Keywords: chemical exchange saturation transfer; molecular and cellular probes; lanthanide complexes; nanoparticles;paramagnetic shift reagents
INTRODUCTION
The concept of transferring saturated magnetization to waterproton resonance by irradiating the absorption of a proton poolof another molecule that is in slow/intermediate exchange withwater protons has been exploited extensively over the years inseveral fields of NMR spectroscopy (1). Its translation to MRIled to magnetization transfer contrast (MTC), which is the resultof selectively observing the interaction of bulk water protonswith the semisolid macromolecular protons of a tissue (2). The‘bound’ proton pool also includes water molecules in the hydra-tion shell of the macromolecules, whose motion is markedlyslowed down by hydrogen-bonding interactions. The T2 valuesof the protons associated with the tissue macromolecules aretoo short for direct MRI signal acquisition, but, on applicationof an off-resonance saturation pulse, one may observe an effecton the NMR signal of the ‘free’ water signal. Thus, MTC is basedon the magnetic interaction (through dipolar and/or chemicalexchange) between water protons and macromolecular protons.The technique has been exploited extensively as it has the abilityto image indirectly semisolids, e.g. immobilized proteins, whosesignal decays too rapidly to be imaged directly. The amount ofMTC depends on the concentration, surface chemistry and bio-physical dynamics of the macromolecules.The success gained by the use of MTC prompted Ward and
Balaban (3) to propose a new class of molecular MRI agentsbased on the same contrast mechanism. They were named‘chemical exchange saturation transfer’ (CEST) agents and, in adecade or so, have acquired great popularity in the field of MRIprobe development. A CEST agent is a system containing oneor more mobile protons in slow/intermediate exchange with wa-ter proton resonances. Thus, on irradiation of the absorption ofthe exchanging protons, saturated magnetization is transferredto the water signal, thereby causing a decrease in its intensity.
CEST agents are therefore classified as ‘negative’ agents becausetheir presence is signalled by a ‘darkening’ of the regions inwhich they distribute. In contrast with semisolid macromole-cules, the absorption of the mobile protons of CEST agents isgenerally relatively sharp and their frequencies are preciselydefined. The latter feature associates each CEST agent with itsdefined frequency-encoding property, thus opening up the fieldto the multiplex detection of different contrast agents in thesame anatomical region (4–6). Moreover, the detection of CESTagents does not require the acquisition of precontrast images,because the contrast is assessed by the comparison between thewater signal measured after the irradiation of the mobile protonsof the CEST agent and that obtained by simply offsetting the irradi-ation frequency far from the absorption of the mobile protons (7).
* Correspondence to: S. Aime, Via Nizza 12, 10126 Torino, Italy.E-mail: [email protected]
a D. D. Castelli, E. Terreno, D. Longo, S. AimeDepartment of Molecular Biotechnology and Health Sciences, Molecular Imag-ing Center, University of Torino, Torino, Italy
b E. Terreno, S. AimeCenter for Preclinical Imaging, University of Torino, Colleretto Giacosa, Italy
The main determinants of the efficacy of CEST agents are:(i) the number of exchangeable protons to be irradiated; (ii) theirexchange rate; and (iii) the T1 value of solvent water protons inthe solution containing the CEST agent. Soon after the seminalpaper of Ward and Balaban (3), who reported a number ofdiamagnetic molecules that act as potential CEST agents, a stepahead in terms of sensitivity enhancement was gained with theintroduction of paramagnetic CEST (paraCEST) agents (8–10).
Indeed, the large chemical shift values of the mobile protonsof a paramagnetic agent allow the selective irradiation of fastexchanging spins, thereby improving the efficiency of saturationtransfer. Clearly, the design of the paraCEST agent takes advan-tage of the extensive background of paramagnetic shift reagents(SRs) developed decades ago by NMR spectroscopists in the pre-high-field superconducting magnet era (11).
However, in spite of the good progress attained, the sensitivityof CEST contrast is still lower (approximately one order of magni-tude for small-sized agents) than the contrast mechanism basedon the paramagnetic effect on the T1 relaxation of water protons.Furthermore, the sensitivity gap increases when the T1 probeis conjugated to a nanoparticle, because of the enhancedrelaxation effect of the paramagnetic center immobilized in thenanosystem.
SUPRAMOLECULAR ADDUCTS
The first approach to increase the number of exchangeableprotons by maintaining the advantage of paramagneticallyshifted resonances was reported by our group in 2003 (12). Therationale relied on the design of noncovalent supramolecularadducts between a paramagnetic SR and a macromolecularsubstrate acting as the provider of a large number of mobileprotons. These systems were dubbed ‘supramolecular CEST’(supraCEST) agents.
As proof-of-concept, the well-known negatively charged SR[TmDOTP]4– [DOTP, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraphosphonic acid; Tm, thulium] (13) and the cationicpolyarginine were selected as the interacting pair. Thepolyaminoacid contains two different sets of mobile protons,both potentially CEST active (i.e. the amide protons of the poly-mer backbone and the guanidine protons of the side chains).However, the Z spectrum of the polymer alone (0.11mM) at pH7.4 did not show any CEST effect (Fig. 1) because the signal ofthe guanidine protons at physiological pH is already coalescedwith the bulk water, whereas the detection of the CEST effecton saturation of the amide protons is largely hampered bythe strong ‘spillover’ effect caused by the relatively intense irradi-ation field (25 mT).
On addition of an excess of the paramagnetic SR (almost20-fold), a supramolecolar adduct is formed, in which almost allthe mobile guanidine protons of the polymer interact with theSR (an affinity constant of 3� 104 was determined for adductformation by relaxometric measurements carried out with theGd3+ complex (Gd, gadolinium) of the DOTP ligand) and, conse-quently, their resonances move away from the bulk waterresonance of about 30 ppm (upfield) (Fig. 1). In addition to theshift effect, the formation of the supramolecular adduct may alsoaffect the exchange rate of the guanidine protons, thereby am-plifying the CEST efficiency. By measuring the CEST effect as afunction of the concentration of the interacting species (keepingthe SR/polyarginine molar ratio of approximately 20 : 1), an
in vitro detection threshold of 30 mM (c. 1.7mM for the polymer) wasdetermined for the SR concentration. A similar effect has beenreported recently by Evbuomwan et al. (14), who highlighted thatthe formation of an ionic pair between an anionic paraCEST agentand protonated amine groups exposed on the surface of modifiedsilica nanoparticles led to a significant acceleration of the exchangeof the metal-bound water protons of the complex.Other studies that have dealt with the formation of noncovalent
supraCEST agents have also been reported. Snoussi et al. (15)reported an interesting study based on diamagnetic interactingpartners, namely the negatively charged polyuridylic acid and acationic G5 polyamidoamine (PAMAM) dendrimer. Bothcompounds show a CEST effect at a similar irradiation frequency.Interestingly, on formation of the supramolecular ionic pair, theCEST effect arising from the RNA-like system decreased, whereasthe CEST response from the dendrimer increased. This findingcan probably be ascribed to mutual effects on the protonexchange rate of the mobile protons caused by the interaction.Still in the field of gene-related CEST agents, Wu et al. (16)
showed that a cationic polymeric paraCEST system can act as a po-tential reporter for gene therapy. Importantly, they demonstratedas proof-of-concept that the positively charged CEST polymer couldact as a transfection agent, showing the ability to successfullydeliver a gene to encode green fluorescent protein into cells.Ali et al. (17) investigated supramolecular systems formed by
human serum albumin and several Ln(III)-based (Ln, lanthanide)paraCEST agents. They found that the set-up of such bindinginteraction causes a two-fold acceleration of the exchange rate ofthe labilemetal-boundwatermolecule of the complexes. However,this effect had little influence on the CEST efficiency, therebymaking such agents of potential interest as vascular probes.
DENDRIMERS
Another route that has been explored to amplify the sensitivityof the CEST agent deals with the covalent linking of paraCESTmolecules on dendrimeric platforms. In 2007, Gruell and
Figure 1. Z spectra of a solution containing 0.11 mM of polyargininealone (open circles) and in the presence of 2 mM of [TmHDOTP]4– [DOTP,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonicacid)] (7 T; 312 K; pH 7.4; saturation scheme: 2-s single continuous wavepulse; saturation intensity, 25 mT). The chemical exchange saturationtransfer (CEST) peak at 30 ppm corresponds to the guanidine protonsof the polymer shifted on interaction with the paramagnetic shiftreagent. [Adapted from ref. (12).]
coworkers (18) described the synthesis of Yb(III)–DOTAM-function-alized [DOTAM, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxyamide; Yb ytterbium] polypropylene imine dendrimers. The applicabil-ity of these dendritic paraCEST MRI agents for pH mapping wasevaluated on a 7-T NMR spectrometer and a 3-T clinical MRI scanner.Based on the different numbers of exchangeable amide protons, thelowest detectable concentration of the first- and third-generationden-dritic paraCEST agents is a factor of about 4 and 16 lower, respectively,than that of a mononuclear reference complex.Along the same line of reasoning, Pagel and coworkers (19)
designed systems in which two different paraCEST agentswere conjugated to a second- and fifth-generation PAMAMdendrimer, respectively. The systems were then tested for anin vivo competitive assay. Injection of both contrast agents intoa mouse model of mammary carcinoma resulted in a temporalincrease in the CEST effect in the tumor, with different kineticsfor the two agents according to the particle size.
LIPOSOMAL VESICLES
lipoCEST
Early work on paraCEST agents showed that water itself could beused as a source of mobile protons, provided that it is in slowexchange on the NMR time scale with the bulk water (20,21).Two NMR signals are in slow exchange whenever their separa-tion in chemical shift (expressed by Δo) is larger than theirexchange rate (expressed by kex). To exploit water as the sourceof the mobile proton pool, paraCEST agents were designed topossess a slow-exchanging water molecule bound directly tothe metal site of a paramagnetic complex. However, thesesystems still suffered from a relatively poor sensitivity, as only
two protons contribute to the saturation transfer process.The need to increase drastically the sensitivity of these probesled to the consideration of systems possessing a very large num-ber of magnetically equivalent water molecules. Large amountsof water molecules can be found in the inner cavities ofnanovesicular systems, such as liposomes. According to the sizeof the nanoparticles (50–300 nm in diameter), the number ofexchangeable protons can reach as high as 106–108. However,this pool cannot be used as such for the CEST experiment,because the Δo separation between the inner water protonsand the ‘bulk’ water protons is too small to exceed the exchangerate. To remove the isochronicity between these two exchangingsignals, it was suggested that a paramagnetic water SR should betrapped in the inner liposomal cavity (Fig. 2). These agents werenamed lipoCEST agents (22).
Analogous to most paraCEST agents, the SRs used are Ln(III)complexes in which the ninth coordination site around the Lnion is occupied by a water molecule; however, unlike paraCESTagents, the bound water must be in fast exchange with theintraliposomal water molecules. The coordinated water moleculeand the intraliposomal water molecules, being in fast exchange,yield a single absorption whose chemical shift value (dintralipo) isthe sum of three contributions:
dintralipo ¼ dDIA þ dBMS þ dHYP [1]
where dDIA is the diamagnetic contribution (almost negligible),dBMS is the bulk magnetic susceptibility (BMS) contribution anddHYP is the hyperfine contribution.
dBMS does not require a direct chemical interaction between thewater molecules and the paramagnetic center, and is dependenton the shape and orientation of the compartment containing theparamagnetic SR, the concentration of the SR and the effectivemagnetic moment (meff) of the paramagnetic center. In a spherical
Figure 2. Schematic representation of a lipoCEST agent. CEST, chemical exchange saturation transfer; SR, shift reagent.
compartment, such as conventional liposomes, the susceptibilityeffects average out to zero, and therefore the only contributionto the intraliposomal water shift is dHYP.
Conversely, dHYP requires a chemical interaction between theparamagnetic center and the water molecule, and can operateeither through bond (contact shift) or space (pseudocontact ordipolar shift). The pseudocontact term is proportional to themolar fraction of metal-bound water molecules and to the shiftof the water protons at the coordination site (dM):
dHYP ¼ bound water½ �q dMð Þ= bulk water½ � [2]
where q is the number of coordinated water molecules, and dM isdirectly proportional to the magnetic anisotropy of the complex(Δw) and a geometric factor (G) defined as follows:
Δw ¼ CJ � A02 r2h i G / 3 cos2y� 1r3
where CJ is Bleaney’s constant which characterizes each Ln(III)ion and can have a positive or negative value. In particular, CJis positive for europium (Eu), erbium (Er), Tm and Yb, andnegative for cerium (Ce), praseodymium (Pr), neodymium (Nd),samarium (Sm), terbium (Tb), dysprosium (Dy) and holmium(Ho). CJ for Gd is equal to zero, thereby clarifying why Gd(III)complexes do not work as water SR, at least in sphericalcompartments.
The term A20< r2> refers to crystal field parameters of the
complex, and can assume positive or negative values, y is theangle between the principal magnetic axes of the complex andthe Ln–Hbound water vector, and r is the distance between theparamagnetic center and the protons of the coordinated watermolecule (Fig. 3).
Hence, the capability of an SR to shift the resonance of waterprotons depends on the Ln ion and the geometry of thecomplex. Figure 4 shows the shifting ability of Dy(III) and Tm(III) complexes of a series of polyamino-polycarboxylate ligands[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),
diethylenetriaminepentaacetic acid (DTPA), 10-(hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (HPDO3A)and DOTMA]. On this basis, Tm(III) and Dy(III) complexes appearto be the candidates of choice because they have high CJ valueswith opposite sign (positive and negative, respectively).For a given ligand, the induced shift depends on the
magnitude of the CJ values of the coordinated Ln(III) ion only,and therefore Dy(III) complexes induce a larger shift than theirTm(III) analogs. However, for a given Ln(III) ion, there is a strongeffect from the ligand because of the different geometriesadopted by the corresponding complexes. On this basis, the C4axially symmetric Tm/Dy–DOTMA complexes exhibit the highestshift because of the maximization of the (3 cos2 y – 1) term.Figure 5 shows the 1H NMR spectrum, Z spectrum and
parametric CEST map of a suspension containing a lipoCESTagent (Tm–DOTMA as SR). lipoCEST agents display a remarkablesensitivity that reaches the picomolar range (expressed as theliposome concentration) and, importantly, they do not requirethe use of an unsafe, high-intensity saturation field.Zhao et al. (23) developed an analytical model to correlate the
CEST efficiency to the size of liposomes. They reported that, for afixed fraction of the intraliposomal water pool in the suspension,the CEST effect increases for smaller liposomes because of afaster water exchange rate across the liposomal membrane,associated with the larger surface-to-volume ratio. Thesetheoretical and experimental tools are expected to benefit theapplications of liposomes to the sensing of the cellular environ-ment, the targeting and imaging of biological processes, andthe optimization of drug delivery properties.The resonance frequency offset of the water protons inside
liposomes may be modulated by changing the nature andconcentration of the entrapped SR. However, the shift of theinner water resonance in this type of lipoCEST is limited by the
Figure 3. Typical structure of a mono-aquo Ln(III) complex with a squareantiprismatic structure in which the metal-coordinated water molecule islocated along the magnetic axis of the complex. Ln, lanthanide.
Figure 4. Normalized chemical shift values for the bulk waterprotons in solutions containing 1 M of the reported shift reagents.DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DOTMA,1,4,7,10-tetraazacyclododecane-a,a’,a’’,a’’’-tetramethyl-1,4,7,10-tetraaceticacid; DTPA, diethylenetriaminepentaacetic acid; HPDO3A, 10-(hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid.
maximum amount of SR that can be entrapped (for in vivo use,the osmolarity of the solution inside the vesicle must be isotonicwith the physiological medium, i.e. 300 mOsm). Hence, using themost efficient mononuclear Ln(III) complexes, the maximumchemical shift separation between the inner and outer waterresonance was c. 4 ppm (downfield or upfield according to thesign of the Bleaney constant of the Ln(III) ion). Therefore, thein vivo application of these agents appears to be limited bythe interference with the overlapping endogenous magnetiza-tion transfer contribution. To overcome this drawback, it isnecessary to explore other routes to attain larger separationbetween the inner water and the bulk water resonances.Equation [1] shows that the shift of the intraliposomal water
protons can be increased by acting on the BMS effect. However,to achieve this goal, the paramagnetic SR must be entrapped ina nonspherical compartment. The liposomal membrane issemipermeable, i.e. water molecules can freely cross thephospholipidic bilayer, whereas ionic solutes or hydrophilic neu-tral small-/medium-sized molecules cannot. This property can beexploited to tailor the shape of liposomes by applying osmoticgradients. When a lipoCEST agent loaded with a hypo-osmoticsolution of SR (e.g. 40 mM instead of 300 mM of a neutral com-plex) is added to a solution isotonic with the physiological fluids,the vesicles shrink and leak water to reach osmotic equilibrium,and this process induces a change in vesicle shape (Fig. 6) (24).The nonspherical lipoCEST agents obtained show large BMS
contributions to the shift of the resonance of the water protonsentrapped in the vesicles. Interestingly, this contribution is largerthan the hyperfine term, thus allowing a dramatic increase(from 4 to 12 ppm) of the chemical shift separation betweenthe two exchanging pools. In addition, the BMS effect depends
on the concentration of the SR entrapped in the nonsphericalliposomes and, again, the maximum payload of SR is limited byosmotic rules. Hence, different strategies have been designedto further increase the concentration of SR without affectingthe osmolarity of the intraliposomal cavity. One consists of theincorporation of amphiphilic SRs into the liposome membrane(25). Another route involves entrapping polynuclear neutralcomplexes into the inner aqueous cavity (Fig. 7) (26).
The sign of dBMS depends on the orientation of thenonspherical vesicle with respect to the external magnetic fieldB0. It is known that phospholipid-containing particles arenaturally oriented in a magnetic field (27), and the driving forcefor the orientation is the sign of the magnetic susceptibilityanisotropy (Δw) of the bilayer. Hence, the orientation may beswitched from parallel to perpendicular by changing the signof Δw. As phospholipidic membranes possess a negative Δwvalue, the orientation change requires a switch of Δw to apositive value. This result can be achieved on incorporation ofan amphiphilic paramagnetic complex with a positive magneticsusceptibility anisotropy into the liposome bilayer. As the signof the anisotropy is dependent on both the Ln(III) ion and thestructure of the metal complex, the shift values of lipoCESTagents loaded with several amphiphilic Ln(III) complexes(basically conjugated to two aliphatic chains of suitable length)can be measured (Fig. 8) (28).
Thanks to the exploitation of the BMS effect, the window ofthe accessible irradiation frequency values can be extendedconsiderably from �4 ppm observed for spherical vesicles to+30/–45 ppm! Several benefits may be envisaged for suchimproved CEST agents. First, it is expected that the increasedseparation from the resonance of bulk water could reduce
Figure 5. (a) 1H NMR spectrum (14.1 T and 39 �C) of a suspension of a lipoCEST agent entrapping Tm–DOTMA (DOTMA, 1,4,7,10-tetraazacyclododecane-a,a’,a’’,a’’’-tetramethyl-1,4,7,10-tetraacetic acid) (inset). The signal at c. 4 ppm downfield from the bulk water corresponds tothe water protons entrapped within the liposome that contains 0.1 M of shift reagent (SR). (b) Corresponding Z spectrum obtained at 7 T (39 �C;liposome concentration, 2.9 nM; irradiation field intensity, 12 mT; irradiation time, 3 s). (c) MRI chemical exchange saturation transfer (CEST) map (matrix,64� 64) of a phantom consisting of eight capillaries containing suspensions of the above-mentioned lipoCEST agent at different concentrations (1–6:1.5 nM, 0.75 nM, 0.32 nM, 0.16 nM, 80 pM, 40 pM; irradiation pulse: train of 120 sinc3 pulses, 25 ms each; B1 field intensity, 12 mT).
Figure 6. Osmotic shrinkage of spherical lipoCEST (CEST, chemical exchange saturation transfer) agents leads to the formation of aspherical vesicles,as confirmed by cryo-transmission electron microscopy characterization.
drastically the artifacts in the MRI CEST images generated by theasymmetry of the bulk water signal and/or the inhomogeneity ofthe imaging coil, which are among the main determinants of theinhomogeneous distribution of the resonance frequency of thebulk water. Second, the extension of the available irradiationfrequency values facilitates the setting up of imaging protocolsaimed at the visualization of multiple lipoCEST probes.
A very elegant demonstration of the relationship between theorientation in the magnetic field and the chemical shift of theintravesicular water protons for aspherical lipoCEST agents wasreported by Burdinski et al. (29). They used a capillary with aninner diameter of 100 mm, coated with a monolayer of cyclodex-trins able to stably host adamantane moieties, which wereexposed on the surface of the lipoCEST agents. Suitablysynthesized adamantane-bearing amphiphilic Dy(III) and Tm(III)complexes (which have Δw values of opposite sign) were incor-porated into the lipoCEST membrane and classical water-solubleSRs were encapsulated in the inner aqueous cavity. Once theliposomes are tightly bound to the capillary’s surface and,depending on the alignment of the capillary (parallel or perpen-dicular) with respect to B0, the expected magnetic alignment-dependent shifts were observed. These results showed that theorientation of nonspherical liposomes bound to a target surface
could be determined by using a routine CEST procedure. As acorollary, these findings may offer unique opportunities inmolecular MRI applications, as bound and unbound lipoCESTagents can be discriminated on the basis of their different CESTresonance frequencies.It was recalled above that one of the major advantages of
CEST methodology deals with the possibility of the visualizationof several probes in the same MR image. For in vivo applications,it is necessary that the involved CEST agents display sufficientlydifferent resonance frequencies of their mobile protons associ-ated with a good sensitivity. As lipoCEST agents possess bothproperties, they have been tested to assess their potential atthe preclinical level. The first in vivo co-localization of twolipoCEST agents was carried out on a muscle of a mouse. TheCEST responses of the two agents did not interfere with eachother, thus allowing the separate visualization of the twosystems present at nanomolar concentrations (Fig. 9) (30).Later, the first in vivo targeting experiment using lipoCEST
agents was reported by Flament et al. (31), who targeted theavb3 integrin receptor, which is one of the most common neo-angiogenesis biomarkers. To this purpose, an RGD-functionalizedlipoCEST agent was formulated and injected intravenously into amouse model of U87 glioma brain tumor. They found an
Figure 7. Schematic overview of the different generations of lipoCEST (CEST, chemical exchange saturation transfer) agents. HPDO3A,10-(hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; SR, shift reagent.
Figure 8. Intraliposomal water shift values (25 �C) for a series of nonspherical lipoCEST (CEST, chemical exchange saturation transfer) agents encap-sulating Tm–HPDO3A [HPDO3A, 10-(hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid] and incorporating the amphiphilic complexesreported at the top. Ln, lanthanide; SR, shift reagent.
enhanced CEST contrast, not only in the tumor, but also in otherbrain regions, probably as a result of nonspecific binding and/ordistribution of the RGD-lipoCEST agent. Clearly, althoughtargeted lipoCEST agents must be improved to match therequired specificity, it is noteworthy that these studies makein vivo detection feasible.Another interesting field of application of lipoCEST agents
deals with the design of smart MRI probes. A representativeexample has been reported by Langereis et al. (32), whodesigned a dual 1H–19F thermosensitive lipoCEST agent able toreport on temperature changes. The inner cavity of the liposomewas filled with both a classical paramagnetic SR (to enable1H CEST detection) and a fluorine-labeled compound (NH4PF6)for 19F detection. At temperatures below the gel-to-liquidtransition phase of the liposome, the 19F NMR signal was notdetectable because of marked signal broadening caused by thestrong T2 paramagnetic effect induced by the SR, whereas agood CEST contrast was observed. Conversely, on approachingthe transition temperature, the two probes were released fromthe nanocarrier, with the consequent disappearance of the CESTcontrast, and appearance of 19F MRI, no longer influenced by theproximity effect of the SR (Fig. 10). Hence, the 19F signal couldbe used to quantify the amount of released drug payload,
and the CEST signal could measure the local nanocarrierconcentration (i.e. to quantify the drug delivery) before release.This study demonstrated the potential of such an agent in cancertheranostic research.
Finally, it has been shown that, in addition to liposomes, otherinnovative nanovesicular platforms, such as polymersomes, cansuccessfully act as highly sensitive CEST agents (33).
Liposomal vesicles entrapping CEST systems
Liu et al. (34) reported in vivo CEST experiments for the multiplevisualization of liposomes by preparing vesicles entrapping dif-ferent diamagnetic CEST agents. They selected three compounds[glycogen (Glyc), L-arginine (L-Arg) and poly-L-lysine (PLL)], whichwere encapsulated in stealth (i.e. pegylated) liposomes havingan approximate diameter of 100 nm. Figure 11 illustrates thecharacteristics of such diamagnetic liposomes (DLs).
Three artificial colors were associated with the mobile protonpools of the system: red for the hydroxyls of Glyc (frequency off-set, 0.8 ppm), yellow for the amino group of L-Arg (1.8–2.2 ppm)and green for the backbone amide protons of PLL (3.6 ppm). Theyapplied this multicolor MRI acquisition scheme for noninvasivelymphatic imaging. Liposomes were injected in the right footpad
Figure 10. Left: 1H chemical exchange saturation transfer (CEST) effect (irradiation field intensity, 4.5 mT) and 19F NMR signal intensity of liposomescontaining Tm-HPDO3A and NH4PF6 as a function of temperature (B0 = 7 T). HPDO3A, 10-(hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid. Right: 1H MRI CEST and 19F MR images of temperature-sensitive liposomes on a clinical 3.0-T MRI scanner. The CEST signal (color scale in per cent)disappeared at 311 K and the fluorine signal appeared at 315 K (overlay with the 1H image for co-localization and clarity). [Adapted from ref. (32).]
Figure 9. Multiple visualization of lipoCEST agents injected in the thigh muscle of a mouse. The chemical exchange saturation transfer (CEST)responses measured on saturation at 3.5 ppm (spherical agent entrapping Tm-DOTMA) and –17.5 ppm (aspherical vesicles entrapping Dy-DOTMA)were rendered in false colors (7 T; saturation time, 2 s; power, 6 mT; block pulse). DOTMA, 1,4,7,10-tetraazacyclododecane-a,a’,a’’,a’’’-tetramethyl-1,4,7,10-tetraacetic acid. [Adapted from ref. (30).]
of vaccinated C57BL/6.SJL mice, and an MR image of an axial slicewas acquired at 24 h post-injection. The measured magnetizationtransfer ratio curves for both the footpad at the injection site andthe contralateral region displayed an offset-dependent shapecaused by the inherent asymmetry of the magnetization transferratio (MTRasym) of the tissue based on competing magnetizationtransfer effects for semisolid components. However, the CEST con-trast in the popliteal lymph node (PLN) was clearly distinguishedfrom the background signal, and was significantly higher than thatdetected for the control PLN (Fig. 12).
The encapsulation of paraCEST agents into liposomes wasexploited by Opina et al. (35) to develop pH-sensitive systems.They encapsulated pH-responsive paraCEST agents [Dy(III) andTm(III) complexes of DOTA–(gly)4 ligand] inside the aqueouscavity of liposomes. Although the Dy(III) complex showed thelargest hyperfine shift, the combination of favorable chemicalshift and amide signal linewidth for the Tm(III) complex madethe latter more promising for future in vivo applications.The pH dependence of the CEST effect differed between the freeand encapsulated agent over the acidic pH regions.
Figure 11. Multicolor spectrum of diamagnetic liposomes (DLs). Left: diagram displaying the components of DLs. DIA-CEST, diamagnetic chemicalexchange saturation transfer. Right: asymmetric magnetization transfer ratio (MTRasym) plot showing the water signal intensity reduction as a functionof frequency for three DLs (~30 nM) in vitro (pH 7.3 and 37 �C), with red assigned to OH in glycogen DL, yellow assigned to NH2 in L-arginine DL, andgreen assigned to NH in poly-L-lysine (PLL) DL. [Adapted from ref. (34).]
Figure 12. Representative color MR images using different diamagnetic liposome (DL) combinations. (a) Unilateral glycogen liposomes. (b) UnilateralL-arginine (L-Arg) liposomes. (c) Unilateral poly-L-lysine (PLL) liposomes. (d) Bilateral L-Arg and PLL liposomes. Arrowheads in (a)–(d) indicate the loca-tion of popliteal lymph nodes (PLNs). For (a)–(c), from left to right: T2-weighted anatomical image; asymmetric magnetization transfer ratio (MTRasym)image at the frequency of interest with chemical exchange saturation transfer (CEST) contrast displayed using the jet color map in Matlab; MTRasym/T2-weighted image overlay with CEST contrast highlighted using a 64-bit scaled single-color (i.e. red, yellow or green) map; mean MTRasym plot. In themean MTRasym plot, the values shown are of DL(+) PLN (full line), DL(–) PLN (broken line) and their difference (colored line). (d) Representative two-color CEST image demonstrating the simultaneous detection and visualization of PLL (green, left PLN) and L-Arg (yellow, right PLN), with the meanMTRasym values of the left (L) and right (R) PLNs plotted in green and yellow, respectively. [Adapted from ref. (34).]
Nevertheless, the use of nanoparticles amplified the CESTsensitivity by a factor of approximately 104 compared with thefree nonencapsulated agent. Hence, this pH-sensitive nanoprobecould prove useful for the mapping of pH in body regions inwhich liposomes accumulate (e.g. tumors).
Other paraCEST nanoparticles
In 2006, Lanza and coworkers (36) synthesized a nanoconstructin which a bifunctional paraCEST chelate was covalentlyconjugated to the surface of perfluorocarbon nanoparticles, withthe motivation of stably incorporating very high payloads ofparaCEST agents onto a nanocarrier. Moreover, the surface ofthe nanoparticle was appropriately functionalized to selectivelybind fibrin (Fig. 13).Recently, Evbuomwan et al. (14) have reported the preparation
and CEST characterization of paraCEST agents conjugated tosilica-modified nanoparticles. Approximately 1200 EuDOTA–(gly)4
– molecules were attached to each nanoparticle via conjuga-tion of agent carboxyl groups to surface amines. Althoughthe CEST properties of the modified nanoparticles showed thatsurface conjugation of the paramagnetic agent resulted incomplete elimination of the water exchange signal, thisapproach is interesting because of the high payload of com-plexes per particle that can be obtained. In future, such systemsmay be prepared with paraCEST complexes exploiting mobilepools different from the bound water protons (14).Vasalatiy et al. (37) implemented a prelabeling strategy for
the conjugation of preformed Ln ligand chelates to adenovirusparticles. Using complexes of Tm3+ with the isothiocyanate ofDOTA-tetraamide, a large number of paraCEST agents wereattached to the surface residues of recombinant adenovirus type5 particles. The potential of such conjugates to act as paraCESTimaging agents was tested using an on-resonance WALTZsequence for CEST activation. A 12% decrease in bulk watersignal intensity was observed relative to controls. This
demonstrates that viral particles labeled with paraCEST-typeimaging agents can potentially serve as targeting agents formolecular imaging.
Wu et al. (38) reported the copolymerization of a DOTA-tetraamide ligand having a single acrylamide side chain (M1)with 2-methylacrylic acid, 2-(acryloylamino)-2-methyl-1-propane-sulfonic acid or N-isopropylacrylamide to create a series of linearrandom copolymers using classical free radical chain polymeriza-tion chemistry. All polymeric agents were found to possesssimilar intermediate-to-slow water exchange and CEST charac-teristics as the parent EuDOTA-tetraamide monomer. Thehighest CEST sensitivity displayed by these systems was 2 mM.
Paramagnetic micellar systems have also been explored, inparticular micelles containing the Eu(III) complexes of threeDOTA-tetraamide ligands having variable alkyl chain lengths(C12, C14 and C16) (39). The three complexes spontaneouslyform micelles of variable size. The complex having the largestCEST effect per unit (the C16 analog) had a detection limit of5.3 mM. This represents an approximate 250-fold increase insensitivity relative to the monomethylamide control (detectionlimit, �1.3 mM). These features highlight the potential of usingmicelle-based systems such as these as paraCEST agents formolecular imaging by MRI (39).
CONCLUSIONS
Nanoparticle-based agents appear to be the candidates ofchoice for several relevant MRI diagnostic and ‘theranostic’applications. In less than a decade, chemistry-based groups haveshown how such systems can be manipulated in order todramatically enhance the sensitivity and specificity of the CESTresponse.
The effect of particle shape on the CEST response yieldsinteresting insights for the design of novel applications that arenot conceivable with molecular systems. In spite of the
Figure 13. (a) Chemical structure of the lipid-conjugated paramagnetic chemical exchange saturation transfer (paraCEST) contrast agent. Eu3+ ischelated to methoxy-benzyl-DOTA (DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;), which is functionalized with a phospholipidmoiety for incorporation into the lipid membrane of perfluorocarbon nanoparticles. (b) Saturation transfer of a two-chambered phantom containingparaCEST nanoparticles (left) and control nanoparticles (right). The signal enhancement in the paraCEST nanoparticle chamber was significantly higher thanthat in the control nanoparticle chamber (*p< 0.05). (c) Images of fibrin-targeted paraCEST (left) and control (right) nanoparticles bound to plasma clots.Images obtained with saturation at –52 ppm (top) show no differences between clots treated with paraCEST or control nanoparticles. Subtraction images(bottom) reveal signal enhancement on the surface of the clot treated with paraCEST nanoparticles, and no enhancement of the clot treated with controlnanoparticles. The Contrast to noise ratio (CNR) calculated at the clot surface was significantly higher with paraCEST nanoparticles relative to controlnanoparticles (*p< 0.05). [Adapted from ref. (36).]
impressive improvements achieved in vitro, one of the majordrawbacks for the in vivo translation of nanoparticles is repre-sented by their sequestration from organs of the mononuclearphagocytic system (MPS), such as the liver and spleen. Thisgeneral issue, which obviously holds for other classes of MRIprobe (macro- and supramolecular), is even more problematicfor CEST particles, as it leads to the degradation of the particlesor to their compartmentalization, both processes convergingto hamper the CEST response. It has been shown recently that,on comparing the dynamics of the contrast of lipoCESTagents with T1 and T2 paramagnetically loaded liposomes inthe tumor region, the CEST effect quickly disappears, whereasthe T1 and T2 effects last for a much longer period of time (40).The observed behavior was ascribed to the sequestration ofliposomes by tumor-associated macrophages, which impliesthe compartmentalization of liposomes in endosomal cavities.Thus, the presence of additional barriers (liposome, endosomeand plasma membrane) slows down the exchange rate betweenthe liposomal inner cavity and bulk water, decreasing theCEST effect.
To overcome this drawback, the design of nanoparticle-basedCEST agents must be revised in the light of the interactions ofthe particles when distributed in a biological environment. Themaintenance of the particles in the extracellular environment(also in the case of targeting procedures) appears to be manda-tory for the in vivo exploitation of the excellent CEST propertiesshown in in vitro experiments. Thus, we remain confident thatthe great potential shown by nanoparticle-based contrast agentswill find appropriate routes to clinical translation. More workappears to be necessary to design ‘stealth’ CEST particles, asthe uptake from MPS organs is a crucial step in the design ofany targeting experiment. Much progress has been made inrecent years in the field of nanocarriers for drug delivery, whichmay be readily translated to the imaging reporters discussedherein. As for most MRI reporters, the targets for CEST particlesare in the vascular and extracellular matrix. The possibility ofthe simultaneous visualization of several biomarkers in the sameanatomical region is a tremendous driving force for investingefforts in this class of MRI agent.
Acknowledgements
This research was funded by the University of Torino (codeD15E11001710003, Project: Innovative Nanosized TheranosticAgents), Regione Piemonte (PIIMDMT and Nano-IGT projects),MIUR (PRIN 2009). It was scientifically supported by ESF COSTAction TD1004 (Theranostics Imaging and Therapy: An Actionto Develop Novel Nanosized Systems for Imaging-Guided DrugDelivery), and CIRCMSB (Consorzio Interuniversitario di Ricercasulla Chimica dei Metalli nei Sistemi Biologici).
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