pubs.acs.org/Langmuir © 2009 American Chemical Society
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Lanthanide Phytanates: Liquid-Crystalline Phase Behavior, Colloidal Particle Dispersions, and Potential as Medical Imaging Agents Charlotte E. Conn,† Venkateswarlu Panchagnula,†, Asoka Weerawardena,† Lynne J. Waddington,‡ Danielle F. Kennedy,† and Calum J. Drummond*,†,§
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† CSIRO Molecular and Health Technologies (CMHT), Private Bag 10, Clayton South MDC, VIC 3169, Australia, ‡CSIRO Molecular and Health Technologies (CMHT), 343 Royal Parade, Parkville, VIC 3052, Australia, §CSIRO Materials Science and Engineering (CMSE), Private Bag 33, Clayton South MDC, VIC 3169, Australia. Current address: Chemical Engineering Division, National Chemical Laboratory, Dr. Homi Bhabha Rd, Pune, India 411 008.
Received October 21, 2009. Revised Manuscript Received November 29, 2009 Lanthanide salts of phytanic acid, an isoprenoid-type amphiphile, have been synthesized and characterized. Elemental analysis and FTIR spectroscopy were used to confirm the formed product and showed that three phytanate anions are complexed with one lanthanide cation. The physicochemical properties of the lanthanide phytanates were investigated using DSC, XRD, SAXS, and cross-polarized optical microscopy. Several of the hydrated salts form a liquid-crystalline hexagonal columnar mesophase at room temperature, and samarium(III) phytanate forms this phase even in the absence of water. Select lanthanide phytanates were dispersed in water, and cryo-TEM images indicate that some structure has been retained in the dispersed phase. NMR relaxivity measurements were conducted on these systems. It has been shown that a particulate dispersion of gadolinium(III) phytanate displays proton relaxivity values comparable to those of a commercial contrast agent for magnetic resonance imaging and a colloidal dispersion of europium(III) phytanate exhibits the characteristics of a fluorescence imaging agent.
Introduction Metal-containing liquid-crystalline mesophases, or metallomesogens, are of interest because of their potential applications in areas as wide-ranging as liquid-crystal displays, porous material templating, electrochemical cells, and recently, as contrast agents in medical imaging.1-6 Commonly encountered metallomesogens are metal-containing salts of long-chain fatty acids, commonly known as “metal soaps”. Alkali and transition-metal soaps have been widely studied.7-9 However, similar research into the lanthanide salts of long-chain surfactants, especially their lyotropic behavior, has received far less attention, although those for lanthanide complexes have been covered extensively.1,10 Interestingly, one of the earliest studies reporting the lyotropic behavior of lanthanide(III) sulfates stemmed from the fact that these were used as starting materials for the synthesis of liquid-crystalline Schiff’s base complexes of lanthanides.11,12 We note that although lanthanide salts of amphiphiles have been reported previously, the discovery of their mesomorphic behavior is of recent origin.13 *Corresponding author. E-mail:
[email protected]. (1) Binnemans, K.; Gorller-Walrand, C. Chem. Rev. 2002, 102, 2303. (2) Bottrill, M.; Nicholas, L. K.; Long, N. J. Chem. Soc. Rev. 2006, 35, 557. (3) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. Rev. 1999, 99, 2293. (4) Raimondi, M. E.; Seddon, J. M. Liq. Cryst. 1999, 26, 305. (5) Rocha, J.; Carlos, L. D.; Ferreira, A.; Rainho, J.; Ananias, D.; Lin, Z. Adv. Mater. Forum II 2004, 455-456, 527. (6) Seddon, J. M.; Raimondi, M. E. Mol. Cryst. Liq. Cryst. 2000, 347, 465. (7) Giroud-Godquin, A. M. Coord. Chem. Rev. 1998, 178, 1485. (8) Binnemans, K. Chem. Rev. 2005, 105, 4148. (9) Giroudgodquin, A. M.; Maitlis, P. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 375. (10) Piguet, C.; Bunzli, J. C. G.; Donnio, B.; Guillon, D. Chem. Commun. 2006, 3755. (11) Binnemans, K.; Van Deun, R.; Bruce, D. W.; Galyametdinov, Y. G. Chem. Phys. Lett. 1999, 300, 509. (12) Galyametdinov, Y. G.; Jervis, H. B.; Bruce, D. W.; Binnemans, K. Liq. Cryst. 2001, 28, 1877. (13) Marques, E. F.; Burrows, H. D.; Miguel, M. D. J. Chem. Soc. 1998, 94, 1729.
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The self-assembly of soap molecules into liquid-crystalline mesophases can be described in terms of the concept of the selfassembly of amphiphiles advanced by Israelachvili et al. and is governed by the effective shape of the molecule, which is strongly influenced by the competing interactions of the polar headgroups and the alkyl chains.14 This can be semiquantified using the concept of the critical packing parameter, CPP = ν/(lca0), where lc is the effective length of an amphiphilic chain, a0 is the effective amphiphilic headgroup area, and ν is the average volume occupied by the amphiphilic hydrophobic chain. Molecules with a packing parameter of 1 (i.e., molecules with an effective reverse wedge shape) will preferentially form inverse phases. A variety of mesophases may be adopted, including a flat bilayer or lamellar structure as well as more highly curved phases of cubic or hexagonal symmetry. The inverse bicontinuous cubic phases (QII) consist of a single continuous bilayer draped over a minimal surface of zero mean curvature that subdivides space into two interpenetrating but not connected water networks. Three types of inverse cubic phases have been identified in lipid systems.15 These are based on the Schwarz diamond (D) and primitive (P) and on the Schoen gyroid (G) minimal surfaces and are denoted as QIID, QIIP, and QIIG, respectively.16 The main structure observed for the lanthanide phytanates studied here is a hexagonal columnar mesophase consisting of long cylinders of polar headgroups with hydrocarbon chains radiating outward and packed onto a 2D hexagonal lattice (Figure 1C). In the presence of water, this type of structure is also known as an inverse hexagonal mesophase (HII). Soap molecules can also form (14) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525. (15) Kaasgaard, T.; Drummond, C. J. Phys. Chem. Chem. Phys. 2006, 8, 4957. (16) Luzzati, V.; Tardieu, A.; Gulikkrz, T; Rivas, E.; Reisshus, F. Nature 1968, 220, 485.
Published on Web 12/29/2009
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Figure 1. (A) Structure of phytanic acid, (B) an energy-minimized molecular model for Gd(III) phytanate (including one water of hydration), and (C) the putative hexagonal columnar structure of Gd(III) phytanate. (B, C) Gd is green, O is red, and C is gray. For clarity, H atoms have been omitted.
inverse micelles where the hydrophilic headgroups are arranged toward water cores with hydrophobic chains radiating outward. These micelles can pack into ordered cubic arrays17 or form an entirely disordered packing resulting in a fluid inverse micellar phase, the L2 phase. Intermediate and swollen spongelike phases have also been identified in non-lamellar-forming lipid systems, although to our knowledge these have not yet been identified for metal-containing liquid crystals.18,19 Among the lanthanide salts of amphiphilic carboxylates and alkanoates, most notable is the published work by Binnemans and co-workers, where the thermotropic behavior and the effects of chain length and lanthanide ion size on the mesophases of straight-chain alkanoates were studied.20,21 It was found that the lanthanide dodecanoates have a lamellar bilayer structure in the solid state and that mesophases formed only for light lanthanide ions having larger ionic radii. It has also been found that various hydrates of the metal soap or the anhydrous analogues can be formed by tuning the pH of the metathesis reaction displacing sodium in the alkanoates with the lanthanide and also by varying the chain length.1 A study by Corkery investigating the effects of curvature on the liquid crystallinity of midchain branched fatty acid salts of transition metals and lanthanide salts showed that the Cu, Zn, Sr, and Ba salts formed a liquid-crystalline hexagonal columnar mesophase at room temperature. An increase in hexagonal cell size with increasing ionic radius was observed.22 Lanthanide salts of amphiphiles have the potential to be efficient medical contrast agents with improved imaging capabilities as well as featuring the ability to carry therapeutic “payloads”. Contrast agents, used to increase the contrast between the target organ and the surrounding tissues, are now used in over 35% of magnetic resonance imaging (MRI) diagnoses.23 Most (17) Seddon, J. M.; Zeb, N.; Templer, R. H.; McElhaney, R. N.; Mannock, D. A. Langmuir 1996, 12, 5250. (18) Conn, C. E.; Ces, O.; Mulet, X.; Finet, S.; Winter, R.; Seddon, J. M.; Templer, R. H. Phys. Rev. Lett. 2006, 96, 108102. (19) Mulet, X.; Gong, X.; Waddington, L. J.; Drummond, C. J. ACS Nano 2009, 3, 2789. (20) Jongen, L.; Binnemans, K.; Hinz, D.; Meyer, G. Liq. Cryst. 2001, 28, 1727. (21) Binnemans, K.; Jongen, L.; Gorller-Walrand, C.; D’Olieslager, W.; Hinz, D.; Meyer, G. Eur. J. Inorg. Chem. 2000, 1429. (22) Corkery, R. W. Phys. Chem. Chem. Phys. 2004, 6, 1534. (23) Aime, S.; Cabella, C.; Colombatto, S.; Crich, S. G.; Gianolio, E.; Maggioni, F. J. Magn. Reson. 2002, 16, 394. (24) Pfeiffer, H.; Speck, U.; Renneke, F.; Hoyer, G.; Weinmann, H.; Rosenberg, D.; Gries, H.; Mutzel, W.; Muetzel, W. NMR, X-ray and Ultrasonic Diagnostic Compsns. - Contg. Salts of Metal Complexes, Including New Cpds; CA Patent 124069, Schering Ag (Schd), 1984.
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contrast agents approved for human use are extracellular fluid (ECF) gadolinium-based agents such as Magnevist.24 The paramagnetic gadolinium(III) ion significantly reduces both the longitudinal and transverse proton relaxation times (T1 and T2) relative to that of pure water.25 Because the free gadolinium(III) ion has been shown to be toxic in both in vivo and in vitro studies,26-29 it is generally sequestered by chelation3 or encapsulation30-32 in biomedical applications. Commercial contrast agents such as Magnevist, which is the gadolinium complex of diethylenetriamine pentaacetic acid,24 are all nonspecific and suffer from the shortcomings of limited resolution and relatively low inherent sensitivity.33 Here we have investigated the use of self-assembled gadolinium(III)-containing systems as MRI contrast agents.34 This potentially allows us to access desirable properties of the selfassembled system, for example, the capacity to encapsulate drugs, along with sustained release and targeted delivery.35,36 In addition, self-assembled phases of lamellar, cubic, and hexagonal symmetry can be dispersed in water to form nanoparticles known as liposomes, cubosomes, and hexosomes, respectively. The aqueous interior of these colloidal particles allows for the movement of water into and out of the framework, and their high surface area can accommodate large numbers of paramagnetic ions. In addition, their specificity can be enhanced by modification with targeting molecules. Such systems could additionally be used as contrast agents for optical fluorescence imaging, for example, for terbium(III) and europium(III) ions, which are luminescent in aqueous solution and generally retain their luminescence when bound to complex ligand systems.37 In this article, we present an investigation of the thermal and lyotropic behavior of lanthanide phytanates, along with an investigation into the utility of nanoparticulate dispersions of lanthanide phytanates as potential contrast agents for magnetic resonance imaging. Both unsaturation and branching are known to promote mesophase formation because of the reduction in van der Waals attraction forces in the chain region,22,38-41 and we have previously investigated lanthanide oleates with unsaturated hydrocarbon chains.34 Here we have extended our research to include the phytanate chain, an isoprenoid-type hydrocarbon chain with four methyl substituents along its saturated hydrocarbon backbone. The structure of phytanic acid is shown in Figure 1A. We also attempt to understand the influence of metal ion size and valency on the mesophase formed. (25) Meade, T. J.; Taylor, A. K.; Bull, S. R. Curr. Opin. Neurobiol. 2003, 13, 597. (26) Biagi, B. A.; Enyeart, J. J. Am. J. Physiol. 1990, 259, C515. (27) Ergun, I.; Keven, K.; Uruc, I.; Ekmekci, Y.; Canbakan, B.; Erden, I.; Karatan, O. Nephrol., Dial., Transplant. 2006, 21, 697. (28) Molgo, J.; del Pozo, E.; Banos, J. E.; Angaut-Petit, D. Br. J. Pharmacol. 1991, 104, 133. (29) Morcos, S. K. Catheterization Cardiovascular Interventions 2006, 68, 812. (30) Bolskar, R. D.; Benedetto, A. F.; Husebo, L. O.; Price, R. E.; Jackson, E. F.; Wallace, S.; Wilson, L. J.; Alford, J. M. J. Am. Chem. Soc. 2003, 125, 5471. (31) Kato, H.; Kanazawa, Y.; Okumura, M.; Taninaka, A.; Yokawa, T.; Shinohara, H. J. Am. Chem. Soc. 2003, 125, 4391. (32) Sitharaman, B.; Kissell, K. R.; Hartman, K. B.; Tran, L. A.; Baikalov, A.; Rusakova, I.; Sun, Y.; Khant, H. A.; Ludtke, S. J.; Chiu, W.; Laus, S.; Toth, E.; Helm, L.; Merbach, A. E.; Wilson, L. J. Chem. Commun. 2005, 3915. (33) Aime, S.; Cabella, C.; Colombatto, S.; Crich, S. G.; Gianolio, E.; Maggioni, F. Magn. Reson. Imaging 2002, 16, 394. (34) Liu, G.; Conn, C. E.; Drummond, C. J. J. Phys. Chem. B 2009, 113, 15949. (35) Malmsten, M. Soft Matter 2006, 2, 760. (36) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4, 449. (37) Richardson, F. S. Chem. Rev. 1982, 82, 541. (38) Attard, G. S.; West, Y. D. Liq. Cryst. 1990, 7, 487. (39) Maldivi, P.; Bonnet, L.; Giroudgodquin, A. M.; Ibnelhaj, M.; Guillon, D.; Skoulios, A. Adv. Mater. 1993, 5, 909. (40) Sagnella, S.; Conn, C. E.; Krodkiewska, I.; Drummond, C. J. Soft Matter 2009, 5, 4823. (41) Sagnella, S.; Conn, C. E.; Krodkiewska, I.; Moghaddam, M.; Seddon, J. M.; Drummond, C. J. Langmuir 2010, 10.1021/la903005q.
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Experimental Section Materials. All reagents and precursor materials were purchased from Sigma-Aldrich. Phytanol (3,7,11,15-tetramethylhexadecan-1-ol) was synthesized from phytol (3,7,11,15-tetramethyl2-hexadecen-1-ol) following reduction over Raney nickel as described in the literature.42 Synthesis of Phytanic Acid (3,7,11,15-Tetramethylhexadecanoic Acid) by the Oxidation of Phytanol. The oxidation of phytanol was carried out according to the established protocols42 with minor modifications. Phytanol (30 g) was dissolved in 60 mL of acetic acid and 1200 mL of acetone. A solution of 24 g of chromium trioxide dissolved in 30 mL of water was added dropwise to the above solution placed in an ice bath. After the addition was complete, the mixture was allowed to stir at room temperature for 90 min. After confirming via 1H NMR that the product was formed, the reaction was allowed to proceed to completion overnight. Thereafter, 500 mL of water was added, followed by powdered sodium bisulfite to quench the oxidation. The precipitated chromium salts were filtered off using a Buchner funnel and Whatman no. 4 filter paper, and the solvent was evaporated. The predominantly acetone mixture that first evaporated was used to wash the chromium salts. The sticky material obtained after evaporation was suspended in 400 mL of water and extracted (overnight) into 500 mL of ether, thereby removing most of the chromium salts. The aqueous layer was washed with ether several times and added to the product, and the solvent was evaporated. After workup and distillation, about 20 g of pure phytanic acid was collected. The purity was assessed using 1H NMR and a reversed-phase HPLC/RI detector. NMR spectra were in accordance with those in the literature and indicated no significant impurity. HPLC analysis indicated a purity of g95%. Known impurities of possible side product phytanic ester and reactant phytanol were ruled out on the basis of the retention times. Synthesis of Lanthanide Salts of Phytanic Acid. The synthesis of the metal soaps was achieved via double decomposition using the methods of Binnemans et al.21 and Corkery.22 The addition of NaOH to obtain the sodium soap of the fatty acid was followed by double decomposition with the lanthanide/transition-metal salt (hexahydrated, either a lanthanide chloride or a nitrate). Lanthanum, cerium, neodymium, samarium, europium, gadolinium, terbium, and dysprosium(III) salts were synthesized. Subsequently, the product was washed with water, ethanol, and acetone. Thereafter, the dried product was recrystallized from a pentanol/water (5:1) mixture before the final freeze drying. FTIR spectroscopy was used to confirm the product formation as evident from the disappearance of the carbonyl stretch at 1700 cm-1. The final metal soaps usually contained very small amounts of unreacted phytanic acid. Elemental Analysis. The carbon and hydrogen contents of the Ln phytanates (Ln = La, Ce, Nd, Sm, Eu, Gd, Tb, and Dy) were determined by C/H elemental microanalysis (combustion analysis) at the University of Otago, New Zealand. The rare earth metal content was determined using a Varian Vista spectrophotometer with inductively coupled plasma atomic emission spectroscopy (ICP-AES). Samples for ICP-AES analysis were prepared by dissolving 0.1 g of Ln phytanate in 1 M HCl (15 mL). The solution was extracted with diethyl ether (3 30 mL) to remove the phytanic acid, made up to 100 mL with Milli-Q water, and used directly for ICP-AES analysis. The phytanic acid content was determined by analytical HPLC (Alltech Alltime, 250 mm 4.6 mm ID 5 μm). Infrared Spectroscopy. FTIR was performed using a BOMEN MB 101 from Extech Equipment Pty. Ltd. Spectra of the neat samples as KBr pellets were obtained at room temperature in the 4000-400 cm-1 range and accumulated for 32 scans at a resolution of 8 cm-1. (42) Burns, C. J.; Field, L. D.; Hashimoto, K.; Petteys, B. J.; Ridley, D. D.; Rose, M. Aust. J. Chem. 1999, 52, 387.
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Thermal Analysis. DSC measurements were performed on a Mettler 3000 system. Solid samples weighing 5-10 mg were sealed in aluminum pans (25 μL) with pierced lids and heated or cooled at scan rates of 2.5, 10, and 22.5 °C min-1. Thermograms were recorded in a nitrogen atmosphere using empty aluminum pans as the reference. An indium standard was used to calibrate the DSC temperature ((0.3 °C) and enthalpy scale. TGA on a microbalance was carried out in the temperature range of 25-600 °C using a Mettler Toledo 851 system under a nitrogen atmosphere. Cross-Polarized Optical Microscopy. Neat lanthanide phytanates were melted between a microscope slide and coverslip and then cooled to room temperature. Water added at the edge of the coverslip was drawn between the two glass surfaces and surrounded the solidified material by capillary action. Optical textures were observed with an Olympus IMT2 cross-polarized optical microscope equipped with a Mettler FP82HT hot stage and an FP90 programmable temperature controller. X-ray Powder Diffraction. XRD patterns were obtained using the powder diffraction beamline at the Australian Synchrotron.43 Samples were contained within a custom-designed sample stage, mounted on the omega axis of the diffractometer, and measured in transmission mode. A 1 mm 0.8 mm 1 A˚ beam was aligned to a reference point on the stage containing an R-alumina standard. X-ray diffraction data from 2 to 82° at ambient temperature were collected on a Mythen II Microstrip detector. Some XRD patterns were obtained using a Bruker D8 advanced X-ray diffractometer. Cu KR radiation (40 kV, 40 mA) was monochromated with a graphite sample monochromator. Each sample was scanned over the 2θ range of 1-40° with a step size of 0.02° and a count time of 4 s/step. Analyses were performed on the collected XRD data for each sample using Bruker XRD search match program EVA. Small-Angle X-ray Scattering. SAXS experiments on bulk Ln phytanates in excess water (Ln = Nd, Sm, Eu, Tb, and Dy) were carried out at the 15-ID-D (ChemMatCARS) beamline of the Advanced Photon Source (Argonne, Illinois).44 The experiments used a beam of wavelength λ = 1.50 A˚ (8.27 keV) with dimensions of 200 μm 100 μm and a typical flux of 1 1012 photons/s. Two-dimensional diffraction images were recorded on a Bruker 6000 CCD detector with an active area of 94 mm 94 mm and a pixel size of 92 μm. The CCD detector was offset from the main beam, allowing analysis in the q range of 0.0187-0.807 A˚-1 at a sample-to-detector distance of 0.6 m. Temperature control was in the range of 7-90 °C. For the Ln phytanates where Ln = La, Ce, and Gd, SAXS experiments were carried out using the SOL beamline at Imperial College London. X-rays were produced using a Phillips PW2213/ 20 generator operating at 40 kV and 30 mA through an AEG-type 50/21 X-ray tube. The X-rays were monochromated using a quartz crystal monochromator that isolates the Cu radiation, and a line focus beam was generated. The diffraction pattern therefore does not consist of a set of concentric circles but instead a series of symmetrical lines that are detected using an X-ray-sensitive photographic negative (Kodak, MR). The spacings range of the beamline was from approximately 105 to 2 A˚, enabling both wide- and small-angle X-ray scattering to be observed. Experiments on dispersed samples were carried out at the SAXS/ WAXS beamline at the Australian Synchrotron. The experiments used a beam of wavelength λ = 0.80 A˚ (15.0 keV) with dimensions of 700 μm 500 μm and a typical flux of 1.2 1013 photons/s. Two-dimensional diffraction images were recorded on a Mar CX165 detector with analysis in the q range of 0.0305-1.047 A˚-1. (43) Wallwork, K. S.; Kennedy, B. J.; Wang, D. Synchrotron Radiat. Instrum., Int. Conf. 2007, 879, 879. (44) Cookson, D.; Kirby, N.; Knott, R.; Lee, M.; Schultz, D. J. Synchrotron Radiat. 2006, 13, 440.
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Conn et al. For all samples, image analysis was carried out using AXcess, an IDL-based software package developed by Dr. Andrew Heron at Imperial College London.45 Dispersion Procedures. The Ln phytanates (Ln = Eu, Gd, Tb, and Dy) were dispersed in water to form particles. First, the lanthanide phytanates were dissolved slowly in the minimum amount of 2-methyl-2 propanol at 60 °C to form a homogeneous solution. This was followed by the addition of 10 wt % of stabilizer Pluronic F127. The mixture of lanthanide phytanate and F127 was added to 50 mL of Milli-Q water with mixing in an ultratarrax (Polytron PT 10-35 GT, Kinematica, Switzerland) for 5 min at a speed of 15 000 rpm at 80 °C. The dispersion was immediately passed through a high-pressure homogenizer (Avestin, Germany) using a pressure of 10 000 psi for six passes at 60 °C. In addition, the Ln phytanates (Ln = Eu, Gd, Tb, and Dy) were dissolved in phytantriol at 10 wt % with the addition of CHCl3 at 60 °C. CHCl3 was later evaporated off using a rotor vapor. This was further dried via a freeze dryer overnight. Five hundred milligrams of this solution (50 mg of the soap sample) was dissolved in 49.5 mL of water (containing 5 mg of F127 stabilizer). The mixture was homogenized in an ultratarrax (Polytron PT 10-35 GT, Kinematica, Switzerland) for 5 min at a speed of 15 000-20 000 rpm at 80 °C and immediately passed through a high-pressure homogenizer (Avestin, Germany) at a pressure of 10 000 psi for four to five passes at 60 °C. All resulting solutions were milky in appearance, indicating the formation of colloidal particles. The particles were sized by dynamic light scattering measurement (Coulter LS230 particle size analyzer). Cryo-TEM. A laboratory-built humidity-controlled vitrification system was used to prepare the samples for cryo-TEM. The humidity was kept close to 80% for all experiments, and the ambient temperature was 22 °C. A 4 μL aliquot of the sample was transferred onto a 300-mesh copper grid coated with a lacy Formvar-carbon film (ProSciTech, Thuringowa, Queensland). After 30 s of adsorption, the grid was blotted manually using Whatman 541 filter paper for 2-10 s. The blotting time was optimized for each sample. The grid was then plunged into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until required. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA) and a Tecnai 12 transmission electron microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. At all times, low-dose procedures were followed using an electron dose of 8-10 electrons/A˚2. Images were recorded using a Megaview III CCD camera and AnalySIS camera control software (Olympus) at magnifications between 70 000 and 110 000. Energy Dispersive X-ray Spectroscopy (EDAX). EDAX experiments were carried out at the Bio 21 Institute, Melbourne using a Tecnai T30F TEM fitted with an EDAX detector.
NMR Relaxation and Concentration Dependence Studies. For particle dispersions of Ln phytanates (Ln = Eu, Gd, Tb, and Dy), the longitudinal and transverse relaxation times, T1 and T2, were measured at 20 MHz (0.47 T) and room temperature with a MINISPEC from Bruker. For T1 measurements, the standard inversion-recovery (IR) method was used as the pulse sequence.46 The recycle delay time was set to 5 times the T1 value. Typically, 20 points were taken for each T1 measurement. For T2 measurements, the Carr-Pucell-Meiboom-Gill (CPMG) method was used.46 The longitudinal relaxivity, r1, and transverse relaxivity, r2, were then determined from the slope of the linear regression fits of 1/T1 and 1/T2 versus the Gd concentration, respectively. To determine the lanthanide ion concentration, 1 mL of each lanthanide phytanate dispersion was freeze dried and the lanthanide ion concentration was calculated from the dried mass. (45) Seddon, J. M.; Squires, A. M.; Conn, C. E.; Ces, O.; Heron, A. J.; Mulet, X.; Shearman, G. C.; Templer, R. H. Philos. Trans. R. Soc. London, Ser. A 2006, 364, 2635. (46) Modern NMR Techniques for Chemistry Research; Derome, A. E., Ed.; Pergamon Press: Oxford, U.K., 1987; Vol. 6, p 86.
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Article Table 1. Elemental Analysis of the Ln Phytanates Ln
%Ca
%Ha
%Lna
La Ce Nd Sm Eu Gd Tb Dy
65.96 (64.95) 10.97 (10.99) 12.9 (11.4) 65.39 (65.41) 10.86 (10.98) 13.3 (13.0) 63.56 (63.61) 10.64 (10.94) 13.4 (11.7) 64.12 (64.29) 10.60 (10.88) 13.8 (14.5) 65.47 (65.24) 10.78 (10.86) 14.0 (13.5) 65.47 (64.93) 10.98 (10.81) 14.4 (14.1) 64.64 (64.84) 10.64 (10.79) 14.5 (13.4) 66.12 (65.69) 10.90 (10.75) 14.8 (12.6) a The calculated values are given in parentheses.
phytanic acid/Ln
(H2O)n
3.05 2.98 2.96 2.93 2.89 2.95 2.85 2.94
2 1.5 3 2 1 1 1 0
Luminescent Behavior of Europium(III) Phytanate. Fluorescence emission spectra for europium(III) phytanate particle dispersions were measured on a Perkin-Elmer model LS-50B fluorimeter. Lanthanide ion concentrations were determined as for the NMR relaxation studies. Molecular Modeling. Avogadro 0.8.1 was used to obtain the energy-minimum conformation of Gd(III) phytanate (including one water of hydration) using the Ghemical force field with the steepest-decent algorithm.
Results and Discussion Elemental Analysis. The elemental analysis results for C, H, the lanthanide metals, and the hydration content of the synthesized lanthanide phytanates are shown in Table 1, together with the molar ratio of phytanic acid and lanthanide metal ions. The C, H, and lanthanide contents were consistent with the expected values from the Ln(C19H39COO)3 stoichiometry. The energyminimized structure for the molecule Gd(C19H39COO)3 (including one water of hydration) is shown in Figure 1B. With the exception of dysprosium phytanate, which is anhydrous, hydration numbers varied between 1 and 3 water molecules per lanthanide phytanate molecule. FTIR Spectroscopy. Infrared spectra for the Ln phytanates (Ln = La, Ce, Nd, Sm, Eu, Gd, Tb, and Dy) and for phytanic acid were recorded in the spectral region from 400 to 4000 cm-1. The absorption spectral frequencies for phytanic acid, lanthanum(III) phytanate, and gadolinium(III) phytanate are provided in Table 2. Because the FTIR spectra for the other lanthanide phytanates are very similar to that of gadolinium(III) phytanate, we focus our discussion on this spectrum. The disappearance of the strong peak corresponding to the carbonyl stretch at 1710 cm-1 in the IR spectrum indicates the successful reaction of the metal with phytanic acid. On ionization, this splits into two peaks at 1600-1500 and 1460-1400 cm-1 corresponding to the symmetric and antisymmetric vibrations, respectively, of COO-.22 In contrast, the absorption maxima corresponding to vibrations within the hydrocarbon chain region remain almost unchanged upon formation of the soap from the acid. These include the absorption bands of the C-H stretching vibrations, viz., the symmetrical stretching vibration of CH2 at 2860-2850 cm-1, the asymmetrical stretching vibrations of CH2 at 2920 cm-1 and of CH3 at 2960-2950 cm-1, and deformation bands (twisting and wagging) of CH3 at 1350-1050 cm-1. Absorption due to OH stretching modes occurs in the range of 3500-2500 cm-1 and was observed for gadolinium(III) phytanate as a broad band of medium intensity in this region. This supports a hydrate structure for gadolinium(III) phytanate or the presence of physisorbed water.47 Thermal Analysis. DSC measurements were carried out on all lanthanide phytanates. Samples were heated and cooled at (47) Binnemans, K.; Jongen, L.; Bromant, C.; Hinz, D.; Meyer, G. Inorg. Chem. 2000, 39, 5938.
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Table 2. Infrared Absorption Spectral Frequencies (cm-1) with Their Assignments for Phytanic Acid, Lanthanum(III) Phytanate, and Gadolinium(III) Phytanate infrared absorption spectral frequencies (cm-1) assignments
phytanic acid
O-H stretch
3600 2926 2569
C-H stretch in CdC-H CH3, C-H asymmetric stretch CH2, C-H asymmetric stretch CH2, C-H symmetric stretch OH stretch CdO stretch COO-, C-O asymmetric stretch CH2, deformation COO-, C-O symmetric stretch CH3, symmetric deformation progressive bands (CH2, twisting and wagging)
Gd phytanate
2920 ∼2961 ∼2864 3340
2927 3340
2926 2854 2674
2961 2920 2864 2722
2962 2921 2856 2722
1710
1746 (vw)
1463
1461 1457 1379 1258
a
1379 1297 1226 1170 C-O stretch 1285 OH, out-of-plane stretch 933 735 CH2, rocking COOH, bending mode 627 COOH, wagging mode 480 a Broad undefined peak in the region from 3000 to 2900 cm-1
Table 3. Transition Temperatures of the Ln Phytanates as Determined by DSC and Cross-Polarized Microscopy Ln
Tg/°C
Tm/°C
La Ce Nd Sm Eu Gd Tb Dy
-81 -81 -82 -81 -74 72 -78 -71
67-79 47-68 69-73 59-110 67-79 43-80 67-97 71-74
scan rates of 2.5, 10, and 22.5 °C min-1 in the range of -140 to 190 °C. All of the phytanates showed a glass transition at approximately -70 to -80 °C (Table 3). Peaks corresponding to a melting transition were not observed. This probably reflects both the broad temperature range over which melting occurs (as determined by visual observations) and the low enthalpy change associated with melting for systems that have disordered hydrocarbon chains. Approximate melting temperatures were therefore determined by visual observation using optical microscopy (Table 3). No trend is observed with ionic radius. We compare this with similar data obtained for the same series of lanthanide oleates where a decrease in the melting point was observed across the lanthanide series and explained by a consideration of the lanthanide ion size.34 However, we note that the highest melting point recorded was for samarium(III) phytanate, which is the only neat salt to form the hexagonal phase. The thermal behavior of the lanthanide phytanates was also investigated by TGA. In the TGA trace of air-stored lanthanide soaps, the dehydration of physisorbed water happens around 100 °C. Decomposition to volatiles such as alkanones and CO2, basic soaps, metal oxycarbonates, and metal oxides usually begins around 200 °C. The TGA traces for the lanthanide phytanates are, in general, very similar. Dehydration of physisorbed water occurs between 100 and 150 °C. At 400 °C, a substantial weight loss of 75-80% is observed for all samples, corresponding to thermal degradation of the system. We note that a weight loss 6244 DOI: 10.1021/la904006q
La phytanate
933 732 656
1718 (vw) 1740 (vw) 1436 1456 1379 1264 1170 935 732 642
peak at approximately 100 °C is observed for dried dysprosium(III) phytanate, which elemental analysis had indicated was anhydrous. This suggests that dysprosium(III) phytanate is hygroscopic. X-ray Diffraction of the Neat Samples. Room-temperature X-ray powder diffraction images were recorded for the Ln phytanates (Ln = La, Ce, Nd, Sm, Eu, Gd, Tb, and Dy). The spectrum for samarium(III) phytanate contained √ √ relatively broad peaks with d spacings in the ratio of 1:1/ 3:1/ 4 characteristic of a hexagonal columnar mesophase of lattice parameter 27.7 A˚ (Figure 2A). For all other metal soaps, the spectrum displayed one broad peak roughly coincident with the first-order hexagonal reflection of samarium(III) phytanate. For some of the lanthanide salts, an additional peak at approximately 4.6 A˚ was observed and is characteristic of molten hydrocarbon chains within the mesophase. We believe that all of the lanthanide phytanates form self-assembled liquid-crystalline mesophases with molten hydrocarbon chains. Although the peak at 4.6 A˚ was not observed for all samples, this may be due to the strong X-ray absorption of the lanthanide(III) ions, which can result in this peak being almost flat.22 A hexagonal columnar phase has previously been observed for Cu, Zn, Sr, and Ba metal soaps containing a monomethylbranched C16 chain.22 Again, for these systems, the ability to form a well-ordered hexagonal mesophase depended on the metal ion with Ca, Al, Ag, La, Ce, Tb, and Lu metal soaps forming a more disordered mesophase characterized by a single broad peak roughly coincident with the first-order hexagonal reflection. X-ray Diffraction of the Hydrated Samples. SAXS measurements were carried out on lanthanide phytanate samples following prolonged equilibration in excess water over a period of approximately 1 month. Note that throughout the text “neat” refers to a sample without water added and “hydrated” refers to a sample that has undergone prolonged equilibration in excess water. SAXS results showed that samarium, europium and terbium(III) phytanate distinct diffraction peaks in √ √ contain √ the ratio 1:1/ 3:1/ 4:1/ 7, again characteristic of a hexagonal columnar phase; see Figure 2B for terbium(III) phytanate. Neodymium(III) phytanate and dysprosium(III) phytanate Langmuir 2010, 26(9), 6240–6249
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Figure 3. (A) Lanthanum(III) phytanate and (B) europium(III) phytanate in excess water viewed with cross-polarized optical microscopy recorded at 25 °C. The lanthanum(III) phytanate sample was viewed 2 weeks after exposure to water.
Figure 2. X-ray diffraction pattern for (A) neat samarium(III) phytanate obtained using powder XRD (B) terbium(III) phytanate in excess water via using SAXS and (C) europium(III) phytanate in excess water via powder XRD. All images were obtained at √ room temperature. For all samples, diffraction peaks in the ratio 1:1/ 3:1/ √ 4 are observed, characteristic of a hexagonal columnar phase.
displayed broadened diffraction peaks; however, these were in a ratio consistent with the formation of a hexagonal phase. The remaining Ln phytanate samples (Ln = La, Ce, and Gd) displayed only one broad peak roughly coincident with the first-order reflection of the more ordered hexagonal phases. It should be noted, however, that these three samples were run using laboratory-based SAXS and a low overall diffraction intensity may have precluded the identification of higher-order diffraction peaks. Langmuir 2010, 26(9), 6240–6249
X-ray powder diffraction images were then recorded for the Ln phytanates (Ln = La, Ce, Nd, Sm, Eu, and Gd) after longer equilibration times in water of approximately 6 months. Images were obtained on the powder diffraction beamline of the Australian Synchrotron as described in the Experimental Section. For these samples, cerium, neodymium, samarium, and europium(III) √ phy√ tanate contain distinct diffraction peaks in the ratio of 1:1/ 3:1/ 4. The spectrum for europium(III) phytanate is shown in Figure 2C. Lanthanum and gadolinium(III) phytanate show two broadened peaks, one coincident with the first-order peak and the √ hexagonal √ second coincident with a combined 3 and 4 peak. We suggest that this represents a hexagonal phase with reduced long-range order and that very long equilibration times may be required for hexagonal-phase formation for lanthanum and gadolinium(III) phytanate. Figure 1C shows the putative hexagonal columnar structure formed with Gd ions arranged down the center of the cylinders and hydrocarbon chains radiating outward. The Gd(III) phytanate molecules have been rotated around the long axis of the cylinder to allow the hydrocarbon chains to fill the available space. Crossed-Polarized Microscopy of Lanthanide Phytanates in Excess Water. A few small, irregular spherulites were observed under crossed polarizers for lanthanum, neodymium, samarium, terbium, and dysprosium(III) phytanate (Figure 3A). We note that the texture is similar to that seen by Corkery for a Zn-branched chain soap that also forms a hexagonal columnar mesophase,22 and the result is therefore in agreement with SAXS and XRD results on hydrated samples. The small overall number of spherulites observed may reflect kinetic difficulties in forming the hexagonal mesophase discussed in the following section: microscopic textures were observed over the course of a week, and XRD results have indicated that the formation times for some of the hexagonal mesophases are on the order of months. In fact, the Zn texture observed by Corkery formed only after 2 years with the sample having previously displayed a homeotropic texture. Cerium, europium, and gadolinium(III) phytanate display a birefringent texture (Figure 3B). Although SAXS and XRD results indicate that these samples also form hexagonal mesophases, relatively short equilibration times necessary for the microscopy technique used may not have allowed the formation of this phase. Hexagonal-Phase Lattice Parameter. The effective lattice parameter of a hexagonal mesophase was calculated from the d spacing of the first-order reflection, obtained using XRD and √ SAXS with the equation a = (2/ 3)d. This is plotted as a function of the lanthanide(III) ionic radius in Figure 4. We use the ionic radii rLn3þ values that are reported in the literature for rare earth complexes with a coordination number of 6.48 Previous research (48) Shannon, R. D. Acta Crystallogr., Sect. A 1976, A32, 751.
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Conn et al. Table 4. Relaxivities of the Lanthanide Phytanates at 0.47 T (20 MHz) and Room Temperature relaxivity (mM-1 s-1)
particle size (nm) dispersions Eu phytanate Gd phytanate Tb phytanate Dy phytanate Magnevist
Figure 4. Plot of HII lattice parameter as a function of ionic radius for both hydrated and neat samples.
has suggested that the lattice parameter for both well-ordered and disordered hexagonal mesophases formed by metal soaps with monomethyl-branched C16 chains displays a linear increase with increasing ionic radius of the metal ion.22 Here we also observe an approximately linear increase in the lattice parameter with ionic radius for both neat and hydrated samples. A notable exception to the general trend is neat gadolinium(III) phytanate, which has an effective hexagonal lattice parameter of approximately 2.5 A˚ less than that expected from the linear trend. We note that gadolinium(III) phytanate was resynthesized and the same effective lattice parameter was obtained in both cases. In general, the lattice parameter of the hexagonal mesophases was found to be in the range 27-31 A˚. Molecular modeling of the structure of the Gd (III) phytanate molecule (shown in Figure 1B) indicates that the distance from the Gd headgroup to the end of the phytanyl chain is approximately 20 A˚. This suggests significant chain interdigitation in the hexagonal packing arrangement. The lattice parameter of the wet samples is 1 to 2 A˚ higher than those of the neat samples, indicating that these samples swell slightly in water. Samples are also more likely to form an ordered hexagonal phase in the presence of water. Although only samarium(III) phytanate forms a hexagonal phase in the absence of water, following prolonged equilibration in excess water cerium, neodymium, samarium, europium, and terbium(III) phytanate show distinct hexagonal peaks and lanthanum, gadolinium, and dysprosium(III) phytanate show broadened peaks consistent with the formation of a less-ordered hexagonal phase. Such behavior is unlikely to reflect changes in interfacial curvature (addition of water to the headgroup should reduce the desire for interfacial curvature with a concomitant decrease in the ease of formation of the hexagonal phase) and may reflect water stabilization of the internal cores of the columnar micelles in the hexagonal array. Note that in the presence of water this phase may also be known as an “inverse” or “reversed” hexagonal mesophase. We note that for many samples the lattice parameter obtained using XRD is higher than that obtained using SAXS and that more samples displayed distinct hexagonal peaks using XRD. Because XRD measurements in excess water were run approximately 5 months after the SAXS measurements, this suggests that the equilibration times for the formation of an ordered hexagonal mesophase in excess water are on the order of months. The formation of a well-ordered hexagonal phase is less likely for lanthanum, gadolinium, and dysprosium(III) phytanate, which were the only samples not to show distinct hexagonal diffraction peaks using either powder XRD or SAXS. For 6246 DOI: 10.1021/la904006q
D10 451 462 458 461
D50 626 645 629 639
D90 876 940 882 911
r1 (mM-1 s-1) r2 (mM-1 s-1) 0.04 2.40 0.02 0.11 4.90
0.42 5.45 1.23 2.05 6.26
lanthanum and dysprosium(III) phytanate, such behavior may reflect the large and small ionic radii of these lanthanides, respectively, which, for geometric reasons, could prevent the adoption of the long cylinders associated with the hexagonal phase. Gadolinium(III) phytanate has a lower-than-expected d spacing in the absence of water. However, it falls within the linear trend for d spacing in the presence of water, so the result is more difficult to explain using geometric arguments. Dispersions of Ln Phytanates. In previous work by us on lanthanide oleates, we have described the suitability of salts containing a paramagnetic lanthanide ion (gadolinium(III), dysprosium(III), and terbium(III)) as a contrast agent for magnetic resonance imaging.34,49 For biomedical applications, such systems must be dispersed into submicrometer-sized particles for delivery within the body. We have previously shown that gadolinium(III) oleate, which forms a gel-like lamellar phase at room temperature (i.e., with disordered headgroups but chains locked into an all-trans conformation), may be successfully dispersed into submicrometer particles.34 It may also be incorporated within a Myverol bicontinuous cubic phase, which not only results in a dramatically increased relaxivity but also incorporates favorable properties of the self-assembled phase, such as biocompatibility and the potential for targeted delivery.49 The formation of a self-assembled hexagonal columnar mesophase by several lanthanide phytanates in the presence of water potentially allows us to directly access these favorable properties of the self-assembled phase without the requirement of doping. We have therefore dispersed europium, gadolinium, terbium, and dysprosium(III) phytanate into submicrometer-sized colloidal particles using the method described in the Experimental Section. Additionally, europium, gadolinium, terbium, and dysprosium(III) phytanate have been doped within phytantriol cubosomes at a loading of 10 wt %. The particle size distributions for these dispersions are given in Tables 4 and 5. Note that for biomedical applications bulk phases must be dispersed in physiological buffers. We have previously observed that such buffers can cause slight structural changes in the dispersed phases of some systems (unpublished data). Cryo-TEM Measurements on Dispersed Samples. CryoTEM measurements were conducted on dispersed samples of europium, gadolinium, terbium, and dysprosium(III) phytanate. Because of the long equilibration times associated with the formation of the hexagonal phase in water for these samples, experiments were run both on freshly prepared dispersions and on samples that had been allowed to equilibrate in water over a period of several months before dispersion. We observed no difference in the dispersed structure of samples prepared using either of the two preparation methods. The dispersed structure of all four samples was similar, consisting mainly of opaque particles and spidery crystals, which appear black on the TEM images (Figure 5). The opaque particles were generally spherical, although some faceted particles were (49) Liu, G.; Conn, C. E.; Drummond, C. J. Langmuir 2009, 10.1021/la902845j.
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Table 5. Relaxivities of the Lanthanide Phytanates in Phytantriol at 10 wt % at 0.47 T (20 MHz) and Room Temperature particle size (nm) dispersions Eu phytanate Gd phytanate Tb phytanate Dy phytanate
D10 461 98 454 447
D50 642 240 616 698
D90 999 449 861 1116
relaxivity (mM-1 s-1) r1 (mM-1 s-1) r2 (mM-1 s-1) 0.534 6.39 0.22 2.33
0.346 5.44 1.79 2.85
observed. A wide size variation was observed from >2 μm down to 50 nm. This distribution is larger than that calculated using DLS, suggesting that particles may aggregate with time. In fact, white precipitated material was visually observed in most samples. The spidery crystals observed were sometimes, but not always, associated with the opaque particles. This tended to be sample-dependent; for europium(III) phytanate, the small crystals observed were not often associated with the opaque particles (Figure 5A), whereas gadolinium(III) phytanate particles were often heavily coated (Figure 5B). The opaque particles were very beam-sensitive, which is common for lanthanide-containing systems.49 However, for some samples some internal structure could be resolved before beam damage occurred (Figure 5C). It is therefore possible that such systems retain some order in the dispersed state. SAXS experiments were carried out on dispersed samples at the SAXS/WAXS beamline of the Australian Synchrotron with no diffraction peaks resolved. We note that this does not necessarily indicate the absence of order in the dispersed systems because the weak scattering intensity associated with dispersed samples, combined with the highly X-ray absorbing nature of the lanthanide ions, could preclude peak identification. The spidery crystals observed are unlikely to be pure lanthanide phytanates, which are noncrystalline even in the nonhydrated state. However, previous research has shown that gadolinium can leach out into aqueous solution, and energy-dispersive X-ray spectroscopy (EDAX) experiments carried out at the Bio21 Institute, Melbourne confirmed the presence of gadolinium in a sample of the crystalline material (Figure 6). We therefore suggest that the spidery crystals observed may be insoluble gadolinium species that have formed in water. Leaching of very toxic free gadolinium would make these systems unsuitable for biomedical applications. However, the fact that the particles remain stable in water for many months and effectively swell very little and do not undergo a phase change means that any lanthanide leaching must be relatively small. We have previously shown that the incorporation of gadolinium(III) oleate within a dispersed bicontinuous cubic phase increases the relaxivity of the system. We have therefore incorporated europium, gadolinium, terbium, and dysprosium(III) phytanate at a loading of 10 wt % within phytantriol, which forms a QIID bicontinuous cubic phase in excess water at room temperature. Pure phytantriol may be easily dispersed into relatively stable, submicrometer cubic-phase particles known as cubosomes.50 Upon addition of 10 wt % Ln phytanate (Ln = Gd, Tb, and Dy), the dispersed systems consist of a mixture of cubosomes, hexosomes, liposomes, and/or large irregular faceted and striated particles (Figure 7A-D, respectively). The doped systems are more difficult to disperse than pure phytantriol; a white precipitate was visually observed in most samples, and particles were “sticky” and often clumped together. Phytantriol doped with 10 wt % gadolinium(III) phytanate contains liposomes, large irregularly faceted and striated particles, and cubosomes with and without bubbles of water around (50) Dong, Y. D.; Larson, I.; Hanley, T.; Boyd, B. J. Langmuir 2006, 22, 9512.
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Figure 5. Cryo-TEM images of dispersed samples of (A) europium(III) phytanate and (B, C) gadolinium(III) phytanate.
the edges. For terbium(III) phytanate-doped systems, we also observe hexosome-like structures in addition to liposomes and cubosomes. The dysprosium(III) phytanate-doped system also contains a mixture of hexosomes and cubosomes. This system displays a very wide variety of particles sizes with cubosomes observed up to a micrometer in diameter. In addition, for this system, many particles have spidery black crystals incorporated within them (Figure 7B). For all samples, the volume of crystalline material observed was much less than that seen for the pure dispersed phase. The structure of dispersed phytantriol doped with 10 wt % europium(III) phytanate differs from that of the other three samples with no obvious hexosomes, cubosomes, or liposomes. Rather, we observe opaque, featureless spherical particles that are mostly
