) agent with an improved safety profile can be delivered safely at the doses required for sensitive cell detection, with reduced concerns of the fate of dissociated free Mn(II) ions. Another strategy for increased cellular sensitivity of OATP-engineered cells is increased transporter targeting of probes, which is what was explored in this work. We hope to continue to assess the OATP uptake of these novel agents in vivo for reporter gene imaging of various OATP-engineered cell types.

Preliminary measurements using this small focused library of manganese-based MR probes show that despite lower agent relaxivity, the choice of lipophilic group can promote significant liver uptake in mice and yield contrast enhancement in vivo that is comparable to high spin Gd(III) probes at equivalent doses. Despite initial investigations on novel probe uptake through human derived OATPs showing only modest uptake through OATP1B1, with a trend towards influence on control cell relaxation rates, further investigation will pave the way for these compounds to be used as MR reporter gene imaging probes.

MATERIALS AND METHODS

Nuclear Magnetic Relaxation Measurements

Relaxivity at 3 T was measured using a fast-spin echo (FSE-IR) pulse sequence with the following imaging parameters: matrix size = 400×400, repetition time (TR) = 5000 ms, echo time (TE) = 19.1 ms, echo train length (ETL) = 4, number of excitations (NEX) = 2, receiver bandwidth (rBW) = 125.00 kHz, inversion times (TI) = 50, 68, 94, 128, 175, 239, 327, 447, 612, 836, 1144, 1564, 2139, 2925, 4000, in-plane resolution = 0.3×0.3 mm², slice thickness = 2.0 mm, acquisition time = 3 min, 45 s per inversion time. A temperature sensing probe was used to maintain a temperature of 37°C throughout the entire imaging session.

In Vivo Magnetic Resonance Imaging

All in vivo experiments were completed in accordance with our animal use protocol (AUP 2020–058), and Western University’s animal use guidelines. Healthy female BALB/c mice were anesthetized using isoflurane gas at a 5% induction and 2% maintenance rate. Mice were positioned on a custom-built heated animal holder with built-in temperature and respiration sensors, with a small-animal nose cone (Kent Scientific Corporation, Torrington Connecticut) used for gas flow and passive isoflurane scavenging. Tail vein catheters were inserted prior to placement of the animals in the scanner. All images were acquired with a clinical 3-Tesla MRI scanner (General Electric Healthcare Discovery MR750, Milwaukee Wisconsin, USA) using a custom built whole-body small animal birdcage coil (inner bore diameter 31 mm, coil length 15.1cm). T₁-weighted images were acquired using a 3D-Spoiled Gradient Recalled Acquisition in the Steady State (3D-SPGR) with the following image parameters: FOV = 12.0 cm, TR = 14.7 ms, TE = 2.456 ms, rBW = 62.5 kHz, Matrix size = 400×400, Flip Angle = 60°, NEX = 1, voxel size = 300-μm³ isotropic, scan time = 2:18 minutes. Pre-contrast images were taken before intravenous injection of 0.1 mmol/kg of one of the novel manganese agents, followed by 40μL wash with sterile saline. For this preliminary study, three of the five agents synthesized were evaluated in vivo: Mn-2 and Mn-3 were selected based on their higher relaxivity, and Mn-5 was selected as it possesses an ethoxy-benzyl group that is known to promote uptake of Gd-EOB-DTPA). Thirty consecutive 3D T₁-weighted images were collected at a temporal resolution of 2 minutes and 18 seconds, with an approximate 60 second delay between contrast agent injection and start of the first acquisition for a total of 70 minutes of dynamic contrast enhancement monitoring.

Image analysis was performed using a custom-written MATLAB app (R2021a, MathWorks, Natick, Massachusetts, United States) to manually segment regions of interest in the liver, bladder, muscle, kidney, blood, brain, and gallbladder at each imaging time point. Dynamic contrast enhancement of the tissues was assessed at each time by comparing signal in the ROI to that of the pre-contrast image, on a voxel-by-voxel basis using Equation 1.

$$\mathit{\text{Voxel}}\mathit{\text{Contrast}}\mathit{\text{Enhancement}}=\frac{\mathit{\text{Voxel}}\mathit{\text{signal}}\mathit{\text{post}}\mathit{\text{contrast}}-\mathit{\text{Voxel}}\mathit{\text{signal}}\mathit{\text{pre}}\mathit{\text{contrast}}}{\mathit{\text{Voxel}}\mathit{\text{signal}}\mathit{\text{pre}}\mathit{\text{contrast}}}$$ Equation 1.

%CE maps were generated for better visualization of enhancing/non-enhancing regions which helped identify homogenous enhancing liver regions (omitting non-enhancing vasculature) to include in % CE analysis (figure 3D–F). Liver, kidney, and blood washout half-lives were calculated by mono-exponential curve fitting, restricted to time values including and beyond the time of peak contrast enhancement, following equation 2, where $\%\mathit{\text{CE}}$ is the percent contrast enhancement of the organ in question at a time t, $t_{1/2}$ is the washout half-life of the organ in question, and $A$ is the amplitude at the peak of the percentage contrast enhancement.

$$\%\mathit{\text{CE}}=A{e}^{-t(\frac{\text{ln}(2)}{t_{1/2}})}$$ Equation 2.

Lentivirus Construction and Production

Lentiviral transfer plasmids co-encoding zsGreen fluorescent protein with either OATP1B1 or OATP1B3 were cloned using the In-Fusion HD Cloning kit (Takara Bio USA Inc, Madison, Wisconsin, United States). Third-generation packaging and envelope-expression plasmids were co-transfected with each of the two transfer plasmids (zsGreen/OATP1B1, zsGreen/OATP1B3) into human embryonic kidney (HEK 293T). Lentivirus-containing supernatants were harvested 24h and 48h post-transfection, filtered through a 0.45-μm filter, and used immediately for transductions¹³.

Cellular Engineering

MDA-MB-231 human breast cancer cells were seeded at 10⁵ cells/well in a 6-well plate and incubated overnight. Culture media was then replaced with 1 ml of fresh culture media lacking FBS and antibiotics but spiked with 8ug/ml polybrene. Viruses generated from plasmid (LV-zsG-OATP1B1 or LV-zsG-OATP1B3) were added to the media and after 6 hours, 1 ml of culture media with 20% FBS was added. The cells were then transferred back to standard culture media on the second day of transduction and flow cytometry was performed 48 hours later to test the transduction efficiency. Engineered cells were reanalyzed for stable zsGreen expression at 70- and 100-days post transduction using flow cytometry where stable zsGreen expression was confirmed.

Western Blot

Cell lysates were extracted by Pierce^™ RIPA buffer treatment with Halt^® protease and phosphatase inhibitor cocktail (Thermo Sci.), and protein (30 μg) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis using Blot^™ 4–12% Bis-Tris Plus and transferred onto a nitrocellulose membrane using the iBlot2 transfer system. Blocking and antibody dilution was done using 3% bovine serum albumin in PBS with 0.05% Tween-20. The membrane was probed with anti-human rabbit polyclonal OATP1B3 antibody (1:500) (Sigma) overnight at 4°C followed by IRDay^® 680 RD goat anti rabbit secondary antibody for one hour at room temperature. Protein expression was visualized using the Odesyss imaging system. GAPDH (1:10000) was used as a loading control.

Cell Culture

Human embryonic kidney 293T (HEK 293T) and human triple negative breast cancer cells (MDA-MB-231) were obtained from American Type Culture Collection, Manassas, Virginia, United States, for use in establishing an engineered cell line. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, at 37C and 5% CO₂. MDA-MB-231 cells were transduced with lentivirus expressing a zsGreen fluorescent reporter and one of two organic anion transporting polypeptide isoforms (either OATP1B1 or OATP1B3). Fluorescent associated cell sorting was performed using a FACSAria III cell sorter (BD Biosciences, Mississauga, Ontario, Canada) to isolate only zsGreen-positive cell population for expansion.

In vitro OATP1B1/OATP1B3 Uptake Measurements

Upon determining the influence of novel manganese contrast agents on murine livers (through endogenous OATPs in murine hepatocytes), we sought to evaluate their uptake (if any) to the human OATP transporters in human hepatocytes (OATP1B1 and OATP1B3). 1×10⁶ control, OATP1B1, or OATP1B3 engineered MDA-MB-231 breast cancer cells were seeded in 15-cm cell culture dishes and expanded until > 90% confluency was reached. Cells were first replenished with fresh culture media before being incubated for 90 minutes with one of the five novel manganese at a concentration of 1.6 mM. Cells were subsequently washed with phosphate buffered saline (PBS) and were then trypsinized, centrifuged, and pelleted into 250 μL polymerase chain reaction (PCR) sample tubes. The sample tubes containing pelleted cells were placed in a sample holder filled with 1% agarose gel and arranged into a single row within the imaging coil. Samples were heated to 37°C, and imaged at 3 Tesla (General Electric Healthcare Discovery MR750) using a fast-spin echo (FSE-IR) pulse sequence with the following imaging parameters: matrix size = 400×400, repetition time (TR) = 5000 ms, echo time (TE) = 19.1 ms, echo train length (ETL) = 4, number of excitations (NEX) = 2, receiver bandwidth (rBW) = 125.00 kHz, inversion times (TI) = 50, 68, 94, 128, 175, 239, 327, 447, 612, 836, 1144, 1564, 2139, 2925, 4000, in-plane resolution = 0.3×0.3 mm², slice thickness = 2.0 mm, acquisition time = 3 min, 45 s per inversion time.

Longitudinal relaxation rates (R₁) were estimated by performing non-linear least-squares fitting of signal intensity at the each of the inversion times listed above, on a pixel-by-pixel basis, using a custom MATLAB application (MATLAB, MathWorks, Natick, Massachusetts, United States), and the following signal equation 3. Where, $M_{\mathit{\text{ss}}}$ is the final steady state magnetization, and $M_i$ is the magnetization at the first value of the inversion recovery curve, which is independent of the final steady state magnetization $M_{\mathit{\text{ss}}}$.

$$S=\left|M_{\mathit{\text{ss}}}-(M_{\mathit{\text{ss}}}-M_i){\bullet e}^{\raisebox{1ex}{$-TI$}\!\left/ \!\raisebox{-1ex}{$T_1$}\right.}\right|$$ Equation 3.

Statistics

A two-way ANOVA with Šídák’s multiple comparison test was used to determine the period of statistically significant mean signal intensity increase from baseline, and the period of significant enhancement, over the 70-minute imaging time course. A one-way ANOVA with Tukey’s multiple comparisons test was used to determine the difference in peak %CE values in the liver, as well as the difference in washout half-lives and area under the curves in the blood, liver, and kidney. A paired one-tailed t-test was used to determine the differences in relaxation rates between the treated control cells and the treated engineered cells, as well for the differences between untreated and treated control cells for all agents.