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Correspondence: Address to Marco A. Zarbin, MD, PhD, Institute of Ophthalmology and Visual Science, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Room 6155, Doctors Office Center, 90 Bergen St, Newark, NJ 07103.
Herein, we discuss recent applications of nanotechnology to ophthalmology, including nanoparticles for drug, gene, and trophic factor delivery; regenerative medicine (in the areas of optogenetics and optic nerve regeneration); and diagnostics (eg, minimally invasive biometric monitoring). Specific applications for the management of choroidal neovascularization, retinal neovascularization, oxidative damage, optic nerve damage, and retinal degenerative disease are considered. Nanotechnology will play an important role in early- and late-stage interventions in the management of blinding diseases.
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Learning Objectives: On completion of this article, you should be able to (1) name areas in which nanotechnology may have an impact on ophthalmology, (2) name some obstacles to incorporation of nanotechnology into ophthalmic practice, and (3) recognize that nanotechnology may help to usher in the era of regenerative medicine in ophthalmology.
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In their editorial and administrative roles, William L. Lanier, Jr, MD, Terry L. Jopke, Kimberly D. Sankey, and Nicki M. Smith, MPA, have control of the content of this program but have no relevant financial relationship(s) with industry.
Dr. Zarbin is a paid consultant for: Calhoun Vision, Inc, Imagen Biotech, Inc, Iridex, Novartis, and Pfizer.
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Date of Release: 12/01/2013
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Nanotechnology involves the creation and use of materials and devices at the size scale of intracellular structures and molecules. The systems and constructs deployed typically are on the order of less than 100 nm. Novel nanosized materials and chemical assemblies, chip-based technologies, and miniaturized devices already provide novel tools that impinge directly on ophthalmology.
and herein we provide an update on recent innovations concerning specific and potential applications of nanotechnology to ophthalmology, including nanoparticles for drug, gene, and trophic factor delivery; regenerative medicine (in the areas of optogenetics and optic nerve regeneration); and diagnostics, (eg, minimally invasive intraocular pressure [IOP] monitoring).
Nanoparticles for Drug, Gene, and Trophic Factor Delivery
Loss of oxygen or its electrons alters the oxidation state of cerium oxide nanoparticles (“nanoceria”) and creates defects in their lattice structure. As their size decreases, nanoceria (3-5 nm in diameter) exhibit more oxygen vacancies in their crystal structure, which can allow them to function as antioxidants. Chen et al
found that intravitreal injection of nanoceria prevents light-induced photoreceptor damage in rodents, even if injected after the initiation of light damage. Vacancy-engineered nanoceria also inhibit the development of and promote the regression of pathologic retinal neovascularization in the Vldlr knockout mouse, which carries a loss-of-function sequence variation in the very low-density lipoprotein receptor gene and whose phenotype resembles a clinical entity known as retinal angiomatous proliferation.
Regression occurs even if intravitreal nanoceria treatment is administered after the mutant retinal phenotypes are established. A single injection has a prolonged effect (weeks) because nanoceria are a catalytic and a regenerative antioxidant. Cai et al
found that a single intravitreal injection of nanoceria can maintain protective effects on the retina for at least 6 weeks in tubby mice. Tubby mice exhibit rapid photoreceptor degeneration starting on day 14, with undetectable retinal function at 2 months of age secondary to a splice site sequence variation in the Tub gene. Tubby mice also exhibit cochlear degeneration, obesity, insulin resistance, and decreased fertility, a constellation of symptoms resembling Usher syndrome. After day 7, tubby mice were intravitreally injected with 172 ng of nanoceria, and retinal structure and function were preserved and only began to decline after the mice reached day 49. Nanoceria inhibit the development of increased vascular endothelial growth factor (VEGF) levels in this model and also up-regulate the expression of genes associated with antioxidant defense and modulated survival vs apoptosis pathways.
Nonviral gene vectors (eg, polymers and lipids) offer low risk of immunogenicity, relatively low cost, and, possibly, greater ease of production compared with viral vectors. In addition, nonviral DNA nanoparticles can accommodate large genes, unlike traditional viral vectors. For example, although gene replacement is a logical strategy to treat autosomal recessive Stargardt disease, which is associated with sequence variations in the photoreceptor-specific flippase ABCA4, the large size of the ABCA4 complementary DNA (6.8 kbp) has hampered progress in the development of genetic treatment. To circumvent the size limitation, Han et al
used nanoparticles to subretinally deliver ABCA4 to AbCA4-deficient mice. Afterward, they identified persistent ABCA4 transgene expression for up to 8 months after injection and found marked correction of structure and function, such as reduced lipofuscin accumulation and improved dark adaptation. Polyplexes, DNA nanoparticles that are complexes of cationic polymers (eg, pegylated polylysine) and DNA, have also been used to restore retinal function in the rds+/– mouse,
which has sequence variation in peripherin, a photoreceptor-specific glycoprotein that is critical for outer segment disc assembly, outer segment orientation, and photoreceptor structural stability. More than 80 different sequence variations in the RDS gene have been identified in humans, and they are associated with multiple retinal diseases, including autosomal dominant retinitis pigmentosa and progressive macular degeneration. The retina in the rds+/– mouse exhibits a classic, well-defined autosomal dominant retinitis pigmentosa phenotype characterized by early-onset rod degeneration and late-onset cone degeneration.
By choosing promoters that are cell specific, one can establish additional specificity in the locus of gene expression. Thus, one can target gene expression to retinal pigment epithelium (RPE) cells, rods, cones, or rods and cones, depending on which promoter is used. Koirala et al
took advantage of this principle by documenting that a plasmid, with enhanced green fluorescent protein driven by the RPE-specific promoter VMD2, could be incorporated into nanoparticles and subretinally delivered into mice. Afterward, expression was detected only in the RPE, without change in retinal architecture or function. Notably, mice that had the plasmid delivered via nanoparticles exhibited expression throughout the RPE, whereas mice injected with free DNA exhibited expression only at the site of injection. Furthermore, expression was greater in nanoparticle vs free DNA delivery at all time points assessed, consistent with sustained cytosolic plasmid delivery via nanoparticles.
Liposomes are nanoparticles that can carry hydrophobic or hydrophilic cargo. Liposomes can be coated with ligands that direct them to specific cell surface receptors for cell targeting as well as with polymers that prolong their half-life in the circulatory system. Polyethylene glycol (PEG) can be conjugated with different molecules to enhance their solubility and stability in plasma and to reduce immunogenicity. Subretinal PEG-substituted lysine peptide nanoparticles containing DNA do not cause tissue damage or inflammation greater than what is observed in normal saline injections.
created a novel functional liposome consisting of arginine-glycine-aspartate motif peptides conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethyleneglycol)-2000] to enhance the uptake of encapsulated siRNA in RPE cells. These investigators found that arginine-glycine-aspartate–PEGylated liposomes had a 4-fold increase in siRNA delivery to RPE cells compared with PEGylated liposomes.
identified increased receptor-mediated endocytosis uptake of novel folate-decorated PEG-b-polycaprolactone nanoparticles compared with standard nanoparticles. In vitro, the folate-decorated nanoparticles were found to deliver triamcinolone steadily to ARPE-19 cells in tissue culture, which resulted in decreased levels of VEGF and increased levels of pigment epithelium–derived factor (PEDF) over 3 weeks.
Of note, the nanoparticles containing triamcinolone resulted in less cytotoxicity compared with direct administration of triamcinolone alone. Pharmacologic blockade of VEGF plays a critical role in treating choroidal neovascularization, which is the major blinding complication of age-related macular degeneration.
developed an intracellular antiangiogenic therapy relying on a 3-component system: (1) plasmids expressing Flt23k intraceptors that consist of the VEGF-binding domains 2 and 3 of Flt (the highest-affinity VEGF receptor), (2) poly(lactic-co-glyocolic acid) biodegradable nanoparticles as a delivery system, and (3) the tripeptide adhesion motif arginine-glycine-aspartate to coat nanoparticles and facilitate selective homing to choroidal new vessels (CNVs) after systemic intravenous injection (Figure 1). They previously found that these antiangiogenic nanoparticles inhibit laser-induced CNVs in rats.
used an argon laser to induce CNVs bilaterally in mice on day 0. On day 10, either verteporfin (Visudyne, Valeant Pharmaceuticals International, Inc.) or verteporfin loaded in cationic liposomes was injected intravenously. For each mouse, one eye was treated with photodynamic therapy and the other served as a control. The specimens were examined histologically 1 week later for response to treatment. Liposome-loaded verteporfin was as efficacious as verteporfin alone in treating neovascularization but resulted in less tissue damage, possibly secondary to cationic liposomes' affinity for active angiogenesis.
Intravitreal injection of nanoparticles containing PEDF into Royal College of Surgeons rats resulted in greater preservation of opsin and photoreceptors with reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling–positive cells over 8 weeks compared with injection of PEDF alone.
found that intravitreal glial cell line–derived neurotrophic factor (GDNF)–loaded biodegradable poly(lactic-co-glycolic acid) microspheres provide sustained retinal ganglion cell (RGC) protection in a rodent model of glaucoma. Microspheres (approximately 8 μm in diameter) containing GDNF were fabricated using a modification of a spontaneous emulsion technique. Moreover, the use of a novel solid-in-oil-in-water emulsion solvent evaporation technique with incorporation of vitamin E into the microspheres has a protective effect on GDNF. Checa-Casalengua et al
found that this revised formulation allowed for release of active GDNF for up to 3 months and improved survival of photoreceptors and RGCs in vitro. Intravitreal injection of these microspheres in a mouse model of glaucoma resulted in improved RGC survival compared with GDNF, vitamin E, or blank microspheres, an effect that was present for up to 11 weeks after injection.
The ability of nanoparticles to deliver siRNA over prolonged intervals and selectively to tissues may improve treatment efficacy and specificity. Du et al
found, for example, that in vivo knockdown of EphB4 (erythropoietin-producing hepatocellular carcinoma receptor B4) levels by short hairpin RNA (shRNA) delivered by a lentiviral system decreased CNVs. Laser-induced CNVs in C57BL/6 mice were treated with phosphate-buffered saline, EphB4 shRNA, or lentiviral empty vector. Fluorescein angiography and histologic analysis revealed that down-regulation of EphB4 resulted in decreased areas of leakage and CNV mean thickness compared with phosphate-buffered saline and empty vector alone.
Albumin-derived nanoparticles that deliver plasmids containing genes for the Flt receptor (VEGFR1), which binds free VEGF, penetrate keratocyte cytoplasm and provide sustained inhibition of injury-induced corneal neovascularization.
Flt1 can be alternatively spliced to produce soluble FLT1, the soluble VEGF receptor 1 that lacks the transmembrane and intracellular kinase domains and, hence, acts as a soluble sink for free VEGF. Owen et al
took advantage of this phenomenon and designed morpholinos directed against splice site targets in FLT1, the hypothesis being that this approach could result in increased levels of soluble FLT1 as opposed to membrane-bound FLT1, thereby decreasing VEGF signaling. As predicted, intravitreal injections of the FLT1 morpholinos increased the soluble to membrane-bound FLT1 ratio 5-fold and decreased laser-induced CNVs by 50%. Similarly, Lai et al
delivered solulable FLT1 via recombinant adeno-associated virus subretinally in a mouse model of retinal neovascularization and found inhibition of abnormal blood vessel growth. Flt23k is an anti-VEGF intraceptor similar to the soluble VEGF binding domain of soluble FLT1. Cho et al
found that nanoparticle delivery of FLT23K-expressing plasmids improved the corneal transplant survival rate and decreased corneal neovascularization in mice. This rescue was enhanced by the addition of topical triamcinolone. Instead of posttranslationally inhibiting VEGF, Qazi et al
created nanoparticles loaded with plasmids expressing shRNA against VEGF-A to decrease the signal pretranslationally. They found that nanoparticles loaded with VEGF-A shRNA reduced corneal neovascularization (as per the CD31 marker) twice as well as solitary plasmid treatment in mice after mechanical alkali injury.
Nanoparticle delivery systems that deliver relevant shRNAs might serve as useful treatment for retinal degenerative disease, diabetic retinopathy, or glaucoma. Regarding retinal degenerative disease, production of the photoreceptor determinant Nrl transcription factor results in rod development, whereas the absence of Nrl causes cones to develop from photoreceptor precursors. Retinitis pigmentosa results in early rod photoreceptor death and loss of vision. Montana et al
found that knocking down Nrl expression partially reprograms rods to adopt the molecular, morphologic, and functional characteristics of cones. Reprogramming rods into cones was sufficient to reduce photoreceptor cell death in the rho–/– mouse model of retinitis pigmentosa. This work suggests that future knockdown of Nrl with shRNA may serve as a useful therapeutic approach to some types of retinitis pigmentosa. Regarding diabetic retinopathy, severe retinopathy can be associated with retinal neovascularization. Connective tissue growth factor is produced by retinal Muller cells in diabetic rats and results in increased deposition of extracellular matrix (ECM) and angiogenesis. Intravitreal injection of shRNA against connective tissue growth factor decreases ECM production, suggesting that this growth factor may be another potential future target of shRNA clinically.
In contrast to the currently available retinal prosthesis, optogenetics has the potential for minimally invasive neuronal stimulation with high spatial resolution. In principle, for example, one can convert a large fraction of the approximately 1 million RGCs into photosensitive neurons vs the number one might stimulate with a 60- or 200-electrode array. Optogenetics may play an important role in ophthalmic regenerative medicine.
Channelrhodopsin-2 (ChR2) and halorhodopsin (HaloR) are naturally occurring light-activated ion channels
In preclinical models, the kinetics of ChR2- and HaloR-mediated light responses is compatible with the retina's temporal information-processing requirements. However, ChR2 and HaloR exhibit low light sensitivity, with threshold activation light intensities approximately 5 to 6 log units higher than those of cones. Furthermore, the light intensity operating range of ChR2 and HaloR is 2 to 3 log units, in contrast to the normal retinal dynamic range of 10 log units. Low light sensitivity may hinder the use of ChR2 and HaloR clinically. Recently, a new ChR2 variant, the calcium translocation ChR, has been identified that has an accelerated response time and a voltage response that is 70-fold more light sensitive than that of ChR2.
Numerous other “next-generation” ChRs have been engineered via site-directed mutagenesis and provide improved parameters for experimental and clinical approaches, and these advances have been well summarized by Lin.
In these mice, the light-activated glutamate receptor restores light sensitivity to the RGCs, reinstates light responsiveness to the primary visual cortex, and restores the pupillary light reflex and natural light-avoidance behavior. Optogenetic recovery of vision also can be achieved with nonviral approaches, such as intravitreal injection of acrylamide-azobenzene-quaternary ammonium (AAQ). The AAQ acts as a synthetic K+ channel photoswitch. Polosukhina et al
reported that intravitreal injection of AAQ restored the pupillary light reflex and light-avoidance behavior in mice lacking retinal photoreceptors by interacting with multiple types of cells in the retina, primarily RGCs (Figure 3). This therapeutic modality does not require exogenous gene delivery or manipulation, suggesting limited hurdles in delivery to patients. This approach is reversible (vs viral therapy), repeatable, and apparently nontoxic.
Biomaterials and Regenerative Medicine
Retinal progenitor cells can be delivered to the eye, migrate to correct regions in the retina, and differentiate into photoreceptors with appropriate markers and morphologic features. It is essential, however, that these photoreceptors can transmit a signal centrally for vision perception. Pearson et al
used Gnat1–/– mice, which lack rod function, to address this issue. They found that transplanted rod precursors could form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina, which were then able to transmit visual signals to higher-order visual processing centers in the brain and induce meaningful optokinetic head tracking and visually guided behavior. Similarly, de Lima et al
found that a combination of methods that synergistically activate RGCs' intrinsic growth state enables these cells to regenerate axons over the entire length of the optic nerve, across the optic chiasm, and into the brain in mature mice that have undergone optic nerve crush injuries. Axons innervated the dorsal lateral geniculate nucleus, the superior colliculus, and other visual target areas, leading to partial recovery of depth perception, the optomotor response, and circadian photoentrainment. In addition, olfactory stem cells can rescue the optic nerve in an animal model of glaucoma.
Nanofabrication techniques and judicious use of various biomaterials will probably play an important role in operationalizing cell-based therapy for regenerative ophthalmic medicine.
Poly(ε-caprolactone) (PCL) is biodegradable, is biocompatible, can be spin-cast to a thin film (5 μm) with controlled microtopography (that favors cell adherence), and promotes the differentiation of retinal progenitor cells. Additionally, these scaffolds can support the adhesion, proliferation, and differentiation of mouse retinal progenitor cells in vitro and migration into mouse retinal explants.
found that PCL scaffolds could be implanted subretinally in porcine eyes and were well tolerated, without signs of tissue disruption or inflammation. Furthermore, this study revealed that PCL short nanowires preserved the overlying outer retina better than PCL electrospun and PCL smooth scaffolds and exhibited the most suitable degree of stiffness for surgical delivery. By sealing protein between two layers of PCL film, Bernards et al
found that PCL scaffolds can act as a reservoir that steadily releases active protein for up to 70 days. This function may be used in the future as an effective means to deliver biological therapies to cells in the scaffold.
Three-dimensional hyaluronic acid–based hydrogels with encapsulated retinal progenitor cells have been delivered to the subretinal space without perturbing retinal architecture and have resorbed, allowing retinal progenitor cells to differentiate and to express the mature photoreceptor marker recoverin within 3 weeks of implantation.
In addition to providing structural and nutrient support, scaffolds and nanoparticles have been shown to secrete a variety of agents that promote survival and integration of retinal precursors, notably chondroitinase ABC,
Although generation of RGCs from embryonic and induced pluripotent stem cells is in an early stage of development, one can envision combining cell-based therapy with composite nanofabricated cell conduits or hydrogel matrices (which not only provide signals to cells but also provide structural support to encapsulated cells and which eventually can be metabolized into nutrients) to provide RGC regenerative therapy for various optic neuropathies, including glaucoma.
Chitosan is a nontoxic polysaccharide with mucoadhesive properties that can increase drug delivery to human corneal epithelium
devised a biodegradable scaffold composed of a 75:25 ratio of chitosan to polycaprolactone that had the mechanical and biocompatibility properties that allowed for growth, differentiation, and production of tight junctions of corneal epithelial cells. With further optimization, such a system may be able to create corneal grafts for transplantation.
Some ways in which nanotechnology has improved diagnostic imaging have been reviewed previously.
demonstrated that cationic (but not neutral) liposomes labeled with indocyanine green could, with high affinity, stain active CNV lesions in C57BL/6 mice when viewed through a scanning laser ophthalmoscope.
Microelectromechanical and nanoelectromechanical systems engineering have been used to manufacture IOP monitors. The IOP exhibits diurnal and instantaneous fluctuations. Nocturnal IOP measurement may be more critical to glaucoma management than the typical daytime measurement in an outpatient setting.
developed a disposable highly oxygen-permeable silicone soft contact lens with an embedded sensor (Triggerfish; Sensimed AG). Microfabricated strain gauges (170-nm platinum, 25-nm titanium) are embedded in the contact lens and measure circumferential changes in the area of the corneoscleral junction that can reflect changes in IOP (Figure 4). Wireless powering and communication between the contact lens and the recording unit are achieved with a microprocessor and a 9-mm mean diameter, 8-μm-thick gold loop antenna integrated into the lens. Wireless power and data transfer occur via a patched periorbital antenna from which a cable is connected to a portable recorder, which is worn around the patient's waist. The silicone contact lens has a diameter of 14.1 mm and central and peripheral thicknesses of 585 and 260 μm, respectively. Three hundred data points are acquired during a 30-second measurement period, which is repeated every 5 minutes.
Variations in central corneal thickness and rigidity and, possibly, motion artifacts may limit the utility of this approach. The contact lens sensor can essentially provide continuous IOP monitoring so that the patient does not need to be awakened from sleep to monitor IOP.
Barriers to Clinical Application
Several obstacles to the incorporation of nanotechnology into medicine are recognized.
Safe bionanomanufacturing techniques also must be identified. This issue is particularly relevant when scaling up production for commercial distribution of products. Clean room processes similar to those used for semiconductor device manufacture may be needed in some cases. Although nanotechnology permits precise targeting of therapeutic modalities and minimizes the doses of drugs delivered in vivo, nanomaterial toxicity may still occur. Test protocols to assess nanomaterial safety have been developed.
Nanomedicine has already had an impact in the areas of biopharmaceuticals (eg, glaucoma drugs and neurotrophic factors), implantable materials (eg, tissue regeneration scaffolds), and diagnostic tools (eg, IOP monitors) in ophthalmology. The examples provided herein demonstrate that nanotechnology will play an important role in early- and late-stage interventions in the management of blinding diseases. Nanotechnology already has been applied to the measurement and treatment of different disease states in ophthalmology, including choroidal neovascularization, retinal neovascularization, oxidative damage, optic nerve damage, and retinal degenerative disease. During the next few years, as these discoveries transition from laboratory experiments to clinical practice, they are likely to have a major effect on the development of sight-preserving and sight-restoring treatments for conditions that currently are among the major causes of blindness.
Nanomedicine-nanoparticles, molecular biosensors and targeted gene/drug delivery for combined single-cell diagnostics and therapeutics.
Grant Support: This study was supported, in part, by Research to Prevent Blindness Inc (M.A.Z., R.R.), the Joseph DiSepio AMD Research Fund (M.A.Z.), and the HRH Prince Khalid bin Abdulla al Saud Research Fund of the New York Eye and Ear Infirmary (R.R.).
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