Can Nanotechnology Improve Treatment of Retinal Disease?

Very small particles could mean very big changes for retina.

Marco A. Zarbin, MD, PhD • Carlo Montemagno, PhD • James F. Leary, PhD • Robert Ritch, MD

Nanotechnology, first described in a seminal paper by Richard Feynman,¹ involves the creation and use of materials and devices at the scale of intracellular structures and molecules, and it incorporates systems and constructs with sizes on the order of <100 nm. Scientists and clinicians have used nanoparticles containing gene transcription factors and other modulating molecules that allow reprogramming of cells in vivo,² as well as nanomaterials to induce selective differentiation of neural progenitor cells³ and to create neural-mechanical interfaces.^4-6

We have reviewed previously some applications of nanotechnology for the treatment of retinal disease,^7-9 and in this article we will highlight the use of nanoparticles for drug and gene therapy, nanoengineering of viral vectors, and optogenetics.

DRUG AND GENE THERAPY

Particles can be internalized by cells via different mechanisms — eg, phagocytosis, macropinocytosis, caveolarmediated endocytosis, or clathrin-mediated endocytosis — which, in turn, results in exposure of nanoparticles to different intracellular environments.¹⁰

One can engineer nanoparticles for a particular mode of intracellular entry, depending on the choice of nanoparticle-targeting molecules, eg, cholesterol favors uptake via caveolin-mediated endocytosis, and transactivating transcriptional activator peptide favors macropinocytosis.^11,12 One can even target nanoparticles to particular subcellular organelles, eg, mitochondria¹³ or the nucleus.¹⁴

Some patients with retinitis pigmentosa have a tyrosine kinase mutation that is also present in Royal College of Surgeons (RCS) rats.^15-17 The retinal pigment epithelium of RCS rats cannot phagocytose photoreceptor outer segments properly, resulting in progressive rod and cone photoreceptor degeneration.¹⁸

Sakai et al.¹⁹ prepared ~585-nm–diameter basic fibroblast growth factor (bFGF) nanoparticles using bovine gelatin and recombinant human bFGF. Eight weeks after intravitreal injection into RCS rat eyes, bFGF nanoparticles were still present in the outer retina. Compared to an intravitreal injection of 2.5 µg bFGF, bFGF nanoparticle–treated animals had better outer nuclear layer preservation and better-preserved electroretinograms (Figures 1 and 2).

Figure 1. Morphologic rescue of the superior retina of RCS rats. Representative photographs eight weeks after injection with bFGF-nanoparticles (NPs) (A, B) and blank NPs (C, D). Note the preservation of the outer nuclear layer (ONL) in the bFGF-NP–treated retina, compared with the blank NP-treated retina. Higher magnification showed the respective differences better. Original magnification, x100. Scale bar, 50 ∞m. Reproduced with permission from Sakai et al.¹⁹

Figure 2. Effects of bFGF-NPs on scotopic ERG in RCS rats six weeks after injection. Amplitudes of a- (A) and b-waves (B) were measured and plotted. Note that bFGF-NP–treated eyes show significantly increased a- and b-wave amplitudes compared with bFGF-treated eyes (a-wave, *P < .005;="" b-wave,="">P < .05).="" data="" represent="" mean="" ±="" sd="" (n="8," each="" group).="" reproduced="" with="" permission="" from="" sakai="" et="">¹⁹

Oxidative damage plays a role in the pathogenesis of many retinal diseases, including age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, and phototoxicity.^20-25 As the size of cerium oxide (CeO2) nanoparticles (“nanoceria”) decreases, they demonstrate formation of more oxygen vacancies in their crystal structure, particularly at 3-5 nm of diameter.^26,27 As a result, nanoceria can scavenge reactive oxygen intermediates.

Zhou et al.²⁸ demonstrated that vacancy-engineered nanoceria promote regression of pathological retinal neovascularization in the Vldlr knockout mouse, which carries a loss-of-function mutation in the very low-density lipoprotein receptor gene and the phenotype of which resembles retinal angiomatous proliferation (Figures 3 and 4).²⁹

Figure 3. Nanoceria inhibit the development of pathologic intra- and subretinal vascular lesions in the Vldlr-/- retina. Photomicrographs of whole mount retinas (A-C) and eye cups (RPE, choroid, and sclera) (D-F) from P28 animals are shown. All retinal blood vessels were labeled green by the vascular filling assay. Wild-type (WT) retinas (A) showed the normal, weblike retinal vasculature, whereas those from the Vldlr-/- mice (B) showed numerous intraretinal vascular lesions or “blebs” (IRN blebs). See white arrows for exam ples. A single injection of nanoceria at P7 inhibited (C) the appearance of these lesions. Eye cups from WT mice (D) showed no subretinal neovascular (SRN) “tufts,” but those from Vldlr-/- mice (E) had many bright SRN tufts. A single injection of nanoceria on P7 inhibited the appearance of these SRN tufts (F). Reproduced from Zhou et al.²⁸ with permission.

Figure 4. Retinal vascular lesions in the Vldlr-/- retinas re quire continual production of excess reactive oxygen spe cies. Vldlr-/- mice were injected at P28 with saline or nanoceria and sacrificed one week later on P35. Analysis of VEGF levels by western blot (A) showed a four-fold reduction (B) within one week of nanoceria injection. The numbers of intraretinal neovascular (IRN) blebs (C), and subretinal neovascular (SRN) tufts (D) were also dramatically reduced. * P <.05; **P <.01. Reproduced from Zhou et al.²⁸ with permission.

This regression occurs even if nanoceria are injected intravitreally after the mutant retinal phenotypes are established. A single injection inhibits neovascularization for weeks because nanoceria are a catalytic and regenerative antioxidant. Nanoceria inhibit development of increased vascular endothelial growth factor levels in this model,²⁸ which may mean this nanoparticle will be effective in treating macular edema in diabetic eyes and choroidal neovascularization–induced retinal edema in AMD eyes.^30-32

Viral vectors deliver genes efficiently (see below) but can be associated with risks, such as immunogenicity and insertional mutagenesis. Nonviral vectors (eg, polymers, lipids) have high gene-carrying capacity, low risk of immunogenicity, relatively low cost, and greater ease of production. ³³

Nanoparticles can deliver genes efficiently to stem cells³⁴ and have been explored as a means for gene delivery in the diagnosis and treatment of ocular disease.^35-38 Complexes of cationic polymers and negatively charged plasmid DNA, termed polyplexes, can have transfection efficiency comparable to adenoviral vectors.³⁹ Polyplexes have nanometer size, have large vector capacity, are stable in nuclease-rich environments, and have relatively high transfectivity for both dividing and non-dividing cells.^38,39

Cai et al.^40,41 used DNA nanoparticles consisting of single molecules of DNA compacted with 10-kDa polyethylene glycol (PEG)–substituted lysine 30-mer peptides containing the wild-type retinal degeneration slow (Rds) gene, peripherin/rds, to induce cone photoreceptor rescue in an animal model (rds+/-) of human retinitis pigmentosa (Figure 5). While these results are promising, this approach may require additional refinement.

Figure 5. Transferred normal mouse peripherin (NMP) leads to structural rescue of the rds+/- phenotype. Light micro graphs (top row) and electron micrographs (bottom row, n = 3–5/group) from the temporal side of rds+/- eyes were examined. After postnatal day-5 injection, moderate ultra structural rescue is detected in the outer segments (OSs) of nanoparticleinjected eyes (arrows) at post-injection day-30 (PI-30); significant ultrastructural improvement in OSs of nanoparticle-injected eyes is apparent by PI-120. OS discs are properly aligned and flattened, and improved OSs do not exhibit the swirl-like structures typical of the rds+/-. IS, inner segment layer; RPE, retinal pigment epithelium. Scale bars = 10 ∞·m. Reproduced with permission from Cai et al.⁴¹

For example, although the immune response to polylysine-based nanoparticles seems to be less than that to viral capsid proteins, the efficiency of gene transfer is not as high (most of these nanoparticles are degraded in the endosomal complexes).⁴² As a result, one may have to use large numbers of nanoparticles, which increases the chance of an immune response.

NANOENGINEERING OF VIRAL VECTORS

Adeno-associated viruses (AAVs) are small (4.7 kilobase carrying capacity), nonpathogenic, single-stranded DNA parvo-viruses that can transduce dividing and nondividing cells.⁴³ Recombinant AAVs (rAAVs) have relatively low immunogenicity, can target many nondividing cells, and can provide sustained efficient therapeutic gene expression after a single treatment.⁴⁴ Recombinant AAVs have been used to treat humans with Leber congenital amaurosis.^45-47 Nanoengineering via site-directed mutagenesis has induced modifications of the virus capsid that may improve its clinical utility.

Epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) can phosphorylate AAV2 capsids at tyrosine residues.^48,49 Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but do not transduce well, in part because the AAV capsids are ubiquitinated and then degraded by the proteasome.^49,50

Zhong et al.⁵¹ demonstrated that site-directed mutagenesis⁵² of the surface-exposed tyrosine residues increases vector transduction efficiency 30-fold in vivo compared to wild-type AAV2. The increased transduction efficiency is due to impaired capsid ubiquitination and improved intracellular trafficking to the nucleus. Petrs-Silva et al.⁵³ demonstrated that tyrosine-to-phenylalanine capsid AAV2 mutants showed 10- to 20-fold higher transgene expression of the entire retina after intravitreal injection, compared to AAV with wild-type capsids.

In clinical studies completed thus far, subretinal injections have been required to transduce photoreceptors satisfactorily.^45,47,54 Subretinal virus delivery requires pars plana vitrectomy and has a higher likelihood of complications (eg,retinal tears) than intravitreal delivery, which can be done in an office setting under topical anesthesia.

Thus, nanoengineering of the AAV capsid may provide an important tool for facilitating gene therapy to photoreceptors. Still, subretinal delivery may have some advantages over intravitreal virus delivery. The subretinal space, for example, is a relatively immune-privileged site,⁵⁵ which may reduce the likelihood of an immune response after repeat treatment with viral vectors.⁵⁶

OPTOGENETICS

Optogenetics refers to the transfection of cells with light-activated ion channels to induce light sensitivity in neural tissue, the capacity of which to respond to light stimuli is absent or severely degraded. This topic has been reviewed extensively.^8,9,57 In the case of patients with retinitis pigmentosa, extensive rewiring of inner retinal circuits and inner retinal neuronal degeneration occur in association withphotoreceptor degeneration.^58,59

Nonetheless, it is possible to create visually useful percepts by stimulating retinal ganglion cells electrically.^60-63 Use of light-sensitive ion channels, rather than electrodes, to stimulate retinal neurons (including ganglion cells, bipolar cells and/or photoreceptors lacking outer segments) has the potential for noninvasive neuronal stimulation with high spatial resolution.^64-76 Naturally occurring, as well as man-made, light-sensitive ion channels have been deployed for this purpose. Some examples are considered below.

Channelopsin-2 is a light-gated ion channel that is sensitive to blue light and is derived from green algae, Chlamydomonas reinhardtii. When its attached chromophore, all-trans retinaldehyde, undergoes reversible photoisomerization, channelopsin-2 undergoes a conformational change that alters its permeability to mono- and divalentcations.⁷⁷ The complex of channelopsin-2 and all-trans retinal is termed channelrhodopsin-2 (ChR2).

Halorhodopsin (HaloR) is a yellow light–activated chloride ion pump from the archea, Natronomonas pharaonis. Results in preclinical models of retinitis pigmentosa indicate that the kinetics of ChR2- and HaloR-mediated light responses are compatible with the temporal processing requirements of visual information in the retina.

Unfortunately, ChR2 and HaloR both exhibit low light sensitivity, with threshold activation light intensities ~5-6 log units higher than those of cones.^68,73 Also, the light intensity operating range of microbial rhodopsins is 2-3 log units, compared to the normal human retinal dynamic range of 10 log units. Thus, transfection of ganglion cells might not be clinically efficacious (even if ON and OFF ganglion cellscan be targeted separately).

Because of the signal convergence from photoreceptors and bipolar cells onto many ganglion cells, one approach to improve light sensitivity with these optogenetic tools is to transfect bipolar cells or photoreceptors. Doroudchi et al.⁷⁸ achieved stable and specific ChR2 expression in ON bipolar cells using a recombinant AAV vector packaged in a tyrosine-mutated capsid. Using this approach, in multiple preclinical models of human retinitis pigmentosa, light levels that elicited visually guided behaviors were within the physiological range of cone photoreceptors.

In typical retinitis pigmentosa, the rod photoreceptors degenerate first, and the cone cell bodies remain for a time after their outer segments are lost.⁵⁹ Because cones normally are depolarized in the dark and are hyperpolarized by light, light-activated opening of the HaloR chloride channel in cone inner segments might mimic normal light-induced hyperpolarization of photoreceptors.

Busskamp et al.⁷⁵ demonstrated that enhanced HaloR expression in light-insensitive cones (via AAV transfection) can restore light sensitivity in several preclinical models of retinitis pigmentosa. These resensitized cones activated all retinal cone pathways, drove directional selectivity, activated cortical circuits and mediated visually guided behaviors.

Synthesis of light-sensitive ion channels has been achieved by coupling naturally occurring ion channels with molecules (eg, azobenzene), the photoisomerization of which results in reversible activation of the ion channel (Figure 6).^{64,66,67,76,79}

Figure 6. Use of molecular engineering for the development of neural prosthetics: design of an allosteric photoswitch. Top left: (a) An agonist (orange) is tethered to a ligand-binding domain (LBD) through an optical switch (red) via linkers (black). In one state of the switch, the ligand cannot reach the binding pocket, whereas in the other state, the ligand docks and stabilizes the activated (closed) conformation of the LBD. (b) Schematic representation of the operating mode of ionotropic glutamate receptor switch (iGluRs). Binding of an agonist (orange) stabilizes the activated (closed) conformation of the LBD and allosterically opens the pore, allowing flow of Na⁺, Ca²⁺, and K⁺. NTD: N-terminal domain; TMD: transmembrane domain. (c) The principle of light-activated glutamate receptor (LiGluR). Reversible opticalswitching of a tethered agonist on the LBD opens and closes the pore. Top right: Structure of a photoswitched agonist. (b) Structure of MAG 4 (which contains a cysteine-reactive maleimide (M), an azobenzene switch (A), and a glutamate head group (G)) in its transstate (dark and 500 nm) and cis state (380 nm). Reproduced from Figures 1 and 2 of Volgraf et al.⁶⁷ Reprinted by permission from Macmillan Publishers Ltd: Nature Chemical Biology, 2:47, copyright 2006.

In the case of azobenzene, one end of the molecule is covalently tethered to the ion channel, and an “active moiety” — eg, an agonist, antagonist, or pore-blocking agent — is attached to the other. Because the thermally relaxed trans isomer is more extended (~0.7 nm longer) than the higher energy cis isomer, light absorption by azobenzene creates a conformational change in the molecule that alters the relationship of its active moiety to the ion channel, which leads to a change in ion movement across the cell membrane. (The active moiety can interact with the ion channel in only one of the isomeric states.)

A genetically and chemically engineered light-gated mammalian ion channel, the light-activated glutamate receptor (LiGluR), has been expressed selectively in retinal ganglion cells of the rd1 mouse.⁸⁰ (These mice have a null mutation in a cyclic GMP phosphodiesterase [PDE6b], a mutation that is present in some patients with retinitis pigmentosa.) In the rd1 mice, the LiGluR restores light sensitivity to the retinalganglion cells, reinstates light responsiveness to the primary visual cortex, and restores both the pupillary reflex and a natural light-avoidance behavior.

Although targeting rod bipolar or photoreceptor cells might permit increased light sensitivity, as well as higher spatial resolution because of signal convergence from these cells onto retinal ganglion cells, these approaches may be compromised by the alterations in synaptic circuitry that accompany photoreceptor degeneration.^58,59,81-83 Thus, there may be value in targeting ganglion cells, particularly if ON and OFF receptive fields can be created.⁷⁴

CONCLUSIONS

Nanotechnology will have an important impact in many areas of medicine, including ophthalmology. The earliest impact is likely to involve drug and gene therapy delivery methods, cell-based therapy and neural prosthetics. Nanotechnology will bring about the development of regenerative medicine (ie, replacement and improvement of cells, tissues and organs), ultrahigh-resolution in vivo imaging, microsensors and feedback devices, and artificial vision. These innovations will have a major impact on the development of sight-preserving and sight-restoring treatments for conditions that currently lead to irreversible blindness. RP

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