Magnetic nanoparticles as intraocular drug delivery system to target Retinal Pigmented Epithelium

Disorders of the retina and RPE tissues, located in the posterior eye chamber, are responsible for the majority of blindness both in childhood and adulthood. Long-term delivery of biologically active molecules to the RPE is problematic and remains a challenge. The cornea/sclera constitutes a static barrier severely limiting ocular bioavailability of surface instilled drugs and retinal-blood barrier prevent ocular drug diffusion after systemic administration. Intravitreal (IVT) and subretinal injections are considered as the most effective ways of delivering material to the back of the eye. In particular, subretinal injection seems to be the only effective option to target RPE but are very invasive with reduced patient compliance compared to IVT injections. IVT injection involves injection of drug in solution directly into the vitreous which is far from optimal for three reasons: short term complications caused by the initial high drug concentration, very short retention time and lack of tissue specificity. In the present study, we provide a method for exclusive and fast localization of drugs to RPE, with the use of magnetic nanoparticles (MNPs), by IVT injection. MNPs form a powerful drug delivery system because their reactive surface can be easily functionalized with biocompatible coatings and bioactive molecules to prevent interaction with healthy tissues and increase their target specificity . In addition to their established role as molecular carriers, MNPs have two other advantages. They can be controlled by noncontact forces and tracked by magnetic resonance imaging (MRI) . Furthermore, different MNPs have FDA approval for clinical use e.g., Endorem® (MRI contrast agent for diagnosis of liver tumors). Although the MNPs have not yet been tested on humans for ocular applications, there are evidences from studies in rats, that the iron oxide MNPs are non-toxic to the ocular structures . We have investigated the ability of MNPs to target RPE by IVT injection, using wild type Xenopus laevis as model system. Xenopus offers favorable features, such as external development, large supply of embryos with each fecundation, a very short early development time (3 days to reach tadpole) and close homology with human genes. A remarkable similarity in the molecular signaling processes, cellular structure, anatomy, and physiology of eye has been observed among Xenopus and other high-order vertebrates, including humans . Their relatively large size (from 1-1.2 mm (zygote) to 1 cm (5 days old)) enables an easy manipulation and IVT microinjection. Finally, the use of this model generates minor ethical issue compared to mammals because these embryos are considered to not be sufficiently sentient or experience nociceptive sensations when subjected to experimental procedures. We used commercial fluorescent MNPs with a negative surface charge and a hydrodynamic size of 252 nm to analyze their biodistribution after intravitreal injection, i.e., in the anterior part of the eye and, specifically, in a region behind the lens surrounded by the vitreous humor, into the left eye of Xenopus embryo. 24 h after the injection, we observed, by Prussian blue staining of paraffin sections, that MNPs are specifically retained in the ocular tissues without any diffusion to the other tissues, including the contralateral eye. Quantitative analysis of the iron content by thiocyanate colorimetric assay was used to confirm that all the MNPs were retained in the injected eye. The average ferric iron content in the injected eyes was significantly higher than in the control eyes. These results, together with the histochemical observations, demonstrate that the MNPs are retained exclusively inside the injected eye. We did not observe any toxic effects on the ocular structures caused by MNPs: no death or embryonic malformations were observed and the injected eye exhibited completely normal development. Even if the particles were injected in the anterior part of the eye they localized preferentially in the posterior segment, in a region corresponding to the RPE. The RPE is a single layer of pigmented cuboidal epithelial cells adjacent to the neural retina. In order to define precisely the MNP localization after one day from the injection, we studied the fluorescence of MNPs on cryostat sections without pigment bleaching, to highlight RPE. In this way we established the precise localization of the MNPs, as red spots, in RPE. The moderate red background is most likely due to a partially degradation of the MNPs linked fluorophore, as confirmed by the histological staining (Prussian Blue) of particles which were found to co-localize only with RPE layer. In order to characterize the kinetics of the migration process, we monitored the localization of MNPs at different time points starting from 5 min to 24 h after injection. Just 5 min after injection, the particles started to spread out from the vitreous chamber (VC), adhering to the neural retina (NR) and only few were in RPE . After the MNPs continue to progressively migrate at RPE from the vitreous chamber and the migration process is completed within 24 h. Another crucial factor for efficient drug delivery is permanence at the intended target site. For this reason, we monitored the retention of MNPs in embryos for periods up to 20 days. The MNP localization follows RPE during its development, included in the later stages (20 days) where RPE microvilli interdigit with the outer segment of photoreceptors. It is known that NPs size and surface charge influence the movement of nanoparticle-based ocular drug delivery systems. We investigated the effect of size and charge on MNP movement by comparing the localization of our MNPs (250 nm, -17 mV) with the localization of particles of similar size but more negatively charged (MNP-), or positively charged (MNP+), or particles with neutral charge but small size (MNPs). Surprisingly, the results were the same with all kinds of MNPs, i.e., they localized in RPE one day after injection with only a small fraction in NR and no particles diffusion to extra-ocular tissues. For the first time we demonstrated that charge surface, beyond the size, does not influence the localization of nanoparticles in RPE. We speculate that MNPs, with different charge and size, can diffuse in the vitreous, infiltrate among retinal neurons without cells engulfing until their reach RPE. These cells have a strong phagocytic activity, required for maintaining constant renewal process of the photoreceptor outer segments. In order to understand if the capability of MNPs to localize in RPE is species specific, we injected MNP in the left eye of zebrafish embryos at 48 hour post fertilization. We found that after 1 day from injection the MNPs localize specifically in RPE also in zebrafish. This datum suggests that the localization of MNPs in RPE is not species-specific. In conclusion, we have developed a protocol for fast and specific localization of magnetic nanocarriers in RPE layer, in an embryo model for the study of vertebrate diseases as a first step for therapeutic proof-of-concept studies, replacing or drastically reducing the use of mammals. We demonstrated that MNPs localize autonomously and specifically in RPE after IVT injection independently by particle size and surface charge. Moreover, this process seems to be not specie-specific. The MNPs have the potential for development as an ocular drug delivery, capable of targeting RPE with sustained controlled drug release providing MRI tracking for a variety of retinopathies. Moreover, the MNPs could be exploited also for magnetic hyperthermia treatments of ocular iper-proliferative diseases. Additionally there are other challenging applications which could be explored on the use of MNPs, such as magnetic targeting of RPE in the treatment of retinal detachment by applying external magnetic forces. REFERENCE Giannaccini M, Giannini M, Calatayud MP, Goya GF, Cuschieri A, Dente L, Raffa V. Magnetic nanoparticles as intraocular drug delivery system to target Retinal Epithelium (RPE). Int. J. Mol. Sci. 2014, 15:1590-1605.