Ion carrier modulated MRI contrast

An ion-responsive MRI contrast agent based on a POPC liposomal scaffold is generated that displays a large amplitude relaxivity switch. Entrapment of MR active Gd-DOTA within cholesterol-doped, i.e., membrane rigidified, liposomes dampens the MR response through diminished water exchange across the lipid bilayer. Relaxivity is re-established by integration of ion carriers in the liposome membrane to mediate solvated ion flux.


Introduction
Magnetic contrast agents, capable of enhancing image sensitivity within a MRI scan, are central to the effective diagnosis (and consequent prognosis) of a very broad range of clinical conditions and disease states. 1 Nanoparticulate contrast agents (NP CAs) offer enhanced signal-to-noise ratios and high associated relaxivity over their molecular counterparts, enriching MR image acquisition, oen with accompanying improved circulation times and lower doses (and thus potential for reduced toxicity). 24][5] In heralding a new era of disease specic reporting, many of these are able to respond to a desired environmental stimulus (such as pH, ions, or biomolecules). 6Paramagnetic liposomes are a potentially potent subclass of these, possessing high biocompatibility, and an associated capacity for payload incorporation within the interior cavity. 7iposomal CAs have been synthesised either through the immobilisation of paramagnetic probes on the membrane surface, or by encapsulation of magnetic probes such as Gdchelates within the intra-vesicular volume of the phospholipid bilayer.These approaches have supported the incorporation of a high associated payload of 10 4 to 10 5 molecular chelates, 8 such as gadoteric acid (Gd-DOTA), that can promote T 1 contrast generation in vivo. 9A number of thermosensitive DPPC liposomes capable of supporting thermally activated T 2 relaxivity have also been reported. 9,102][13] The inuence of water exchange (limited and/or thermally activated) across the lipid bilayer is highlighted in this prior work; contrast is limited by membrane water permittivity and, thus, potentially responsive to any bilayer fragmentation or change in rigidity (thermally or chemically imposed, such as through the integration of cholesterol). 14,15Where DPPC liposomes have been loaded with both a therapeutic agent (Camptosar) and IONPs, thermally induced irreversible fragmentation enables both agent delivery and direct monitoring of this by T 2 weighted MRI. 16on channel mis-regulation is directly causative of several diseases; an ability to monitor exogeneous ion imbalance in vivo would be benecial for enhancing our understanding of these processes. 17Ion carriers capable of mediating transmembrane transport may offer valuable therapeutic intervention, 18,19 and their development is an area of intense interest.Numerous synthetic ion transporters are known, including some that exhibit stimulus responsive behaviour. 20Since ions are heavily solvated in aqueous solution, 21 we hypothesised that recognition mediated transport would be accompanied by at least some associated exchange of water across the bilayer.We expected that this, in turn, would support a mechanistically novel platform for which image contrast switches in response to exogenous ions.

Results and discussion
MR active 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes were here generated by a typical extrusion process, using a polycarbonate membrane with a pore size of 100 nm.POPC lipid membranes with incorporated cholesterol are highly biologically relevant, as they represent an idealised and highly tuneable model of the biomembrane. 22All liposomal samples exhibited high levels of colloidal stability (PDI < 0.3 for all samples), with an associated hydrodynamic size of 109.50 ± 4.51 nm and z-potential of −6.94 ± 0.71 mV for non-cholesterol modied POPC liposomes (POPC-C 0% -N) measured immediately aer synthesis (as resolved by dynamic light scattering (DLS) measurements, ESI 9(a) †).Gd-DOTA was incorporated within the intra-vesicular cavity at the point of liposome formation, by dispersing the desired concentration of chelate in water and adding to the dried lipid lm (ESI †).Resuspension, freeze-thawing and subsequent extrusion steps were conducted to afford the desired MR active vesicles (see the ESI † for procedure details).The liposomes were shown to be stable for 72 h, sufficient for MR analysis, with no signicant differences in size or surface charge (ESI 9(a) and 9(b) †), observations of translational importance. 23A range of Gd-DOTA loading densities were investigated (ESI 10 †), with 75 mM ultimately added at the point of synthesis, to bias water exchange as rate limiting in relaxivity/contrast.These liposomes exhibited longitudinal relaxivity values (r 1 = 4.70 ± 0.02 mM −1 s −1 at 1.41 T and 298 K, in water) characteristically indicative of effective diffusive water access across the lipid membrane.Notably, this water ux could be controllably "tuned down" signicantly (at 1.41 T, associated r 1 = 2.52 ± 0.09 mM −1 s −1 , ESI 11 †) by doping the bilayer membrane with 45 mol% cholesterol, (POPC-C 45% -N), a process associated with a negligible change in hydrodynamic size (99.35 ± 3.87 nm) and z-potential (−9.93 ± 1.12 mV) with a transmission electron microscopy (TEM) resolved diameter equal to 98.2 nm (ESI 12 †).Good colloidal stability over 90 hours was also observed upon incubation of POPC-C 45% -N and POPC-C 45% -T in serum (ESI 13 †) with no signicant variation in number mean.Osmotic shock experiments using DLS additionally conrmed the role of cholesterol in gating water ux (ESI 14 †), whereby liposomes composed solely of POPC exhibited a ∼20% reduction in the hydrodynamic diameter between incubation in pure water and 20 mM KCl solution.In comparison, liposomes doped with a high concentration of cholesterol (i.e., >40%) 24 displayed no signicant variation in size when subjected to the same conditions, conrming bilayer rigidication.
It is known that gramicidin-A (GramA) peptide channels can be reliably integrated into lipid bilayers, including those presented by liposomes, and are able to mediate water ux by a Grotthuss hopping mechanism at rates exceeding 10 −6 cm −2 s −1 (Fig. 2(a)). 25,26The integration of GramA (2 mol% w.r.t.POPC) within the cholesterol modied liposomes was observed to support a very signicant (>30%) boost in MR relaxivity for liposomes possessing 45-50 mol% cholesterol loading (Fig. 2(b)).Signicantly, these experiments demonstrate that initially low liposomal contrast could be switched on if water ux across the bilayer is promoted.
We then sought to control (ion associated) water ux by integration of selective ion carriers within the liposomal Fig. 2 (a) A schematic highlighting the transport of water molecules from the exterior solution to the interior pool of the liposomes, mediated by GramA (PDB ID: 1MAG). 26The pore diameter and the length of GramA are 0.4 nm and 26 nm respectively, with the diffusion rate of water highlighted. 25,26The diameter of the pore channel is narrow and therefore it enables a single chain of 7-9 water molecules to align within it. 25

(b)
The influence of GramA integration (2 mol% w.r.t.POPC) on the relaxivity (obtained at 1.41 T, 298 K) of pre-formed Gd loaded vesicles, across a range of cholesterol doping levels, to highlight the increased rigidification at higher cholesterol loading.Error bars for all data points were obtained from a linear regression analysis on linear plots of 1/T 1 versus [Gd(III)] to obtain a value for the gradient, which is equal to r 1 ± 1 s d.
membrane.8][29][30] This K + ionophore, along with a tripodal thiourea anionophore developed by Busschaert et al. capable of efficiently transporting Cl − , 31 were co-incorporated into the membrane of cholesterol-modied lipid vesicles (POPC-C 45% -T) by addition of a 1 mol% (w.r.t.POPC) DMSO stock solution to the liposome suspension.Extensive prior work supports non-destructive transporter integration by this method. 19Some water transport is known to be associated with the resultant ion transfer across the lipid bilayer. 32The rate of diffusion of water across an unmodied lipid membrane has been theoretically estimated to be 2 × 10 −4 to 50 × 10 −4 cm s −1 and is greatly reduced upon incorporation of cholesterol into the membrane (conrmed using stopped ow osmotic analyses). 33,34Given that the associated ion transport rates for the K + and Cl − transporters used in this work are in the region of 10 2 to 10 4 s −1 , we anticipated a marked increase in water ux in their presence. 27,31,32Osmotic analyses herein conrm the coupling of anion/cation symport exchange across the bilayer to an enhanced water ux (see ESI 16 †).Carrier mediated KCl symport (co-transport) and the associated increased water ux specically correlate with an observable hydrodynamic size modulation as resolved by DLS (ESI 15 and 17 †).Real time analyses of this (ESI 18 †) enable an estimation of some 750% increase in net water ux when both mobile carriers are present (ESI 19 †).Pleasingly, this carrier mediated transmembrane water ux leads to substantial (60%) relaxivity change; notably this is observed only in the dual integration of both transporters and in the presence of physiologically relevant levels of KCl i.e., through the activation of net neutral K + /Cl − symport transport. 35Integration of either the cation or anion transporter alone in the presence of the same levels of KCl results in no detectable change in relaxivity (ESI 20(a) and (b) †).The requirement for KCl symport was also conrmed through ion transport experiments in liposomes using a uorescence assay, which demonstrated that efficient Cl − net ux in the presence of K + requires both the cationophore and the anionophore to be present (ESI 21 †). 36 1 was independent of the KCl concentration across the physiological range (0.1-5.0 mM), consistent with the high activity of both ionophores (see ESI 22, † measured at 1.4 T). 27,31,37 Assessing the anion selectivity of this we noted relative enhancements in r 1 of 83% and 67% were observed experimentally in the presence of 1.08 mM Br − and NO 3 − respectively (see Fig. 3(b)), seemingly correlating with the ease of anion desolvation and hence transport efficiency.31 Cation selectivity trends were entirely consistent with the known Vln binding selectivities (see Fig. 3(a)).38 Since molecular Gd chelates are used in approximately 40% of all MRI examinations, 39 we then demonstrated that this ion transport could generate signicant image contrast change within T 1 weighted images obtained on a clinical MRI scanner (at 1.5 T and 3 T, at 298 K; Fig. 4).T 1 itself predictably increases for both POPC-C 45% -N and POPC-C 45% -T liposomes as magnetic eld strength (B) increases (ESI 23 †).
Building on the observations of ion ux mediated contrast switching with Gd-DOTA carrying liposomes, we integrated Gd-   doped mesoporous silica nanoparticles (Gd-MSNs) into functional POPC liposomes (POPC-C 45% -MSN-N) through fusion (ESI 7 and 8 for characterisation, and ESI 24-27 †). 40Characterisation by TEM and DLS conrms that these Gd-MSNs are consistently and effectively encapsulated within lipid bilayers, as expected. 41,42Since paramagnetic MSNs support much higher levels of relaxivity than molecular chelates, 4 we hypothesied that the initial water transmembrane transport (and the small internal water pool) would be more limiting.Pleasingly, ion transport MR relaxivity turn-on is not only fully consistent with that observed with Gd-DOTA, but also indeed of a greater turn on magnitude; for both 0 and 45 mol% cholesterol doped lipid bilayers, relaxivity switches close to 200% were observed (Fig. 5) with Gd-MSN doped liposomes containing 1 mol% of both cation and anion transporters (POPC-C 45% -MSN-T) in the presence of a 1.08 mM KCl gradient.

Conclusions
This work, then, demonstrates that the general principal of ion mediated water ux across the membrane of MR active liposomes is associated with signicant modulation of MR relaxivity and associated image contrast.A "switch off" of paramagnetic liposome MR contrast can be established through lipid membrane rigidication and selectively re-established through water associated ion transport using synthetic ionophores.This proof of concept lays the groundwork for potentially imaging ion imbalance and the associated diseases thereof.Derivations which are both ratiometric (by, for example, integrating a cholesterol-anchored 19 F probe) 43 and enzyme activated 44 can be envisaged.

Fig. 1 A
Fig. 1 A schematic illustrating the transfer of electrostatically bound K + to Vln and Cl − by tripodal thiourea across the lipid bilayer of MRactive liposomes, accompanied by associated water transport which switches on MR image contrast.

Fig. 3
Fig. 3 Longitudinal relaxivity values obtained at 1.41 T, 298 K for 45 mol% cholesterol-doped liposomes in both the absence and presence of KCl (1.08 mM) and transporters (1 mol% each).Error bars represent 1 s d. on either side of the mean r 1 value across two independent samples.Inset (a) shows the cation selectivity (1.08 mM MCl) with the greatest response observed for RbCl (∼120%).Analogous Li and Na responses were <10%.Inset (b) displays the anion selectivity trend (1.08 mM KX).

Fig. 4 T
Fig. 4 T 1 weighted MRI maps, recorded at 1.5 T and 3 T (298 K) for the cholesterol doped lipids in the absence and presence of the transporters and KCl salt (1.08 mM).

Fig. 5 r
Fig. 5 r 1 values obtained at 1.41 T, 298 K for the POPC-MSNs (for 0 mol% and 45 mol% cholesterol modified bilayers), in both the absence and presence of the ionophores (1 mol% each), with the associated relative percentage switch in r 1 highlighted.Error bars are calculated as described in Fig. 2(b).