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Supramolecular Aggregates of Amphiphilic Gadolinium Complexes as Blood Pool MRI/MRA Contrast Agents: Physicochemical Characterization Mauro Vaccaro,†,‡,| Antonella Accardo,§ Diego Tesauro,§ Gaetano Mangiapia,†,‡ David Lo¨f,| Karin Schille´n,| Olle So¨derman,| Giancarlo Morelli,*,§ and Luigi Paduano*,†,‡ Department of Chemistry, UniVersity of Naples “Federico II”, Via Cynthia, 80126 Naples, Italy, CSGI (Consorzio per lo SViluppo dei Sistemi a Grande Interfase), Department of Biological Sciences, CIRPeB UniVersity of Naples “Federico II” & IBB CNR, Via Mezzocannone 16, 80134 Naples, and Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund UniVersity, P.O. Box 124, SE-221 00 Lund, Sweden ReceiVed December 27, 2005. In Final Form: May 5, 2006 In this paper, we present the development of a new potential blood pool contrast agent for magnetic resonance imaging applications (MRA/MRI) based on gadolinium complexes containing amphiphilic supramolecular aggregates. A novel amphiphilic unimer, containing the DTPAGlu chelating agent covalently bound to two C18 alkylic chains, has been synthesized. DTPAGlu is a well-known chelating agent for a wide number of ions such as the paramagnetic metal ion Gd3+ used as contrast agent in MRA/MRI. The wide aggregation behavior of this surfactant, as free base or as gadolinium complex, has been studied and compared by means of dynamic light scattering, small-angle neutron scattering and cryogenic transmission electron microscopy techniques. Near neutral pH in both cases, the dominant aggregates are micelles.The high negative actual charge of the surfactant headgroup causes a strong headgroups repulsion, promoting the formation of large and high curvature aggregates. By decreasing pH and less markedly increasing the ionic strength, we observe a micelle-to-vesicle transition driven by a decreased electrostatic repulsion. A straightforward switch between different aggregation states can be particularly useful in the development of pHresponsive MRA/MRI contrast agents.
1. Introduction Magnetic resonance imaging (MRI) is an imaging technique and is one of the most widely used diagnostic tools in clinical practice. Its main advantage is that it allows rapid in vivo acquisition of images, and under specific conditions, it makes imaging at cellular resolution possible. The technique has proven very valuable for the diagnosis of a broad range of pathologic conditions in all parts of the body.1 Currently, stable Gd3+-poly(aminocarboxylate) complexes are widely used as contrast agents in MRI. These agents are intravenously administered to patients, and by reducing the relaxation time of water protons present in the effected tissues, they help to produce a higher quality (higher contrast) image. The efficacy of a contrast agent is commonly expressed by its proton relaxivity r1, defined as the paramagnetic longitudinal relaxation rate enhancement of the water protons by unity concentration (mM) of the agent.2 Although MRI gives very resolved images, due to its very low sensitivity, it needs an elevated concentration of contrast agent (10-4 M). To reach the required local concentration, many carriers have been developed such as liposomes3 or other microparticulates:4 micelles,5 dendrimers,6 water-soluble fullerenes,7 linear * Corresponding author. Tel.: +39081674229. Fax: +39081674090. E-mail:
[email protected]. † University of Naples “Federico II”. ‡ CSGI. § CIRPeB University of Naples “Federico II”, & IBB CNR. | Lund University. (1) Weissleder, R.; Mahmood, U. Mol. Imaging Radiol. 2001, 219, 316. (2) Aime, S.; Botta, M.; Fasano, M.; Terreno, E. Chem. Soc. ReV. 1998, 27, 19. (3) Glogard, C.; Stensrud, G.; Rovland, R.; Fossheim, S. L. Intern. J. Pharm. 2002, 233, 131.
polymers,8 or proteins,9 all of them derivatized with a high number of metal complexes. Among these carriers, micellar and vesicular aggregates have recently drawn much attention owing to their easily controlled properties and good pharmacological characteristics.10 For example, the self-assembling of Gd(III)(DOTA) or Gd(III)(DTPAGlu) complexes derivatized with a lipophilic tail allows high relaxivity MRI contrast agents to be obtained.5,11 In other words, gadolinium complexes containing amphiphilic supramolecular aggregates present two contemporary and interesting properties: an enhanced ability to change solvent proton relaxation rates (through the occurrence of a long molecular retention time τR) and an increased lifetime of the contrast agent in the circulating blood by avoiding the extravasation typical of the small-sized Gd3+ complexes commonly employed in MRI investigations.12,13 Their high in vivo stability, short T1 spin lattice relaxation time, and long vascular retention time make these contrast agents interesting in other magnetic resonance imaging applications (4) Morel, S.; Terreno, E.; Ugazio, E.; Aime, S.; Gasco, M. R. Eur. J. Pharm. Biopharm. 1998, 45, 157. (5) Accardo, A.; Tesauro, D.; Roscigno, P.; Gianolio, E.; Paduano, L.; D’Errico, G.; Pedone, C.; Morelli, G. J. Am. Chem. Soc. 2004, 126, 3097. (6) Wiener, E. C.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.; Gansow, O. A.; Tomalia, D. A.; Lauterbur, P. C. Magn. Res. Med. 1994, 3, 1. (7) Toth, E.; Bolskar, R. D.; Borel, A.; Gonzalez, G.; Helm, L.; Merbach, A. E.; Sitharaman, B.; Wilson, L. J. J. Am. Chem. Soc. 2005, 127, 799. (8) Aime, S.; Botta, M.; Garino, E.; Geninatti Crich, S.; Giovenzana, G.; Pagliarin, R.; Palmisano, G.; Sisti, M. Chem. Eur. J. 2000, 6, 2609. (9) Schmiedl, U.; Ogan, M.; Paajanen, H.; Marotti, M.; Crooks, L.; Brito, E.; Brasch, R. C. Radiology 1987, 162, 205. (10) Torchilin, V. P. AdV. Drug DeliVery 2002, 54, 235. (11) Andre, J. P.; Toth, E.; Fischer, H.; Seelig, A.; Macke, H. R.; Merbach, A. E. Chem. Eur. J. 1999, 5, 2977. (12) Anelli, P. L.; Lattuada, L.; Lorusso, V.; Schneider, M.; Tournier, H.; Uggeri, F. Magn. Res. Mater. Phys., Biol. Med. 2001, 12, 114. (13) Geva, T.; Greil, G. F.; Marshall, A. C.; Landzberg, M.; Powell, A. J. Circulation 2002, 106, 473.
10.1021/la053500k CCC: $33.50 © 2006 American Chemical Society Published on Web 06/22/2006
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Figure 1. Schematic representation of the (C18)2DTPAGlu(Gd) unimer employed to formulate aggregates.
such as in angiography. Magnetic resonance in angiography (MRA) is the technique in which blood vessels are imaged by magnetic resonance. Contrast-enhanced MRA provides a fast, reliable, noninvasive method for imaging large vascular territories, allowing delineation of pulmonary blood supply in patients with complex pulmonary stenosis and atresia14 visualization and determination of the patency of arterial and venous coronary grafts,14 transplanted renal artery stenosis, and detection of renal artery stenosis.15,16 In this paper, we present the development of a new potential blood pool contrast agent based on gadolinium complexes containing amphiphilic supramolecular aggregates for MRA/ MRI application. A new amphiphilic unimer (C18H37)2CONHLys(DTPAGlu)CONH2 (Figure 1), containing the DTPAGlu chelating agent covalently bound to two C18 alkylic chains, has been synthesized. DTPAGlu is considered as a well-known chelating agent for a wide number of ions such as the radioactive metals (111In, 67Ga, 90Y, 177Lu, and 68Ga), used for diagnostic or therapeutic nuclear medicine applications, or paramagnetic metal ions (Gd3+) for applications in magnetic resonance imaging (MRA/MRI). This molecule is able to form a variety of aggregates, such as micelles, bilayer planes, and vesicles. The purpose of this report is to present a detailed physicochemical characterization of supramolecular compounds formed by (C18)2DTPAGlu, in the presence and absence of Gd3+, in order to highlight their potential use as contrast agents in MRA/MRI. The study has been performed by monitoring the effect of pH and ionic strength on the chemical structure of the aggregates and on their properties by meaning of dynamic light scattering, small-angle neutron scattering, and cryogenic transmission electron microscopy techniques. 2. Materials and Methods 2.1. Materials. Fmoc-Lys(Mtt)-OH amino acid derivative, coupling reagents, and Rink amide MBHA resin were purchased from Calbiochem-Novabiochem (Laufelfingen, Switzerland). The DTPAGlu pentaester, N,N-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid1-(1,1-dimethylethyl) ester, and N,N-dioctadecylsuccinamic acid were synthesized according to the procedures reported in the literature.17,18 All other chemicals were commercially available from SigmaAldrich, Fluka (Bucks, Switzerland), or LabScan (Stillorgan, Dublin, Ireland) and were used as received unless otherwise stated. All solutions were prepared by weight using doubly distilled water. Samples to be measured by SANS technique were prepared using heavy water (Sigma-Aldrich, purity >99.8%). (14) Boehm, D. H.; Wintersperger, B. J.; Reichenspurner, H.; Gulbins, H.; Detter, C.; Kur, F. Heart Surg. Forum. 1999, 2, 222. (15) Luk, S. H.; Chan, J. H.; Kwan, T. H.; Tsui, W. C.; Cheung, Y. K.; Yuen, M. K. Clin. Radiol. 1999, 54, 651. (16) D’arceuil, H. E.; de Crespigny, A.; Pelc, L.; Howard, D.; Alley, M.; Seri, S.; Hashiguchi, Y.; Nakatani, A.; Moseley, M. E. Magn. Res. Imaging 2004, 22, 1243. (17) Anelli, P. L.; Fedeli, F.; Gazzotti, O.; Lattuada, L.; Lux, G.; Rebasti, F. Bioconjugate Chem. 1999, 10, 137. (18) Schmitt, L.; Dietrich, C.; Tampe, R. J. Am. Chem. Soc. 1994, 116, 8485.
2.2. Molecule Synthesis. Synthesis of the (C18H37)2CONHLys(DTPAGlu)CONH2 unimer was carried out in solid phase under standard conditions using Fmoc/tBu strategy.19 Rink-amide MBHA resin (0.78 mmol/g, 0.5 mmol scale, 0.64 g) was used as a polymeric support. The Fmoc-R-NH2 on the resin was removed by a N,Ndimethylformamide/piperidine (80/20) mixture. Fmoc-Lys(Mtt)-OH residue (1.248 g, 2.0 mmol) was activated by 1 equiv of PyBop (benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium) and HOBt (1hydroxybenzotriazole) and 2 equiv of DIPEA (N,N-diisopropylethylamine) in DMF and coupled on the resin stirring the slurry suspension for 1 h. The solution was filtered and the resin washed with three portions of DMF and three portions of DCM (dichloromethane). The Mtt-protecting group (4-methyl-trityle) was removed by treatment with the DCM/TIS/TFA (94:5:1) mixture. The peptide resin was stirred with 5.0 mL of this solution for 2 min. This procedure was repeated several times until the solution became colorless. The resin was washed 3 times by DCM and 3 times by DMF. At this point, the DTPAGlu-pentaester chelating agent was linked, through its free carboxyl function, to the -NH2 of the lysine residue. The coupling was performed in DMF using 2 equiv of DTPAGlupentaester, 2 equiv of HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium), and 4 equiv of DIPEA with respect to the synthesis scale. The coupling time, compared with the classical solidphase peptide synthesis protocol, was increased up to 2 h, and the reaction was checked to see if was completed by using Kaiser test. Then, the N-terminal Fmoc protecting group of the lysine residue was removed, and 1.244 g (2.0 mmol) of N,N-dioctadecylsuccinamic acid was condensed twice for 1 h in the DMF/DCM (1/1) mixture. The lipophilic moiety was activated in situ by the standard HOBt/ PyBop/DIPEA procedure. The coupling was monitored by the qualitative Kaiser test. The resin was washed 3 times by DMF, 3 times by DCM, and 3 times by ethyl ether. The cleavage from the resin and deprotection of the five tBu protecting groups from the DTPAGlu chelating agent were obtained stirring for 2 h with an acidic solution of TFA containing triisopropylsylane (2.0%) and water (2.5). The crude product was slowly precipitated at 0 °C by adding water dropwise, washed several time with small portions of water, and lyophilized in order to remove the solvent. The white solid was recrystalized from MeOH/H2O and recovered in high yield (>85%). The product was identified by MS (ESI+) spectra, obtained using a Finnigan Surveyor MSQ single quadrupole electrospray ionization mass spectrometer (Finnigan/ Thermo Electron Corporation San Jose, CA). (C18)2DTPAGlu: MS (ESI+): m/z (%): 1194.1 (100) [M-H+] The complexation of the gadolinium complex (C18)2DTPAGlu(Gd) was carried out by adding a slight excess of GdCl3 (1.1 mmol) to the 1 mM aqueous solution of the (C18)2DTPAGlu ligand (1.0 mmol) at neutral pH and room temperature. The excess of uncomplexed Gd3+ions was removed by centrifugation of the solution brought to pH 10 with NaOH. The absence of free Gd3+ was checked by use of xylenol-orange indicator.20 2.3. Sample Preparation. All solutions were prepared by weight, buffering the samples at a defined pH value in the 3.0-7.4 range. Two different buffer solutions were used: for physiological condition, pH 7.4, a 0.10 M phosphate buffer was used, whereas for lower pH (19) Chang, W. C.; White, P. D.; Oxford Univ Press: New York, 2000. (20) Brunisholz, G.; Randin, M. HelV. Chim Acta 1959, 42, 1927.
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values, a 0.10 M citric acid/phosphate buffer, when mixed at different ratio allowed moving from pH 7.0 to pH 3.0. pH measurements were made by using a pH meter MeterLab PHM 220. The pH meter was calibrated with three standards at pHs of 3.00, 7.00, and 10.00. To prepare vesicles, DTPAGlu unimers were dissolved in buffer solution and successively placed in a bath sonicator for 3-4 h. In general, acidification of the solution produces a slight decrease of the solubility of the solute. Samples to be analyzed were prepared at concentrations well below the solute solubility limit, and the measurement were carried out on samples where no presence of precipitate was evident. The resulting aggregate containing solution was extruded by passing it 10 times through a polycarbonate membrane with a pore size diameter of 100 nm in order to obtain aggregates of a uniform size distribution. Aggregate solutions containing NaCl (0.9 wt %) were prepared in a similar way. 2.4. Dynamic Light Scattering (DLS). The setup for the dynamic light scattering measurements was an ALV/DLS/SLS-5000F, CGF8F based compact goniometer system from ALV-GmbH, Langen, Germany. A detailed description of the instrumentation can be found in the literature with the difference that cis-decahydronaphthalene was used instead of toluene as a refractive index matching liquid.21 Analysis of the DLS data was performed by fitting directly to the experimentally measured time correlation function of the scattered intensity G(2)(t) often presented as the normalized function, g(2)(t) - 1.22 The models used in the fitting procedures are expressed with respect to the normalized time correlation function of the electric field, g(1)(t), which is related to g(2)(t) by the Siegert relation 23,24 g(2)(t) - 1 ) β|g(1)(t)|2
(1)
where β (e1) is the coherence factor, which accounts for deviation from ideal correlation and depends on the experimental geometry. g(1)(t) can either be a single-exponential or a multiexponential decay with corresponding relaxation times, τ, depending on the system investigated. A distribution of relaxation times, A(τ), can be obtained by an inverse Laplace transformation of a multiexponential g (t) ) (1)
∫
+∞
-∞
t τA(τ) exp - d ln τ τ
( )
dσ dΣ (q) ) npP(q)S(q) + dΩ dΩ
( )
inch
(4)
where np represents the number density of the scattering objects present in the system, P(q) and S(q) are respectively the form and the structure factors of the scattering particles, whereas (dΣ/dΩ)inch takes into account the incoherent contribution to the cross section measured, mainly due to the presence of hydrogenated molecules. The form factor contains information on the shape of the scattering objects, whereas the structure factor accounts for interparticle correlations and is normally important for concentrated or charged systems. Provided that solutions are quite dilutes (c < 10-3 mol kg-1), the structure function S(q) can be approximated to the unity, and the scattering cross section is reduced to dΣ dσ (q) ) npP(q) + dΩ dΩ
( )
inch
(5)
(2)
where τ ) Γ-1 and Γ is the relaxation rate that is used to calculate the diffusion coefficient D. The relaxation time distribution τA(τ) is obtained by regularized inverse Laplace transformation (RILT) of the measured intensity correlation function using the calculation algorithms REPES24-26 and that incorporated in the ALV-5000/E software. The relaxation rate Γ is obtained from the first moment of the relaxation time distribution, and from its value is estimated the apparent translational diffusion coefficient D, by this relation Γ D ) lim 2 qf0q
Meitner Institut, Berlin. In the first case, neutrons with an average wavelength λ of 6.2 Å and a wavelength spread ∆λ/λ < 0.1 were used. A two-dimensional array detector at three different sampleto-detector distances (2, 8, and 20 m) detected neutrons scattered from the samples. These configurations allowed for the collection of the scattering cross section in an interval of transferred momentum q ranging between 0.002 and 0.18 Å-1. Concerning the scattering data collected at the V4 instrument, a neutron beam of λ ) 7 Å with a spread {0.08 < ∆λ/λ < 0.18} was used. A 2D detector allowed for the collection of the data in an interval of transferred momentum q ranging between 0.003 and 0.15 Å-1. Raw data, collected from the two facilities, were corrected in order to get absolute scattering cross sections as already described.27 The scattering cross section dΣ/dΩ containing information on interactions, size and shapes of aggregates present in the system can be expressed for a collection of monodisperse bodies as28
(3)
where q is the absolute value of the scattering vector (q ) 4πn0 sin (θ/2)/λ), n0 is the refractive index of the solvent, λ is the incident wavelength, and θ is the scattering angle. Thus, D is obtained from the slope of Γ as a function of q2, where Γ is measured at different scattering angles. 2.5. Small-Angle Neutron Scattering (SANS). Small angle neutron scattering (SANS) measurements were performed at the KWS2 instrument located at the FRJ-2 reactor of the Forschungszentrum of Ju¨lich and at the V4 facility sited at the Hahn and (21) Jansson, J.; Schille´n, K.; Olofsson, G.; Cardoso da Silva, R.; Loh, W. J. J. Phys. Chem. B 2004, 108, 82. (22) Berne, B. J.; Pecora, R. Dynamic Light Scattering: with Application to Chemistry, Biology and Physics; Dover Publication: Mineola, NY, 2000. (23) Siegert, A. J. F. MIT Rad. Lab. Rep. 1943, 465. (24) Stepa´nek, P., Brown, W., Ed.; Oxford University, 1993; p 177. (25) Johnsen, R.; Brown, W.; Harding, S. E.; Sattelle, D. B.; Bloomfield, V. A., Eds.; Royal Society of Chemistry, Cambride, 1992; p 77. (26) Schille`n, K.; Brown, W.; Johnsen, R. Macromolecules 1994, 27, 4825.
Microstructural parameters of the aggregates have been obtained by applying the appropriate model to the experimental SANS data. Indeed experimental data have shown in the analyzed systems the existence of cylindrical micelles and/or the presence of vesicular aggregates. Scattering from cylindrical structures is characterized by a region where the dΣ/dΩ ∼ q-1 power law dependence holds. The single particle form factor for such micelles can be written as29
P(q) ) (Fc - F0) πR l 2
2
∫
π/2
0
1 sin2 q cos φ 9[j (qR sin φ)]2 2 1 sin φ dφ (6) 2 1 (qR sin φ)2 q cos φ 2
(
(
)
)
where l is the length of the cylinders, R is the radius of the base, j1 is the first-order Bessel function, and Fc - F0 is the scattering length density difference between the cylinders and the solvent. Vesicular aggregates cannot be observed in their completeness since the Guinier region of such objects falls almost completely in the USANS domain. As a consequence, the SANS region is characterized by a power law dΣ/dΩ ∼q-2 due to the scattering of the vesicular double layer. Indeed, the q range spanned by the SANS measurements is such to allow the vesicles to be regarded as randomly (27) Wignall, G. D.; Bates, F. S. J. Appl. Crystallogr. 1987, 20, 28-40. (28) Kotlarchyk, M.; Chen, S. H. J. Chem. Phys. 1983, 79, 2461-2469. (29) Triolo, R.; Magid, L. J.; Johnson, J. S., Jr.; Child, H. R. J. Phys. Chem. 1982, 86, 3689-3695.
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Table 1. Diffusion Coefficients and Hydrodynamic Radii Obtained from DLS Measurements for the Systems Studied at Different pH Valuesa systems
pH
Dfast×1011 (m2s-1)
RH (Å)
Dslow×1012 (m2s-1)
RH (Å)
(C18)2DTPAGlu (0.0001 mol kg-1)-water (C18)2DTPAGlu (0.0001 mol kg-1)-water (C18)2DTPAGlu (0.0001 mol kg-1 )-water (C18)2DTPAGlu (0.0001 mol kg-1)-water (C18)2DTPAGlu (0.0001 mol kg-1)-water (C18)2DTPAGlu(Gd) (0.0001 mol kg-1)-water (C18)2DTPAGlu(Gd) (0.0001 mol kg1)-water (C18)2DTPAGlu(Gd)(0.0001 mol kg-1)-water (C18)2DTPAGlu(Gd) (0.0001 mol kg1)-water (C18)2DTPAGlu(Gd) (0.0001 mol kg1)-water
7.4 6 5 4 3 7.4 6 5 4 3
3.03 ( 0.04 3.01 ( 0.01 2.91 ( 0.03
66 ( 1 66 ( 1 68 ( 1
2.64 ( 0.05 2.64 ( 0.03 2.50 ( 0.05
76 ( 1 76 ( 1 80 ( 2
3.2 ( 0.5 2.81 ( 0.11 3.1 ( 0.3 5.0 ( 1.0 2.7 ( 1.0 4.1 ( 0.3 3.2 ( 0.4 4.3 ( 0.2 5.6 ( 0.3 6.3 ( 0.2
623 ( 91 712 ( 36 645 ( 61 402 ( 83 742 ( 27 492 ( 46 623 ( 84 461 ( 23 362 ( 25 324 ( 17
a
The terms fast and slow refer to micelles and bilayer structures, respectively.
Table 2. Diffusion Coefficients and Hydrodynamic Radii Obtained from DLS Measurements for the Systems Studied in Condition of Physiological Ionic Strengtha systems
pH
(C18)2DTPAGlu (0.0001 mol kg -1)-NaCl 0.9 wt %-water (C18)2DTPAGlu(Gd) (0.0001 mol kg -1)-NaCl 0.9 wt %-water
7.4 7.4
a
Dfast×1011 (m2s-1)
RH (Å)
Dslow×1012 (m2s-1)
RH (Å)
3.20 ( 0.12
62 ( 2
2.9 ( 0.3 8.1 ( 0.7
691 ( 73 251 ( 22
The terms fast and slow refer to micelles and bilayer structures, respectively.
Table 3. Structural Parameters of the Aggregates at Different pH Values Determined by Small-Angle Neutron Scattering (SANS)a systems
pH
Nagg
R (Å)
l (Å)
d (Å)
(C18)2DTPAGlu (0.00079 mol kg-1)-D2O (C18)2DTPAGlu (0.00079 mol kg-1)-D2O (not extruded) (C18)2DTPAGlu (0.00063 mol kg-1)-D2O (C18)2DTPAGlu (0.00063 mol kg-1)-D2O (C18)2DTPAGlu(Gd) (0.00063 mol kg-1) -D2O (C18)2DTPAGlu(Gd) (0.00063 mol kg-1) -D2O (C18)2DTPAGlu(Gd) (0.00063 mol kg-1) -D2O
7.4 7.4 4.5 3 7.4 4.5 3
125 ( 15 102 ( 10 771 ( 80
39 ( 3 38 ( 4 36 ( 2
198 ( 21 108 ( 39 710 ( 60
72 ( 9
110 ( 10 723 ( 65
35 ( 3 34 ( 2
233 ( 30 741 ( 82
61 ( 8 47 ( 5 52 ( 5 53 ( 8 51 ( 6
a The terms N , R, and l refer to the aggregation number, the radius, and the length of the micelles, respectively, whereas d refers to the thickness agg of the bilayer structures.
oriented planar sheets for which the form factor can be expressed by30
P(q) ) 2π(Fc -
1 F0) Sd2 2 2
q
(qd2 ) (qd2 )
(CEVS) system to avoid water evaporation and to ensure cryofixation of the specimen at a controlled temperature (25 °C).31,32
3. Results and Discussion
sin2
(7)
2
where d is the plane thickness and S is its surface per unit volume. Scattering from solutions containing cylindrical structures has been analyzed by using eqs 5 and 6, whereas in the systems containing vesicles aggregates, eqs 5 and 7 have been used. Systems containing both of the objects have been treated assuming each kind of aggregate scattered independently from the other and expressing the cross section as the sum of the form factors weighted for two scale factors (Kcyl and Ksheets) the relative number density of the objects dΣ dσ (q) ) npKcylPcyl(q) + KsheetsPsheets(q) + dΩ dΩ
( )
inch
(8)
and treating the scale factors as adjustable parameters. The parameters (radius of the cylinders and the thickness d of the bilayer structure) extracted by the fitting procedure are reported in Table 3. 2.6. Cryo-Transmission Electron Microscopy (Cryo-TEM). Cryogenic-transmission electron microscopy (cryo-TEM) images were carried out at the Center for Chemistry and Chemical Engineering in Lund, Sweden, on a Philips CM120 BioTWIN Cryo electron microscope operating at 120 kV. A small drop of the sample solution was applied to a copper EM grid with a holey carbon film, and excess solution was blotted with a filter paper, leaving thus a thin sample film spanning the holes in the carbon film. Sample preparation was carried out in a controlled environment vetrification (30) Ma, G.; Barlow, D. J.; Lawrence, M. J.; Heenan, R. K.; Timmins, P. J. Phys. Chem. B. 2000, 104, 9081-9085.
3.1. Dynamic Light Scattering. pH Effect. Figure 2a shows the relaxation time distributions for 1 mM (C18)2DTPAGlu at different pH values. At pH 7.4, the distribution is clearly bimodal with well-separated modes, and the fast mode has a higher amplitude than the slow mode. The relaxation rates (Γ ) τ-1) for the fast and the slow modes were measured at different q values. The linear relation of the relaxation rates confirms that both modes are due to translational diffusion processes, attributed to two different complexes, with apparent translational diffusion coefficients Dfast ) (30.3 ( 0.4) × 10-12 m2/s and Dslow ) (3.2 ( 0.5) × 10-12 m2/s respectively, see Table 1. The StokesEinstein equation may be used to evaluate the hydrodynamic radius, RH, at infinite dilution
RH )
kBT 6πη0D0
(9)
where D0 is the translational diffusion coefficient at infinite dilution, kB is the Boltzmann constant, T is the absolute temperature, and η0 is the solvent viscosity. Due to the high dilution (10-4 mol kg-1) and high ionic strength of the systems, we have approximately D ≈ D0, so eq 9 can be reasonable used to estimate the hydrodynamic radius of the aggregates. (31) Talmon, Y. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 364. (32) Almgren, M.; Edwards, K.; Gustafsson, J. J. Curr. Op. Colloid Interface Sci. 1996, 1, 270.
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Figure 3. Relaxation time distributions at θ ) 90° for 1 mM (C18)2DTPAGlu(Gd) solution as function of pH.
Figure 2. (a) Relaxation time distributions at θ ) 90° for 1 mM (C18)2DTPAGlu solution as function of pH. (b) Intensity correlation functions at θ ) 90° for 1 mM (C18)2DTPAGlu solution as function of pH.
The RH values obtained for the slow and fast modes are 623 ( 91 and 66 ( 1 Å respectively, see Table 1, and they are compatible with bilayer structures, such as vesicles and micelles with an elongated shape.33 The relaxation time distribution of the (C18)2DTPAGlu solution at physiological pH collected in Figure 2a reveals the contemporary presence of micelles and bilayer structures or vesicles and that the dominant aggregates present in the system are micelles. The magnitude of the peak of this latter is sensibly higher than that corresponding to the vesicles. Variation of pH has a drastic effect on the size distribution of the aggregates. An overall view of the aggregation behavior may be obtained by plotting the intensity correlation functions or the relaxation time distributions obtained from RILT analysis of the former as a function of pH at a fixed scattering angle (θ ) 90°) (Figure 2b). Figure 2b shows that the time correlation function of scattered intensity g(2)(t) - 1 translates to longer (33) Jonstromer, M.; Johnsson, B.; Lindman, B. J. Phys. Chem. 1991, 95, 3293.
decay time as pH decreases, indicating a growth in the size of the aggregates. In particular in the pH range between 7.4 and 5.0, the relaxation time distributions (Figure 2a) are substantially unvaried; the distributions are bimodal, and the high amplitude peak at faster relaxation times corresponds to the diffusion of micelles. As the pH decreases from 5.0 to 4.0, the picture changes: the distribution becomes almost monomodal and shifts toward slower relaxation times as expected for larger aggregates. Thus, subsequent acidification results in a further shift to slower times of the relaxation time. At pH ) 3.0, the aggregates present in the systems have been evaluated to have an average hydrodynamic radius of about 742 ( 27 Å, confirming that large particles such as vesicles are present. The results from the DLS measurements clearly show that the aggregation behavior is sensitive to pH because of the presence of five carboxylic groups in the surfactant headgroup. Owing to the high negative actual charge of the surfactant headgroup, which causes a strong headgroup repulsion, at physiological pH, the tendency to form bilayer structures is sensible reduced. At lower pH (pH 5.0-4.0) the size distributions change as expected in light of the carboxylic acids pKa ∼ 4.5.34 In fact around this pH, the DTPA group should be probably in its uncharged form, supporting the formation of large and low curvature aggregates, such as bilayer aggregates or vesicles. DLS measurements were also carried out on the systems containing the gadolinium. The relaxation time distributions at θ ) 90° are presented for (C18)2DTPAGlu(Gd) in water at different values of pH in Figure 3. At physiological pH, the relaxation time distribution is bimodal with two peaks of similar amplitude; both modes were found to have a linear dependence with respect to q2. The diffusion coefficients are Dfast ) (26.4 ( 0.5) × 10-12 m2/s and Dslow ) (4.1 ( 0.3) × 10-12 m2/s, whereas the hydrodynamic radii are 76 ( 1 and 492 ( 46 Å for the fast and slow modes, respectively, as seen in Table 1. These results suggest that micelles, possibly with an elongated shape, likely rodlike type, are coexisting with bilayer structures at pH 7.4. Below pH 6, the distribution becomes monomodal; the relative amplitude for the slow mode increases on the expense of the fast (34) Durham, E. J.; Ryskiewich, D. P. J. Am. Chem. Soc. 1958, 80, 4812.
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Figure 4. Relaxation time distributions at θ ) 90° for 1 mM (C18)2DTPAGlu solution at pH 7.4 in the presence and absence of salt and at pH 3.
mode yielding to a single peak positioned at a slower relaxation time at pH 4.0. Finally, the comparison of the systems as free base and as Gd3+ complex suggests that in both cases cylindrical micelles and double layer structures are formed. Although in the latter systems there is a sensible tendency to form bilayer structures, the presence of Gd3+ reduces the actual charge of the surfactant from -5 to -2. When the gadolinium is complexed by the DTPAGlu moiety, the slow mode has an amplitude similar to that of the fast mode. As it is well-known, we are considering an intensity-weighted distribution and not a number distribution. This leads to a dominance of the larger objects in the distribution even if they are in fewer amounts. This means that even in the presence of Gd3+ at pH 7.4 the system may be constituted predominantly of micelles although the light scattering from the bilayer aggregates is considerable due to their scattering power. Ionic Strength Effect. The effect of ionic strength on the shape and size of the aggregates formed by the novel amphiphilic molecule has also been investigated. With this aim, DLS measurements were performed on (C18)2DTPAGlu and (C18)2DTPAGlu(Gd) aqueous solutions at physiological pH containing sodium chloride NaCl at 0.9 wt %, the value corresponding to the physiological ionic strength condition. The addition of the salt to the (C18)2DTPAGlu solution resulted in a much weaker variation in the size distribution with respect to what we observed by varying pH, as can be observed in Figure 4. The relaxation time distribution is substantially unvaried compared with that obtained without salt at the same pH value. The distribution is bimodal and dominated by the fast mode. The average diffusion coefficients obtained from the moments of the distribution, using eq 3, are Dfast ) (32.0 ( 1.2) ×10-12 m2/s and Dslow ) (2.9 ( 0.3) ×10-12 m2/s, respectively, and they are similar to the values obtained in the salt-free case (see Table 2). In the case of (C18)2DTPAGlu(Gd) aggregates, a change in the ionic strength seems to effect the structure of the aggregates. In Figure 5, the effect of ionic strength on aqueous solutions of (C18)2DTPAGlu(Gd) with respect to pH is shown. At pH ) 7.4 in the absence of salt, the distribution is bimodal, whereas the addition of salt yields a unimodal distribution with a quite broadened peak, shifted toward slower relaxation time. Although the relaxation time distribution at pH 7.4 in the presence
Vaccaro et al.
Figure 5. Relaxation time distributions at θ ) 90° for 1 mM (C18)2DTPAGlu(Gd) solution at pH 7.4 in the presence and absence of salt and at pH 3.
Figure 6. Scattering intensity profile for (C18)2DTPAGlu-D2O at pH 7.4 (0), (C18)2DTPAGlu-D2O at pH 4.5 (O), (C18)2DTPAGluD2O at pH 3 (4), and (C18)2DTPAGlu-D2O at pH 7.4 not extruded (b). (s) Fitting curve to the experimental data through the model reported in the text.
of NaCl appears to be similar to that at pH 3, it does not necessarily indicate an increase of the bilayer structures population as mentioned before. The distribution obtained could be due either to a polydispersity of larger aggregates or to two unresolved peaks similar to those observed in the bimodal regime. It is thus difficult to draw any final conclusions about the effect of the ionic strength, but it appears to be of a less importance than compared to the pH effect on the size and shape of the aggregates. 3.2. Small-Angle Neutron Scattering. SANS measurements were carried out on samples at selected pH values and physiological ionic strength conditions on the basis of the experimental results obtained by using the DLS, and in addition the effect on the preparation procedure has been investigated. pH Effect. Figure 6 shows clearly the evolution of the supramolecular aggregates with the pH from micelles to vesicles or bilayer aggregates, as highlighted by DLS results. At pH 7.4, the scattering profile (empty squares) reveals the coexistence of bilayers and micelles. At low q values, a q-2 decay typically of
Blood Pool MRI/MRA Contrast Agents
Figure 7. Scattering intensity profile for (C18)2DTPAGlu(Gd)D2O at pH 7.4 (O). Fitting of the data according to the respective model gives the solid dot line shown for the sample, the dash line refers to bilayer structures and the short dash dot line to cylindrical micelles.
double layer scattering is observed, whereas at intermediate q range (0.02 < q/Å-1 < 0.06), a rising peak originating by the presence of micelles appears. We note that when (C18)2DTPAGlu is simply dissolved in water the micelles are the only species present in solution, as the q-1 intensity decay in the q range (0.0035 < q/Å-1 < 0.025) suggest, solid circles in Figure 6. The scattering intensity of the sample at pH ) 4.5 (empty circles) is substantially similar to that observed at pH ) 7.4. Actually, due to the lower solubility of the solute in the sample at pH ) 4.5 with respect to that at pH ) 7.4, the scattering intensity is markedly reduced; thus, at the former pH, a fewer number of micelles is expected. At pH 3.0, the cross-scattering section (empty triangles) is a clear q-2 power law in the whole q range analyzed, indicating the presence of only bilayer structures in the system. At this pH, (C18)2DTPAGlu is substantially uncharged, all the carboxylic groups present in the DTPA moiety are protoned, and the formation of vesicles or in general bilayers is advantaged, as detailed discussed in the DLS section. SANS measurements were also performed on the aggregates obtained by using the gadolinium complex of (C18)2DTPAGlu unimer in function of the pH. Samples in complexed and uncomplexed form exhibit a quite similar cross scattering profile when compared at the same pH. Structural parameters of the aggregates (as free base or as Gd3+ complex), obtained by fitting experimental data trough appropriate models, are reported in Table 3. In particular, bilayer structures have been modeled through eqs 5 and 7, whereas for rodlike micelles, eqs 5 and 6 have been used. The length of the cylinders cannot be always extracted from the scattering data in the accessible range of the scattering vector q (q > 0.002 Å-1) as indicated by the missing of the Guinier region at small q. As result of that, in the reported tables, large values are taken as the lower limit for the real length. Systems containg both aggregates micelles and bilayer structures have been modeled through eq 8, i.e., assuming small superposition of the scattering cross-section of each kind of aggregate. The reliability of using the sum of the two contributions bilayer structures (dashed line) and rodlike micelles (dashed dot line) in eq 8 has been supported by the small superposition of the two terms as shown in Figure 7. At qmax (∼0.03 Å-1) where
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Figure 8. Scattering intensity profile for (C18)2DTPAGlu(Gd)D2O at pH 7.4 (0), (C18)2DTPAGlu(Gd)-D2O at pH 4.5 (O), and (C18)2DTPAGlu(Gd)-D2O at pH 3.0 (2). (s) Fitting curve to the experimental data through the model reported in the text.
the micelle peak is present, the bilayer structures contribute only 1% of the entire scattering intensity. Summarizing, at pH 7.4, rodlike micelles with a length of ∼200 Å and a radius of ∼40 Å are present. Micelles increase their length as the pH is reduced to 4.5 (∼700 Å length), and in both cases, they coexist with bilayer structures that are the dominant structures at pH 3.0 (see Figure 8). The thickness of bilayer structures varies from 70 to 40 Å as the pH is reduced. We note that the radius of vesicles cannot be evaluated from the SANS data because the cross-section at lower q would be needed. This could be obtained only performing USANS measurements. It is likely that the loss of the electrostatic repulsions, due to the complete neutralization of the carboxylic groups at low pH values, allows a more compact structure. Ionic Strength Effect. The effect of ionic strength on the systems of (C18)2DTPAGlu in the absence and presence of Gd3+ has been investigated at pH ) 7.4. In both cases, any substantially effect of the salt addition has not been revealed. In fact, the scattering profiles of the systems containing NaCl (see Figures 1 and 2 in the Supporting Information) are nearly quite similar to those observed for the salt free case. In other words, the salt addition does not produce any marked change in the aggregates population, which remains unvaried with respect to those observed at pH 7.4. The structural parameters extracted from the SANS fitting procedure reveal a slight increase in aggregation number of the rodlike micelles containing Gd3+ and a substantially independence of the thickness of the bilayer structures from the ionic strength, see Table 4. These latter are substantially the same as those evaluated for the system in the absence of NaCl. 3.3. Cryo-Transmission Electron Microscopy. Cryo-TEM measurements were performed to obtain images of the aggregates studied by DLS and SANS techniques. The images collected in Figure 9 give a clear indication of the structural evolution of the aggregates formed by (C18)2DTPAGlu as a function of the pH. The cryo-TEM results confirm the conclusions drawn from the scattering techniques: a gradual transition by decreasing pH, from small rodlike micelles (pH ) 7.4), to threadlike micelles (pH ) 4.5), and finally to bilayer aggregates or vesicles (pH ) 3.0). At physiological pH, micelles are visualized in the image as dark dots, because objects with
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Table 4. Structural Parameters of the Aggregates in Condition of Physiological Ionic Strength Determined by Small-Angle Neutron Scattering (SANS)a systems
pH
Nagg
R (Å)
l (Å)
d (Å)
(C18)2DTPAGlu (0.00079 mol kg-1)-NaCl 0.9 wt %-D2O (C18)2DTPAGlu(Gd) (0.00063 mol kg-1)-NaCl 0.9 wt %-D2O
7.4 7.4
195 ( 35 201 ( 23
37 ( 3 34 ( 2
206 ( 23 226 ( 28
72 ( 9 53 ( 4
a The terms N , R, and l refer to the aggregation number, the radius, and the length of the micelles, respectively, whereas d refers to the thickness agg of the bilayer structures.
Figure 9. cryo-TEM image for 1 mM (C18)2DTPAGlu at different pH values: (a) at pH 7.4 micelles appear as black dots which are difficult to distinguish from the background; (b) at pH 4.5 threadlike micelles are observed (circular stains are artifacts); (c) at pH 3 vesicles appear unilamellar with a radius in the range of 700-1000 Å.
a size of about 5-6 nanometers are in the limit of the resolution of electron microscopy technique used (see Figure 9a). At pH 4.5, micelles change their shape and appear in the vitrified sample as threads with slightly swollen end caps (Figure 9b).We note that the aggregates seen in the image are characterized by a low contrast, which is typical of micelles. At pH 3, the images are dominated by the presence of vesicles, as foreseen from the DLS and SANS measurements. Most of the vesicles are unilamellar, see Figure 9c, with a mean number-averaged radius in the range of 700÷1000 Å. This value is in agreement with the apparent z-averaged hydrodynamic radius estimated by the DLS experiments. Cryo-TEM images were also carried out on the system containing the gadolinium, confirming the results collected by the scattering techniques. At physiological pH, the cryo-TEM micrographs show open bilayer structures and elongated and thick structures similar to fibers, corresponding to cylindrical micelles, see Figure 10a. With decreasing pH, the images are dominated by the presence of bilayer structures as predicted by DLS and SANS results. The images collected for the system at pH 3.0 show clearly the presence in solution of vesicles (Figure 10b) and open bilayers, similar to those visualized at pH 7.4. The number-averaged radius of vesicles ranges between 500 and 600 Å. The latter structures, although, appear as hollow tubes having a thickness of around 60 Å with well marked edges. The edges appear quite dark probably because of the presence of the gadolinium, which is oriented toward the aqueous phase. These aggregates, as confirmed by recent DSC experiments, represent gel phases containing bilayers with crystallized hydrocarbon chains separated by a liquidlike solvent whose gel to liquid-crystalline phase transition temperature Tm is around 70 °C.35 (35) Evans, D. F.; Wennerstro¨m, H. The Colloidal Domain: Where Physics, Chemistry, Biology, and Technology Meet, 2nd ed.; Wiley-VCH: New York, NY, 1999.
Figure10. Selectedcryo-TEMimagesfor1mM(C18)2DTPAGlu(Gd) at different pH values: (a) at pH 7.4 open bilayers and cylindrical micelles similar to fibers appear (circular stains are artifacts); (b) at pH 3 vesicles with a radius in the range of 500-600 Å.
4. Conclusions In this report, we have presented amphiphilic supramolecular aggregates with potential application in the therapeutic and diagnosis in clinical medicine. Such aggregates are formed by a novel molecule constituted basically by the DTPAGlu moiety bound to a hydrofobic double-tail (18 carbon atoms). The amphiphilic molecule synthesized behaves as an anionic surfactant, and it is capable of forming aggregates of different sizes and shapes (rodlike micelles, threadlike micelles, and vesicles) in aqueous solution by varying the method of preparation and the environmental conditions such as pH and ionic strength. The aggregation behavior of the surfactant, as free base and as gadolinium complex, has been studied by means of dynamic light scattering (DLS), small-angle neutron scattering (SANS), and cryo-transmission electron microscopy (cryo-TEM). Fur-
Blood Pool MRI/MRA Contrast Agents
thermore, we have also measured the relaxivity values of Gd3+ in such aggregates. For (C18)2DTPAGlu-water binary system, we have observed a micelle-to-vesicle transition, which confirms the three-stage Lichtenberg model.36,37 We have found the following sequence of aggregation states as the solution pH was decreased from pH ) 7.4 to 3.0:
rodlike micelles f threadlike micelles f vesicles The micelle formation at physiological pH is explained by the high negative charge of the surfactant headgroup, which causes strong headgroup-headgroup repulsions. A decrease of the pH causes protonation of the carboxylic groups in the surfactant headgroup and a decrease of the electrostatic repulsion between the headgroups, favoring the formation of large and low curvature aggregates such as bilayer structures or vesicles. The increase of the ionic strength has a less significant effect compared to the pH effect, salt addition produces slight tendency of the system to form more compact aggregates. In the (C18)2DTPAGlu(Gd)-water system, double layer structures over the entire pH range and in the absence or presence of chloride sodium are generally observed. The presence of Gd3+ produces a mild effect as above-discussed for the pH reduction. Thus, for this system, formation of low and large curvature aggregates such as open bilayers is supported and this tendency is further enhanced by lowering the pH rather than adding an electrolyte. Looking at cryo-TEM micrographs, these structures appear as hollow tubes that probably correspond to gel phases, where the alkyl chains are crystallized and liquidlike solvent is still present between the bilayers. This hypothesis has been confirmed by DSC measurements that show a well-defined peak at 70 °C corresponding to gel-liquid-crystalline phase transition temperature Tm.38 In this case, the vesicles formation can be enhanced by heating the solution containing the amphiphile molecule over the Tm. The new amphiphilic molecule presented in this report provides novel and interesting opportunities to investigate surfactant
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aggregation properties, that may be exploited in several applications such as in drug and gene delivery39 or in medical diagnostic, where a straightforward switch between different aggregation states can be particularly useful. Regarding this latter point, recent studies have revealed that the extracellular fluid of tumors cells has a lower pH than that for healthy tissues, so that it has been an increasing demand in current clinical diagnostics for pH-responsive MRI/MRA contrast agents7 for the in vivo pH mapping of tissues in tumor diagnosis. In particular, our aggregates appear to be good candidates for blood pool MRI/MRA contrast agents in tumor diagnosis. In fact, relaxivity measurements performed on (C18)2DTPAGlu(Gd) aggregates at pH 7.4 in the presence and absence of NaCl at physiological ionic strength have shown interesting results: r1 ) 21.5 and 24.0 mM-1 s-1 (at 20 MHz and 25 °C), respectively. To date, these values are higher than those measured for our previous micellar aggregates5 and are among the highest values ever reported in the literature for contrast agents in MRA /MRI applications. We are at present continuing the search for a more detailed study of the influence of temperature on the aggregation behavior in different conditions of pH and ionic strength, and we are further investigating the effect of these parameters on the relaxivity values of the supramolecular aggregates. Acknowledgment. The authors thank the European Molecular Imaging Laboratories Network (EMIL) for financial support. The authors are grateful to Gunnel Karlsson for the cryo-TEM imaging. Some of the authors (M.V., G.M., and L.P.) thank the Institut fu¨r Festko¨rperforschung and the Hahn Meitner Institut for the provision of beam time. The SANS experiments have been supported by the European Commission, NMI3 Contract RII3-CT-2003-505925. We also thank Dr. Aurel Radulescu and Dr. Astrid Brandt for the insightful discussion on the smallangle neutron scattering data. We thank Dr. Eliana Gianolio, University of Turin, for relaxivity measurements and helpful discussions on the compound relaxivity behavior. Supporting Information Available: Scattering profiles of the systems containing NaCl. This material is available free of charge via the Internet at http://pubs.acs.org. LA053500K
(36) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 740. (37) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285. (38) Manuscript in preparation.
(39) Bell, P. C.; Bergsma, M.; Dolbnya, I. P.; Bras, W.; Stuart, M. C. A.; Rowan, A. E.; Feiters, M. C.; Engberts, J. B. F. N. J. Am. Chem. Soc. 2003, 125, 1551.
