Conflict/competing interest: No stated conflict of interest.
Funding sources: We gratefully acknowledge funding support from the National Eye Institute (EY-017971 to JLG, and P30 EY014801 to UM), the Department of Defense (W81XWH-09-1-0674), Hope For Vision and the Seigal Foundation (JLG), and an unrestricted grant from Research to Prevent Blindness (University of Miami).
Dr Jeffrey L Goldberg, University of Miami Miller School of Medicine, 1501 NW 10th Avenue, BRB 826, Miami, FL 33136, USA. Email: email@example.com
Background: Magnetic nanoparticles may be used for focal delivery for cells, plasmids or drugs, and other applications. Here we asked whether magnetic nanoparticles could be detected in vivo at different time points after intravitreal injection by magnetic resonance imaging.
Methods: Adult Sprague-Dawley rats received intravitreal injections of 50-nm or 4-µm magnetic particles into the left eye, with an equal volume of phosphate-buffered saline into the right eye (as controls). Animals were examined by magnetic resonance imaging at 1 h, 1 day and 5 weeks after injection. Eyes, brain, liver, spleen and kidney were also imaged with high-resolution ex vivo magnetic resonance imaging scanning.
Results: In vivo magnetic resonance imaging at the 1 h and 1 day time points more clearly detected magnetic particles in the 4 µm group compared with the 50-nm group, although 50-nm magnetic nanoparticles were easily visualized with high-resolution magnetic resonance imaging ex vivo. Five weeks after intravitreal injection magnetic resonance imaging clearly detected 4-µm particles inside the eye, but by this time point the 50-nm magnetic nanoparticles could not be detected by either in vivo or ex vivo high-resolution magnetic resonance imaging. No magnetic particles were detected in any other organ.
Conclusions: Magnetic resonance imaging could be used to track magnetic nanoparticles in the eye with the dosing selected for this study. Clearance varies by size, with 50-nm magnetic nanoparticles cleared more quickly than 4-µm particles. Thus, nanoparticles may provide advantages over micron-scale particles when considering risks associated with long-term persistence.
There has been increasing interest in magnetic nanoparticles as delivery vehicles for drugs, genes, cell replacement therapies, or as magnetic resonance imaging (MRI) tracking agents in such therapies. For example, nanoparticles have been used for sustained drug delivery to the retina for treatment of macular oedema1 or experimental autoimmune uveoretinitis in animal models;2 or for DNA delivery for treatment of neovascularization,3–6 retinitis pigmentosa3,7 or light-induced degeneration of photoreceptor cells.2 Furthermore, a use for magnetic nanoparticles has previously been explored, for example, for ocular neuroprotection by local magnetic hyperthermia with the induction of head shock proteins8 and for efficient transfection of adult retinal endothelial cells9 Magnetic nanoparticle-based contrast agents can be visualized by MRI, for example, when conjugated to antibody targeted for tumour angiogenesis in rat and mouse models10 or when loaded into mesenchymal stem cells,11 bone marrow cells12 or neural stem cells,13 which can then be traced in vivo.
Drug delivery, gene delivery and cell therapy to the ocular structures has been a persistent goal in ophthalmology. For example, drug delivery to the retina is challenged by the increased infection risk from repeated intravitreal injections. Cell therapies are limited by the inadequate methods both to deliver cells to the appropriate region and then to retain the cells. Hence, nanoparticle-based therapeutics may provide more accurate and controllable delivery mechanisms.
It is not known, however, whether magnetic nanoparticles (i) are detectable in the eye with conventional MRI as a tracking agent; (ii) persist in the eye; or (iii) egress the eye but accumulate in other organs. It is difficult to track magnetic nanoparticles in histology sections, either using haematoxylin & eosin or iron staining, and histological studies do not address magnetic nanoparticle detectability in vivo. We therefore explored the use of MRI to visualize magnetic nanoparticles in vivo.
All procedures were conducted in accordance with institutional review and the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research. Adult female Sprague-Dawley rats, each weighing 200–250 g, were anesthetized using an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (7.5 mg/kg) preoperatively, and received subcutaneous buprenorphine (0.05 mg/kg, Bedford Lab, Bedford, OH, USA) for postoperative analgesia. Eye ointment containing erythromycin was applied to protect the cornea.
Intravitreal injections were performed just posterior to the pars plana with a pulled glass pipette connected to a 50-µL Hamilton syringe. Care was taken not to damage the lens. Rats with any significant postoperative complications (e.g. retinal ischemia, cataract) were excluded from further analysis. Animals were allocated to three experimental groups: a control group received no intravitreal injections; the other two groups received a in their right eyes a 3-µL single injection of phosphate-buffered saline (PBS), and in their left eyes either 3-µL PBS containing 4-µm-diameter (1.69 mg total) superparamagnetic, polystyrene-coated particles (Dynabeads M-450, tosyl-activated, 37% iron oxide w/v; Invitrogen Dynal, Oslo, Norway), or 50-nm-diameter (1.65 mg total) superparamagnetic, dextran-coated nanoparticles (goat anti-mouse IgG-conjugated, 55–59% iron oxide; Miltenyi Biotec, Auburn, CA, USA). These magnetic nanoparticles were chosen based on non-toxicity found in our previous study.14 The chosen injected volume (3 µL into a 52-µL rat vitreous)15 of particles is similar to what could be used in a human eye (50–300 µL into a 4-mL human vitreous) as is done clinically. Animals were kept for 1 h, 1 day or 5 weeks before MRI scanning.
The MRI datasets were collected on a 4.7-Tesla (200 MHz) 40-cm bore magnet with a Bruker Avance (Bruker, Billerica, MA, USA) console using an actively shielded gradient set. Optimized radio frequency coils were used to image the different subjects and samples; a dual coil transmit/receive setup was employed for the imaging of the head of the subjects with a saddle shaped quadrature surface coil used for signal reception. A 72-mm linear birdcage radio frequency coil was used for the body MRI experiments and a linear birdcage radio frequency coil with a diameter of 14 mm was used for the imaging of the excised tissue samples in order to increase sensitivity of signal detection. It was anticipated that the iron-based contrast agent would primarily affect T2 and T2* contrast mechanisms, therefore high-resolution T2 weighted Rapid Acquisition Relaxation Enhanced (RARE) acquisitions were acquired along with three-dimensional T2* weighted Gradient Echo Fast Imaging acquisitions as a reference. The T2 RARE acquisitions were collected in the sagittal, coronal and axial orientations. The T2 RARE acquisitions of the excised organ studies employed the following settings: a time of repetition of 3000 ms, an echo time of 16 ms, an effective echo time of 34 ms, a field of view of 2.8 cm by 1.6 cm, a matrix size of 232 × 136 and an acquisition time of 2 h 0 min. The sagittal and coronal acquisitions employed a slice thickness of 0.3 mm and an in-plane resolution of 78 µm by 63 µm. The T2 RARE axial acquisition was collected with a slice thickness of 0.50 mm and an in-plane resolution of 63 µm by 63 µm. The three-dimensional T2* weighted Gradient Echo Fast Imaging acquisitions had the following settings: a time of repetition of 80 ms, an echo time of 10 ms, a flip angle of 30 degrees, a field of view of 3.0 × 0.8 × 0.8 cm, a matrix size of 512 × 128 × 128, a resolution of 59 µm by 63 µm by 63 µm and an acquisition time of 8 h 22 min. Following acquisition the three-dimensional datasets were reconstructed into sagittal, coronal and axial orientations for later analysis. For detection of nanoparticles at the 1-week time point or in the brain at 1 day, we used a Siemens 3T Trio MRI scanner (Siemens, Munich, Germany). Similar settings were employed for all experiments (head, body and excised tissues studies) only the settings associated with sample dimensions changed in any appreciable way, the image resolution settings are listed in the Figure legends.
After scanning the full body, the eyes, brains, liver and spleen were dissected from killed rats, washed in PBS, placed in an 8-mm crystal nuclear magnetic resonance tube filled with PBS and scanned. This process reduced air bubbles around the sample and provided a susceptibility-matched environment to improve the line width of the sample, reducing susceptibility issues and improving image quality to allow for better visualization of the iron particles.
Magnetic nanoparticles are detectable after intravitreal injection
To demonstrate detection of magnetic particles in vivo, we first injected the animals with 3 µL of 4-µm or 50-nm iron oxide core magnetic particles and scanned animals 1 h post injection. In PBS-injected or uninjected control eyes, ocular structures (lens and vitreous) were clearly identified and intact with no additional MRI signal or dropout (Fig. 1a; uninjected animal data not shown). In 4-µm magnetic particle-injected eyes, a large dark (hypointense) area was observed at the back of the eye reflecting the signal dropout because of the iron oxide particles (Fig. 1a). In 50-nm magnetic nanoparticles-injected eyes, a hypointense area was also detected when scanning the eye in situ (arrow in Fig. 1b). Thus, both magnetic micro- and nanoparticles are detectable by MRI after intravitreal injection.
Magnetic nanoparticles are detectable 1 day but not 5 weeks after injection
When we scanned the animals 1 day after injection, we observed MRI signal in 4-µm particle-injected animal eyes (3/3 animals; Fig. 2a). We were unable to detect signal from animals injected with 50-nm nanoparticles when scanning the whole animal in vivo (Fig. 2b), but after enucleation to facilitate the use of higher-resolution MRI coils, the 50-nm magnetic nanoparticle signal was detected in injected eyes (Fig. 3). By 5 weeks, however, we only detected MRI signal hypointensity in animals injected with 4-µm (3/3 animals) but not 50-nm (0/3 animals) particles, using either in vivo or ex vivo high-resolution MRI (Figs 2c,d,3 and Table 1). In a limited additional study we looked in the closest organ, the brain, at 1 day post injection and we found that neither the microparticles nor the nanoparticles were detected in the brain after 1 day. We also found at 1-week time point for animals injected with magnetic nanoparticles, two of three animals had no evidence of magnetic nanoparticles, and only one animal had magnetic nanoparticles in the vitreous, where they were observed to be more broadly distributed and less punctate. The MRI signal from magnetic (or generally, metal) particles yields a diffuse hypointensity much greater than the actual particle size. Our data show that the distribution of both the 50-nm and 4-µm particles is contiguous in nature and does not form multiple clumps in different areas of the eye. Furthermore, the location of the particle ‘cloud’ is mainly in the vitreous and along the retina (Fig. 4). Therefore, MRI methods can detect magnetic nanoparticles at 1 day and 1 week after injection.
Table 1. Summary of detection of magnetic nanoparticles by magnetic resonance imaging
‘++’ detectable in vivo; ‘+’ detectable ex vivo (high-resolution scan); ‘−’ undetectable.
Magnetic particles are not detected in other organs at 5 weeks
Finally, we sought to determine whether magnetic nanoparticles accumulated in other organs. We did not detect any signal from 50-nm magnetic particles in the brain, liver, spleen or kidney at 5 weeks after intravitreal injection, using ex vivo high-resolution MRI of the explanted organs (Fig. 5a–d). There was no hyperintense signal observed with 4-µm magnetic particles in the abdomen (Fig. 5e–g) at 5 weeks, detected using in vivo MRI images. To confirm that our MRI setup would have been able to detect the 50-nm magnetic nanoparticles, we scanned control dissected organs immediately after injecting them directly with 50-nm magnetic nanoparticles, and found hypointense signal in the injection sites (Fig. 6). Therefore, our MRI methods can detect 50-nm magnetic nanoparticles in these explanted organs after direct injection, confirming that these nanoparticles did not accumulate focally in these organs to a detectable level at 5 weeks after injection into the eye.
Recently there has been an increasing interest on using nanoparticles to assist in the delivery of drugs, genes, as well as cells. Most previous studies in this area have investigated the biodistribution of magnetic nanoparticles after systemic intravenous administration, and show that serum iron levels gradually increase for up to 1 week then slowly decline thereafter, with accumulation and degradation observed in the liver and spleen but not in the brain, heart, kidney and lung.16,17 With systemic administration, nanoparticle clearance lasted over 3 weeks.16
To our knowledge, this study is the first to investigate the biodistribution of magnetic micro- and nanoparticles after intraocular injection. The difficulty in tracking nanoparticles in histology sections, either using haematoxylin & eosin or iron staining, has pushed us to explore the possibility of using other tracking methods such as MRI to dynamically monitor nanoparticles in vivo. In this study, we have examined the persistence of magnetic micro- and nanoparticles after intravitreal injection. We found that 4-µm diameter microparticles were still detectable in the eye 5 weeks after injection, but the 50-nm nanoparticles were not detectable at 5 weeks even by employing an ex vivo high-resolution scan of the eyes, although these results may be limited by smaller sample sizes. No magnetic particles were detected in other organs systemically, although the total dose of particles used in the eye is considerably less than what has been studied systemically, because of the eye's small volume. In our previous study,14 we found that the magnetic nanoparticles were safe for intraocular injection at a single injection dosage volume of 3 µL (1.65 mg) and we found no toxicity of magnetic nanoparticles at the histology level. Whether coatings or surface functionalization of particles will change their biodistribution and clearance from the eye is still an interesting question that needs further study, although data suggest that surface functionalization is certainly sufficient to direct nanoparticles to specific tissues.18,19
Nanoparticles have also been used as tracking agents to label and trace cells in vivo.20,21 After labelled cells die, however, such nanoparticles can be engulfed by macrophages, making interpretation of results difficult.22 We did not explore in this study at a histological level whether particles were engulfed by macrophages, although our prior data suggested that at least microparticles remain free after intravitreal injection.14
Overall, we showed that MRI could be used to track nanoparticles in vivo in the eye, and that particle size is a determining factor in their ocular persistence. These data emphasize the importance of considering particle size and detection method when used in vivo in the design of particles for therapeutics or diagnostics in the eye. Nano-scale particles may be preferred to micron-scale particles if longer-term persistence is not preferred.