Manufacturing and in vivo inner ear visualization of MRI traceable liposome nanoparticles encapsulating gadolinium
- Jing Zou1Email author,
- Rohit Sood2,
- Sanjeev Ranjan†2,
- Dennis Poe†1,
- Usama A Ramadan3,
- Paavo KJ Kinnunen2 and
- Ilmari Pyykkö1
© Zou et al; licensee BioMed Central Ltd. 2010
Received: 10 August 2010
Accepted: 18 December 2010
Published: 18 December 2010
Treatment of inner ear diseases remains a problem because of limited passage through the blood-inner ear barriers and lack of control with the delivery of treatment agents by intravenous or oral administration. As a minimally-invasive approach, intratympanic delivery of multifunctional nanoparticles (MFNPs) carrying genes or drugs to the inner ear is a future therapy for treating inner ear diseases, including sensorineural hearing loss (SNHL) and Meniere's disease. In an attempt to track the dynamics and distribution of nanoparticles in vivo, here we describe manufacturing MRI traceable liposome nanoparticles by encapsulating gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) (abbreviated as LPS+Gd-DOTA) and their distribution in the inner ear after either intratympanic or intracochlear administration.
Measurements of relaxivities (r1 and r2) showed that LPS+Gd-DOTA had efficient visible signal characteristics for MRI. In vivo studies demonstrated that LPS+Gd-DOTA with 130 nm size were efficiently taken up by the inner ear at 3 h after transtympanic injection and disappeared after 24 h. With intracochlear injection, LPS+Gd-DOTA were visualized to distribute throughout the inner ear, including the cochlea and vestibule with fast dynamics depending on the status of the perilymph circulation.
Novel LPS+Gd-DOTA were visible by MRI in the inner ear in vivo demonstrating transport from the middle ear to the inner ear and with dynamics that correlated to the status of the perilymph circulation.
In the process of optimizing the functionalization of each type of nanoparticle, it is essential to be able to trace their passage through the cochlea. However, it is problematic to track the MFNPs in the inner ear by histological means. First, the appearance of autofluorescence from lipofuscin granules, which are found in the cochleae of humans, chinchillas, guinea pigs and rats, impairs the detection of the fluorescence signals from the MFNP targets [5–10]. Second, the inner ear is composed of fluids, soft tissue, and bone  (Figure 1). This results in signal loss in histological study, which is incapable of retaining the inner ear fluids. Finally, it is inconvenient to observe the dynamics of MFNPs in the inner ear in histological studies.
MRI is an excellent tool to trace MFNPs that are labeled with a contrast agent through the inner ear by detecting their signal changes in both the inner ear fluids and soft tissue in vivo. The critical regions of the cochlea, including the lateral wall, spiral ganglion cells, and cochlear nerve, are also demonstrated with the resolution of a 4.7 T MRI [11, 12]. Gadolinium- tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) was shown to be efficiently taken up into rat inner ears and detected at 60 min post-transtympanic injection with greater signal intensity seen in the scala vestibuli than in the scala tympani and no uptake into the endolymph after four hours . In the present study, amphiphilic MRI-traceable liposome nanoparticles were first developed from 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt) [DMPE-DTPA (Gd)] (LPS-DTPA-Gd). The contribution of effective Gd (water accessible Gd) to MR signal was evaluated by disassembling Gd chelate lipid nanoparticles with 5% sodium dodecyl sulfate (SDS). Finally, effective MRI traceable liposome nanoparticles were developed by encapsulating Gd-DOTA in the nanoparticles. These nanoparticles, which are abbreviated LPS+Gd-DOTA, were finally visualized in vivo in the rat inner ears with MRI.
Contribution of effective Gd to MR signal intensity of LPS-DTPA-Gd
Relaxation times of different liposome nanoparticles at certain concentrations.
Relaxation times (mS)
(mean ± SD)
1897 ± 370
273 ± 4
1697 ± 380
455 ± 5
1566 ± 324
447 ± 5
1980 ± 11
620 ± 7
184 ± 13
102 ± 1
Transportation of LPS+Gd-DOTA through the middle-inner ear barriers
Distribution of LPS+Gd-DOTA in the inner ear after intracochlear injection
In the present work, we demonstrated that multifunctional liposome nanoparticles, which were tagged with gadolinium, were visualized by MRI. T1 signal enhancement correlates to the amount of water accessible to the gadolinium, shown by the phenomenon that SDS treatment induced T1 relaxation time of LPS-DTPA-Gd equivalent to that of Gd-DOTA at the same concentration. It was reported that Gd-DOTA and Gd-DTPA showed similar relaxivities . This suggested that each LPS-DTPA-Gd molecule became water accessible upon dissolution by SDS. Because the ratio of Gd to lipid is nonadjustable in LPS-DTPA-Gd, there was difficulty in generating sufficient MR signal. Alternatively, encapsulation of Gd-DOTA into the liposomes was an improved technique to increase the amount of gadolinium available for MR imaging. With this method it was possible to encapsulate 500 mM Gd into the liposomes compared to LPS-DTPA-Gd where the maximum concentration of incorporated Gd was 0.5 mM. Moreover, highly monodisperse NPs were obtained when Gd-DOTA was encapsulated into the liposomes. This is critical for quality control in pharmaceutics, and correlates with treatment efficacy. It is important to note that the gadolinium within LPS+Gd-DOTA nanoparticles is carried in the aqueous core and the shell can be manufactured to be identical to that of the treatment nanoparticles. Therefore, this MRI study is directly translatable for predicting the destination of these liposome nanoparticles in clinical use to carry genes or drugs.
Gd-DOTA-tagged liposome nanoparticles were demonstrated within the inner ears of rats in vivo using MRI. After intracochlear injection, nanoparticles diffused throughout the inner ear efficiently and immediately reached the modiolus, which includes the spiral ganglion cells. Therefore, the multifunctional liposome nanoparticle is a promising vector to deliver treatment agents to inner ear targets from a cochlear implant, where the spiral ganglion cells are a major concern. After intracochlear injection, distribution of nanoparticles in distal destinations, such as the semicircular canals, within one hour indicated that liposome nanoparticle can also be applied in drug delivery to the vestibular organ. The efficacy of delivery was enhanced by injecting a larger volume, likely due to the increased driving force, or by opening a second window into the scala vestibuli to avoid the pulsatile egress of cerebrospinal fluid (CSF) from the scala tympani. This reduced distribution with lower injection volumes suggests that diffusion of the hydrophobic liposome nanoparticles in the hydrophilic perilymph may be problematic. In the clinic practice of cochlear implantation, the constant release of nanoparticles from an advanced drug delivery electrode could resolve this diffusion issue.
Application of therapy into the middle ear, as a minimally invasive technique for inner ear drug delivery, is a favorable approach for the treatment of inner ear diseases that are not suitable for cochlear implantation. The outcome of intratympanic treatment would depend in a large part upon the efficacy of transportation from the middle ear to the inner ear. The existence of at least two pathways, including the round window membrane and the oval window, from the rat middle ear to the inner ear were recently supported by an in vivo MRI study . There are a number of additional studies concerning round window membrane passage in the literature. The round window membrane appears well adapted for active transport. There are microvilli on the surface of outer epithelium and abundant organelles, such as mitochondria, rough endoplasmic reticulum, and a well-developed Golgi complex, that provide metabolic support and mechanisms for transport activity . Our own unpublished data showed the appearance of clathrin in the round window membrane of rats, which suggests a specific pinocytosis. There is discontinuity in the basement membrane of the inner epithelium and loose junctions between inner epithelial cells that provide potential openings to support diffusion.. Both the surface characteristics and the size of the nanoparticles are important factors that affect the passage from the middle ear to the inner ear. One μm latex microspheres were reported to pass through the round window membranes of chinchillas and Rhesus monkeys . In that study, the quantity of latex microspheres that passed through the round window membrane was unknown, but this is an important factor to define in studies of therapy directed toward inner ear diseases. In the present observations, liposome Gd nanoparticles of 130 nm passed the middle ear-perilymph barriers in quantities sufficient for MR imaging in 3 h and reached a signal intensity of above 1/5 of that in the middle ear cavity. Our preliminary histological study demonstrated augmented distribution of LPS+Gd-DOTA into the utricle of the vestibule than that in the spiral ligament capillary and spiral ganglion cells of the cochlea at 8 h following transtympanic injection of TRITC labeled LPS+Gd-DOTA in specimens taken from the rat after MRI study (data not shown). This implies the involvement of oval window in transporting LPS+Gd-DOTA. The oval window pathway was proved by abolish of immediate Gd-DOTA uptake in the vestibule and scala vestibuli induced by oval window sealing (data not shown). Potential uptake of TRITC labeled LPS+Gd-DOTA in other organs such as the brain, liver, and kidney should be investigated in a future study. These same advanced MFNPs showed targetability of their distribution in rat cochleae when functionalized with TrkB ligand  after intratympanic administration.
In the present study, novel MRI visible multifunctional liposome nanoparticles were designed by encapsulating Gd-DOTA inside the hydrophilic core of the nanoparticles. Acceptable r1 values were realized in the final products. The Gd-DOTA-tagged liposome nanoparticles were visualized in the rat inner ear in vivo after both intracochlear and transtympanic injections. The intracochlear approach induced stronger signals on T1-weighted images. MRI also proved to be an excellent tool to monitor the purity of the Gd-DOTA-tagged liposome nanoparticles. There are potentially broad applications for these novel types of nanoparticles in future biomedical investigations.
Reagents and animals
Sphingosine (Sph), egg phosphatidylcholine (EggPC), and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) [DSPE-PEG2000], 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt) [DMPE-DTPA (Gd)], were from Avanti polar lipids (Alabaster, AL). Gd-DOTA (DOTAREM) was from Guerbet, Cedex, France. Hepes, and EDTA, were from Sigma. The purity of lipids was checked by thin-layer chromatography on silicic acid coated plate (Merck, Darmstadt, Germany) developed with a chloroform/methanol/water mixture (65:25:4, v/v/v). Examination of the plates after iodine staining, and when appropriate, upon UV illumination revealed no impurities. Lipid concentrations were determined gravimetrically with a high precision electrobalance (Cahn, Cerritos, CA). The other chemicals were of analytical grade and from standard sources.
Grouping of animals and time of MRI measurement post-LPS+Gd-DOTA administration
MRI time post-LPS+Gd-DOTA administration
40 min, 90 min
40 min, 90 min, 3 h, 6 h, 24 h, 48 h
0 min, 20 min, 30 min, 40 min, 1 h
Preparation of liposomes containing Gd-DTPA-DMPE (LPS-DTPA-Gd)
Preparation of liposomes encapsulating Gd-DOTA (LPS+Gd-DOTA)
A 4.7 T MR scanner with bore diameter of 155 mm (PharmaScan, Bruker BioSpin, Germany) was used in both phantom and in vivo MR measurements. The maximum gradient strength was 300 mT/m with an 80-μs rise time. A dedicated rodent head coil (linear bird cage coil) with diameter of 38 mm was used for the phantom and animal studies. To evaluate T1 and T2 relaxation times of LPS-DTPA-Gd nanoparticles and analyze the contribution of water-accessible Gd-DTPA to MR signal, the nanoparticle solutions were prepared with physiological saline at a concentration of (1 mM containing 0.5 mM Gd). 5% SDS was applied to disassemble the nanoparticles and expose all the lipid-bond Gd-DTPA to the water. This was done to detect MRI signal from all of the Gd-DTPA associated with the nanoparticles, including that amount within the nanoparticles that could be shielded by the lipid from proton interaction. Gd-DOTA diluted with physiological saline at a concentration of 0.5 mM was used as a positive control. Plastic phantom tubes (400 μl, AgnTho's AB, Sweden) were filled with the solutions of Gd-DOTA, LPS-DTPA-Gd nanoparticles, or LPS-DTPA-Gd nanoparticles plus 5% SDS.
In order to measure relaxivities, r1 and r2, of LPS+Gd-DOTA, solutions of various concentrations were placed into plastic phantom tubes arranged concentrically within a 50 ml syringe. Dilutions were made with artificial perilymph containing 145.5 mM NaCl, 2.7 mM KCl, 2.0 mM MgSO4, 1.2 mM CaCl2, 5.0 mM HEPES, pH adjusted to 7.4 . Negative controls were prepared using either plain artificial perilymph or HEPES buffer. Each sample was prepared in duplicate.
T2 relaxation time was determined using a multi-slice multi-echo sequence (MSME), based on CPMG (Carr-Purcell Meiboom-Gill) spin echo (SE) [repetition time (TR) 1500 ms, echo time (TE) 7-229 ms, 32 echo times, matrix size 128 × 128, single slice, slice thickness 2.0 mm, field of view (FOV) 5.0 cm, resolution 0.098 × 0.130 mm2, number of excitation (NEX) 3]. T1 relaxation times were determined by rapid acquisition with relaxation enhancement (RARE) sequence with variable TR (TR 100, 432, 859, 1458, 2472, 7500 ms, TEeff 8.7 ms, RARE factor 2, NEX 1, matrix size 128 × 128, FOV 5 cm, single slice with slice thickness 2.0 mm).
Transtympanic injection of LPS+Gd-DOTA
LPS+Gd-DOTA nanoparticles were administered through transtympanic injection under an operating microscope as previously reported . An incision of approximately 1 mm length was made in the anterior superior quadrant of the tympanic membrane by a 25 gauge needle to release any potential air bubble. Either purified or unpurified 0.04 ml LPS+Gd-DOTA (1 mM LPS+Gd-DOTA containing 500 mM Gd-DOTA) were injected into the middle ear cavity through the posterior superior quadrant of the tympanic membrane. As a positive control, Gd-DOTA (100 mM) was transtympanically injected as above.
Intracochlear administration of LPS+Gd-DOTA to open perilymphatic compartments
The bulla was exposed through a post-auricular approach. Working under the operating microscope, a hole was drilled through the bulla with a 2 mm diameter burr. The round window membrane was identified superior to the stapedial artery as reported previously . The scala tympani of the basal turn was opened with a 0.5 mm cutting burr inferior to the stapedial artery and the round window. A second opening was made in the scala vestibuli of the basal turn. After wicking away the perilymph that egressed through the opening, two pieces of gelatin sponge soaked in LPS+Gd-DOTA (the size of each piece was proximately 3 mm3 after soaking in the LPS+Gd-DOTA solution) were placed against the openings in the middle ear cavity. The bulla opening was covered by muscle and the wound was sutured closed.
Intracochlear injection of LPS+Gd-DOTA into closed perilympatic compartments
After drilling into the scala tympani of the basal turn as above, polyimide tubing (MicroLumen Inc., Tampa, USA) was inserted intraluminally into a polyethylene tubing (I.D. 0.28 mm, O.D. 0.61 mm, Becton Dickinson, Franklin Lakes, USA) and fixed with nail polish. The tubing was filled with LPS+Gd-DOTA solution. After opening the scala tympani of the basal turn with a 0.5 mm cutting burr, the high performance polyimide tubing tip (MicroLumen, Tampa, USA), which was connected to polyethylene tubing (I.D. 0.28 mm, O.D. 0.61 mm, Becton Dickinson and Company, USA) and primed with LPS+Gd-DOTA solution was inserted into the scala tympani and was sealed circumferentially with Histoacryl (enbucrilate) glue (Aesculap AG, Tuttlingen, Germany). LPS+Gd-DOTA nanoparticles were initially applied by bolus injection of 5 μl, but they failed to demonstrated any MRI detectable signal for uncertain reasons. Although it is a physiologically excessive volume, 50 μl was injected and produced satisfying MR signal. The remaining observations were performed with 50 μl LPS+Gd-DOTA. The bulla opening was covered by muscle and the wound was sutured closed.
In vivo MRI observation of LPS+Gd-DOTA in the inner ear
The body temperatures of the rats were maintained by circulating warm water and their respirations were recorded with Physio Tool-1.0.b.2 program (Bruker, Germany). Rats were placed in the magnet with the ears positioned at the isocenter. 2D and 3D MRI measurement was performed. T1-weighted 2D images were acquired with RARE sequence (TR/TEeff 500/10 ms, RARE factor 4, matrix size 256 × 192, slice thickness 0.5 mm, FOV 2.5 × 2.5 cm2, resolution 0.098 × 0.13 mm2, NEX 33). T1-weighted 3D images were acquired with RARE sequence (TR/TEeff 500/12 ms, RARE factor 16, matrix size 64 × 64 × 64, FOV 0.89 × 0.89 × 0.89 cm3, resolution 0.139 × 0.139 × 0.139 mm3, NEX 2).
For the groups who had either intracochlear administration of LPS+Gd-DOTA with open perilymph circulation or transtympanic injection of LPS+Gd-DOTA, MRI scanning commenced immediately after the procedure. After setting up the geometry with T2-weighted 2D imaging, T1-weighted 2D, and T1-weighted 3D images were acquired at different time points. In the group of intracochlear injection of LPS+Gd-DOTA with closed perilymph circulation, LPS+Gd-DOTA nanoparticles were injected after setting up the MRI geometry. Then T1-weighted 2D, and T1-weighted 3D images were acquired as above. MRI measurements were followed up to 48 h in the group of transtympanic injection of LPS+Gd-DOTA and for one hour in the group of intracochlear administration of LPS+Gd-DOTA with open perilymph circulation (Table 2).
3D volume rendering of the inner ear MRI
OsiriX v3.3.2 (OsiriX Foundation, Geneva, Switzerland) software was applied for 3D rendering of the 3D raw stacked MR images of the inner ear. 3D volume renderings were created for visualisation of the inner ear structures with the ability to rotate the images into various orientations and to create photographs.
Quantification of signal intensity and statistics
ParaVision PV 4.0 (Bruker, Germany) software was used for post-processing of MR images and calculations of T1 and T2 relaxation times in phantom and signal intensity measurements in the inner ear compartments. Signal intensities in the region of interest were measured in the middle ear cavity, the vestibule, the scala vestibuli and scala tympani in the higher basal turn, and the brainstem using ParaVision 4.0. Differences in the signal intensities between 3 h and 6 h time points among these compartments were compared with student t-test. Signal intensity ratio of the vestibule, the scala vestibuli, and scala tympani over the middle ear was defined as the indicator of transport efficacy and compared between LPS+Gd-DOTA and Gd-DOTA groups with student t-test. A difference was considered to be statistically significant at P < 0.05. Photoshop CS3 software was used for labeling and demonstration of the inner ear anatomy in MR image.
The authors gratefully acknowledge financial support from the European Community 6th Framework Program on Research, Technological Development and Demonstration (Nanotechnology-based Targeted Drug Delivery. Contract number: NMP4-CT-2006-026556, Project acronym: NANOEAR).
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