Insights on the relaxation of liposomes encapsulating paramagnetic Ln-based complexes
G. Mulas,a,c G. Ferrauto,a W. Dastrù,a R. Anedda,c S. Aime,a,b E. Terrenoa,b,* a Department of Molecular Biotechnology and Health Sciences, University
of Torino, Via Nizza 52, 10126 – Torino, Italy
b Center for Preclinical Imaging, University of Torino, Via Ribes 5, 10010 –
Colleretto Giacosa (TO), Italy
c Porto Conte Ricerche Srl, Tramariglio, Alghero (SS), Italy
* Corresponding author: Prof. Enzo Terreno
Center for Preclinical Imaging, University of Torino
Via Ribes, 5
10010 – Colleretto Giacosa (TO), Italy Phone: +39-0125-538942
Fax: +39-0125-538664
e-mail: enzo.terreno@unito.it
Running title:
Keywords: MRI, Magnetic Susceptibility, Lanthanides, Liposomes, Words count: 2794
Abstract
Purpose: to describe and quantify the different relaxation mechanisms operating in suspensions of liposomes that encapsulate paramagnetic lanthanide(III) complexes
Theory and Methods: the paramagnetic contribution to the measured transverse relaxation rate of lanthanide-loaded liposomes (R2plipo) can be
expressed as the sum of magnetic susceptibility effects and the dipolar contribution. Phospholipids vesicles encapsulating different Ln(III)-HPDO3A complexes (Ln= Eu, Gd, or Dy) were prepared using the conventional thin film rehydration method. Relaxation times (T1, T2 and T2*) were measured
at 14 T and 25°C. The effect of compartmentalization of the paramagnetic agent inside the liposomal cavity was evaluated by means of an IRON-modified MRI sequence.
Results: NMR measurements demonstrated that Curie spin relaxation is the dominant contribution (> 90%) to the observed transverse relaxation rate of paramagnetic liposomes. This was further confirmed by MRI that showed the ability of the liposome entrapped lanthanide complexes to generate IRON-MRI positive contrast in a size dependent manner.
Conclusion: The Curie spin relaxation mechanism is by far the principal mechanism involved in the T2 shortening of the water protons in
suspension of paramagnetic liposomes at 14 T. The access to IRON contrast extends the potential of such nanosystems as MRI contrast agents.
Introduction
In the last decade, liposomes loaded with paramagnetic lanthanide complexes have received much interest as highly sensitive contrast agents for MR-molecular imaging applications. An interesting peculiarity of such nanoagents deals with the ability to generate different types of MRI contrast (T1, T2, CEST, or 19F-MRI) according to the magnetic properties of
the loaded lanthanide metal ion.1,2
Gd(III) is by far the best choice for T1-based contrast due to the strong
dipolar interaction between the unpaired electrons of the metal (long electronic relaxation time) and the protons of the exchangeable water molecule(s) bound to the paramagnetic center. Conversely, lanthanide ions that induce large paramagnetic shift effects on the intraliposomal water protons without affecting too much their transverse relaxation rate (e.g. Tm(III)) are well suitable for the preparation of CEST agents.3
Paramagnetic liposomes have been also proposed as T2/T2* agents as
potential surrogates of superparamagnetic iron oxide particles, and it was claimed that magnetic susceptibility is the dominant contribution to the observed transverse relaxation rate enhancement.4,5 Such effects does not
require a direct access of the water molecules to the paramagnetic ion, and occurs when paramagnetic complexes are segregated in compartmentalized systems.6,7
Though some theoretical aspects of the proton water relaxation of Gd(III)-loaded liposomes have been already published,8 the aim of this note is to
provide an experimental base to describe and quantify the contribution of relaxation mechanisms operating in a suspension of paramagnetic liposomes, extending the study to other lanthanide ions than Gd(III).
Methods - Chemicals
1,2-dipalmitoyl-sn-glycerophosphocoline (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Avanti Polar Inc. (Alabaster, AL, USA). Gd-HPDO3A ligand was kindly provided by Bracco Imaging
(Colleretto Giacosa (TO), Italy), whereas the corresponding Ln(III) complexes were synthesized according the published procedure.9 All other
chemicals were purchased from Sigma Aldrich.
- Liposomes preparation
Liposomes encapsulating Ln(III)-HPDO3A complex (Ln= Eu, Gd, or Dy) were prepared using the conventional thin film rehydration method (temperature 55°C, hydration solution Ln-HPDO3A 300 mM, total lipid content 40 mg/ml). The bilayer was composed by DPPC/DSPE-PEG2000 (95/5 in moles). Liposomes were obtained by extrusion through polycarbonate filters or by sonication (to reduce size). Not encapsulated complex was eliminated by exhaustive dialysis (4°C against isotonic HEPES/NaCl buffer at pH 7.4). Mean hydrodynamic liposome diameter was determined by dynamic light scattering (Zetasizer NanoZs, Malvern instruments, UK) and the concentration of Ln(III)-complexes encapsulated into liposomes was estimated measuring bulk magnetic susceptibility by NMR.10
- MRI measurements
MRI experiments were performed on a Bruker Avance 300 MHz. An IRON modified sequence was used consisting of a spectrally-selective preparatory scheme with on-resonance saturation of the bulk water protons (RF saturation pulse intensity=3 µT; total saturation module duration=1000 ms) followed by a conventional RARE spin-echo imaging sequence (TE=5.637 ms, TR=5 s, rare factor=8, FOV=3 cm, matrix =64 x 64, slice thickness=1 mm). The same sequence was acquired without the saturation module as control.
MRI phantoms were prepared by mixing the liposome suspension with agar gel (1%) to a final volume of 100 µl. To prevent interface artifacts, no plastic or glass tubes were used. Accordingly, mixtures were simply poured into preformed agar holes. MRI phantoms consisted of free and liposome entrapped Ln(III)-HPDO3A complex.
Relaxation times measurements were performed at 14 T and 25°C on a Bruker Avance 600 spectrometer. T1 was measured by the Inversion
Recovery sequence, whereas T2 was determined by
Curr-Purcell-Meiboom-Gill sequence. T2*values were directly determined from the bandwidth at
half height of the NMR water signal.
- Theory
The R2 of liposomes entrapping paramagnetic lanthanide(III) complexes
receives a significant contribution from magnetic susceptibility effects. To a first approximation, the paramagnetic contribution to the measured R2 (R2plipo=R2measlipo-R2measdia) can be considered as the sum of two terms:
[1]
where the superscript terms dip e susc refer to the contribution that requires (dip) or not (susc) a direct access of water protons to the paramagnetic center. Thus, the quantification of the susceptibility contribution requires the assessment of the dipolar term. In analogy to what is commonly done for R1,4,11 R2dip depends on: i) the relaxation rate of
the intraliposomal protons (R2intralipo), ii) the exchange rate of the water
protons (intralipo) across the liposome membrane, and iii) the volume
fraction of the intraliposomal water over the total volume (f, usually ≤ 10 %, Eq. 2).
[2]
R2intralipo is the product between the intraliposomal millimolar concentration
of the paramagnetic complex ([LnL]intralipo) and the millimolar transverse
relaxivity of the complex entrapped in the liposome (assumed to be the same of the free complex, r2free ):
[3]
When R2intralipo>>kintralipo, the dipolar contribution is controlled by kintralipo and
weighted by f (that can be determined as reported elsewhere4,11). Thus, R
2p
may be estimated once kintralipo is known.
The same equations also apply for the longitudinal relaxation rate (R1p),4,11
thereby providing a simple and direct access to kintralipo. In summary, kintralipo
can be obtained measuring R1 if the condition R1intralipo>>kintralipo applies.
This condition can be easily met entrapping in liposomes with a low kintralipo
value a relatively high amount (tens to hundreds mM) of a Gd(III) complex .11
Furthermore, as kintralipo is not dependent on the chemical nature of the
entrapped Ln-complex, such a value can be used to calculate the R2dip
value (and consequently the R2susc contribution) for liposomes loaded with
any paramagnetic Ln-complexes regardless of the fulfillment of the R2intralipo>>kintralipo condition.
Results
To assess and quantify the different relaxation mechanisms operating in a suspension of paramagnetic liposomes, three samples of phospholipids vesicles were prepared differing in the encapsulated lanthanide complexes: Eu-, Gd-, or Dy-HPDO3A (Chart 1).
The composition of the liposomal bilayer was chosen to ensure a low water permeability,11 thereby increasing the contribution from relaxation
mechanisms that are not mediated by the water proton exchange across the vesicle membrane.
Table 1 reports the 1 mM normalized relaxation rates (longitudinal r1, and
transverse r2) for free or liposome-encapsulated complexes (14.1 T and
25°C).
For Gd-loaded liposomes (Lipo-Gd-DPPC) with a size around 100 nm, the observed r1 value (0.2 mM-1s-1) is strongly “quenched” (92 %) by the
occurrence of a low kintralipo value. Such a r
1 reduction can be used to
estimate the water permeability of the liposome membrane,9,11 which was
ca. 0.1 m/s, i.e. slightly lower than the value previously reported for the same liposome formulation (but loaded with 10 mM of Gd-HPDO3A).11
Conversely, the transverse relaxivity of this sample was ca. one order of magnitude higher than the free complex (53.1 s-1mM-1 vs. 4.3 s-1mM-1).
To evaluate whether this contribution was correlated to the paramagnetism of the lanthanide ion (i.e. μef), liposomes with the same
size and composition, but loaded with Eu- or Dy-HPDO3A, were prepared, and their relaxometric properties were tested in the same conditions of the previous sample (Table 1).
Analogously to Lipo-Gd-DPPC, both Eu- and Dy-based liposomes displayed an exchange-limited longitudinal rate (in case of Lipo-Eu-DPPC, r1 values
were too small to be determined), and a similar r2 enhancement (r2lipo vs. r2free) of ca. one order of magnitude.
To quantify the magnetic susceptibility effect for the three samples, the dipolar contribution due to the water exchange across the bilayer (r2dip)
was calculated from the kintralipo value obtained from the analysis of the
longitudinal rate for Lipo-Gd-DPPC sample. The results obtained indicated that such a contribution (r2susc in Table 1) is by far the predominant one (>
90%) to define r2lipo.
Figure 1 clearly indicates that r2susc values displayed an excellent linear
correlation with (μef)4.
Since it has been reported that T2/T2*-shortening magnetic susceptibility
effects can be turned on positive MRI contrast by specific pulse sequences,12 the ability of paramagnetic liposomes to act as positive
contrast agents was assessed in vitro using the formulation with the highest T2-effect (i.e. Lipo-Dy-DPPC).
Figure 2 reports the MRI images obtained for two agar phantoms containing a dispersion of Lipo-Dy-DPPC (top row) and free Dy-HPDO3A complex (bottom row) at the same concentration of paramagnetic ion (12.6 mM). The samples were subsequently subjected to RARE (T2
-weighted) and IRON-MRI sequences.
The result highlighted the role of the compartmentalization of the paramagnetic agent in the liposomal cavity that generated not only a marked loss of signal in the RARE-T2w images, but also a positive IRON
contrast, similarly to what observed for SPIO-based sample.13,14 The
contrast is clearly visible and distinguishable both in the equatorial (negative) and polar (positive) lobes nearby the sample containing the paramagnetic liposomes. Conversely, when the same amount of paramagnetic agent is freely dispersed in agar, only a very noisy positive
effect was detected in the images, but no long-range IRON effects were observed around the sample.
Figure 3 displays the the MRI-IRON coronal images of two Dy-DPPC-based liposomes with different size (110 nm/PDI 0.1 vs. 85 nm/PDI 0.16) at the same concentration of Dy(III) (12.6 mM).
The images indicated that the IRON contrast was directly proportional to the size of the vesicles. To get a quantitative assessment of the IRON contrast, the total area of positive signal, as well as the average signal intensity (normalized to the background), were determined. The total area was calculated adding up the area of the different bright lobes (equatorial and polar) surrounding the sample (Table 2).
The IRON effect correlates well to the vesicle size. In fact, a size increase of 30% led to a total enhancement of 44 % as positive area, and 40 % as normalized signal intensity.
Discussion
The formulation of liposomes based on DPPC (known to have a low water permeability) and prepared entrapping 300 mM of the clinically approved neutral Gd-HPDO3A, fulfills the R2intralipo>>kintralipo condition. This formulation
has been already proved to have an exchange-limited R1p value. The kintralipo
value obtained for this formulation at 14 T was in good agreement to what previously obtained at 0.2 T (0.1 vs. 0.3 m/s).11
The data reported in Table 1 for the three examined samples clearly show that r2lipo values are essentially dominated by a long-range relaxation
mechanism in which the paramagnetic metal complex influences the relaxation of the bulk water protons without receiving a contribution from the intraliposomal protons.
Plotting the normalized r2susc values as a function of the paramagnetism of
the Ln(III) ion (expressed by the eff value) an excellent linearity as a
function of (ef)4 was observed (Figure 1). Very interestingly, Pereira et al.
reported the same behavior for zeolite-type silicates doped with lanthanide ions.15 In analogy with paramagnetic liposomes, the relaxation
coordinated to the metal center, and it was demonstrated that r2 of that
systems was dominated by the outer-sphere Curie spin mechanism, which is actually directly proportional to the (μeff4× B02) term. However, an
important difference between the two systems relies upon the difference between R2 and R2* values. In fact, in the paramagnetic zeolite-like
material, R2*>>R2,13 whereas for the paramagnetic liposomes investigated
here r2*≈r2 (as example the suspension of Lipo-Dy-DPPC displayed R2* =
25.9 s-1 and R
2 = 26.0 s-1).
Such a difference can be accounted for in terms of the different motional behavior of the two particulates: in case of liposomes, the r2*≈r2 condition
indicates the occurrence of a motional narrowing condition in which the water diffusion around the nanovesicle is faster than the difference in the resonance frequency (caused by the susceptibility effect) between the water protons of the bulk and those surrounding the nanoparticle.16
As the Curie spin contribution is proportional to B02, the susceptibility
effect significantly increases with the magnetic field.
To assess the detection limit of paramagnetic liposomes as T2-agents,
simulation curves were calculated (Figure 4), where the R2p value (top),
and the percentage of the Curie spin contribution (bottom) were plotted as a function of the magnetic field strength.
The simulations were obtained considering that the dipolar term (R2pdip) is
only controlled by kintralipo, itself unaffected by the applied magnetic field
strength. The validity of this assumption was confirmed by the observation that the dipolar contribution for the three examined samples is nearly constant (ca. 0.2 s-1), despite the very different paramagnetism of the
entrapped lanthanide ion.
The Curie spin term (R2CS) was calculated according to the following
equation:15
[4]
where C is a lump parameter including proton gyromagnetic ratio, Bohr magneton, Boltzmann constant, temperature, water protons-metal ion distance, diffusional correlation time of the nanoparticles, and the concentration of the metal complex.15 The linear regression of the data
reported in Figure 1 allowed the estimation of C (5.6×10-5 s-1T-2), from
which it was possible to correlate R2SC to B0 for different ef values.
The curves reported in Figure 4 clearly outline the remarkable B0
dependence of the Curie spin contribution. Considering an overall metal concentration of 1 mM in the liposomal suspension, and arbitrarily choosing a threshold value of 2 s-1 for the contrast detection, the Dy-based
agent can be already detected at 1.5 T, the Gd-analogue at 2 T, whereas the much less efficient Eu-loaded liposomes could be visualized at 14 T only. Analogously, the percentage contribution of the two mechanisms to R2plipo is much higher for Dy- and Gd-loaded liposomes than for the
Eu-based system. The Curie contribution becomes the prevalent beyond 0.5 T, 0.9 T, and 4.5 T for liposomes entrapping Dy, Gd, and Eu, respectively. Though nanoparticulate T2*/T2 agents (e.g. iron oxide-based particles)
have demonstrated an excellent MRI detection sensitivity, the signal hypointensity they induce may make difficult the discrimination between the signal loss generated by the agent with respect to other sources of hypointense signal such as tissue interfaces, air or motion artifacts,17 as
well as tissue/organs with intrinsic low signal (e.g. lungs) or short T2 (e.g.
liver, muscles). Therefore, it was deemed useful to take advantage of the magnetic field susceptibility created by the particles to develop acquisition modes leading to a positive contrast detection.18 In this context, an
interesting approach was proposed by Stuber et al., who developed a sequence, named Inversion Recovery with ON resonant water suppression (IRON), which provides positive contrast through the application of a spectrally-selective on-resonant radio frequency (RF) saturation pulse before applying the imaging sequence. In such a way, the signal that originates from on-resonant water protons is suppressed by direct saturation, but off resonant protons are not (or minimally) influenced, thus leading to a positive contrast detectable in close proximity of the particles.19
The results illustrated in Figure 2 and Figure 3 highlight the ability of paramagnetic liposomes to generate a positive MRI contrast.
This observation substantiates that the T2 MR contrast generated by the
encapsulation of a paramagnetic agent in the liposome inner core is actually caused by the Curie spin mechanism. To further prove that the IRON-MRI contrast is a measure of susceptibility effects, samples containing different T2 agents (iron oxide nanoparticle, Dy-HPDO3A free
and encapsulated in liposomes), but prepared in order to have the same T2
value, were subjected to the MRI-IRON sequence. Only nanoparticulate systems for which susceptibility effects are expected were able to generate an IRON positive contrast (data not shown). As it has been demonstrated that the R2 enhancement observed for paramagnetic
liposomes was directly correlated with the particle size20 (likely because
the number of paramagnetic molecules inside the vesicle increases with the volume of the liposome), we considered of interest the search for a correlation between IRON contrast and vesicles size. This point is highly relevant for in vivo applications because the behavior of nanosystems in biological environments (e.g. biodistribution and cellular uptake) is strongly affected by the particle size.
Actually, the data reported in Table 2 indicate that when the IRON contrast (expressed as the average signal intensity in the area of positive contrast over the background signal (SI/BS)) was normalized to the micromolar concentration of the liposomes (keeping constant the total concentration of the paramagnetic complex), the efficiency of the bigger nanovesicle was about three-fold higher than the smaller one.
Conclusions
This work was aimed at evaluating the relaxation mechanisms involved in the T2 shortening of the water protons in suspensions of liposomes
encapsulating paramagnetic lanthanide(III) complexes. It has been demonstrated, for the first time, that Curie spin mechanism is by far the dominant (> 90 % at 14 T) relaxation mechanism contributing to the measured R2 value. Furthermore, the close similarity between r2 and r2*
indication of the occurrence of a motional narrowing regime. Concerning the positive contrast, the results obtained highlighted that MRI-IRON contrast can be detected when a high amount of the paramagnetic agent is confined in the vesicular compartment. Moreover, it has been emphasized that the change of liposomes size may significantly affect the magnitude of the IRON contrast. To conclude, after the use of paramagnetic liposomes as T1, T2, and CEST agents, the access to IRON
contrast able to generate a positive contrast further expands the potential of such nanosystems in proton MRI-based biomedical applications.
Acknowledgements
The financial support of the ‘‘Compagnia di San Paolo’’ ( project ‘‘Validazione dimolecole di tipo VHH e Aptameri per il rilascio tumore-specifico di farmaci e la valutazione contestuale della risposta mediante imaging funzionale mirato’’), University of Torino (project “Innovative Nanosized Theranostic Agents”) is gratefully acknowledged.
The work has been carried out under the framework of the ESF EU-COST Action TD1004 (Theranostics Imaging and Therapy: An Action to Develop Novel Nanosized Systems for Imaging-Guided Drug Delivery).
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Table1. Normalized r1 and r2 values measured at 14 T and 25°C for the listed samples. Sample r1free s-1mM-1 r1 lipo s-1mM-1 r2 free s-1mM-1 r2 lipo s-1mM-1 r2 susc * s-1mM-1 Lipo-Gd-DPPC 2.70±0.15 0.20±0.01 4.3±0.3 53.1±2.6 52.9±2.5 Lipo-Eu-DPPC nd nd 0.20±0.01 2.0±0.1 1.8±0.1 Lipo-Dy-DPPC 0.40±0.02 0.020±0.001 13.1±0.6 142.5±7.0 142.3±6.8 * values calculated as described in the text
Table 2. Size dependence of liposome concentration and MRI-IRON signal Sample d nm [Lipo] M Positive area cm2 SI/BSa SI/BSnormb M -1 Lipo-Dy-DPPC 110 0.13 0.36±0.03 33.3±1.5 256.1±10.2 Lipo-Dy-DPPC 85 0.3 0.25±0.02 23.9±1.2 79.7±4.1
a Average signal intensity (SI) within the positive area normalized to the background signal (BS)
Chart 1. Ln-HPDO3A. Liposomes were encapsulated with Ln = Eu3+, Gd3+,
Figure 1. Correlation between r2susc (14 T, 25°C) values and (ef)4 for the
Figure 2. Coronal and axial MRI images acquired with: i) IRON sequence (on the left, RF pulse intensity = 3 µT, total saturation module duration = 1000 ms), or with RARE sequence (right). Images of phantom with Dy-HPDO3A complex loaded liposomes (Lipo-Dy-DPPC, diameter 110 nm) are shown on the top, whereas the free complex phantom images are shown on the bottom. Residual positive signal in the image borders are due to B1
Figure 3. Coronal MRI images of phantoms prepared with Dy-HPDO3A complex loaded liposomes of different diameter (a: 110 nm, b: 85 nm), and acquired with an IRON MRI sequence (RF pulse intensity = 3 µT, total saturation module duration = 1 s). Residual positive signal in the image borders are due to B1 and B0 inhomogeneity.
Figure 4. Simulation of r2lipo (top) and percentage of Curie spin contribution
(bottom) as a function of the magnetic field strength B0. Black:
Lipo-Dy-DPPC, Red: Lipo-Gd-Lipo-Dy-DPPC, Blue: Lipo-Eu-DPPC (curve were simulated as described in the Discussion section).
