Langmuir 2008, 24, 4347-4351
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Paramagnetic Liposomes: Inner versus Outer Membrane Relaxivity of DPPC Liposomes Incorporating Lipophilic Gadolinium Complexes Sophie Laurent, Luce Vander Elst, Coralie Thirifays, and Robert N. Muller* Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, UniVersity of Mons-Hainaut, B-7000 Mons, Belgium ReceiVed January 16, 2008 Proton relaxometric properties of unilamellar DPPC liposomes embedding an amphiphilic paramagnetic chelate (Gd-DTPA-BC14A) in both layers of the phospholipid membrane or only in the external one are compared. The results show that the membrane’s water permeability is able to quench the effect of the paramagnetic complexes located in the internal layer of DPPC liposomes, leading thus to an apparent lower global relaxivity.
Introduction Unilamellar liposomes consist of a bilayered membrane, made of phospholipid molecules, entrapping an internal aqueous compartment. The properties of these nanovesicles can be controlled, and good biocompatibility and biodegradability can be achieved. Liposomes can be sterically protected against opsonization and macrophage uptake by coating with amphiphilic derivatives like poly(ethylene glycol) (PEG) or a PEG-like polymer which, therefore, prolong their vascular retention time.1 Furthermore, their size, charge, and surface can be modified to allow a specific in vivo delivery.2 The potential of paramagnetic liposomes as magnetic reporters for magnetic resonance imaging (MRI) has been reported.3 Paramagnetic liposomes can contain the paramagnetic centers either in the aqueous core or in the membrane. Amphiphilic gadolinium complexes, mimicking phospholipids, can in fact be incorporated in the lamella, thus becoming an integral part the liposome structure. When the paramagnetic complex is encapsulated in the cavity, the core water protons relax much faster than those of the bulk. If the water exchange rate through the lipidic bilayer is very slow as compared to the inner proton water relaxation rate (R1inner ) 1/T1inner), the relaxation regime is biexponential: a smaller T1 characterizes the internal water and a larger one the bulk.4 On the other hand, if the water exchange rate through the membrane is comparable or faster than R1inner, the global paramagnetic relaxation behavior (R1obs) is monoexponential and, for diluted liposomal aqueous systems, is given by eq 1
R1obs ) Pinner
1 + τex
inner
T1
(1)
where Pinner is the molar fraction of the water inside the liposome, T1inner is the relaxation time of the intravesicular water, and τex is the residence time of the water molecules inside the liposome. Equation 1 clearly suggests that the water exchange rate (kex ) 1/τex) through the membrane can decrease the paramagnetic contribution arising from the inner compartment. Such a behavior * Corresponding author. Tel/Fax: +32-65-373520. E-mail: robert.
[email protected]. (1) Torchilin, V. P.; Babich, J.; Weissig, V. J. Lipos. Res. 2000, 10, 483-499. (2) Torchilin, V. P. Nat. ReV. Drug DiscoVery 2005, 4 (2), 145-160. (3) Gore, J. C.; Sostman, F. D.; Caride, V. J. J. Microencapsul. 1986, 3 (4), 251. (4) Vander Elst, L.; Pierart, C.; Fossheim, S. L.; Raux, J. C.; Roch, A.; Muller, R. N. Supramol. Chem. 2002, 14 (5), 411-417.
has been reported for various types of liposomes entrapping Gd-DTPA or Gd-HPDO3A.5,6 If the paramagnetic center is embedded only in the external layer of the membrane, it is in close contact with the bulk water. The reduction of its translational and rotational mobilities may induce an enhancement of the relaxivity that, then, is mainly governed by the motion of the molecular segment carrying the paramagnetic head, the electronic relaxation rates, and the rate of exchange between the water molecules coordinated to the gadolinium ion and the bulk. If the paramagnetic complex is solely embedded in the internal layer, the situation can be compared to the first one where the liposome water core contains a solubilized hydrophilic paramagnetic complex. The vesicular water molecules relax faster than those of the bulk and the overall paramagnetic relaxation rate becomes dependent on the exchange rate of the water through the bilayer. When the paramagnetic complex is embedded in both layers, the situation is more complicated, since the number of paramagnetic complex molecules embedded in the inner (Cin) and outer (Cout) layers could be different, with Cin < Cout, and two contributions arising respectively from the complexes in the inner and in the outer layers have to be considered. If the water exchange rate through the membrane is very slow, the main relaxation effect is expected to be due to the complex in the outer layer. On the contrary, if this water exchange is extremely fast (faster than R1inner), complexes in the inner and in the outer layer will contribute to the observed paramagnetic relaxation rate.7 The present work aims at evaluating the respective contributions of each liposomal layer by comparing two types of paramagnetic liposomes incorporating an amphiphilic paramagnetic complex: either on both sides of the phospholipidic membranes (type 1) or only in the external layer (type 2, see Figure 1). The liposomes made of 1,2-sn-glycero-3-phosphatidylcholin (DPPC) and GdDTPA-BC14A (Figure 2) were characterized by photon correlation spectroscopy (PCS) and proton relaxometry. Type 2 liposomes were prepared by transmetalation with Gd3+ of type 1 liposomes containing La-DTPA-BC14A, a diamagnetic analogue of the paramagnetic Gd-DTPA-BC14A. (5) Koenig, S. H.; Ahkong, Q. F.; Brown, R. D., (III); Lafleur, M.; Spiller, M.; Unger, E.; Tilcock, C. Magn. Reson. Med. 1992, 23, 275-286. (6) Fossheim, S. L.; Fahlvik, A. K.; Klaveness, J.; Muller, R. N. Magn. Reson. Imaging 1999, 17, 83-89. (7) Alhaique, F.; Bertini, I.; Fragai, M.; Carafa, M.; Luchinat, C.; Parigi, G. Inorg. Chim. Acta 2002, 331, 151-157.
10.1021/la800148a CCC: $40.75 © 2008 American Chemical Society Published on Web 03/14/2008
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Figure 1. Structure of both types of paramagnetic liposomes.
Figure 2. Structure of Gd- and La-DTPA-BC14A.
Materials and Methods Relaxometry. Longitudinal and transverse relaxation times were measured at 310 K on Bruker Minispec systems pc120 and mq60 (Karlsruhe, Germany) working at 20 MHz (0.47 T) and 60 MHz (1.41 T), respectively, and on a Bruker AMX spectrometer at 300 MHz (7.05 T). NMRD profiles were obtained at 310 K on a Stelar field cycling relaxometer (Mede, Italy) over a range of magnetic fields extending from 0.24 mT to 0.24 T (0.01-10 MHz). The theoretical adjustment of the NMRD profiles was performed with the classical relaxation models.8-10 Size Measurements. The average hydrodynamic diameter of the liposomes was measured by photon correlation spectroscopy on a Brookhaven system equipped with a goniometer, a photomultiplier BI-PMT/9863, and a BI-9000 AT correlator (Brookhaven Corp. Instruments). The light source is a He-Ne laser working at a wavelength of 632.8 nm. Synthesis of the Complexes. The ligand DTPA-BC14A was obtained by reaction of DTPA bisanhydride with the tetradecylamine, as described by Kimpe et al.11 Equimolar amounts of ligand (2.5 mmol) and GdCl3‚6H2O or LaCl3‚7H2O, respectively dissolved in 100 mL of methanol and 2 mL of distilled water, were mixed. The pH was adjusted to 5 with pyridine and the solution was stirred overnight. The solution was then filtered and the solvent was evaporated under vacuum. A xylenol orange test confirmed the absence of free lanthanide ion.12 The product was treated with 50 mL of distilled water and lyophilized. Synthesis of the Liposomes. A. Synthesis of Liposomes Type 1. DPPC (1 equiv, 0.12 mmol) and Gd complex (0.1 equiv, 0.012 mmol) were dissolved in a mixture of chloroform (25 mL) and methanol (25 mL). The solvent was evaporated under vacuum to obtain a homogeneous film. This film was dispersed in 4 mL of distilled water. The solution was heated at 55 °C and stirred during 45 min. The stirring was stopped and the heating continued for 1 h. Liposomes were then extruded 10 times at 55 °C on two polycarbonate filters with pores of 400 nm in diameter using a T001 (8) Solomon, I. Phys. ReV. 1955, 99, 559-565. (9) Bloembergen, N. J. Chem. Phys. 1957, 27, 572-573. (10) Freed, J. H. J. Chem. Phys. 1978, 68, 4034-4037. (11) Kimpe, K.; Parac-Vogt, T. N.; Laurent, S.; Pie´rart, C.; Vander Elst, L.; Muller, R. N.; Binnemans, K. Eur. J. Inorg. Chem. 2003, 3021-3027. (12) Barge, A.; Cravotto, G.; Gianolio, E.; Fedeli, F. Contrast Med. Mol. Imaging 2006, 1, 184-188.
Figure 3. Typical PCS data of type 1 (top) and type 2 (bottom) liposomes. Lipex extrudor (Lipex Biomembranes, Vancouver, BC, Canada). Biomembranes had a capacity of 10 mL, and the driving gas was nitrogen. B. Synthesis of Liposomes Type 2. First, diamagnetic liposomes incorporating the lanthanum complex analogue in their external and internal layers (type 1) were prepared using the procedure described above. In a second step, a GdCl3 solution (pH 5.15) was added to the suspension of liposomes to exchange external lanthanum by gadolinium. Determination of the Gd Concentration Incorporated in the Liposomes. Relaxometric Technique. Triton X-114 (150 µL; Acros, Geel, Belgium) was added to 250 µL of the liposomes solution to destroy the bilayer structures. Then, 2.5 mL of HNO3 was added to release the gadolinium ion from the complex and the volume was adjusted to 5 mL with distilled water. The relaxation rate was measured and, with the known relaxivity of aquoions (11.76 s-1 mM-1 at 20 MHz and 310 K), used to calculate the concentration of Gd3+ ions. ICP (InductiVely Coupled Plasma) Technique. A solution of 100 µL of liposomes, 300 µL of H2O2, and 300 µL of HNO3 was prepared. This mixture was digested in a microwave oven (Milestone, Analis, Namur, Belgium) and the volume was adjusted to 5 mL with distilled water. The sample was then analyzed by ICP (Jobin Yvon JY70, Lonjumeau, France).
Results and Discussion Synthesis of the Liposomes. Three samples of unilamellar liposomes containing the gadolinium complex in both layers of the membrane were prepared from DPPC and Gd-DTPABC14A (ratio 10:1) using the classical extrusion method. Their hydrodynamic diameter, as measured by photon correlation spectroscopy (mean diameter 171 ( 9 nm, monodisperse
Inner Vs Outer Membrane RelaxiVity of Liposomes
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Table 1. Concentration of Gd Complex and Hydrodynamic Diameter of the Three Samples of Paramagnetic DPPC Liposomes Incorporating Gd-DTPA-BC14A in Both Sides of the Bilayer (Type 1)
initial
final
mean hydrodynamic diameter (nm)
3.06 3.09 3.06
1.72 1.61 1.83
161 176 176
Gd complex concn (mM)
sample 1 sample 2 sample 3
Table 2. Hydrodynamic Diameter of the Three Samples of Paramagnetic Type 2 DPPC Liposomes
sample 1 sample 2 sample 3
initial La complex concn (mM)
concn of GdCl3 added to the solution (mM)
mean hydrodynamic diameter (nm)
3.12 3.07 3.10
0.3731 0.3718 0.3718
147 160 152
distribution, Figure 3), and the amount of complex measured in the final solution (56 ( 4%) were reproducible (Table 1). The preparation of liposomes with the Gd complex located only in the external part was performed as follows. First, three samples of liposomes containing the diamagnetic analogue LaDTPA-BC14A in both membrane layers were prepared as described above and characterized by PCS (monodisperse distribution, mean diameter 153 ( 6.6 nm, Figure 3, Table 2). Subsequently, they were submitted to a transmetalation process with Gd3+ ions. This procedure was based on relaxometric data showing that the apparent relaxivity of a solution of La-DTPABMA added with Gd3+ ions rapidly decreased to reach the expected value of Gd-DTPA-BMA (Figure 4). As expected considering the larger stability constants of Gd complexes as compared to La complexes,13 La ions of La-DTPA-BMA are thus substituted by Gd ions to form Gd-DTPA-BMA. The transmetalation of the liposomes was carried out in similar conditions (pH ) 5.15) with two different GdCl3 solutions (0.247 and 0.381 mM). These amounts of Gd3+ were chosen considering (i) an averaged final concentration of the lanthanide complex of ∼55% (see the data reported for the gadolinium analogue in Table 1), (ii) a nearly equal quantity of complex on both sides of the membrane, and (iii) the necessity to avoid the presence of free GdCl3 at the end of the transmetalation process. The evolution of the transmetalation was followed by proton longitudinal and transverse relaxometry at 60 MHz and 310 K (Figure 5). A fast decrease of the R1 and R2 of both solutions and a stabilization after approximately 2 h (Figure 5) were observed. The corresponding longitudinal and transverse relaxivities calculated after 200 min are similar for both samples (r1 ) 10.1 and 10.2 s-1 mM-1 and r2 ) 18.9 and 18.1 s-1 mM-1 for samples added with 0.247 and 0.381 mM of GdCl3 respectively). To check the complete complexation of Gd3+ ions and thus the absence of free Gd3+ ions, the solution obtained after transmetalation was ultracentrifugated to separate the liposomes from the water. The arsenazo test performed on the supernatant confirmed the absence of free lanthanum or gadolinium ions. The absence of free gadolinium in the supernatant was confirmed by relaxometry. During the transmetalation process, free lanthanum ions were expected to be released in the solution. Their absence in the solution seems a priori surprising. However, one should bear in mind that lanthanide ions can interact with the phosphate groups of phospholipids like those of DPPC. To (13) Cacheris, W. P.; Nickle, S. K.; Sherry, A. D. Inorg. Chem. 1987, 26, 958-960.
Figure 4. Evolution of the apparent relaxivity of a sample containing La-DTPA-BMA (1.045 mM) and GdCl3 (0.933 mM) as a function of time (T ) 310 K, pH 5.15, proton frequency ) 60 MHz). The upper dashed line corresponds to the relaxivity of the Gd3+ aquoion (from GdCl3) and the lower one to the relaxivity of Gd-DTPABMA.
Figure 5. Evolution of the longitudinal (A) and transverse (B) relaxation rates with time at 60 MHz and 310 K of one sample of DPPC/La-DTPA-BC14A liposomes to which 0.247 mM of GdCl3 (closed circles) or 0.381 mM of GdCl3 (open squares) was added and DPPC liposomes to which 0.381 mM of GdCl3 (closed triangles) was added.
confirm this hypothesis, pure DPPC liposomes were prepared and then a solution of gadolinium chloride was added (final concentration 0.381 mM). Their longitudinal and transverse relaxation rates were measured at 60 MHz (Figure 5). At the end of the experiment, the solution was ultracentrifuged and the arsenazo test revealed the absence of free gadolinium in the supernatant. Figure 5 shows that, compared to the relaxation rate
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Figure 6. Proton NMRD profiles of three different samples of type 1 paramagnetic liposomes incorporating Gd-DTPA-BC14A.
Figure 7. Proton NMRD profiles of three samples of type 2 liposomes incorporating Gd-DTPA-BC14A.
Table 3. Longitudinal and Transverse Relaxivities of the Three Samples of Paramagnetic Type 1 and Type 2 DPPC Liposomes Incorporating Gd-DTPA-BC14A 20 MHz
60 MHz
r1 (s-1 mM-1) r2 (s-1 mM-1) r1 (s-1 mM-1) r2 (s-1 mM-1) sample 1 sample 2 sample 3
9.69 8.92 9.86
Type 1 10.74 10.32 10.90
8.59 8.01 8.51
11.60 10.82 11.55
sample 1 sample 2 sample 3
13.13 14.69 14.11
Type 2 15.61 17.77 16.98
9.97 11.37 10.80
18.74 21.88 20.80
of aqueous GdCl3 solution in similar experimental conditions (R1obs ) 4.1 s-1), the value obtained for this liposome solution was much larger (R1obs > 10 s-1). No significant variation of R1 (and R2) with time was observed and the measured values agree with those extrapolated to time zero for the DDPC/La-DTPABC14A liposomes submitted to transmetalation with Gd3+ ions. It seems, therefore, that Gd ions do interact first with the phosphates of DPPC before substituting La3+ in the complex. Nuclear Magnetic Relaxation Dispersion (NMRD) Profiles. The proton NMRD profiles at 310 K of the different samples of DPPC liposomes with Gd-DTPA-BC14A located on both sides of the membrane reach a maximum between 10 and 100 MHz, indicating a slow mobility of Gd ions (Figure 6). The theoretical adjustment of the NMRD profiles is usually performed with the classical relaxation models of internal sphere (innersphere)8,9 and external sphere (outersphere)10 magnetic dipolar interactions. Some parameters are fixed: d, the distance of closest approach (d ) 0.36 nm); r, the distance electronnucleus (r ) 0.31 nm); and D, the relative diffusion coefficient, fixed to the value of 3 × 10-9 m2 s-1.14 The number of water molecules in the first coordination sphere can be assumed to be equal to 1 by analogy to other derivatives of Gd-DTPA. The rotational correlation time, τR; the residence time of the coordinated water molecule, τM; the electronic relaxation time at low field, τS0; and the correlation time, τV, that modulates the electronic relaxation are then adjusted simultaneously. The experimental data of the individual samples were fitted (Figure 6). The mean τR value (5.4 ( 0.8 ns) was much smaller than expected for a Gd complex immobilized in the liposome membrane (the τR calculated from the Stokes-Einstein theory (14) Vander Elst, L.; Sessoye, A.; Laurent, S.; Muller, R. N. HelV. Chim. Acta 2005, 88, 574-587.
Figure 8. Comparison between the mean proton NMRD profiles of the liposomes incorporating Gd-DTPA-BC14A in both layers (type 1, open circles) and only in the external layer (type 2, closed circles).
should be larger than 400 µs). This difference is to be related to a local motion of the Gd complex within the bilayer, as already observed on micellar structures incorporating the same complex.11 A theory taking into account both the global and the local motions could be applied to fit the experimental data,15 but it lies outside the context of this study. The mean τM value (2.1 ( 0.12 ms) agrees with the τM values reported for other paramagnetic liposomes or micelles with similar lipophilic Gd-DTPA bisamides.16 It is worth noting that the simple theoretical model used here is inappropriate since, first, it does not distinguish between the water exchange rate across the membrane and the water exchange rate of the water molecule coordinated to the gadolinium complex and, second, it does not accurately describe slowly rotating systems. The proton NMRD profile of the liposomes with the paramagnetic complex located only in the external part of the membrane was recorded 5 h after the beginning of the transmetalation process in order to reach the equilibrium (Figure 7). It is to be noticed that some Gdcomplex could be transferred to the inner layer if a flip-flop process occurs. However, since the temperature of the measurement is lower than the phase transition of DPPC, we assumed that this process is not significant. This assumption is corroborated by the larger relaxivity of type 2 liposomes as compared to type 1 liposomes (Table 3). (15) Dunand, F. A.; Toth, E.; Hollister, R.; Merbach, A. E. J. Biol. Inorg. Chem. 2001, 6, 247-255. (16) Parac-Vogt, T. N.; Kimpe, K.; Laurent, S.; Pierart, C.; Vander Elst, L.; Muller, R. N.; Binnemans, K. Eur. Biophys. J. 2006, 35, 136-144.
Inner Vs Outer Membrane RelaxiVity of Liposomes
The averaged relaxivities of both types of liposomes incorporating Gd-DTPA-BC14A inside and outside the phospholipidic membrane (Table 3, Figure 6) and only in the external layer (Table 3, Figure 7) are summarized in Figure 8. The results clearly indicate that the complex located in the internal layer contributes less to the global relaxivity. For DPPC/DPPG liposomes of similar size, a water exchange time close to 10 ms has been reported.6 The relatively slow water exchange through the bilayer membrane of DPPC liposomes at 310 K is thus likely to be responsible for the low contribution of the Gd complex in their internal layer (Table 3). The contribution of these complexes to the total relaxivity can be estimated to be lower than 20%. It can be expected that the difference between type 1 and type 2 liposomes will be reduced at temperatures near or above the phase transition of the phospholipids, as well as for liposomes with a higher water permeability, such as liposomes prepared with unsaturated phospholipids or of a smaller diameter.6
Conclusions Syntheses of unilamellar liposomes incorporating Gd complexes either in their external and internal layers or only at the
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external one show a good reproducibility in terms of proton longitudinal relaxivity as well as of liposome size and monodispersity. The latter ones were successfully obtained by transmetalation of La3+ by Gd3+ of DPPC/La-DTPA-BC14A liposomes. At 310 K, the gadolinium relaxivity of these liposomes is increased as compared to that of liposomes containing the complex in both sides of the membrane. Paramagnetic complexes located in the internal part of the membrane of liposomes with low water permeability thus contribute less to the global relaxivity than complexes exposed on the external surface. Acknowledgment. The authors thank Mrs. Patricia de Francisco for her help in preparing the manuscript. This work was supported by the FNRS and the ARC Program 00/05-258 of the French Community of Belgium. The support and sponsorship concerted by COST Action D18 “Lanthanide Chemistry for Diagnosis and Therapy” and of the EMIL NoE of the FP6 of the EC are kindly acknowledged. LA800148A
