Article pubs.acs.org/Langmuir
Evaluation of Gd-DTPA-Monophytanyl and Phytantriol Nanoassemblies as Potential MRI Contrast Agents Abhishek Gupta,†,‡ Liliana de Campo,†,§ Beenish Rehmanjan,† Scott A. Willis,‡ Lynne J. Waddington,∥ Tim Stait-Gardner,‡ Nigel Kirby,⊥ William S. Price,‡ and Minoo J. Moghaddam*,† †
CSIRO, Manufacturing Flagship, P.O. BOX 52, North Ryde, New South Wales 1670, Australia Nanoscale Organisation and Dynamics Group, School of Science and Health, University of Western Sydney, Penrith, New South Wales 2751, Australia § Bragg Institute, ANSTO, Lucas Heights, New South Wales 2234, Australia ∥ CSIRO, Manufacturing Flagship, 343 Royal Parade, Parkville, Victoria 3052, Australia ⊥ Australian Synchrotron, 800 Blackburn Rd, Clayton, Victoria 3168, Australia ‡
ABSTRACT: Supramolecular self-assembling amphiphiles have been widely used in drug delivery and diagnostic imaging. In this report, we present the self-assembly of Gd (III) chelated DTPA-monophytanyl (Gd-DTPA-MP) amphiphiles incorporated within phytantriol (PT), an inverse bicontinuous cubic phase forming matrix at various compositions. The dispersed colloidal nanoassemblies were evaluated as potential MRI contrast agents at various magnetic field strengths. The homogeneous incorporation of Gd-DTPA-MP in PT was confirmed by polarized optical microscopy (POM) and synchrotron small-angle X-ray scattering (SAXS) of the bulk phases of the mixtures. The liquid crystalline nanostructures, morphology, and the size distribution of the nanoassemblies were studied by SAXS, cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS). The dispersions with up to 2 mol % of Gd-DTPA-MP in PT retained inverse cubosomal nanoassemblies, whereas the rest of the dispersions transformed to liposomal nanoassemblies. In vitro relaxivity studies were performed on all the dispersions at 0.54, 9.40, and 11.74 T and compared to Magnevist, a commercially available contrast agent. All the dispersions showed much higher relaxivities compared to Magnevist at both low and high magnetic field strengths. Image contrast of the nanoassemblies was also found to be much better than Magnevist at the same Gd concentration at 11.74 T. Moreover, the GdDTPA-MP/PT dispersions showed improved relaxivities over the pure Gd-DTPA-MP dispersion at high magnetic fields. These stable colloidal nanoassemblies have high potential to be used as combined delivery matrices for diagnostics and therapeutics.
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INTRODUCTION Magnetic resonance imaging (MRI) is one of the most prominent imaging modalities in diagnostic clinical medicine. It is a noninvasive technique which offers excellent spatial resolution and superior soft-tissue contrast without using any harmful ionizing radiation. In MRI, the three parameters which largely conduce to image contrast are the spin density, longitudinal relaxation time (T1), and transverse relaxation time (T2).1−3 To enhance tissue contrast, the relaxation times of the water protons within the tissue can be altered by injecting chemicals known as contrast enhancement agents or contrast agents (CAs). The extent to which a CA modulates the relaxation times of the water protons is termed relaxivity (r1 or r2) and is given by the following: © 2015 American Chemical Society
ri =
Δ(1/Ti ) ; [CA]
i = 1, 2
(1)
where [CA] is the concentration of the contrast agent. Most of the commercially available CAs are polyaminopolycarboxylic complexes of Gd (III) ions which induce positive contrast in a T1-weighted image by increasing the signal intensity. These low molecular weight CAs are small, fast reorientating molecules with restricted specificity and targeting ability and relatively moderate relaxivities at both low and high Received: November 20, 2014 Revised: January 9, 2015 Published: January 12, 2015 1556
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Langmuir magnetic field strengths. This has led to the development of new high molecular weight CAs, which can extravasate from the leaky vasculature in tumors and be retained there due to their ineffective lymphatic drainage, an effect known as the enhanced permeation and retention (EPR).4 Along with improved specificity, high molecular weight CAs also provide superior contrast (better relaxivities) compared to their low molecular weight counterparts.5,6 The high molecular weight CAs are made either by attaching the low molecular weight Gd-chelates to large molecules such as proteins and polymers7,8 or by imparting the amphiphilic character to the Gd-chelates (headgroup), by attaching one or more alkyl or phospholipid chains to them, and letting them self-assemble.9,10 The high molecular weight CAs formed by the latter method are called supramolecular CAs. In general, lipid-based amphiphilic molecules, when dispersed in an aqueous solution, can self-assemble to form a variety of lyotropic liquid crystalline structures ranging from micelles, liposomes to highly ordered lamellar (1D), hexagonal (2D) and cubic mesophases (3D).11,12 These highly ordered liquid crystalline structures, inverse bicontinuous cubic phases (V2) in particular, have attracted a lot of attention as stable drug delivery systems and thus also possess the potential to be used as carriers for MRI CAs.13−15 These structures offer high payloads of the coordinated Gd (III) ions and their extensive water channels may allow efficient water exchange between the inner sphere and outer sphere surrounding the Gd (III) ions, resulting in better relaxivities. To this end, we have previously reported on the synthesis, characterization and self-assembly of the mono/bis oleyl and phytanyl conjugates of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) chelated to the Mn (II) and Gd (III) metal ions.9,10,16,17 Also, we recently reported on the supramolecular nanoassemblies of Gd-DTPAmonooleyl (Gd-DTPA-MO) incorporated within the inverse bicontinuous cubic phase based on glycerol monooleyl (GMO).18 It was shown that >5 mol % Gd-DTPA-MO disrupted the cubic phase geometry of GMO. Also, when dispersed in an aqueous solution, only the dispersions with ≤1 mol % Gd-DTPA-MO retained the colloidal inverse bicontinuous cubic phase nanoassemblies, called cubosomes. However, the relaxivities of all the Gd-DTPA-MO/GMO nanoassemblies were found to be higher than Magnevist, a commercially available contrast agent, at both low (0.54 T) and high (≥ 7 T) magnetic field strengths. Here we extend our investigation to the nanoassemblies of Gd-DTPA-MP (containing a branched alkyl chain) incorporated within 3,7,11,15tetramethyl-1,2,3-hexadecanetriol or phytantriol (PT), a cubic phase forming amphiphile (Figure 1). Phytantriol is a common active ingredient used in the cosmetics industry to improve moisture retention of skin and hair and enhance penetration of amino acids and vitamins. Since the phase behavior of PT was reported to be very similar to that of GMO by Barauskas and Landh in 2003,19 this amphiphile has gained a lot of interest in biomedical research. PT, like GMO, can be dispersed in an aqueous solution to form stable cubosomal nanoassemblies, thus allowing the incorporation of both hydrophobic and hydrophilic molecules. However, due to the absence of the ester bond in PT (compared to GMO), it is more stable at low and high pH and therefore has replaced GMO in many applications including drug delivery.20−22 Also contrary to GdDTPA-MO, which was shown to form mostly rod shaped micellar nanoassemblies,18 Gd-DTPA-MP was recently re-
Figure 1. Chemical structures of (a) Gd-DTPA-MP and (b) phytantriol.
ported to form uniform unilamellar liposomal nanoassemblies, which shows that the latter induces less positive curvature (i.e., less curvature towards the hydrophobic chains). This suggests that a higher concentration of Gd-DTPA-MP should be able to be incorporated in PT without disrupting its cubic phases. Furthermore, Gd-DTPA-MP was shown to possess low (in vitro) cytotoxicity (lower than Gd-DTPA-MO) and high relaxivity at low magnetic field strength.17 The aim of this study was to prepare nanoassemblies with varying concentrations of Gd-DTPA-MP in PT, characterize their nanostructures, and investigate their potential as MRI CAs at various magnetic field strengths. Water penetration studies using cross-polarized optical microscopy were employed to examine the phase behavior of the bulk samples and check the highest concentration of Gd-DTPA-MP that can be incorporated in the PT matrix without disrupting its cubic nanostructure. The lyotropic phase behavior of the GdDTPA-MP/PT mixtures and dispersions was studied by synchrotron small-angle X-ray scattering (SAXS). The morphology, shape, size, and internal structures of the nanoassemblies were studied using cryogenic transmission electron microscopy (cryo-TEM). The size distributions of the nanoassemblies were measured by dynamic light scattering (DLS). The in vitro relaxivities were measured at low (0.54 T) and high magnetic field strengths (9.40 and 11.74 T) and compared to Magnevist. In vitro imaging was performed to view and compare the contrast enhancement in T1- and T2weighted MRI images provided by Gd-DTPA-MP/PT nanoassemblies with that of Magnevist.
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EXPERIMENTAL SECTION
All chemicals were used as obtained without further purification. Phytantriol (96%) was provided by DSM Nutritional Products, Grenzach-Wyhlen, Germany. Pluronic F127 was purchased from BASF, Ludwigshafen, Germany. Magnevist was a gift from Bayer HealthCare Pharmaceuticals, Sydney, Australia. Organic solvents (of analytical grade) were purchased from Merck Australia. H2O was sourced from a Milli-Q Plus Ultrapure water system (Millipore, Australia). Gd-DTPA-MP was synthesized as previously reported.16 Preparation of Gd-DTPA-MP and PT Mixtures. Appropriate amounts of phytantriol and Gd-DTPA-MP were weighed, dissolved in t-butanol and thoroughly mixed by vortexing and ultrasonication. The mixed samples were then freeze dried overnight to obtain binary Gd1557
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Figure 2. Water penetration scans of the (a) 1, (b) 10, and (c) 20% Gd-DTPA-MP/PT mixtures viewed without (top) and with (bottom) crossed polarizers at 25 °C, magnification ×100. In the 20% mixture, the formation of cubic phase was disrupted at high dilutions as shown by the formation of the lamellar texture and Myelin tubules at the water−amphiphile interface. DTPA-MP/PT mixtures as clear oils. The mixtures investigated in this study contained 1, 2, 3, 5, 10, and 20 mol % Gd-DTPA-MP in PT. Water Penetration Studies Using Polarized Optical Microscopy. Water penetration studies using polarized optical microscopy were employed as an initial assessment of the lyotropic phase behavior of the mixtures. Small amounts of Gd-DTPA-MP/PT mixtures were placed between a microscope slide and coverslip, which was placed onto a Linkam LTS-120 hot stage (Linkam Scientific Instruments Ltd., Surrey, England). Water was added to the edge of the coverslip which penetrated by capillary action between the two glass surfaces, thus creating a concentration gradient from 100% water at the edge of the coverslip to 100% amphiphile in the middle of the sample. The mesophases were identified by their characteristic textures observed using an Olympus GX51 inverted optical microscope (Olympus Australia Pty. Ltd., Melbourne, Australia) in the presence or absence of cross polarizing lenses. Images were captured with a Nikon DS-Ri1 camera (Coherent Scientific Pty. Ltd. Inc., South Australia). Dispersion of Mixed Amphiphiles to Nanoparticles. Colloidal dispersions of the Gd-DTPA-MP/PT mixtures were prepared by the addition of Pluronic F127 (Poloxamer 407) (10 wt % of the GdDTPA-MP/PT mixture), a non ionic polymeric stabilizing agent, in excess Milli-Q water followed by vortexing, homogenizing with a CAT X120 handheld homogenizer with T6 shaft (CAT Scientific Inc., California, U.S.A.) at 33 000 rpm for ∼1−2 min and further bathultrasonication. The particle sizes were measured by dynamic light scattering measurements using a Malvern Zetasizer Nano ZS (Malvern Instruments; Worchestershire, U.K.) at 25 °C. Synchrotron Small Angle X-ray Scattering. The lyotropic liquid crystalline structures of the bulk phases in excess water and dispersions were determined by SAXS at the Australian Synchrotron Radiation Source, Melbourne. The diffraction patterns were transmission corrected and background subtracted. Sample-detector distance calibration was performed using silver behenate as the standard. The q (scattering vector) range was 0.01−0.47 Å−1 for both bulk measurements and dispersions. For bulk phases, 70 wt % (excess) water was added to the mixtures and equilibrated for 24−48 h. The equilibrated bulk phases were then placed into button sample cells designed and made at the Australian National University for small sample volumes with high water sealing capacity. For the analysis of dispersions, a multicapillary holder designed and built at the Synchrotron was used. The button cells and capillary holder interchangeably fit into a setup that was temperature controlled with both water and air. The temperatures of the sample during analyses were monitored by inserting a thermocouple in the metal block very close to the samples (for bulk) or in the water filled capillary in the
sample holder (for dispersions). 2-D scattering images were radially averaged to conventional scattering plots using the Scatter Brain program. Scattering intensities I were plotted as a function of q using OriginPro 9.1 (Originlab Corporation, Massachusetts, U.S.A.), and manually indexed. Cryogenic Transmission Electron Microscopy. For cryo-TEM, a 4 μL aliquot of the sample was applied to a 300-mesh copper grid coated with lacey formvar-carbon film (ProSci Tech, Thuringowa, Queensland, Australia). After 30 s adsorption time, the grid was blotted for 2−10 s and immediately plunged into liquid ethane. The humidity and temperature in the plunging device were 80% and 22 °C, respectively. All the frozen grids were stored in liquid nitrogen until required. The frozen grids were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, U.S.A.) and Tecnai 12 Transmission Electron Microscope equipped with a FEI 4kx4k Eagle CCD camera (FEI, Eindhoven, Netherlands) at an operating voltage of 120 kV. Relaxivity Studies. NMR relaxation time measurements were performed at 25 °C on three different spectrometers: a Maran Ultra 23.4 MHz (0.54 T) benchtop spectrometer (Oxford Instruments Molecular Biotools Ltd, Oxon, U.K.) and Bruker Avance 400 (9.40 T) and 500 MHz (11.74 T) spectrometers (Bruker Biospin Corporation, Ettlingen, Germany). T1 measurements were performed by using the inversion recovery method23 and the CPMG technique24,25 was used for T2 measurements. Samples were placed in Sigma-Aldrich NMR tubes (Sigma-Aldrich, 10 mm precision 513-1PS-8) for the 23.4 MHz spectrometer and straight capillary tubes (Wilmad LabGlass, 529-D) in Wilmad NMR tubes (Wilmad Lab Glass, 5 mm 528-PP-7) for other spectrometers. The samples were equilibrated for 5 min at the set temperatures before conducting the measurements. The measurements were carried out without locking the field-frequency. The recycle delay was set to ≥5T1 and the signal was averaged over four scans for both inversion recovery and CPMG measurements. All data fitting for T1 and T2 measurements was performed using OriginPro 9.1. For each dispersion, three to four dilutions were prepared by adding Milli-Q water, and the relaxivity was calculated from the slope of the inverse relaxation time versus Gd concentration. The exact concentrations of Gd (III) ions in the dispersions were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) using a Varian Vista MPX (Varian, California, U.S.A.) according to the method described before.17 In Vitro MRI Studies of the Aqueous Dispersions. All the dispersions were diluted to have the same Gd concentration (0.4 mM) and added to straight capillary tubes, which were placed in 5 mm Wilmad NMR tubes. MRI studies were performed on these samples at 25 °C on the Bruker Avance 500 MHz spectrometer with a Micro5 1558
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Langmuir probe and 5 mm 2H/1H insert. A Gradient-echo Fast Low Angle SHot (FLASH) MRI pulse sequence was used with one scan using a 30° excitation pulse.26 The acquisition parameters were: a repetition time (TR) of 100 to 500 ms, an echo time (TE) of 20 to 100 ms, a slice thickness of 0.3 mm, and a field of view of 15 × 15 mm2 digitized into a 180 × 180 matrix resulting in an in-plane resolution of 83 μm per pixel. Images were processed and exported using Bruker Paravision 5.1 software.
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RESULTS AND DISCUSSION Structure of the Bulk Phases (Mixtures). Water Penetration Studies Using Polarized Optical Microscopy. Polarized optical microscopy was employed as a preliminary test to determine the highest concentration of Gd-DTPA-MP that can be added to PT without disrupting its cubic nanostructure. Under crossed polarizers, isotropic mesophases (e.g., micellar and cubic) appear dark and show no birefringence, whereas anisotropic hexagonal and lamellar phases change the polarization of the polarized light and therefore show characteristic textures. As shown in Figure 2a,b, mixtures with up to 10 mol % Gd-DTPA-MP displayed isotropic dark bands with a smooth interface between the amphiphile and water, thus indicating that the inverse bicontinuous cubic phase structure of PT was retained. The uniformity of the large single phase region, without indication of any regional birefringence on the slide, confirmed the homogeneous incorporation of Gd-DTPA-MP within the cubic phases of PT. In the 20 mol % Gd-DTPA-MP/PT mixture, however, the formation of V2 phases was disrupted as indicated by the formation of the lamellar Myelin tubules (Figure 2c), suggesting a phase transformation at higher concentration of Gd-DTPA-MP in PT.27 Therefore, mixtures (and dispersions) with ≤20 mol % Gd-DTPA-MP in PT were used for further characterization and analysis. As expected because Gd-DTPA-MP forms structures with relatively less positive curvature, the retainment of cubic phases in mixtures with up to 10 mol % of Gd-DTPA-MP in PT is an improvement over the Gd-DTPA-MO/GMO system, in which mixtures with only up to 5 mol % Gd-DTPA-MO retained the cubic phases.18 Synchrotron Small Angle X-ray Scattering of the Bulk Phases. Synchrotron SAXS analysis was performed on the bulk phases of the Gd-DTPA-MP/PT mixtures to explore the liquid crystalline structures more precisely and to check whether the two amphiphiles had been homogeneously mixed into one lyotropic mesophase, as indicated by polarized optical microscopy. The results are shown in Figure 3. The plots were stacked by manually offsetting the intensities by a constant factor to better visualize the data. For pure phytantriol, seven Bragg diffraction peaks in the ratio √2:√3:√4:√6:√8:√9:√10 were observed corresponding to the inverse cubic phase of Pn3m space group. The lattice parameter a was calculated to be 66.0 ± 0.5 Å, which is consistent with the literature.19 The addition of 1 mol % GdDTPA-MP resulted in coexisting Pn3m (dotted arrows) and Im3m (solid arrows) mesophase structures as clear from eight peaks (dotted arrows) of the ratio √2:√3:√4:√6:√8:√9:√10:√12 and six peaks (solid arrows) of the ratio √2:√4:√6:√8 (missing): √10:√12:√14, respectively. The lattice parameter of the Pn3m cubic space group increased to 75.0 ± 0.5 Å and for Im3m a was calculated to be 98.8 ± 0.5 Å respectively. More than 1 mol % Gd-DTPA-MP in PT induced a complete
Figure 3. Synchrotron SAXS data of the bulk phases of phytantriol and different Gd-DTPA-MP//PT mixtures saturated with 70 wt % (excess) water at 25 °C. Phytantriol displayed a Pn3m cubic phase, the 1% Gd-DTPA-MP mixture displayed coexisting Pn3m (dotted arrows) and Im3m (solid arrows) cubic phases, and the 2−5% mixtures displayed Im3m cubic phases. (Note: Intensities are offset by a constant factor to better display the data).
transition from Pn3m to Im3m cubic phases, and the lattice parameters were found to strongly increase with increasing concentration of the negatively charged Gd-DTPA-MP. The lattice parameters of the 2, 3, 4, and 5% mixtures, calculated from at least six peaks, were found to be 114.8 ± 0.5, 125.2 ± 0.5, 165.3 ± 0.5, and 196.0 ± 0.5 Å respectively. The 10% mixture displayed an Ia3d cubic phase (data not shown), which indicates that 70% water was not sufficient to fully hydrate the lipid mixture. Similar trends (phase transition and increasing lattice parameters) were observed in our previous study on the Gd-DTPA-MO/GMO binary mixtures18 and also have been reported for other anionic additives in the phytantriol/F127 system.28 The presence of a single phase and the systemic increase in the lattice parameter with increasing Gd-DTPA-MP concentration further supports that Gd-DTPA-MP is incorporated homogeneously within the PT cubic phase with no phase segregation. Structure of the Dispersions. The above results confirmed that Gd-DTPA-MP/PT mixtures self-assemble to form cubic nanostructures. However, to investigate their potential as MRI CAs, they need to be dispersed in aqueous solutions to submicron sized nanoassemblies. This was achieved as described in the Experimental Section. The dispersions with up to 2 mol % Gd-DTPA-MP appeared milky, 3−5 mol % were slightly translucent and 100 mol % was clear. All the dispersions were found to be stable for months at room temperature, both visually and as determined by SAXS analysis (below). Only slight “creaming” was noticed for ≤2% Gd-DTPA-MP/PT dispersions, which was reversible on shaking. The size distributions and polydispersity indices (PDIs) of the dispersions, as determined by DLS, are given in Table 1. The average sizes (radii) were found to be comparable (around 100 nm) for all the dispersions. However, the PDI was found to increase from PDI < 0.3 (typical of samples with low polydispersity) for the dispersions with ≤2 1559
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Table 1. Longitudinal and Transverse Relaxivities (in mM−1 s−1), Average Hydrodynamic Radii and Polydispersity Indices (PDI) of the Dispersions with Varying Gd-DTPA-MP Concentrations in PT at 25 °C 0.54 T
a
9.40 T
11.74 T
Mol% of Gd-DTPA-MP in PT
r1
r2
r2/r1
r1
r2
r2/r1
r1
r2
r2/r1
z avg (nm)
PDI
1 2 3 5 10 20 100 Magnevist
14.9 13.9 14.2 15.1 15.5 15 17 3.8a
17.8 16.4 16.1 17 17.5 15.9 20.1 4.2a
1.2 1.2 1.1 1.1 1.1 1.1 1.2 1.1
5.7 5.7 5.8 6.0 5.8 5.0 5.5 3.9
28.6 17.1 19.2 24.3 26.7 20.2 34.9 5.0
5.0 3.0 3.3 4.1 4.6 4.0 6.3 1.3
5.3 5.1 5.1 5.1 5.0
33.8 21.9 19.8 22.1 25.5
6.4 4.3 3.9 4.3 5.1
4.6 3.8
40.2 4.9
8.7 1.3
93.4 108.5 103.6 102.4 110.6 89.6 53.0
0.19 0.29 0.43 0.48 0.5 0.49 0.27
Values taken from literature (at 0.47 T).34
mol % Gd-DTPA-MP to PDI ≈ 0.5 for the rest of the dispersions (indicating a polydisperse sample). The lyotropic liquid crystalline structures of the dispersions and their morphology (size, shape, and internal structures) were analyzed by Synchrotron SAXS and cryo-TEM as discussed in the following sections. Small Angle X-ray Scattering of the Dispersions. Synchrotron SAXS data of the dispersions at 25 °C are shown in Figure 4. Phytantriol formed inverse cubosomes of
in the dispersion. The lattice parameter of the resulting Im3m phase was found to increase to 102.2 ± 0.5 Å, which is a little smaller than the one observed in the bulk phases (114.8 ± 0.5 Å). At higher Gd-DTPA-MP concentrations, the dispersions did not show any sharp peaks, but only a steep decay at low q and a broad shoulder at higher q values, consistent with the formation of liposomal particles. The above results demonstrate that at small Gd-DTPA-MP content (≤ 2 mol %), the cubic phases were retained (consistent with the bulk phases). Slight variations observed in the lattice parameters of the dispersions compared to the bulk phases are likely due to the counteracting effects of the negatively charged Gd-DTPA-MP and the neutral Pluronic F127, which was used as the steric stabilizer, within the membranes. Also, Pluronic F127 is well-known to make membranes less negatively curved for glycerol monooleate.29,30 More than 2 mol % of Gd-DTPA-MP induced a phase transition from cubosomes to liposomes, while the bulk phase with up to 10% Gd-DTPA-MP had retained cubic symmetry in excess water, albeit with strongly increasing lattice parameter and therefore water channel size. We believe that at a higher Gd-DTPA-MP content, the water channels were swollen enough to incorporate significant amounts of the stabilizer which lead to the transition from cubosomes to liposomes. These results are in agreement with cryo-TEM data on the dispersed samples presented below. To check the stability of the dispersions, SAXS analyses were repeated on all the dispersions after eight months and the results showed no noticeable change in the structures or lattice parameters (data not shown). Cryogenic Transmission Electron Microscopy. Cryo-TEM was used to directly observe the nanostructures in the dispersions without any staining, fixation, or drying of the sample, as explained in the Experimental Section. Cryo-TEM analyses were conducted on the 1, 2, 3, 5, and 10% Gd-DTPAMP dispersions. The micrographs of 1, 2, 5, and 10% are shown in Figure 5. The dispersions with up to 2% Gd-DTPA-MP in PT displayed mostly cubosomes with occasional unilamellar liposomes, which is consistent with the SAXS analysis. The rest of the dispersions lacked cubosomes and only polydisperse unilamellar liposomes were observed. The average sizes of the nanostructures are in agreement with the DLS results (Table 1). Moreover, the polydispersity was found to increase with increasing Gd-DTPA-MP, also consistent with the DLS results. SAXS and cryo-TEM results for the pure Gd-DTPA-MP dispersion, presented in an earlier publication,17 showed the formation of unilamellar liposomes as well.
Figure 4. Synchrotron SAXS data of the dispersions of different GdDTPA-MP/PT mixtures at 25 °C. The diffraction peaks ratio of the 1% dispersion corresponds to coexisting Im3m (solid arrows) and Pn3m (dotted arrows) cubic phases, the 2% dispersion displayed Im3m cubic phases and the rest showed no notable sharp peaks. (Note: Intensities are offset by a constant factor to better display the data).
Pn3m symmetry with a lattice parameter of 69.4 ± 0.5 Å, which is slightly larger than the bulk phase lattice parameter. For the 1% dispersion, coexisting inverse bicontinuous cubic nanoassemblies with Pn3m and Im3m cubic symmetries were observed. The peak intensities of the six peaks (dotted arrows) of the Pn3m cubic space groups were much higher than the three peaks (solid arrows) of Im3m. Their respective lattice parameters were found to be 74.9 ± 0.5 and 98.8 ± 0.5 Å, which are similar to the bulk phases of 1% mixture. For 2 mol % Gd-DTPA-MP in PT, the full transition from Pn3m to Im3m cubic symmetry, which was observed in the bulk, also occurred 1560
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DTPA-MO/GMO dispersions at low magnetic field strengths.18 For transverse relaxivities, the 100% Gd-DTPAMP dispersion showed higher r2 values than other Gd-DTPAMP/PT dispersions at both low and high magnetic field strengths (Figure 6b). All the dispersions showed better relaxivities (both r1 and r2) than the commercially available, low molecular weight and non-assembling contrast agent, Magnevist, at both low and high magnetic field strengths. However, no notable difference in relaxivities was observed between the nanoassemblies with cubosomes and liposomes. r1 was found to decrease and r2 increase with increasing magnetic field strength, resulting in an overall increase in the r2/r1 ratio from ∼1.1 at 0.54 T to ≥4 at 11.74 T for all the dispersions (Table 1). It should be noted that for Gd-based (positive) MRI contrast agents, a low r2/r1 ratio is desired because otherwise increasing the dose of the contrast agent to enhance T1weighted contrast will have limited effect due to the large counteracting r2 effect.31 To explain the above trends according to the structures of the dispersions, it is important to consider the mechanisms for the relaxation enhancement by paramagnetic contrast agents. The dominant mechanism is the “through space” dipole−dipole interactions between the unpaired electron spins of the Gd(III) ion and the nuclear spins of the directly coordinated water protons. These coordinated water protons (from the first coordination sphere around the metal ion) are then exchanged with the bulk solvent protons to propagate this effect further.32 The dipole−dipole interactions, as given by the so-called Solomon−Bloembergen−Morgan (SBM) equations, are affected by the reorientation of the molecule (characterized by the reorientational correlation time, τR), electron spin relaxation time (Te) and exchange rate of the protons (1/τm, where τm is the residency time of the water proton).33 At low magnetic field strengths, longitudinal relaxivities increase with increasing τR (long τR would suggest a high molecular weight, slow tumbling molecule), however this effect diminishes with increasing magnetic field strength, resulting in the r2/r1 ratio increasing rapidly. Similarly, there is an optimal range of τm which gives high relaxivities at different magnetic fields.31 On the basis of the above discussion, much higher relaxivities of the Gd-DTPA-MP/PT nanoassemblies as compared to Magnevist at 0.54 T can be attributed to their larger sizes, resulting in longer reorientational correlation times. Also, the highly ordered liquid crystalline structures of the nanoassemblies possess extensive continuous water channels which
Figure 5. Cryo-TEM micrographs of the (a) 1, (b) 2, (c) 5, and (d) 10% Gd-DTPA-MP/PT dispersions. 1 and 2% Gd-DTPA-MP/PT dispersions displayed cubosomes (insets show the fast Fourier transforms) and the rest exhibited unilamellar liposomes.
Synchrotron SAXS analysis and cryo-TEM images also confirm that the Gd-DTPA-MP/PT system can form stable colloidal cubosomal nanoassemblies at a higher concentration of the paramagnetic amphiphiles than the Gd-DTPA-MO/ GMO system (which only formed cubosomes at ≤1 mol % GdDTPA-MO).18 Relaxivity Studies and In Vitro Imaging. The longitudinal, r1, and transverse, r2, proton relaxivities of the 1, 2, 3, 5, 10, 20, and 100% Gd-DTPA-MP/PT dispersions were measured at three magnetic field strengths: 0.54, 9.40, and 11.74 T at 25 °C and compared to Magnevist (Table 1). The incorporation of Gd-DTPA-MP in PT improved the longitudinal relaxivities at high magnetic field strengths (9.4 and 11.7 T) when compared to pure Gd-DTPA-MP, whereas the pure Gd-DTPA-MP dispersion showed highest r1 at 0.54 T (Figure 6a). Similar trends were also observed for the Gd-DTPA-MO/ GMO dispersions. However, the Gd-DTPA-MP/PT dispersions showed higher longitudinal relaxivities than the Gd-
Figure 6. (a) Longitudinal and (b) transverse relaxivities of different Gd-DTPA-MP/PT nanoassemblies at different magnetic field strengths. Inset is a zoomed in view of the region from 9.40−11.74 T. 1561
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with up to 2 mol % Gd-DTPA-MP retained their cubic symmetry, while the mixtures with higher Gd-DTPA-MP content were transformed to more flattened planar bilayers of liposomal nanoassemblies. All the dispersions showed much better relaxivities and image contrast than Magnevist at both low and high magnetic field strengths. Finally, PT has been confirmed to be a successful carrier of MRI CAs.
may allow efficient water exchange, resulting in higher relaxivities at high magnetic fields. Comparable relaxivities of the cubosomal and liposomal nanoassemblies could suggest that both the nanoassemblies possess similar τR. However, to completely understand the effect of different structures on the relaxivity, it is imperative to determine the correlation times from relaxometric and 17O measurements. These experiments are ongoing and will be reported in the near future. To directly observe the contrast enhancement, in vitro MRI imaging was also performed on some of the Gd-DTPA-MP/PT dispersions and compared to Magnevist at 11.74 T and 25 °C. In MRI, contrast based on one parameter can be enhanced by carefully selecting the repetition (TR) and echo (TE) times. For example, to observe the positive contrast of the Gd-based contrast agents, T1-weighting can be achieved in the image by setting short TR and TE values.35 Both T1- and T2-weighted images were obtained for the Gd-DTPA-MP/PT and Magnevist phantoms as shown in Figure 7. As expected, Gd-
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Part of this research was undertaken at the SAXS/WAXS beamline of the Australian Synchrotron. We gratefully acknowledge the facilities and the scientific and technical assistance of the National Imaging Facility, University of Western Sydney Node. We also thank Tim Sawkins for the design of the solvent-tight button cells for the bulk sample SAXS measurements and Chris Sheedy for conducting ICP-OES measurements. The financial support from a UWS International PostGraduate Research Scholarship (A.G.) and CSIRO Top-up Scholarship (A.G.) is greatly appreciated.
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Figure 7. In vitro (a) T1- and (b) T2-weighted MRI of various GdDTPA-MP/PT dispersions and Magnevist at the same Gd concentration (0.4 mM) at 11.74 T and 25 °C. Slice thickness = 0.3 mm, field of view =15 × 15 mm2, matrix size = 180 × 180 and TR/TE = (a) 100 ms/20 ms and (b) 500 ms/100 ms. Gd-DTPA-MP/PT dispersions showed much better contrast (brighter in T1-weighted and darker in T2-weighted image) than Magnevist at the same Gd concentration.
DTPA-MP/PT dispersions showed much better contrast than Magnevist in both, owing to their higher relaxivities. No notable difference in signal intensity was observed between the cubosomal and liposomal nanoassemblies in the T1-weighted image. However, in the T2-weighted image, the dispersions with cubosomes showed better contrast (darker image). In this study, we have demonstrated the potential of GdDTPA-MP/PT nanoassemblies as MRI CAs. Although the dispersions with cubosomes and liposomes showed similar relaxivities, the former are still preferred due to the higher payloads of Gd (III) ions they offer per nanoassembly. Note that the relaxivities reported here are per Gd (III) ion and not per nanoassembly, which would be much higher. Further studies on the in vivo contrast enhancement of these nanoassemblies need to be performed and will be addressed in future work.
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CONCLUSIONS Gd-DTPA-MP has been incorporated within the inverse bicontinuous cubic phases of phytantriol. POM and SAXS on the bulk phases of the hybrid mixtures indicated the homogeneous incorporation of Gd-DTPA-MP in PT. The bulk phases in excess water showed inverse bicontinuous cubic mesophases up to 10 mol % of Gd-DTPA-MP. These hybrid mixtures were successfully dispersed in water in the presence of Pluronic F127 as a steric stabilizer. Upon dispersion mixtures 1562
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