Nitroxide-Loaded Hexosomes Provide MRI ... - ACS Publications

Jun 30, 2014 - Waddington, L. J.; Monaghan, P.; Muir, B. W. Bicontinuous cubic phase nanoparticle lipid chemistry affects toxicity in cultured cells. ...
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Nitroxide-Loaded Hexosomes Provide MRI Contrast in Vivo Nicole Bye,*,† Oliver E. Hutt,‡ Tracey M. Hinton,§ Durga P. Acharya,‡ Lynne J. Waddington,‡ Bradford A. Moffat,∥ David K. Wright,⊥,# Hong X. Wang,⊥ Xavier Mulet,‡ and Benjamin W. Muir*,‡ †

National Trauma Research Institute, Alfred Hospital and Department of Surgery, Monash University, Melbourne 3000, Australia CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, VIC 3168, Australia § CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, 5 Portarlington Road, East Geelong, VIC 3219, Australia ∥ Department of Radiology and #Department of Anatomy and Neuroscience, The University of Melbourne, Parkville 3010, Australia ⊥ The Florey Institute of Neuroscience and Mental Health, Parkville 3010, Australia ‡

ABSTRACT: The purpose of this work was to synthesize and screen, for their effectiveness to act as T1-enhancing magnetic resonance imaging (MRI) contrast agents, a small library of nitroxide lipids incorporated into cubic-phase lipid nanoparticles (cubosomes). The most effective nitroxide lipid was then formulated into lower-toxicity lipid nanoparticles (hexosomes), and effective MR contrast was observed in the aorta and spleen of live rats in vivo. This new class of lowertoxicity lipid nanoparticles allowed for higher relaxivities on the order of those of clinically used gadolinium complexes. The new hexosome formulation presented herein was significantly lower in toxicity and higher in relaxivity than cubosome formulations previously reported by us.

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INTRODUCTION The development of novel magnetic resonance imaging (MRI) contrast agents continues to be of importance in addressing the side effects of heavy metals associated with traditional gadolinium (Gd)-based agents. Although clinically used Gd contrast agents are chelated, the metal has been known to leach in some products with less-stable Gd complexes, causing adverse reactions in patients such as nephrogenic systemic fibrosis.1 Any new contrast agents should have low toxicity and should be able to be delivered intravenously without complication. Generally, contrast agents are used when poor contrast of diseased tissue is observed during MRI scanning. These agents produce bright contrast, and the tissue “lightens” in which they are located. Non-heavy-metal-based contrast agents reported in the literature are usually based on 19F- and nitroxide-containing compounds.2−4 Nitroxides are stable organic free radicals with an unpaired (paramagnetic) electron. The use of nitroxide-based compounds as possible MR contrast agents is growing in the literature as their paramagnetic nature enables the shortening of the longitudinal (T1) and transverse (T2) relaxation rates of protons. However, once inside the body, the nitroxide is prone to degradation through reduction to the hydroxylamine, and the rate of this reduction is dictated by the oxygen and redox status of the target tissue.4 Thus, recent efforts have focused on decreasing the rate of reduction through steric hindrance around the nitroxide or through conjugation to a larger scaffold.5 Although these approaches address the degradation problem, the primary focus when © 2014 American Chemical Society

developing improved MR contrast agents is to provide a strong enhancement of the longitudinal (spin−lattice, T1) proton relaxation rate.6 In the current context, this involves the optimization of the interaction of the nitroxide with the surrounding water protons. One possible approach to T1 enhancement is to use the discrete water channels in lyotropic mesophase liquid crystal nanoparticle formulations to finely tune the spin−lattice relaxation time. Thus, we recently reported that cubic-phase nanoparticles (cubosomes), derived from phytantriol and Myverol (mainly composed of glycerol monoolein (GMO)), that contained a myristic acid nitroxide lipid (MyrNox), provided T1 contrast in vivo and also protected the nitroxide from reduction (Figure 1).7 Cubosomes are complex 3D structures interwoven with highly defined water channels resulting in a high surface area and porosity whereas hexosomes result in the production of a 2D structure. Recent work has shown that Gd-containing lipids can be added to cubic-phaseforming lipids such as phytantriol to potentially be used as MR contrast agents.8 We have found that the diameter of the water channel has a significant effect on the MyrNox magnetic resonance relaxation rate.7 Moreover, with increased percentages of MyrNox in the nanoparticles, we observed a phase transition from cubic to hexagonal, which led to a decrease in Received: February 27, 2014 Revised: June 23, 2014 Published: June 30, 2014 8898

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copolymer, was used as a stabilizer to produce dispersed nanoparticles and was purchased from Sigma. Additionally, tricaprylin (glyceryl trioctanoate) from TCI, Japan was used to induce a cubic to hexagonal phase transition in GMO.11 Nitroxide Lipid Synthesis. Cholesterol Nitroxide (CNox). The structures of all nitroxide lipids synthesized are shown in Figure 1. For the synthesis of the cholesterol nitroxide, tempamine (100 mg, 0.6 mmol) was dissolved in dry dichrolomethane (6 mL) and the reaction was cooled to 0 °C. Cholesterol chloroformate (0.6 mmol, 270 mg) and triethylamine (0.9 mmol, 120 μL) were then obtained, and after 10 min at room temperature, TLC analysis indicated that the tempamine had been consumed. The solvent was removed, and the residue was dissolved in a small amount of dichloromethane and chromatographed on silica gel (petroleum spirit 60-80/ethyl acetate 9:1) to give the product (300 mg, 86%) as a red oil which solidified to a reddish-white solid on drying in a vacuum oven at 40 °C for 24 h. 1H NMR (CDCl3, 400 MHz) δ 0.70 (s, 4H), 0.92−1.59 (m, 46H), 1.86− 2.05 (m, 7H), 2.31−2.41 (m, 3H), 4.53 (s, 1H), 5.42 (s, 1H). LC− MS: Retention 12.8 min. ESI (m/z) 606.5 (M + Na). HRMS EI (m/z) Found 583.4808; C37H63O3N2 requires 583.4833. Oleyl Acid Nitroxide (ONox). Tempamine (334 mg, 2.0 mmol) was dissolved in dry dichloromethane (10 mL), and olelyl chloride (2.0 mmol, 600 mg) and triethylamine (2.0 mmol, 300 μL) were added at 0 °C. After 10 min, TLC analysis indicated that the tempamine had been consumed. The solvent was removed, and the residue was dissolved in a small amount of dichloromethane and chromatographed on silica gel (petroleum spirit 60-80/ethyl acetate 9:1) to give the product (430 mg, 49%) as a red oil, which was dried in a vacuum oven at 40 °C for 18 h. 1H NMR (CDCl3, 400 MHz) δ 0.91 (m, 5H), 1.31−1.34 (m, 33H), 1.69 (m, 3H), 2.04 (m, 6H), 2.19 (m, 2H), 5.34 (m, 3H). LC− MS: Retention 12.8 min. ESI (m/z) 458.4 (M + Na). HRMS EI (m/z) Found 435.3947; C27H51O2N2 requires 435.3945. Linoleyl Nitroxide (LNox). Linoleic acid (1.3 g, 4.7 mmol), tempamine (4.8 mmol, 800 mg), hydroxybenzotriazole (7 mmol, 951 mg), and N-methylmorpholine (11 mmol, 1.3 mL) were dissolved in dry dichloromethane (10 mL). EDCI (5.6 mmol, 1.1 g) was then added, and the reaction was stirred at room temperature for 18 h. The solvent was removed, and the residue was dissolved in a small amount of dichloromethane and chromatographed on silica gel (petroleum spirit 60-80/ethyl acetate 9:1) to give the product (580 mg, 28%) as a red oil, which was dried in a vacuum oven at 40 °C for 18 h. 1H NMR (CDCl3, 400 MHz) δ 0.87−1.30 (m, 31H), 1.59−1.87 (m, 6H), 2.03− 2.17 (m, 6H), 4.10 (m, 1H), 5.33 (m, 3H), 5.55 (m, 1H), 5.96 (m, 1H), 6.30 (m, 1H). HRMS EI (m/z) Found 433.3779; C27H49O2N2 requires 433.3789. 3,7-Dimethyl Octanyl Nitroxide (DONox). 3,7-Dimethyl octanoic acid (650 mg, 3.8 mmol), tempamine (3.8 mmol, 650 mg), hydroxybenzotriazole (5.7 mmol, 769 mg), and N-methylmorpholine (9.5 mmol, 1.05 mL) were dissolved in dry dichloromethane (10 mL). EDCI (4.6 mmol, 870 mg) was then added, and the reaction was stirred at room temperature for 18 h. The solvent was removed, and the residue was dissolved in a small amount of dichloromethane and chromatographed on silica gel (petroleum spirit 60-80/ethyl acetate 9:1) to give the product (310 mg, 25%) as a red oil, which was dried in a vacuum oven at 40 °C for 18 h. 1H NMR (CDCl3, 400 MHz) δ 0.95−1.04 (m, 17H), 1.25−1.41 (m, 12H), 1.62 (m, 2H), 2.02−2.26 (m, 6H), 5.37 (m, 1H). LC−MS: Retention 10.4 min. ESI (m/z) 348.2 (M + Na). HRMS EI (m/z) Found 325.2843; C19H37O2N2 requires 325.2850. Nuclear Magnetic Resonance (NMR). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Av400 spectrometer. 1H NMR were recorded at 400 MHz. Due to the paramagnetic nature of the nitroxide, the NMR spectra were poor in terms of resolution, sensitivity, and integration. High-resolution mass spectrometry experiments were carried out using a ThermoQuest MAT95XP employing electron impact at 70 eV, and perfluorokerosene was used as a reference standard. LC−MS analyses were run on a Shimadzu LC-2010 single quadapole mass spectrometer scanning 100−1000 Da at 1 s/scan using a 10 to 100% MeOH gradient (0.1%

Figure 1. Amphiphile chemistries used to make lyotropic mesophase liquid crystal nanoparticle formulations (top structures) and MyrNox reported previously7 (bottom structure).

relaxivity. Thus, the nature of the mesophase nanostructure and the resulting water channel appears to be critical in determining the degree of MR contrast. In light of this observation, the primary aim of the present study was to investigate more thoroughly the cubic phase and lattice parameter effects of various nitroxide lipids on MRI relaxivities. Here we report the synthesis of four novel nitroxide lipids and the use of phytantriol (Phyt) as the cubic-phaseforming lipid to explore the effect of the different lipid tails on the physical and MRI characteristics of the lyotropic mesophase liquid crystal nanoparticle formulations (Figure 2). The first

Figure 2. Nitroxide lipids used in this study.

part of the study involved the use of phytantriol (Phyt) as we have observed that it is able to accommodate larger amounts of additive material (other lipids and various drugs) than are GMO-based cubosomes.9 The second part of the study involved the formulation of low toxicity lipid nanoparticles for testing in vivo. Recent work by us has shown that cubosomes are highly hemolytic and lipid nanoparticles based on phytantriol are significantly more toxic than those made from GMO.10 Therefore, for the in vivo study, once a suitable nitroxide lipid was discovered (3,7-dimethyl octanyl nitroxide (DONox)) it was formulated into hexosomes made from GMO as they are significantly less toxic.10 These novel GMO lipid hexosomes are demonstrated by us to exhibit excellent MR contrast in vivo.

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MATERIALS AND METHODS

Self-Assembly of the Lyotropic Liquid Crystal NitroxideContaining Nanoparticles. Two cubic-phase-forming lipids were used in this work, phytantriol (DSM Nutritional Products, GmbH) and glyceryl monooleate (GMO) from Nu Check Prep (USA). Pluronic F-127, a poly(ethylene oxide)-polypropyline oxide block 8899

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HCOOH) on a VisionHTC18, 50 × 4.6 mm2, 3 μm column at a flow rate of 1 mL/min with a PDA detector collecting data at 254 nm. Preparation of Cubosome and Hexosome Solutions. Samples for screening the longitudinal and transverse relaxation rates were prepared individually by sonicating a mixture of phytantriol or GMO with the required amount of additive such as nitroxide lipid and Pluronic F-127 as stabilizers in Milli-Q water. In each sample vial, the total lipid content was 100 mg/mL and the F-127 concentration was 15 wt % in the mixture. The lipid and F-127 were mixed in a ratio of 85/15 by weight, respectively. The final concentration of F-127 in solution was around 1.75 wt %. F-127 was homogeneously solubilized in the lipid by heating to 70 °C and vortex mixing. To this mixture, water was added and the lipid was dispersed at 70 °C via ultrasonication (Misonix Sonicator S-4000 attached with a Misonix microtip 418) for 3 min in pulse mode (2 s sonic pulses interrupted by 3 s breaks to maintain the temperature) at 50% of the maximum amplitude of the equipment. This resulted in the formation of homogeneous milky dispersions which were stored at 25 °C prior to further experimentation. All measurements were made on nanoparticles dispersed in Milli-Q water. The nanoparticles were added to phosphate-buffered saline prior to injection in animals or use in cell culture experiments at pH 7.4. Cryo-Transmission Electron Microscopy (Cryo-TEM). A laboratory-built humidity-controlled vitrification system was used to prepare the samples for cryo-TEM. Humidity was kept close to 80% for all experiments, and the ambient temperature was 22 °C. Copper grids (200 mesh) coated with a perforated carbon film (lacey carbon film, ProSciTech, QLD, Australia) were glow discharged in nitrogen to render them hydrophilic. Aliquots (4 μL) of the sample were pipetted onto each grid prior to plunging. After 30 s of adsorption time, the grid was blotted manually using Whatman 541 filter paper for approximately 2 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, USA) 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/Å2 for all imaging. Images were recorded using an FEI Eagle 4kx4k CCD camera at magnifications ranging from 15 000× to 50 000×. Dynamic Light Scattering Analysis. Particle size measurements were performed using a Zetasizer Nano instrument (Malvern, U.K.). Particle sizes were measured in Milli-Q water using samples that were appropriately diluted. The analysis was performed at 25 °C, and for each sample, the mean diameter and polydispersity index (PDI) of six measurements were calculated. Synchrotron SAXS Measurements. Small-angle X-ray scattering (SAXS) experiments were performed on the SAXS/WAXS beamline at the Australian Synchrotron. Special glass capillaries (1.5 mm) containing sample solutions were placed in a temperature-controlled sample holder maintained at 37 °C by a recirculating water bath. Samples were exposed to the 12 keV X-ray beam with dimensions of 2500 μm × 130 μm and a typical flux of 5 × 1012 photons/s, and diffraction patterns were recorded using a Pilatus 1 M detector (Dectris, Switzerland). A silver behenate standard was used to calibrate the reciprocal space vector for analysis. Data reduction (calibration and integration) was performed using AXcess, a custom-written SAXS analysis program written by Dr. Andrew Heron from Imperial College, London.12 The bicontinuous cubic phase with Pn3m symmetry (diamond) and the hexagonal phase observed in this study were identified from the positions of diffraction peaks at 21/2, 31/2, 41/2, 61/2, 81/2, and 91/2 and at 1, 31/2, and 41/2, respectively. MRI Relaxivity Measurements. A high-throughput MRI screening technique was used to evaluate the NMR relaxation properties of the nanoparticle solutions at 3 T using a method similar to that previously reported. For MRI relaxivity measurements, 1 mL of each lyotropic liquid crystal nanoparticle formulation was placed in a 96well plate and serially diluted to measure T1 and T2 and calculate

relaxivities at 23 °C in a Siemens (Germany) 3 T TRIO MRI scanner using a body transmitted radio frequency coil and a 12-channel radio frequency receiver coil. To quantify the R2 relaxivity, we used a Carr-Purcell Meiboom and Gill multiecho spin echo sequence to acquire 32 images at echo times (TE) ranging from 11.5 to 310.5 ms with a repetition time (TR) of 3 s. To quantify R1, a spin echo inversion recovery imaging sequence was used to acquire images at 9 different inversion times (TI) ranging from 20 ms to 5 s (TR/TE = 10 000/10 ms). All images were acquired with a 3 mm slice thickness, 100 mm FOV, 192 × 154 matrix size, and 2 averages. Zero filling was used to reconstruct all images to a matrix size of 256 × 256. To calculate R2, we averaged and plotted as a function of TE the signals from regions of interest (>40 voxels) centered within each well. The R2 values were then calculated (as the decay constants) by numerically fitting (using a nonlinear least-squares algorithm (Matlab, MA, USA)) the data to a monoexponential equation S = S0 exp(− TE R 2)

(1)

where S0 is the signal when TE ≪ 1/R2. A similar approach was used to calculate R1 except the data was plotted as a function of TI and fitted to the following equation:

S = S0(1 − 2 exp(− TI R1)

(2)

The respective longitudinal and transverse relaxation rates, R1 and R2, were plotted as a function of the nitroxide lipid concentration. The relaxivities of selected samples were also measured by serially diluting samples. R1 and R2 values were plotted as a function of nitroxide concentration, and linear least-squares analysis (Matlab) was used to quantify the R1 and R2 relaxivities. Cytotoxicity Testing of the Lyotropic Liquid Crystal Nitroxide Containing Nanoparticles. Cell Lines. Chinese Hamster Ovary cells (CHO: ATCC CCL-061) were grown in MEMα modification, and human alveolar-basal epithelial cells (A549; ATCC CCL-185) and human hepatocarcinoma cells (Huh7; kindly supplied by VIDRL, Australia) were grown in DMEM. Both base media were supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 mM Hepes, 1.5 g/L sodium bicarbonate, 0.01% penicillin, and 0.01% streptomycin. Cells were grown at 37 °C with 5% CO2 and subcultured twice weekly. Toxicity Assay. CHO and A549 cells were seeded at 1 × 104 cells per well, and Huh7 was seeded at 2 × 104 cells per well in 96-well tissue culture plates and grown overnight at 37 °C with 5% CO2. The lipid formulations were serially diluted from 500 to 0 μg/mL in water and added to 3 wells in the 96-well culture plates for each sample and incubated for 72 h at 37 °C in 200 μL of standard media. Toxicity was measured using the Alamar Blue reagent (Invitrogen USA) according to the manufacturer’s instructions. Briefly, the medium was removed, cells were washed once with PBS and replaced with 100 μL of a standard medium containing 10% Alamar blue reagent, and cells were then incubated for 4 h at 37 °C with 5% CO2. The assay was read on an EL808 absorbance microplate reader (BIOTEK, USA) at 540 nm (background correction at 620 nm). Obtained data were analyzed in Microsoft Excel. Results are presented as a percentage of untreated cells, and the presented data are representative of three separate experiments in triplicate ±SEM. Hemolysis Assay. Mouse blood in EDTA was obtained from C57BK6 mice from the AAHL small animal facility according to AEC committee approval. The blood was washed in PBS three times with centrifugation at 2000 rpm for 5 min. Cells were then counted and resuspended at a concentration of 7.5 × 106 cells/mL. An aliquot of 100 μL was added to 100 μL of PBS containing the serial dilution of DONox-hex, GMO, or Phy cubosomes in a 96-well microtiter plate and incubated for 1 h at 37 °C with constant shaking. After the removal of the unlysed erythrocytes by centrifugation (1000g, 5 min), 100 μL of the supernatant were transferred to a new microtiter plate, and hemoglobin absorption was determined at 450 nm (background correction at 750 nm) on an EL808 Absorbance microplate reader (BIOTEK, USA). One hundred percent lysis was determined by adding 5 μL of a 0.1% Triton X-100 solution prior to centrifugation. 8900

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Table 1. Physical and Relaxivity Values of Selected Phytantriol and Glycerol Monoolein Lyotropic Mesophase Liquid Crystal Nanoparticle Formulations Containing Various Different Nitroxide Lipids at Differing Concentrations Nox lipid MyrNoxa MyrNoxa MyrNoxa MyrNoxa MyrNoxa MyrNoxa CNox ONox LNox DONox

Nox lipid concentration (wt %)

lipidb

liquid crystal phase

SAXS lattice parameter (Å)

DLS (nm)c

PDId

r1 (m M−1 s−1)e

r2 (mM−1 s−1)f

r1/r2

4 10 14.5 2 10 14.5 2 10 2 10 2 10 2 10 10

Phyt Phyt Phyt Myverol Myverol Myverol Phyt Phyt Phyt Phyt Phyt Phyt Phyt Phyt GMO

cubic hexagonal cubic hexagonal cubic cubic hexagonal cubic hexagonal cubic cubic cubic cubic cubic cubic cubic cubic hexagonal

64.2 48.0 47.0 84.0 57.4 56.2 67.8 66.9 68.12 67.1 68.5 68.6 68.2 66.1 52.9

225.7 205.8 217.8 190.6 195.7 210.4 223.9 193.4 182.2 195.7 234.6 195.6 196.7 225.2 185

0.15 0.14 0.16 0.17 0.14 0.16 0.15 0.16 0.18 0.15 0.16 0.15 0.16 0.17 0.13

0.59 0.29 0.27 0.14 0.11 0.08 1.08 0.59 0.85 0.61 0.46 0.13 0.85 0.49 0.5

1.35 0.68 0.43 0.32 0.2 0.17 2.21 1.23 1.98 0.86 1.05 0.41 1.60 0.76

0.43 0.42 0.43 0.44 0.55 0.49 0.49 0.48 0.43 0.71 0.43 0.33 0.53 0.64

a

MyrNox data included for comparison.7 bPhyt, phytantriol; GMO, glycerol monoolein (the major component of Myverol. cDynamic light scattering (intensity). dPolydispersity index. eLongitudinal (spin−lattice) relaxivity. fTransverse (spin−spin) relaxivity. (delivered at a rate of 10 μL/s) separated by approximately 20 min. The animal received a total final dose of 400 mg/kg (infusions of 150, 150, and 100 mg/kg). Continuous scanning was performed during infusions, and multiple postinjection scans were acquired. Image Analysis. To account for physiological movement between FLASH image frames, the aorta was masked on a template image and the resulting region of interest was warped back into individual frames. Template construction and warping was performed using Advanced Normalization Tools (www.picsl.upenn.edu/ANTS) software.13,14 Further image analysis was performed using MATLAB (The MathWorks, Natick, MA, USA). To account for small variations in the effective TR due to respiratory gating, the T1-weighted images were normalized by the mean intensity from an ellipsoidal ROI in the muscle superior to the lungs. The percent signal change for six different organs was calculated by placing ellipsoidal ROIs on these normalized T1-weighted images and subtracting the baseline signal.

Results are presented as the percentage of hemoglobin release compared to the Triton X-100 control, and the presented data are representative of three separate experiments in triplicate ±SEM. In Vivo MRI. Scanning Protocol. All experimental procedures were approved by the Florey Neuroscience Institutes animal ethics committee (12-101 FNI). Imaging experiments were performed on two rats, and the results were consistent for both animals. Data from one animal is shown for clarity. An adult female Sprague Dawley (SD) rat weighing 343 g was obtained from Florey Animal Services. The rat was housed with food and water ad libitum under a 12 h light/dark cycle. The rat was anesthetized with 5% isoflurane in a 1:1 mixture of medical grade air and oxygen and prepared for surgery. Anesthesia was maintained throughout the insertion of a tail vein catheter and subsequent imaging, with 1−2.5% isoflurane delivered through a nose cone placed over the animal’s snout. A 22 gauge IV catheter was inserted into the lateral tail vein and flushed with a small volume of heparinized saline (10 IU/mL) before being connected to medical grade vinyl tubing preloaded with hexosome solution. The animal was immediately prepared for MRI. The anesthetized animal was laid supinely in an animal holder with respiration continuously monitored throughout the experiment using a pressure-sensitive probe positioned under the rat’s diaphragm. Arterial oxygen saturation, heart and breath rates, and pulse distention were monitored with a Mouse Ox pulse oximeter (Starr Life Sciences Corp., Pittsburgh, PA, USA). The cradle was inserted into a transmit/receive coil fixed inside a BGA12S gradient set for imaging with a 4.7 T Bruker Avance III scanner. The scanning protocol consisted of a three-plane localizer sequence followed by multislice axial, coronal, and sagittal scout images to accurately determine the position of the kidneys and liver. A fast low-angle shot (FLASH) sequence was used to acquire rapid images during contrast agent administration with the following imaging parameters: repetition time (TR) = 18 ms, echo time (TE) = 3.5 ms, flip angle = 60°, number of repetitions (NR) = 160, field of view (FOV) = 8 × 8 cm2, matrix size = 192 × 192, slice thickness = 2 mm, and total scan time = 6 min 10 s. T1-weighted images were also acquired using a rapid acquisition with relaxation enhancement (RARE) sequence with respiratory gating and the following imaging parameters: TR = 400 ms, effective TE = 10 ms, RARE factor = 2, number of averages = 4, FOV = 8 × 8 cm2, matrix size = 192 × 192, and slice thickness = 2 mm. Intravenous Administration of Contrast Agent. After a baseline scan was acquired, hexosome solution (90 mg/mL in phosphatebuffered saline, freshly filtered with a 0.45 μm nylon filter) was administered intravenously via the tail vein catheter as three injections

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RESULTS AND DISCUSSION Synthesis of Nitroxide Lipids and Lyotropic Liquid Crystal Nanoparticles. Previous work by us reported the use of a MryNox lipid produced from the reaction of 4-hydroxyTEMPO and myristol chloride. It was observed that cubosomes containing phytantriol at 4 wt % MryNox had a longitudinal relaxivity of r1 = 0.59 mM−1 s−1. However, at 15 wt % a MryNox a hexagonal phase change occurred in phytantriol with a corresponding longitudinal relaxivity decrease by half to r1 = 0.27 mM−1 s−1 (Table 1). Although a similar trend was observed in Myverol, the overall longitudinal relaxivities were lower.7 In order to identify nitroxide lipids that could be better tolerated in the cubic phase of lipids such as phytantriol, four new nitroxide lipids were synthesized (Figure 2). Thus, the reaction of readily available tempamine with cholesterol chloroformate and oleyl chloride gave the cholesterol nitroxide (CNox) and oleyl nitroxide (ONox) in 86 and 49% yields, respectively. The linoleyl nitroxide (LNox) and 3,7-dimethyl octanyl nitroxide (DONox) were synthesized by employing standard amide coupling procedures. Thus, the coupling of tempamine with linoleic acid and 3,7-dimethyl octanoic acid with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI) gave LNox and ONox in 28 and 25% yields, respectively. As anticipated, the NMR spectra were broad and diffuse. The purity and identity were confirmed by liquid 8901

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chromatography mass spectrometry (LC−MS) and highresolution mass spectrometry. The incorporation of an amide bond into the four new lipids, as opposed to an ester bond in MyrNox, was anticipated to be more stable toward esterases in the blood in vivo. Moreover, although there is literature to guide the design of self-assembling amphiphiles, an accurate prediction of phase behavior is notoriously complex. Therefore, the cross sections of “kinked” lipid tails in CNOx, ONox, LNox, and DONox were chosen as the most likely to be readily incorporated into the curvature of a cubic phase with minimal disruption of the membrane surface. To test this hypothesis, the four Nox lipids, with formulations containing 0 to 10 wt % Nox lipid, were incorporated into Phyt and synthesized. Using Phyt enabled us to do a direct comparison with our early work with MyrNox. Lyotropic liquid crystal nanoparticle dispersions were easily generated from these lipids via the addition of a pluronic (F127) polymer in water acting as a steric stabilizer followed by sonication of the solution. The average particle size measured by dynamic light scattering (DLS, intensity vs size) of all nanoparticle formulations used in this work was found to be consistently around 210 ± 40 nm in dimension and to have polydispersity indices of between 0.14 and 0.18 (Table 1). The nanoparticle sizes and polydispersity indices did not vary significantly between the Nox lipids or their concentration, and no significant trend in size or polydispersity index was observed. Relaxivities of the Lyotropic Liquid Crystal Nitroxide Containing Nanoparticles. Synchrotron SAXS and relaxivity measurements (Materials and Methods) were made to investigate the change in the lattice parameter and possible phase changes with increasing Nox lipid incorporation. These techniques allowed a detailed investigation of the possible internal lamellar, cubic, and hexagonal phase symmetries of the dispersed nanoparticles and their effect on enhancing the longitudinal relaxation rates in water. The relaxivities were calculated by measuring the gradient of the relaxation rate (s−1) versus the Nox lipid concentration (mM) in solution. The Nox lipids studied in this work are paramagnetic due to the fact that they contain a stable unpaired electron. Previous work has shown that liposomes produced with a Nox lipid may induce a slight enhancement in the longitudinal relaxation rate.15 While studies have postulated that liposomes with a nitroxide lipid could enhance the longitudinal relaxation rates, there are limited reports on the observed relaxivities of the these nanoparticles.16 The use of liposomal systems is limited by the fact that the nitroxyl group is rapidly reduced in vivo, producing diamagnetic N-hydroxy compounds.15,16 The use of a fatty nitroxide lipid inside a cubic- or hexagonal-phase nanoparticle enables a significant increase in relaxivity compared to that of the free nitroxyl group. This is due to rotational correlation constant effects from the reduced tumbling of the nitroxide group inside the nanoparticle and potential water confinement/ exchange effects in cubosomes and hexosomes. Figure 3 shows the relaxivity of the various Nox lipid nanoparticle formulations produced with increasing Nox lipid concentration. A decrease in relaxivity is observed with increasing Nox lipid loading from 0 to 10 wt %. Interestingly, as has been observed by us and other researchers, the highest relaxivities were found at the lowest loading of the Nox lipids. The highest relaxivity observed (r1 = 1.1 mM−1 s−1) using the CNox lipid is nearly twice that of the cubosomes previously reported by us containing a MyrNox lipid (r1 = 0.59 mM−1 s−1) (Table 1).7 As a comparison, the relaxivity of small-molecule

Figure 3. Relaxivity (mM−1 s−1) plotted as a function of wt % nitroxide lipid incorporated into phytantriol cubosomes.

nitroxyl contrast agents used in functional MRI imaging are on the order of 0.1 mM−1 s−1 (4.7 T) with half-lives of a few minutes.17 There is a strong effect of the particular lyotropic mesophase upon the various nitroxide lipid nanoparticle relaxivities.7 As the nanoparticles have similar size distributions and polydispersities, rotational correlation constant effects should not be a significant factor in the enhanced relaxivity observed in these Nox lipid nanoparticles. Therefore, the nanostructure and accessibility of the nitroxide lipid headgroup to water within or surrounding the lyotropic mesophase nanoparticles must play an important role in affecting the longitudinal relaxation rate. To further elucidate these effects, the longitudinal relaxation rate was plotted as a function of the lattice parameter in Figure 4. Table 2 shows the cubic-phase lattice parameters and 1/T1 and 1/T2 values of the data presented in Figure 4.

Figure 4. Longitudinal relaxation rate (s−1) plotted as a function of the synchrotron source measured SAXS lattice parameter (Å) of phytantriol cubosomes with increasing wt % of nitroxide lipid.

Using these new Nox lipids at all wt % concentrations in phytantriol, only cubic phases were detected, indicating that they are well incorporated into the nanoparticles. As no phase or phase separation is evident in these formulations with higher loadings of the Nox lipids, we are confident that the new Nox lipids produced are incorporated well into phytantriol cubosomes. The lattice parameters of the cubic (Q2) phases detected were calculated from the scattering curves at different loadings. The SAXS patterns indicated an inverse bicontinuous 8902

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cubic phase with a Pn3m space group (double diamond) typical of phytantriol cubosomes. The longitudinal relaxation rate (R1) shows a clear trend when plotted against the lattice parameter in these systems (Figure 4). As more Nox lipid is incorporated, the lattice parameter decreases and R1 increases. It is clear that the Nox lipids drive an increase in curvature in phytantriol which results in the reduction in lattice parameter observed. This decrease in lattice parameter with increasing additive loading has been observed for other systems and is typical of aliphatic additives. The results indicate that these new Nox lipids are more readily incorporated into phytantriol than the myristic Nox lipid previously reported by us as no hexagonal phase change was observed. This clearly demonstrates the importance of the structure of the Nox lipid tail in influencing the overall phase behavior of the nanoparticles. Moreover, it circumvents the decrease in R1 in the MyrNox system at higher Nox loading, which is attributed to the hexagonal phase change and a limit to amount of MyrNox that can be incorporated into the lipid. In summary, the relaxivity and SAXS data demonstrate that the addition of small amounts of the Nox lipids results in significant nanostructural changes resulting in a decrease in the lattice parameter and an increase in R1 with increasing Nox loading. Cytotoxicity Study of MR Visible Lyotropic Liquid Crystal Nitroxide Containing Nanoparticles in Vitro. The second phase of this study was conducted to produce lyotropic liquid crystal nanoparticles with enhanced relaxivities from the addition of one of the most effective Nox lipids discovered that

Table 2. Lattice Parameters, 1/T1 and 1/T2, of Selected Phytantriol Lyotropic Mesophase Liquid Crystal Nanoparticle Formulations Containing Various Different Nitroxide Lipids at Differing Weight % Concentrations as Shown in Figure 4 Nox lipid CNox

ONox

LNox

DONox

Nox lipid concentration (wt %)

SAXS cubic phase lattice parameter (Å)

1/T1 (s−1)

1/T2 (s−1)

2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10

67.79 67.22 66.84 66.90 66.85 68.15 67.77 67.70 67.43 67.13 68.49 68.83 68.20 68.71 68.61 68.19 67.92 67.19 66.34 66.13

1.81 2.93 3.99 4.12 4.99 1.95 2.93 4.46 5.41 7.02 1.06 1.38 1.53 1.38 1.54 2.62 4.26 7.06 7.17 7.47

3.72 5.88 7.52 7.86 10.37 4.55 5.55 11.00 10.13 9.83 2.43 3.43 3.94 3.51 4.68 4.93 7.11 10.21 11.34 11.63

Figure 5. CryoTEM images of DONox-hex (hexosomes) in water. The insets show hexosomes imaged in transverse view and a Fourier transform showing hexagonal symmetry. (The scale bar is 200 nm.) 8903

dx.doi.org/10.1021/la5007296 | Langmuir 2014, 30, 8898−8906

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(Figure 7). The hemolysis assay was used as an indication of the ability of nanoparticles to disrupt red blood cell

could be delivered in vivo to act as an effective MR visible contrast agent. To advance this work, the DONox lipid was chosen as it was observed to incorporate extremely well into the cubic-phase gel. None of the other nitroxide lipids were studied in the second phase of this work; however, it is important to note that they too may be effective at producing MRI-active hexosomes with GMO. In this second phase of the study, the cubic-phase-forming lipid glycerol monoolein (GMO) was chosen as Myverol is better tolerated in animals upon bolus delivery when compared to phytantriol,10 the reason being that phytantriol-based cubosomes are extremely effective at disrupting cellular membranes and are highly hemolytic.18,19 GMO-based lyotropic liquid crystal nanoparticles are significantly less toxic in vitro and in vivo.7 Recent unpublished work by us has shown that hexosomes are considerably less toxic and hemolytic to cells than cubosomes. Therefore, in the second stage of this research, the DONox lipid was formulated into GMO at 10 wt % containing tricaprylin at 10 wt % in total lipid to induce a hexagonal-phase change and produce MR-active, low-toxicity hexosomes. Imaging by cryoTEM of the hexosome sample (DONox-hex) containing 81 mg of GMO, 9 mg of DONox, 10 mg of tricaprylin, and 15 mg of F-127 per mL of solution revealed a mixture of predominantly hexosomes with a smaller population of liposomes (Figure 5). The hexosomes ranged in size from 50 to 200 nm, with some merged particles. Occasionally liposomal structures were merged with hexagonal-phase particles. Synchrotron SAXS analysis at 37 °C confirmed the presence of hexagonal-phase nanoparticles with a calculated lattice parameter of 52.9 Å. DLS analysis of this material showed particles with an average size of 185 ± 60 nm and a PDI of 0.13 consistent with the cryoTEM observations (Table 1). The longitudinal relaxivity of the 10 wt % DONox-hex nanoparticles was measured to be 0.5 mM−1 s−1. For comparison, the hexosomes containing 15 wt % MyrNox lipid reported in our previous publication when formulated using Myverol had r1 = 0.08 mM−1 s−1 (Table 1). This new formulation is significantly more effective. The DONox-hex sample was then tested in an Alamar blue assay (Figure 6) for cell viability and in a hemolytic assay

Figure 7. Hemolysis assay. DONox-hex (hexosomes) were serially diluted in PBS before addition to mouse red blood cells. Lysis was allowed to continue for 1 h at 37 °C. Intact red blood cells were removed by centrifugation, and released hemoglobin was measured at 450 nm. Experiments were repeated in triplicate three times. Results are presented as the percentage of lysis compared to that of Triton X 100. The presented data is representative of three separate experiments in triplicate ±SEM.

membranes, thereby releasing hemoglobin. For the delivery of nanoparticles via IV bolus, one desires a low hemolytic activity. Reports on the toxicity of lyotropic mesophase nanoparticles are limited despite a large number of in vitro and in vivo studies published.18−20 The results of an Alamar blue cytotoxicity assay on DONox-hex nanoparticles are shown in Figure 6. The results shown from testing in three cell lines is that the DONox-hex nanoparticles have different toxicity profiles depending on the cell lines used. CHO-GFP cells are most sensitive with toxicity observed at 15.6 μg/mL. DONox-hex was toxic at 250 μg/mL in A549 and Huh7 cells with no toxicity at 62.5 μg/mL. The hemolysis assay showed that at 125 μg/mL approximately 30% of the red blood cells were lysed while at 62.5 μg/mL 15% were lysed (Figure 7). This is substantially less membrane disruption than previously observed with GMO and phytantriol cubosomes.10 This finding correlates well with the toxicity assay, indicating that the higher the percentage of lysis of the red blood cells, the greater the toxicity. The difference observed among the various cell lines also indicates that different cells throughout the body will be more susceptible to the toxicity of the hexosomes than others. Therefore, the lowest-toxicity profile is preferred. Other researchers such as Shen et al.20 have reported a similar cytotoxicity of phytantriol-containing cubosomes in L929 fibroblast cells at concentrations greater than 40 μg/mL using an MTT assay. Lyotropic mesophase lipid nanoparticles when dosed at toxic concentrations may interact with the lipid bilayer of cells and result in membrane fusion and lipid exchange that can disrupt membrane integrity and cause cell lysis.10 In another study, Murgia et al.19 reported that GMO cubosomes stabilized with Pluronic F-127 are more toxic than liposomes produced with GMO when the lauroylcholine chloride stabilizer is used. Tilley et al.21 report that phytantriol cubosomes can transfer their lipid material between lamellarand hexagonal-phase nanoparticles. This process occurs via compositional ripening. It has been shown that the direction of lipid exchange is dependent on the chemical potential between

Figure 6. Toxicity of DONox-hex (hexosomes) by serial dilution in CHO-GFP, Huh7, and A549 cells. DONox-hex was serially diluted in water before addition to cells. Cells were incubated for 72 h and then analyzed for toxicity with the Alamar blue reagent for 2 h at 37 °C (Life Technologies, USA). Results are presented as % cell viability of untreated control cells. The presented data is representative of three separate experiments in triplicate ±SEM. 8904

dx.doi.org/10.1021/la5007296 | Langmuir 2014, 30, 8898−8906

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the two lipid systems. Other researchers have reported similar results in equivalent systems.22−24 Barauskas et al.18 reported an investigation of the interaction of various cubic- and hexagonalphase-forming lipids with model cell membranes. They showed that cubic-phase nanoparticles are more effective at disrupting model lipid bilayers than lamellar- and hexagonal-phase nanoparticles. When GMO cubosomes were incubated with blood, they saw significant hemolysis of red blood cells while negligible effects were observed with hexagonal phase nanoparticles (hexosomes). They attributed this hemolytic activity of GMO cubosomes to their ability to destabilize cell membranes, resulting in phase segregation of the lipid bilayers within cellular membranes introducing pore structures and the leakage of cellular content. In our study, we did see the hemolysis of blood cells incubated with the DONox-hex nanoparticles. The difference in findings from that of Barauskas et al. can be attributed to differences in the exact composition of our DONox-hex (hexosomes) studied. One challenge with the approach we have taken in this work is that the concentration of Nox lipid in solution incorporated within the DONox-hex nanoparticles is around 0.017 M, which is an order of magnitude less than what is typically delivered in vivo when using Gd chelates.25 This means that larger doses of our DONox-hex nanoparticles will need to be administered to provide an enhanced contrast effect in vivo. This requirement is aided by the lower toxicity of these hexosome nanoparticles when compared to cubosome formulations. Previous studies by us demonstrated that the DONox-hex nanoparticles are tolerated at up to 600 mg/kg in live rats.7 DONox-hex Nanoparticles Provide Effective MRI Signal Enhancement in Vivo. The DONox-hex nanoparticles produced from GMO were tested in live rats via bolus delivery through a cannula in the tail vein (Materials and Methods). The choice of GMO as the lipid rather than phytantriol was due to its lower toxicity. Its use was further justified by the fact that GMO can readily undergo lipolysis in vivo due to its ester bond whereas phytantriol cannot be easily biodegraded. The DONox lipid, when incorporated into hexosomes, has the potential to function as an MR contrast agent by shortening T1 relaxation times, which will result in the brightening of T1-weighted MR images as long as the nitroxide radical is not reduced rapidly in the body after injection. It was assumed based on results of previous work by us that after bolus delivery the nanoparticles would be rapidly taken up by the MPS, which would then accumulate these nanoparticles in organs such as the liver and spleen. In a study by Nilsson et al., they reported that hexosomes made from phytantriol were observed at small concentrations in the liver, kidneys, and spleens of animals after subcutaneous injection.26 As can be seen in Figure 8, the MRI signal enhancement was ∼300% in the aorta and ∼370% in the spleen. Interestingly, after IV injection only a small enhancement in MR contrast was observed in the liver (∼20%) and kidneys (∼7%) when using the DONox-hex nanoparticles. Immediately after injection, contrast is observed in the aorta (bloodstream) of the animal and then the nanoparticles are observed to leave the blood and gather in other organs, particularly the spleen. The rapid biphasic nature of the MRI signal time curve (Figure 8D) in the aorta and the accumulation of MRI signal in the spleen support the rapid MPS uptake of the DONox hexosomes. This indicates that the hexosome nanoparticles are displaying a different pharmacokinetic pattern when compared to the myristic Nox nanoparticles reported in previous work by us when using

Figure 8. In vivo MRI results. (A) False color T1-weighted MR images before intravenous injection of DONox-hex (hexosomes) in a rat. The large arrow highlights the stomach/kidney, and the small arrow highlights the aorta (B) False color T1-weighted MR images 40 min after the intravenous injection of DONox-hex (hexosomes) in a rat. (C) T1-weighted MR image 40 min after the intravenous injection of DONox-hex (hexosomes) in a rat, highlighting the regions of interest selected to measure signal enhacement. (D) Signal enhancement time curves from six ellipsoidal ROIs placed in different organs: the aorta (red), kidney (yellow), liver (magenta), muscle (dark blue), spleen (cyan), and stomach (green). Contrast enhancement was mostly observed in the aorta and spleen. The three arrows indicate the timing of three IV injections with DONox-hex (hexosomes).

Myverol as the lipid material.7 The myristic Nox particles gave only a maximum enhancement in the liver of